The strain of bovine herpes virus type 1 (ВНv-1) Сncm no. 1-1213, vaccine, diagnostic serological reagent, a method of vaccination, the way serological differences

 

(57) Abstract:

The invention relates to the field of vaccination and diagnosis of diseases K. R. S. , caused by pathogenic bacteria, in particular to a live weakened and inaktivirovannye vaccines to protect animals against herpes virus type 1. The vaccine is created on the basis of mutant herpes virus BHV-I, having a deletion in de gene glikoproteid, with the gene having the appropriate location in the genome BHV-I, and this deletion allows the mutant to differ serologically from BHV-I wild type. The invention also concerns a diagnostic kit for detecting nucleic acid BHV-I antibodies specific for BHV-I, protein BHV-I and method for determining infection with BHV-I in animals. Developed preparations and method are characterized by high efficiency and safety. 5 C. p. f-crystals, 24 ill. table 2.

The scope of the invention

The invention relates to the fields of vaccination and diagnosis of diseases, which are caused by pathogenic bacteria, and includes the use of classical methods of obtaining a live attenuated vaccine or inactivated vaccines and modern methods based on recombinant DNA technology.

More Conques is particularly cattle, against herpes virus type 1 in cows /BHV-1/, and these vaccines are that they are not only safe and effective, but also provide an opportunity to distinguish infected from non-infected animals in a vaccinated population.

Diagnostic systems and procedures that can be used for such a test to distinguish infected and non-infected animals in a vaccinated population, are also an aspect of the present invention.

Background of the invention

BHV-1, including infectious rhinotracheitis virus /IBPV/ infectious pustular vulvovaginitis virus /IPVV/, plays an important role in the development of respiratory diseases and fertility in cows. After acute infection BH-1 often remains with the carrier in a latent form. Latent virus may again be activated under stress - which may be accompanied by clinical manifestations, and then excreted from the body. As a consequence, infected cattle can be considered as a potential carrier BHV-1. BHV-1 often becomes endemic in approximately 75% of Dutch farms. Especially serologically positive to him old animals.

There are a number of inactivated /"killed"/ vaccines and many of the m virus BHV-1, for example, heat treatment, irradiation or treatment with ethanol or formalin. However, this does not always provide sufficient protection. Attenuated vaccine prepared a large number of subcultures on the homology /cow/ or heterologous cells, such as cells of a pig or dog, and then sometimes viruses are also processed physically or chemically. Thus, the viral genome evolve unknown mutations/deletions, which often reduce the properties of the virus that cause disease. Attenuated live vaccines provide better protection than inactivated vaccines, because they give more viral antigens to the immune system of the host. Another important advantage of live vaccines is that they can be assigned through the nose, i.e., at the site where the first replication of the wild-type virus after infection. And still live vaccines need to be improved. Some live vaccines still have abortogenic ability, which manifests itself especially after intramuscular destination. In addition, probably all live vaccines are present in vaccinated cow in a latent state. And there is a possibility that if the vaccine is not very different from the wild-type virus, possible vozmu virus wild type. In the vaccinated cattle can also post BHV-1 wild-type.

For a good control program BHV-1 must have an effective and safe vaccine that can be distinguished from wild-type virus, as the application of an effective vaccine can significantly reduce the circulation of BHV-1, and a test that can distinguish between vaccine and wild-type virus, makes it possible to detect /and then the/ remove the infected cattle in vaccinated populations.

Were developed by BHV-1 vaccines, which are represented as safer than traditional vaccines, and different from the wild-type virus. Was isolated a mutant with a deletion of timedancing, which has a lower aboriginally becomes latent and cannot reaccelerates. In addition, using techniques of recombinant DNA was created BHV-1 vaccine, which had a deletion in the gene for glycoprotein gIII, making this vaccine is featured from BHV-1 wild type by serological techniques. However, there are still objections to these vaccines. On the one hand, gene timedancing involved in viral replication and reducing replication leads to a decrease in protection. On the other hand, glycoprotein gIII important refers to the fact that that intranasal introduction, which gives the best protection in the case of recombinant vaccines is not allowed in some countries. Accordingly, there is a need to create a vaccine that would be as safe, as effective, and, in addition, distinguishable from the BHV-1 wild type, yet it is also desirable that at least one of these vaccines was based on the virus, weakened by using traditional techniques, rather than the virus, created by the technology of recombinant DNA.

Now, with the help of subcultures in cell cultures was obtained strain of BHV-1, which lacks the gene for glycoprotein gE. The first results of our research show that this gene is very useful in serological differentiation with BHV-1 wild type and that it is involved in the expression of virulence. Therefore its deletion promotes security and can make use of the dystrophin timedancing unnecessary. Glycoprotein gE is less important for the induction of protection than glycoprotein gIII. Weakened the traditional way the strain of BHV-1, which can be serologically distinguished from the wild-type virus, is unique. The location and DNA sequence of the gene gE, described here for the first time, were not previously known. Were not also the case distinction on the basis of the gE gene is also unique.

An important advantage of this "traditional" mutant with a deletion of the gE /"traditional" is the traditional method for isolation of attenuated virus/ lies in the fact that his intranasal purpose permitted in the countries where it is prohibited in respect of recombinant vaccines. However, given the different views on security, in addition to this traditional vaccine with a deletion of the gE were also created specific recombinant variants. These recombinant vaccines also had a division of gE and may or may not have a deletion of the gene timedancing, and can also be used as vectors for the expression of heterologous vectors. All these recombinant vaccines can be distinguished from wild-type virus by using the same gE-specific test. The use of a standard test for a number of different vaccines may be a great advantage in the fight against BHV-1 on an international scale. This approach has not been previously described in the field of BHV-1 vaccines.

Serological analysis of the response to BHV-1 in cattle showed that an important fraction of anti-gE antibodies directed against the complex formed by the glycoprotein gE and other glycoprotein BHV-1 glycoprotein gI. Therefore, serological tests that can /also/ demon is only to detect anti-gE antibodies. Cattle vaccinated with a single mutant with a deletion of the gE can develop anti-gI antibodies, which may interfere with detection of anti-gI/gE antibodies. Therefore, this invention also includes a vaccine with double deletion gI/gE.

The content of the invention

First, this invention provides a mutant with a deletion of the BHV-1, which has a deletion in the gE glycoprotein gene. The word "division" includes the deletion of the gene as a whole.

A preferred variant of the invention involves the mutant BHV-1 with a deletion, which has a deletion in the gE glycoprotein gene, which is caused by the procedure of weakening, such as a mutant with a deletion of the Difivac-1, described below.

Other preferred variants of the invention include mutant with a deletion of the BHV-1, comprising a deletion in the gE glycoprotein gene, which was created by recombinant DNA technology, such as mutants with a deletion of the IB7 or IB8, described below.

Another preferred variant of the invention consists of the mutant BHV-1 double deletion, including a deletion in the gE glycoprotein gene and a deletion in the gI-glycoprotein gene, such as a mutant with a double deletion gI/gE Difivac-1E, described below.

In addition, considering the maximum security, under Shane timedancing. The invention also includes a mutant with a deletion of the BHV-1, which has a deletion in the gE gene for the glycoprotein gI-glycoprotein gene, and the gene timedancing.

The invention provides a vaccine composition for vaccinating animals, in particular mammals, more specifically cows to protect them against BHV-1, including a mutant with a deletion of the BHV-1, as described above, and a suitable carrier or stimulator. This composition may be a composition of live or inactivated vaccines.

Further, the invention provides mutant BHV-1, which has a deletion in the gE glycoprotein gene and contains a heterologous gene, introduced by recombinant DNA technology. Preferably, this relates to mutant BHV-1, which contains a heterologous gene, introduced by recombinant DNA technology, the location of the gE glycoprotein gene, and the heterologous gene is under the control of regulatory sequences of the gE gene and may be associated with a part of the gE gene, which encodes the signal peptide. Such heterologous gene may also be under the control of another promoter BHV-1, or under the control of a heterologous promoter. If the mutant BHV-1 has other deletions in addition to deletions in the gE glycoprotein gene, such as the deletion of the location of this additional deletions /th/. Multiple insertions - another opportunity or place in one location deletions, or distributed over several locations deletions.

Introduced heterologous gene preferably encodes for immunogenic protein or peptide of another pathogen or cytokine, which stimulates an immune response. Examples of suitable cytokines are interleukin 2, interferon-alpha and interferon-gamma.

The invention also provides /live or inactivated/ composition of the vaccine for the vaccination of animals, in particular mammals, more specifically cows to protect them against /of various/ of pathogenic bacteria, including mutant BHV-1, which has in itself the encoding heterologous gene for immunogenic protein or peptide that other pathogen, and a suitable carrier stimulator. Of course, the defence may apply to more than one pathogen, i.e. multivalent vaccine, in which the mutant contains a variety of heterologous genes.

Further, the invention relates to compositions comprising a recombinant nucleic acid containing gE-glycoprotein gene BHV-1, part of this gE-glycoprotein gene or nucleotide sequence, derived from the success of recombinant nucleic acids, which includes gE-glycoprotein gene BHV-1, part of this gE-glycoprotein gene or nucleotide sequence, derived from this gE-glycoprotein gene.

The invention also includes a composition comprising a glycoprotein gE BHV-1, part of this glycoprotein gE, a peptide derived from this glycoprotein gE, or a complex of glycoproteins gE and gI, and a composition comprising an antibody that is specific for glycoprotein gE BHV-1, the parts of the glycoprotein gE, peptide, derived from this glycoprotein gE, or complex of glycoproteins gE and gI. "Antibody" means a preparation of polyclonal antibodies and monoclonal antibodies preferred for most applications. The terms "part of the glycoprotein gE" and "peptide derived from the glycoprotein gE" is a designation sequence gE-specific amino acids, which typically have a length of at least about 8 amino acids.

Further, the invention relates to diagnostic systems and procedures to detect nucleic acid BHV-1 in the sample, biological sample, such as blood or serum, blood cells, milk, body fluids such as tears, the washing fluid of the lungs, nasal fluid, semen, capitalsho, specifically cows, comprising a probe or primer nucleic acid having a nucleotide sequence obtained from gE-glycoprotein gene BHV-1, and detection means suitable for the reaction to the detection of nucleic acids.

Further, the invention relates to diagnostic systems and procedures to detect antibodies that are specific for BHV-1 in a sample, particularly a biological sample, such as blood or serum, saliva, sputum, body fluids such as tears, the washing fluid of the lungs, nasal fluid, milk, or tissue of an animal, specifically cows, including glycoprotein gE BHV-1, part of this glycoprotein gE, a peptide derived from this glycoprotein gE, or a complex of glycoproteins gE and gI, and detection means suitable for the reaction to the detection antibodies. Such diagnostic procedures may also contain one or more antibodies that are specific for the glycoprotein gE BHV-1 or specific for a complex of glycoproteins gE and gI BHV-1.

The invention also relates to diagnostic procedures for the detection of protein BHV-1 in a sample, particularly a biological sample, such as blood or serum, blood cells, milk, body fluids Takata or cloth in particular, neural tissue, animal, mammal, specifically cows, comprising one or more antibodies that are specific for the glycoprotein gE BHV-1 or specific for a complex of glycoproteins gE and gI BHV-1, and detection means suitable for analysis for the detection of protein.

The invention further provides a method for determining contamination of BHV-1 animal, particularly a mammal, specifically cows, including a sample from the animal, in particular a biological sample such as blood or blood serum, blood cells, sperm, in particular the seminal fluid, saliva, body fluids such as tears, the washing fluid of the lungs, nasal fluid, milk, or tissue, in particular the nervous tissue, the presence of a nucleic acid containing gE-glycoprotein gene BHV-1, or the presence of the glycoprotein gE BHV-1, or antibodies, which are specific for the glycoprotein gE BHV-1 or specific for a complex of glycoproteins gE and gI BHV-1. The sample may be taken from an animal that has not previously been vacciniavirus the vaccine composition according to the invention or the animal, which was previously vacciniavirus the preparation of a vaccine according to the invention.

Detailed description of the invention

These materials are in accordance with the invention can be used for:

1/ vaccination of cattle against diseases caused by BHV-1, so that it is possible to distinguish infected with BHV-1 animals and vaccinated animals, along with the traditional you can use the generated vaccine;

2/ vaccination of cattle against diseases BHV-1, and diseases caused by other pathogenic bacteria, coding sequences for the protective antigen is La cattle on serological determination or by the technique of detection of nucleic acid /for example, PCR (PCR - polymerase chain reaction)/, animals were infected with BHV-1 wild type or were vaccinated with mutant with a deletion of the gE.

Synthesis of oligopeptides, polypeptides and glycoproteins derived from the coding sequence of the glycoprotein gE-gene and the glycoprotein gI gene of BHV-1.

The results of the analysis of DNA sequences described in the examples, the glycoprotein gE-gene /Fig. 3A/ and isolated DNA fragments that encode for this gene, making it possible, using standard molecular biological procedures, how to synthesize peptides gE protein /oligo - or polypeptides/ and to Express gE protein is completely or large parts through the prokaryotic path /in bacteria or a eukaryotic path /for example, in murine cells/. Through these ways you can get a gE-specific antigen, which may, for example, be used to develop a gE-specific monoclonal antibodies /MAT/. In addition, gE-specific antigen /and gE-specific MAT/ can be used in serological tests to make a distinction between animals vaccinated with vaccine BHV-1 deletions gE and animals infected with a virus BHV-1 wild-type.

The results of the partial sequence analysis DNA glycoprotein aritime cells, expressing the glycoprotein gE, allow the expression of gI/gE complex in eukaryotic cells /Cm. Fig. 13 and 14/. This glycoprotein complex can be used to obtain gI/gE-specific monoclonal antibodies. gI/gE complex can also be used as antigen in serological assays for differentiation between animals vaccinated with one gE BHV-1 mutant with a deletion or double gI/gE BHV-1 mutant with a deletion, and animals infected with a virus BHV-1 wild-type.

gE-specific peptides

Based on the known coding sequence of the protein using an automatic synthesizer can be obtained polypeptides not less than 40-50 amino acids. Now, when identified, the coding sequence of the protein gE glycoprotein strain Lam BHV-1 /Fig. 3A/, it is possible to synthesize polypeptides that BHV-1 gE glycoprotein. Such polypeptides by standard methods it is possible to immunize experimental animals such as mice or rabbits, to develop a gE-specific antibodies. In addition, using this gE-specific peptides, can also determine the location where the anti-gE antibodies react with gE proteins /epitopes/, for example, by way PEPSCAN /Geysen and others , 1984, Proc. Natl, Acad. Sci., USA 81, 3998-4002/. gE-specificity is A.

Prokaryotic expression gE

For the synthesis of gE protein in bacteria /ie prokaryotic expression gE/, fragments of DNA that encode for glycoprotein gE or its parts must be cloned into vectors for prokaryotic expression. The vectors for prokaryotic expression are circular DNA molecules that can keep itself in bacteria as a separate reproducing molecule /plasmid/. These expression vectors contain one or more marker genes that encode for resistance to the antibiotic, and thus allow selection for bacteria with the expression vector. In addition, the expression vectors include /often controlled/ region promoter, for which DNA fragments can be ligitamate and which can then be expressed under the influence of the promoter. In many of these vectors for prokaryotic expression of the desired protein is expressed in the state of Mergence with the so-called protein carrier. In the vector for promoter is the coding sequence for a protein carrier, directly to where you can ligitamate the desired DNA fragment. Proteins mergers are often more stable and/or easier to detect and/or isolate. The level of stability condition, which can be defined is s combination.

Eukaryotic expression of the glycoprotein gE-gene

Although the prokaryotic protein expression offers some advantages, proteins do not have modifications, such as glycosylase and others that take place in eukaryotic cells. As a result of this eukaryotically expressed protein is often more stable antigen. For heterologous expression of proteins in eukaryotic cells, such as mouse cells, use vectors eukaryotic expression. These vectors are plasmids, which are not only able to multiply in the cells of E. coli, but also stably exist in eukaryotic cells. In addition to prokaryotic selective marker they also include eukaryotic selective marker. Similarly, the vectors prokaryotic expression vectors for eukaryotic expression contains a plot of the promoter, which can be ligitamate desired genes. However, the sequence of the promoter in eukaryotic vectors specific for eukaryotic cells. Moreover, in eukaryotic vectors fusion protein-carriers is used very rarely. These vectors are introduced into eukaryotic cells by a standard method transfection /Graham and A. I. van der Eb, 1973, Virology, 52, 456-467/. In addition to eukaryotic plasmid vectors the dog and cow pox/. Therefore, eukaryotic cells can be infected with recombinant viruses.

In General, it is impossible to predict what is the vector and the cell type most appropriate for a particular gene product. For the most part try several combinations.

Eukaryotic expression and glycoprotein gE and glycoprotein gI

The final structure, which reaches a protein depends on the sequence of the amino acids, its styling, its post-translational modifications, etc. is an Important factor, which determines the structure of a protein is its relationship with one or more other proteins. We showed that BHV-1 glycoprotein gE forms a complex with at least one other glycoprotein: BHV-1 glycoprotein gI. The first indication of such a complex emerged from our results with the candidate anti-gE MAT 1, 51, 67, 75 and 78 /Cm. table 2/. These MABS did not react with any Difivac-1, nor with Lamg E-and did not recognize the glycoprotein gE-expressing ZTZ cells. However, these MABS did not react with gE-expressing ZTZ cells after infection Difivac-1, showing that the necessary complementary factors to give the glycoprotein gE desired antigenic conformation for these MATE. In some of our experiments on radio-immunoplate herpes simplex forms a complex with the protein of comparable molecular weight /HSVI glycoprotein gI/, we concluded that BH-1 glycoprotein gE forms a complex with BHV-1 homolog of glycoprotein gI. To explore this BHV-1 gE/gI complex and get the gE antigen with antigenic structure, we expressed both glycoprotein in a single eukaryotic cell. For this purpose we applied the same procedure as described for eukaryotic expression of the same glycoprotein gE. The only additional condition was the use of expression vectors with different eukaryotic breeding markers.

Serological reactions

Serological methods to distinguish between cattle vaccinated Defivac-1 of cattle infected with BH-1 wild-type on the basis of antibodies against gE, preferably based on the use of monoclonal antibodies directed against gE.

They are used as follows:

a/ In accordance with the principle described by Van Oirschot, etc. /Jorn. of Virological Methods, 22, 191-206, 1988/. In this ELISA (ELISA - blocking enzyme-immunosorbent test) for the detection of gI antibodies against the virus of the disease Aujeszky's, antibodies appear to its blocking action on the reaction of the two MAT having two different epitope on the gI. Test or the reaction is carried out as follows. Microtiter tablets covered with a MAT 1 in techtubemedia antigen in a separate uncoated microtiter tablets, for example, 2 hours at 37oC. the Tablets covered with a MAT-1, washed, say, 5 times, after which these tablets are added MAT 2 associated with the horseradish peroxidase /HRPO/. Then preincubation mixture of serum-antigen is transferred to the plates, which are two MAT, followed by incubation, for example, for 1 hour at 37oC. the Tablets are washed and substrate is added to each well. After approximately 2 hours at room temperature tablets is read spectrophotometrically. Each tablet contains the four negative control sera and four serial dilution of positive sera. Serum optical density /OP/ less than 50% of the average OD 4 negative control sera, which were studied on the same tablet, is considered positive.

b/ In accordance with the principle of sandwich indirect double antibody /IDAS/. Here microtiter tablets covered with a MAT or a polyclonal serum directed against gE protein. Incubation of the preparation of the gE antigen leads to the fact that gE is associated with the coating. Antibodies specifically directed against gE in the studied bovine serum, consistently contact CTE covalently bound to the enzyme peroxidase. Finally, the bound conjugate is visible when adding a chromogenic substrate. The specificity of the reaction is checked by carrying out the same procedure with gE-negative control drug instead of the drug of the gE antigen. On each microtiter tablet has a positive and negative control sera. The test is considered valid if positive sera give a positive result in a dilution. The serum is positive if the OD is higher by 0.2 than the standard negative control sera.

c/ In accordance with the principle of IDAS described under 2, but after incubation of the test serum instead conjugate anti-bovine immunoglobulin used anti-gE MAT/HPPO. You can use the serum anti-gE peptide or anti-gE polyclonal serum instead of anti-gE MAT. Tablets are washed to each well is added chromogenic substrate. After approximately 2 hours at room temperature tablets is read spectrophotometrically. Each tablet includes four negative control sera and four serially diluted positive serum. Serum value OP less than 50% of the value of OP 4 negative control sera, which is the dominant enzyme immunosorbent assay /ELISA/, by which the virus antigen, which can be purified or unpurified, is applied by coating on microtiter plate overnight. These tablets analyzed serum is incubated for approximately one hour or longer at 37oC. After the washing procedure for tablets added anti-gE MAT followed by incubation for approximately 1 hour at 37oC. Instead of anti-gE MAT, you can use the serum anti-gE peptide or anti-gE polyclonal serum. Tablets are washed to each well is added chromogenic substrate. After about 2 hours at room temperature tablets is read spectrophotometrically. Each tablet contains the four negative control sera and four serially diluted positive serum. Serum value OP less than 50% of the average OD 4xnegative control sera, which were studied on the same tablet, is considered positive.

All the above procedures can be used traditional cultured virus antigen, which contains gE, and gE-antigen, which is expressed in prokaryotes or eukaryotes. Alternatively, instead of the traditional antigen in these diagnostic samples should be used to develop the so-called "mad cow" test in accordance with the principle described in article Kemp and others, Science, 241, 1352-1354, 1988. This test will be based on communication antigenic sequence of oligopeptides antibodies directed against gE present in infected animals. For such a test oligopeptides should be associated with MABS directed against red blood cells of cows.

Analysis of nucleic acid using the polymerase chain reaction

Oligonucleotides /probes and primers can for example be used in the polymerase chain reaction to distinguish between vaccinated and infected animals. Polymerase chain reaction /PCR/ - technological method by which a nucleic acid of a pathogen in a short time be multiplied billions of times /De polymesase kettingreactie. P. F. Hilderiuk, J. A. Wagenaar, J. W. B. van der Giessen u B. A. M. van der Jeijst, 1990, Fijdschrift voor Dierdeneeskunde deel 115, 1111-1117/. gE oligonucleotides can be chosen in such a way that gE positive genome produces a different product than in gE negative genome. The advantage of this is that an animal that has been vaccinated with a vaccine with a deletion of the gE, gives a positive signal in the test for PCR. However, this approach depends on the presence of nucleic acids of the virus in the sample, for example, blood from the animal, which Provadiya acid can be demonstrated in the blood, but still undecided, will also be demonstrated in the blood of nucleic acid BHV-1 during the latent period.

Use V-1 as a vector

For the expression of heterologous genes in BHV-1 genome is necessary to have accurate information about the plot, which must be inserted heterologous gene. The main sequence should not disrupt the structure, and for the expression of heterologous gene should be a regulatory sequence. The glycoprotein gE-gene is a suitable place for the expression of heterologous genes. gE gene is not essential, therefore, cannot be objected to for the replacement of the gE gene is a heterologous gene. As a consequence, the heterologous gene may be that he will be influenced by the regulatory sequences of the gE gene. However, there is no need to use the regulatory sequence of the gE gene. Expression of heterologous genes can be controlled by others, i.e., a stronger regulatory sequences of different genes. It is also possible to ligitamate heterologous gene to /export/ signal peptide gE gene in order to affect the secretion product of a heterologous gene. Airways way. Developed vectors can be serologically distinguished from the wild type. Creating V-1 mutants, which excrescent heterologous genes can be implemented in the same way as creating mutants with a deletion of the gE shown in the examples. But then fragments deletions should be replaced by fragment, on which the heterologous gene is located at the location of the deletion.

Examples

1/ the Isolation and identification of natural mutant with a deletion of the gE.

a/ Insulation natural /natural/ mutant

Genomic DNA was isolated from several weakened by traditional vaccines in accordance with standard methods and analyzed using restriction enzymes. In particular, we were looking for deviations of the genome that would come to distinguish it from virus BHV-1 wild-type.

The weakening was aimed, in particular, for Usthe region of the genome BHV-1, because in this phase by analogy with the virus herpes simple - probably has a number of genes coding for minor glycoproteins /Definition gene glikoproteid of herpes simplex virus 1 within a cluster of genes dispensable for growth in cell culture, R. Longnecker S. Chatterjee, R. J. Whitley u B. Roijrnan (1987) Proc. Natl 241-245, 1985/, after a large number of subcultures on embryonic kidney cells cows and embryonic tracheal cells cows /Abtr/, possessed a plot of the deviation Usbesides having the normal plot Us. Moreover, this vaccine was formed and large and small spots on Ebtr cells. From this mixed population of three phases of limited cultivation was isolated virus from the site Usdeviant forms, with small patches in each case. The virus isolated in this way was investigated further and was named Difivac-1. He was deposited in the Pasteur Institute, Paris, France, on 27 may 1992, the number of Deposit 1-1213.

b/ Definition deletion of the gE gene in Difivac-1

For further analysis of this deviation in the Usthe plot genomic DNA Difivac-1 was isolated in accordance with standard methods and subjected to blot analysis /Fig. 1A/. Hereditaria this blot fragment Hind III K wild-type labeled 32p confirmed that this fragment located in the center section of the Usshorter 1.0 thousand base pairs /etc., ad/ Difivac-1. Moreover, this analysis it was possible to roughly determine the position of the missing part /Fig. 1B/. For further analysis of this deletion Usplot BHV-1 strain Lam on today genomic DNA of strain Lam /Fig. 2A was isolated and cloned into the vector pUC18, rsus and pBP322 /Fig. 2B/. Was compiled physical map of the area around the estimated position deletions /Fig. 2C/. Based on this physical maps, vectors pKUN 19 and pUC18 /Fig. 2/ were konstruktivny the subclones, suitable for determination of a nucleotide sequence of this region. Using these subclones, by the method of Sanger was determined nucleotide sequence of the two chains throughout the area /shown in Fig. 2C/. This nucleotide sequence was analyzed using the program PC/gene. From a conceptual translation, it appears that the nucleotides /NT/ 168-1893 encode open reading frames 575 amino acids /Fig. 3A/. Further analysis showed that this amino acid sequence has the characteristics of a transmembrane glycoprotein, as shown in Fig. 3B. The fact is that these first 26 amino acids /AK/ enumerated as typical eukaryotic export signal and the area between AK 423 and AK 450 is identified as the transmembrane region. In addition, in this sequence are three potential site of N-linked glycosylation. This predicted amino acid sequence shows a close resemblance to the gE glycoprotein gene by vigorosa BHV-1. For this reason, the gene is called gE. To determine the extent to which this BHV-1 gE gene is absent in Difivac-1, was isolated p318 fragment. Fragment p318 begins with AluI site 55 HT to the desired open reading frame BHV-1 gE and ends 133 NT for it. Genomic DNA Difivac-1 was analyzed these p318 fragment using blot hybridization. It is revealed that Difivac-1 does not contain p318 find sequences /Fig. 5/. This experiment confirmed that Difivac-1 contains a deletion and clearly demonstrated that this deletion takes place around the gE gene.

To determine the size and position of the plot deletions, genomic sequences covering Usthe region Difivac-1 were cloned in prokaryotic vectors (See. Fig. 11C), a fragment with 14.5 etc., ad was cloned into pACYC vector and named p775. Hindlll fragment in 7.4, etc. ad was independently cloned in pUC18 vector and named p728, were isolated two subclone: > PST fragment from 1.4 etc., called clone p737 and AluI fragment - > PST in clone p754 350, etc., N. Analysis of restriction enzyme and the block of the analysis of these clones and data not shown/ demonstrated that deletion of the gE in Difivac-1 is the length 2,7 T. p. H., starting immediately 5' to gE gene and ending on the boundary of Usplot. These 2.7 T. p. N. were substituted doublerow the results of this analysis and to determine the exact point of recombination, the nucleotide sequence of the majority of clone inserts p754 was determined and compared with sequences of the wild type. Cm. Fig. 12. This analysis showed that the recombination is located at 77 base pairs /in/ up from the initial codon gE gene.

c/ assess the safety and efficacy of Difivac-1

Difivac-1 was tested on seven calves free of BHV-1 seronegative specific pathogen. Eight calves were vaccinated through the nose 105TCID502 ml, of which 1 ml was sprayed into each nostril. Eight seven calves free of BHV-1 seronegative specific pathogen were placed in isolation, they were introduced in the nose 2 ml of medium culture, and they served as unvaccinated control animals. Five weeks after vaccination, vaccinated and control calves were dripping nose 107TCID50very virulent strain of Iowa BHV-1. Six weeks later, after controlling for the infection all calves were injected intramuscularly dexamethasone for 5 days to reactivate the estimated latent virus. Clinical signs, rectal temperature and the growth of the organism was under control. The virus was isolated from nasal swabs and serum of temperature and growth rate of calves remained normal, but in vaccinated calves was observed some serious nasal discharge and excessive salivation. Irregularities in the nasal mucosa were observed. After vaccination of nasal smears were allocated Difivac-1 /Fig. 17/. All vaccinated calves had developed neutralizing antibodies against BHV-1. After control of infection all unvaccinated control calves showed apathy, loss of appetite, discharge from nose and eyes, redness of the gums of the lower jaw, strong disturbances in the nasal mucosa within 14 days after control of infection and stunting in 4 days. The vaccinated calves were small, fast healing of damaged mucosa of the nose and did not stop growth. Daily clinical manifestations, rectal temperature and the development of growth are given in Fig. 18, 19 and 20. After control of infection all calves had isolated the virus through the nose, but the number and the period of virus excretion were significantly reduced in vaccinated calves (Fig. 21). The secondary antibody response developed in vaccinated and unvaccinated calves, have developed antibodies after infection.

After the reactivation of the virus control of infection was isolated from one vaccinated tryout, what Difivac-1 did not cause any sign of the disease in young calves and are not susceptible to reactivation. Difivac-1 significantly reduced the effect of the disease and the amount of shedding virus after control of infection.

In conclusion, we can say that Difivac-1 is a safe and effective vaccine for use in the vaccination of animals against infections BHV-1.

2/ Construction of recombinant mutants with a deletion of the gE virus BHV-1

To have BHV-1 vaccines with the ability of differentiation, which are defined molecular better than Difivac-1, and which optionally may contain a deletion, for example, in the gene timedancing in addition to the division of the gE gene, as a Supplement to Difivac-1, were constructed recombinant mutants with a deletion of the gE. Starting from a certain position of the glycoprotein gE-gene and using cloned DNA fragments that flank the gE-gene, it is possible to build a fragment deletions gE. Using standard equipment /F. L. Graham u van der Eb. 1973, Virolgy, 52, 456-467/, you can recombine the fragment deletions in the genome of strain BHV-1 wild-type, which gives a mutant with a deletion of the gE.

a/ building a fragment with deletion gE

To build the fragment with deletion of the gE was chosen fragment, which, with onerous sequence, to allow recombination with the genome of the wild type. Was selected gene at the 5' /up/ side, fragment 1.2, etc., > PST - AsuII, which ends on 18 NT to the starting codon gE. 3' /down/ slice was selected fragment 1.2, etc., N. EcoNI-DcaI, which starts from 2 NT to stop codon gE gene /Fig. 6/.

To build the fragment of the gE deletion fragment in 1.4, etc., N. > PST -SmaI from 8.4 T. p. N. HindIII K fragment of strain Iam BHV-1, located on the 5' side of the gE gene, was subcloned into the SmaI and > PST site of plasmid pUC18. This clone was named p515. EcoNI-SmaI fragment located at the 3' - side gE and outbound from 4.1, etc., N. HindIII-EcoRI clone was cloned into the unique AsuII site p515. Thus the construction of the fragment with a deletion of the gE was completed, and built the clone was named p519. Although in principle the entire insert > PST -SmaI p5I9 can be used as a fragment with a deletion of the gE, this is undesirable. The fact that > PST -SmaI has a length of approximately 100-150 base pairs /by/ in a repeating sequence that flanks the Usa plot. This piece 100-150 can still recombine with a repeating sequence on the other side Usarea where there is gE gene, and thus can give undesirable products of recombination. For this reason, inacia fragment with deletion of the gE with the genome BHV-1 wild-type

In order to carry out recombination between the build section with a deletion of the gE and genome BHV-1 wild-type, microgramme number two DNA molecules together transfairusa in the embryonic tracheal cells cows /Abtr/ in accordance with standard method F. L. Graham u of A. J. van der Eb. /1973, Virology, 52, 456-467/. The mechanisms of cell recombination provide recombination small percentage of DNA molecules /2-4%/, which are included in the cells. To select reconstituted mutants with a deletion of the gE, viral mixture formed after transfection, scatters on a fresh cell culture Abtr. In most cases, the individual populations of the virus, developing /spots/, originate from the same virus. For isolation of mutant BHV-1 strain Lam with a deletion of the gE were isolated 230 of these patches /plaques/ and investigated in accordance with standard immunological methods using specific monoclonal antibodies to BHV-1 /MAT/, which do not react with infected cells Difivac-1. These MABS directed against the glycoprotein gE. Five of the 230 plaques did not react with these MAT. DNA from these 5 plaques were studied further.

c/ DNA analysis of the constructed mutants with a deletion of the gE strain Lam BHV-1

DNA preparations 3x/1B7, 1B8, 2H10/ above the mbrook and other 1989/. Double digestion of these DNA preparations using > PST and DraI and subsequent gel electrophoresis and blot hybridization fragment deletions > PST -DraI 2.3, etc., called as a probe showed that the gE gene of the viral genome populations 1B7 and 1B8 just removed as desired; see Fig. 7A and 7B. Population 2H10 has > PST -DraI fragment deviations. Blot-hybridization with gE-specific probe show that none of the three DNA preparations are not gE sequence /results not shown/. Population 1B7 and 1B8 virus BHV-1 are estimated by recombinant mutants with a deletion of the gE. Population 1B7 virus BHV-1 was tested for the properties of the vaccine.

d/ Construction of double mutants with a deletion of timedancing/gE

Because BHV-1 recombinant mutants with a deletion of only one gene can be sufficiently reduced virulence, deletions also provided in the gene timedancing /TC/ BHV-1 strains Lam and Harberink. These mutants were constructed similar to those used for the above-mentioned mutants with a deletion of the gE /results not shown/. These mutants with a deletion of the TK was used for construction of double mutants with a deletion of the TK/gE.

e/ Construction of double mutants with a deletion of the glycoprotein gI/glycoprotein gE

Porkolt the detection of anti gI/gE antibodies /see below/, we also invented the vaccine with double deletion gI/gE. This mutant with double deletion gI/gE can be constructed using the same procedures that were used to construct a mutant with a single deletion of the gE. Analysis of partially nucleotide sequence of the upper end of a fragment of 1.8 T. p. N. > PST, which covers the 5' end of the gE gene revealed an open reading frame with significant homology with respect to the gI homologues found in other herpes viruses. Cm. Fig. 13 and 14. Using SmaI - > PST fragment from 350, which covers the expected 5' end of the gI gene and a fragment of EcoNI-SmaI located down from the gE gene, it is possible to build a fragment deletions gI/gE. This fragment can recombine with the genome of the wild type, to obtain a mutant with a deletion and gI/gE virus BHV-1. Cm. Fig. 16. 80-90 amino acids that can theoretically be obtained, will not be able to identify antibodies that can interfere with the detection of anti-gI/gE antibodies. Further sequence analysis of the gI gene will allow you to create a deletion gI, which covers the entire region coding gI. This mutant with double deletion gI/gE was named Difivac-IE.

e/ assess the safety and efficacy of mutants Lam gE-and Lam gE-, TK-< / BR>
Properties of mutant vaccine strain is specific pathogens calves. Each mutant strain was sprayed in the nose 6 calves. Each calf was given a total dose of 105TCID50in 2 ml of medium culture, of which 1 ml was sprayed into each nostril. 6 calves in the nose was sprayed environment culture, free of the virus, and these were unvaccinated control calves. After 5 weeks after vaccination, all calves vaccinated and control, was contracted through the nose 107TCID50highly virulent strain of Iowa BHV-1. After vaccination and after control of infection was controlled clinical manifestations, rectal temperature and body weight. Samples were also taken from the nose to determine the number of days of shedding virus from the nose.

After vaccination behavior, appetite, rectal temperature and growth of calves remained normal. All vaccinated calves showed a strong nose and a small selection of violation of the nasal mucosa. The virus can be isolated from the nose vaccinated calves for approximately 7 days /table 1/.

After control of infection all unvaccinated control calves showed apathy, loss of appetite, discharge from nose and eyes, redness of the gums of the lower jaw, strong violations of the nasal mucosa and reduced growth rate. In calves, the HAC is CA. Not all calves vaccinated Lam gE-developed nasal discharge or violation of the mucosa. The vaccinated calves were observed apathy, loss of appetite or other clinical symptoms. Rectal temperature, growth and clinic after control of infection is shown in Fig. 22, 23 and 24. Unvaccinated calves were isolated virus from the nose 2 times longer vaccinated /table 1/.

These results demonstrate that the mutant strains Lam gE-and LamgE-, TK-BHV-1 practically does not cause any clinical signs of disease in young calves. Both mutant strain stopped manifestation of the disease after control of infection and reduced the period of excretion of the virus through the nose by 50%.

Mutant strains Lam gE-and Lam gE-, TK-virus BHV-1 safe and effective for use as vaccines for animals against infections BHV-1.

3/ the Prokaryotic expression gE

For prokaryotic expression gE-glycoprotein gene BHV-1 is still used expression vectors pGEX /D. B. Smith and K. S. Johnson, Gene 67 /1988/ 31-40/. Code vectors pGEX to glutathione S-transferase /GST/ protein carrier from Schistoma japonicum, which is influenced by promoter, SPS construct pGEX-2T600S3 /Fig. 8A/. In this design using standard molecular biological techniques /Sambrook and others 1989/ for CST genome was Legerova SmaI fragment of 600, which encodes for the N-terminal stretch of 200 amino acids gE protein. This design is intended to be arranged in form, and each time to GST legasuite fragment 600 on different reading frame. All these constructs were introduced into strain DH5 Escherichima coli, induced by IPTG and formed proteins were transferred to nitrocellulose after electrophoresis polyacrylamide gel by blokirovaniya /wester blatteng/. Immunological detection with anti-GST showed that only the correct reading frame /N 3/, which encodes for a site of gE protein, leads to the clear expression of the protein of the proposed merger size 27K /GST/ + 20K /gE/ = 47 K. Three of MAB isolated us that do not react with Difivac-1, identify 47 KD GST - gE protein fusion in the band; see Fig. 8B.

4/ Eukaryotic expression of the glycoprotein gE-gene

For eukaryotic expression of the glycoprotein gE-gene pre-selected vector pEVHIS. Vector pEVHIS as eukaryotic marker is the encoding of the HISD gene for histidinolovorans [EC 1.1.1.23] /C. Hartmann and R. Mulligan 1988, Proc. Natl Acad. Sci USA, 85, 8047-8 egion promoter of the immediate early gene of human cytomegalovirus /HCMV/, with a unique restriction enzyme sites located behind it. To create the expression vector pEVHIS/gE used the fragment including the entire coding region of the glycoprotein gE-gene. It begins on the AluI site 55 according to the proposed open reading frame gE and ends at 133 on her. This region was cloned for HCMV promoter vector pEVHIS, which produced a design pEVHIS/gE /Fig. 9/. pEVHIS/gE was amplified in the cells of E. coli DH5 and purified via gradient of cesium chloride /Sambrook and others 1989/. This purified DNA was transliterowany on BaLb/C-3T3 in accordance with the method of Graham and van der Eb. Transformed cells were selected by histidinol in order to isolate then twenty-resistant histidinol colonies. These colonies were studied MAT 81 by an immune reaction of the peroxidase monolayer /IPMA/. In the four colonies was expressed gE protein, of these four colonies 3T3 gE clone 9 was used to isolate subclan with high gE expression. The clone isolated by this method /named 3T3 gE 9.5/ was used for characterization of candidate anti-gE monoclonal antibodies.

5/ Eukaryotic expression and BHV-1 glycoprotein gE, and BHV-1 glycoprotein gI in the same cage

To expr is ositio gene of BHV-1 gI. Since the gene gI glycoprotein of herpes simplex virus is located directly above the gE gene glikoproteid, it was hypothesized that the gene of BHV-1 will be located at the corresponding position. To test this, we determined the sequence of the region 283 nucleotide located at 1, etc., called up from the beginning of the gene gE BHV-1. Conceptual translation of this region showed that the second reading frame encodes for a sequence of 94 amino acids that is homologous to glycoprotein gI of herpes simplex virus /Fig. 13 and 14/. Because homologous segment is a length of 80 amino acids from the start codon, the estimated start of the open reading frame gI gene of BHV-1 is approximately 250 NT upstream from the region of the sequence. From this it follows that the SmaI fragment of 1.7 T. p. H., which starts from 400 HT up sequence region, and ends in the gE gene, contains the complete coding region of the gI gene of BHV-1. This SmaI fragment of 1.7 T. p. N. was cloned into the eukaryotic vector MSV - neo /Cm. Fig. 15/. This vector contains the strong promoter of the virus murine sarcoma and selector neo gene, which encodes for resistance to the antibiotic G-418 sulfate Geneticin. The resulting design MSV neo GI would be the. rangitiratanga cells were selected using environment culture 400 mcg geneticin/ml, and resistant colonies were isolated and tested as a candidate anti-gE MAT, which did not react with 3T3 gE 9.5 cells. From this we selected a clone, which is reacted, for example, with MAT 66, reacts as BHV-1 wild-type.

6/ Feature candidate anti-gE MAT

Were obtained MAT against BHV-1 wild-type and selected based on their inability to respond with embryonic tracheal cells cows (Abtr), infected Difivac-1. These MABS were tested for their reactivity with

a/ a mutant with a deletion of the Lam gE-;

b/ the above product prokaryotic expression in band;

c/ the above gE-expressing Balb/c-3T3 cells;

d/ cells mentioned under c/ and infected Difivac-1, and

e/ Balb/c-3T3 cells expressing gE/gI complex.

To test for reactivity under a, c, d and e were used immune response of monolayer peroxidase /IPMA/. The results in Table 2 show that we received the MAT, which is directed against E /rooms 2, 3, 4, 52, 66, 68, 72 and 81/ and MAT /1, 51, 53, 67, 75 and 78 which may be directed against conformational antigenic domains on gE/gI complex. Completion of IPMA for mapping and is a glycoprotein gE and that one domain, probably formed by the complex gE/gI. Table 2.

Detection of anti-gE antibodies in cattle infected with BHV-1

To determine whether the serum of infected cattle antibodies against gE, was held indirect block IPMA 16 candidates gE-MAT and the following 8 selected sera:

- 2 sera of cows vaccinated Difivac-1 and infected control virulent strain of Iowa, who got 14 days after control of infection;

- 2 sera of cows experimentally infected with subtype 1 virus BHV-1, which was taken 20 months after infection. One of the cows were infected by exposure to the contact;

- 2 sera of cows experimentally infected with subtype 2 virus BHV-1, which was taken 20 months after infection. One of the cows were infected from contact;

- calf serum, free from specific pathogen, which was vaccinated with ts mutant vaccine and after 3 weeks were infected with subtype 2b virus BHV-1, serum was taken at 7 weeks after control of infection;

- serum gnotobiotics calf vaccinated with mutant vaccine and infected 3 weeks later, subtype 2b virus BHV-1, serum climbed cherestal against antigenic domains III and IV on gE and against antigenic domain 1, which is probably located on the complex gE/gI. We can conclude that gE is appropriate serological marker for distinguishing between infected with BHV-1 and vaccinated cattle.

7/ Detection of nucleic acids BHV-1 by PCR procedure using a gE-specific primers BHV-1

Starting with a specific nucleotide sequence of the gE gene of BHV-1 was selected pair of primers suitable for PCR using the program selection primer Lowe and others /Rowe, J. Sharefkin. S. Ai, Jand and C. W. Dieffenbach, 1990, nucleic Acids Res. 18, 1757-1761/. These primers were named P3and P4and shown in Fig. 10. The primers are located at 159 HT in both directions and provide amplification of the fragment of 200 NT. Using the primers P3and P4and isolated DNA BHV-1 were optimized conditions for PCR procedure. They included, in particular, varying the concentration of MgCl2concentrations of glycerol and conditions cycles. The optimal selected buffer for use with P3and P4for DNA amplification BHV-1 - 10 mm Tris pH 8.0, 50 mm KCl, 0.01% gelatin, 2.6 mm MgCl2and 20% glycerol. Optimal cyclic conditions /Perkin Member Cetus DNA Thermal Cyclek/ for cycles 1-5: 1 min 98oC, 30 sec 55oC and 45 sec 72oC for cycles 6-35: 30 sec 9632p dCTP used for blot analysis was TagI fragment 137, which was located between the binding sites of the primer. /Fig. 10/. After autoradiography hybridized filters you can watch the band 200. Using this path amplification only 10 BHV-1 genome /approximately 1.5 1015µg DNA/ leads to a well detectable signal /result not shown/. Similarly, a procedure was developed PCR using primers based on the coding sequence glikoproteid gIII BHV-1 /D. R. Fitzpatrick, Babink, Zamb, 1989, Virology, 1973, 46-57/. To distinguish between DNA BHV-1 wild-type and vaccine mutant with a deletion of the gE DNA samples were both analyzed gE-specific PCR and gIll-specific PCR. In the sample preparation of DNA Difivac-1 was identified as gIII positive and gE is negative.

Since the discovery of DNA BHV-1 in bovine semen, it would be important for BHV-1 specific PCR procedure, an attempt was made to conduct a gE-specific PCR for bovine semen infected with BHV-1. However, the unknown components in the sperm have a strong inhibitory effect on chain reaction polymerase /CRP/. So was recoverables 1 mg/ml proteinase K /PC/ total volume of 300 μl of 0.15 NaCl, 0.5% Na-Sarkosil and 40 mm DTT at 60oC. After 1 hour the sample was cooled to room temperature and was added 300 μl of 6M NaI and the sample was incubated for 5 minutes. From this mixture of DNA isolated by standard extraction chloroform/isoamylalcohol and was deposited in 1 volume of isopropanol. The precipitate was filtered 2.5 M NH4Ac/70% ethanol and re-suspendible in 10 mm Tris pH 7.4, 1 mm etc, 0.5% Tween 80 and 0.1 mg/ml PC for a second incubation for 1 hour at 60oC. This DNA preparation was directly subjected to the polymerase chain reaction.

Description of drawings

Fig. 1

Blot-analysis /Sodthern blot/ strains BH-1 Difivac-1 and Iowa.

A. Autoradiogram blot Defivac-1 and Iowa genomic DNA.

In lanes 1 and 3 DNA Difivac-1 was applied after digestion with restriction enzymes HindIII and > PST, respectively. In lanes 2 and 4 DNA Iowa was used after digestion with restriction enzymes HindIII and > PST, respectively. The fragment size is specified in thousands of base pairs /etc. N./.

Viral DNA was isolated by centrifugation environment culture /70 ml/ roller bottle 450 cm2/ with Ebtr cells infected with virus for 2 hours in 25% /weight/weight/ sucrose cushion in 10 mm Tris pH 7.4. 150 mm NaCl and 1 mm ATDC at 20 thousand rpm accordance with standard methods /J. Sanbroon, Fretsch and T. Maniatis, 1989, Molecular Cloning: a Lab. Man, 2nd ed. Cold Spring Harbor Laboratory Press, Naw York). This DNA restriction enzyme digestion is carried out by enzymes from Boehringer Mannheim in SURE /discontinuous buffers supplied by the manufacturer.

After separation on a 0.7% agarose gel horizontal electrophoresis blokirovaniya on nitrocellulose filter /Shleicher &Schuell/ filter previously hybridisable 6 hours at 42oC in 50% formamide, 3 x SSC /1 SSC = 0.15 M NaCl and 0.015 M Na-citrate, pH 7.4/ DNA 50 ál of denatured salmon sperm /Sigma/ ml and 0.02% bovine serum albumin, 0.02% polyvinyl pyrrolidone and 0.02 ficoll and 0.1% and-dodecyl sulfate /SDS/. Then hybridization was carried out by adding to the same solution HindIII K fragment marked by32p dCTP /Selection HindIII K fragment was based on cloning and the cleavage site on the map DNA from bovine herpes virus I /Strain Cooper/ Lohn F. Wayfield, Peter L. Good, Holly J. Vanoort, Aephonso R. Campboll and Dand A. Reed, Lournl of Virology N 1983/ 259-264/. After 12-14 hours of hybridization, the filter was washed for 2 hours in 0.1% SDS and 0.1% SSC /chloride solution and sodium citrate/ at 60oC. HindIII K fragment was cloned in pUC18 vector in accordance with standard procedures cloning /Sambrook, Fritsch Maniatis 1989, Moleculal Cloning: a Lab. Manual, Inc. 2nd ed. Cold Spring Habor Laboratory Press, N.-Garonna with a low temperature melting /BRL, Life Technologies / gel and isolated from the agarose by standard phenol extraction and ethanol precipitation. Isolated HindIII K fragment was marked arbitrary Ostrovki DNA Kit 1004.760 from Boehringer Mannheim. The autoradiograph hybridisierung filters was carried out with the exposure time of 36 hours film Kodak XAR at -70oC using a reflective screen.

B. Physical maps HindIII K fragment of Iowa in 8.4 so SC and Hindlll fragment in Difivac-1 in 7.4, etc., N. Because of comigration > PST fragments in 6 T. p. N. and lack of > PST fragment at 1.8 T. p. N. in Didivac-I deletion must occur in a shaded area.

Fig. 2

Subclavian fragments BHV-1 wild-type around the region without Difivac-1

In A shows the components of the genome BHV-1: Unique long region /Uv/; Unique short region /Us/ and two b /Ivand Tv/. This map is based on the published analysis of strain Cooper /John F. Wayfield P. Y. Good, Holly J. Vanoort, A. R. Campboll and David A. Reed, Journal of Virology /1983/ 259-264/.

In B is shown the fragments from the region Usthat were cloned in prokaryotic vectors include EcoRI fragment in 15.2, etc., N. in pACYC, Hindlll fragment in 8.4, etc. ad in pUC18 and the fragment EcoRI-HindIII 2.7 and 4.1, etc., in N. pBP322. Isolation of viral DNA fragments was performed in accordance with the camping in accordance with standard procedures /J. Sambrook, E. F. Fritsch and T. Waniatis 1989. Molecular Cloning: a laboratory Protocol, 2nd ed. Cold spring Habor Laboratory Press, N. York/.

In C shows the physical map of the region where localized the putative deletion in Difivac-1.

D identifies some subclones of this region, which was used for further analysis. Two > PST fragment was cloned in pKUN19 and other fragments into pUC18.

Fig. 3

A: Nucleotide sequence 2027 nucleotides of the Usregion of strain Lam BHV-1 around the proposed location, which was delawana in Difivac-1, as shown in Fig. 2C [from site identification ALuI on the left to spot the HincII recognition on the extreme right. The nucleotide sequence of the inserts subclones shown in Fig. 2, was determined by analysis on two strings using the method of dideoxy sequence Sanger and others /F. Sanger, S. Nicklen, and Coulson, 1977, Proc. Natl. Acad. Sci, 74, 5463-5467/. This purpose was used treatments T7 sequence Pharmacia in accordance with the procedure specified by the manufacturer. For radioactive tagging was used [35S] ATP /Amersham/. Sequence analysis of the regions with gas chromatography /GC/ with compression artifacts was repeated with 7-deaza-dG option procedures Pharmacia. Specified is inability open reading frame 575 residues AK, which was revealed after the conceptual translation of the nucleotide sequence. This translation is based on a universal code and was determined using the computer program PC /Gene/ version 1.03 PC/gene, Nov. 1987/. This open reading frame 575 AK begins with methionine at HT 168 and ends stopping codon at nucleotide 1893.

Structural analysis of the open reading frame 575 residues AK was also carried out by a computer program Pc/gene. The first 26 AK form a eukaryotic export signal, shown in Fig. "signal peptide". With a score of 6.2 splitting sequence of this signal is assumed between AK 26 and 27. The sequence 575 AK has 3 potential site of N-linked glycosylation /NXT/S/, indicated by the line under the remnants of amino acids. In accordance with the method of Pao and Argos between AK 423 and AK 450 has a transmembrane region, shown in Fig. "transmembrane round". Sequence recognition /regions/ for restriction enzymes AsuII, SmaI, HindIII and Econi underlined. The calculated molecular weight of this polypeptide is 61212.

B: Schematic representation of the structural characteristics of the open reading frame above 575 AK.

F is islote gE gene of herpes simplex virus /HSV/ and other E homologous genes gI virus and other pseudoralies I varicella - zoster (VZV).

Sequences used for this comparison were taken from the following publications: HSV: Seguence determination and geneti content od the short unique region in the genome of herpes Simplex virus type I D. Y. Me Geoch, A. Dolan, Donald S. and F. Y. Rixon (1985) Jornal Mol. Biol 181, 1-13, vsv: DNA sequence of the Uscomponent of the varicella - zoster virus genome. A. J. Davidson (1983), EMBO Journal 2.2203-2209. PRV: use of Agt II to usalate genes for 2 ps. rabus v. grey coprotoid, Petnovskis and others (1986). J. of Virology GO 1985-193].

These sequences were compared using the program sequence analysis Miltalin/Corpet, 1988, Nucl. Acids Res 16, 10881-10890/.

On A shows a diagram in which all sequences of four amino acids is shown schematically. Here the predicted transmembrane part /TM/ shown under each other. In addition to the predicted export signal sequences and potential N-linked sites of glycosylation /I/ shows two conserved region in which the relative position of cysteine residues often remain unchanged /CCC/.

In B shows the results Multalin compare the centrally located region rich in cysteine, four versions of gE. Asterisks indicate identical amino acids and colon similar amino acids.

Fig. 5

Figure photos, obtained the d BstI /1, 2/, EcoPI /3, 4/ and Hindlll /5, 6/, separated on 0.7% agarose gel, blokirovanie on the nitrocellulose and hybridized HindIII K fragment marked with32p strain Lam BHV-1, in accordance with the procedures set out in the label of Fig. 1A.

Panel B: nitrocellulose blot of the same gel as in A, hybridized gE-specific probe p318 BHV-1. This probe includes a AluI-HincIII region shown in Fig. 2C.

Fig. 6

Building fragment of BHV-I gE deletion

In A shows the position of the gE gene and used clones. Components of the genome BHV-I are: Unique long /Ui/ region; Unique short /Us/ region and two repeat /IP and TP/. To get a region located at the 5' side of the gE gene, PST -SmaI fragment of 1.4 T. p. H. from the HindIII K fragment V-1 in 8.4, etc., N. strain Lam was subcloned into the SmaI site and > PST plasmids PuC18. This clone was named p515, and it is shown in B. EcoNI-SmaI fragment located at the 3' - side gE coming from the HindIII-EcoRI clone at 4.1 T. p. N., was cloned in the unique AUSII plot p515. In order to allow ligation of the rest of EcoNI to the residue ASUII clone p515 was digested ASUII, then processed by the enzyme maple /Boehringer Mann/ and dCTR to get one casinoby the remainder in the rest of the ASUII /Sanbrook and others 1989/. This stage is you can ligitamate in this vector. Such a clone was named p519.

Fig. 7

A. figure photos received blot analysis of DNA preparations IB7, IB8 and 2H10. Isolation of DNA, restriction enzyme digestion, blokirovanie and hybridization were carried out in accordance with the procedures described in Fig. 1A. After double digestion of DNA preparations 1B7, 1B8 and 2H10 using > PST -DraI fragments were separated on 0.7% agarose gel and then batrouny on nitrocellulose filter. This filter was hybridisable32p dCTP labeled as a probe fragment deletions > PST -DraI 2.3, etc., N. In rows 1-3, respectively, were separated samples VG, 1B8 and 2H10. The number 4 was applied DNA BHV-1 wild-type strain Lam and in the range of 5 - fragment deletions in 2.3, etc., N.

B. Physical map of the EcoRI fragment of strain Lam BHV-I 15,2, etc., N. the Map shows the position of the plots up > PST, DraI and HindIII and the position of the probe hybridization referred to in 7A.

Fig. 8

Prokaryotic expression of gE BHV-I

For prokaryotic expression gE BHV-I fragment SmaI gE gene 600 has been merged in the three reading frames with the coding region of the gene glutathione S-transferase from Schistosoma japonicum in the vector pGEX-2T/ D. B. Smith, K. Johnson, Gege 67 /1988/ 31-40/. Recombinant molecules with the desired orientation (Syn) fragment ops this build merge were named pGEX-2T600SI, PGEX-2T600S 2 and G-2T600S3.

A. Diagram of one of the constructs pGEX-2T600S. On NH2side of the region that encodes for the product of the merger of GST-gE, is the region of the tac promoter, induced by isopropylthioxanthone /IPTG/.

B. Image photographs obtained in blot-analysis of all protein drugs DH5 cells , transformed with pGEX-2T600S. Night cultures of DH5 cells , transfetsirovannyh constructs pGEX-2T600S1, pGEX-2T600S2 and pGEX-2T600S3, was 1/10 in the medium Luria - Bertani /LB/ 50 mcg/ml ampicillin and after 1 hour of growth induced IPTG - 5 hours. These induced cultures were centrifuged for 5 minutes at 6000 x g and included 1 x mixed layer /2% SDS /sodium dodecyl sulphate/, 10% glycerol, 5% mercaptoethanol and 0.01% bromophenol blue/ [1.5 ml of culture was included in 500 ál of the mixed layer and was heated at 95oC for 5 minutes. Then 50 µl per lane was separated on a vertical 12.5% polyacrylamide gel according to standard procedures and then in semi-dry condition was lateralis on nitrocellulose filter using LKB-miltiphor II Nova Blot under the conditions specified by the manufacturer.

In lanes M was applied pre-painted marker protein /BRL Life Techol. Inc. K, K, K, K, K, 18X-2T600S1, pGEX-2T600S2 and pGEX-2T600S3.

In panel A we can see the result blot analysis of antisera. To this end, the filter was incubated in accordance with standard procedures /E Hallow, Lane, 1986, Antibodies: Cold Spring Harbor Laboratory, N. York/ in blocking buffer /PBS + 2% milk powder and 0.05% Twecn 20/, and then a polyclonal anti-GST serum of the rabbit. Then the filter was washed and incubated with goat-anti-rabbit immunoglobulin serum conjugated horseradish peroxidase. Then linked goat antibodies immunohistochemistry showed chromatogram /diaminobenzidin, chlorinator and H2O2/. The product of the merger of GST, which is indicated by the arrow, has a predicted size of approximately 47 to frame 3.

In panel B we can see the result blot analysis with a monoclonal antibody MAT 4, which recognizes gE protein. To this end, the hollow filter as in panel A, was blocked, incubated MAT, washed and incubated with goat-anti-rabbit immunoglobulin serum conjugated horseradish peroxidase /Origin/. Then connected the rabbit antibodies immunohistochemistry showed a Chromogen. The band, which is visible in the track 3 /frame 3/, is 47 tons in size and arrow.

Fig. 10

The position of the gE-specific primers and probes for PCR procedures for the detection of DNA BHV-1

The diagram shows the sequence of nucleic acids gE glycoprotein gene BHV-1 from nucleotide 1272-2027 [sequence was taken from Fig. 3] . The primers ISP P3and P4are underlined. The nucleotide sequence of P3-5'-AGC-TGG-TGG-TGC-CAG-TTA-GC-3' /PEFC. ID N:2/. The nucleotide sequence of P4/in addition to the binding sequence of the primer defined above/ -5'-ACC-AAA-CTT-TGA-ACC-CAG-AGC-G-3' /PEFC. ID N: 3/. The probe used for hybridization blot for the detection of PCR amplified DNA, represented TagI fragment 137, located between the binding sites of the primer, the ends of this fragment indicated. For comparison with Fig. 3 shows the sites HindIII and EconI.

Fig. 11

Mapping gE deletions Difivac-1

A shows the physical map of the EcoRI fragment in 15.5, etc., N. strain Lam BHV-1 wild type. B shows the physical map of the EcoRI fragment in 14.5 T. p. N. Difivac-1. Both EcoRI fragment covering the full unique short regions of the genomes of the respective viruses. The position of the gE gene and the estimated position of the gI gene are indicated by open boxes. Maps A and B are arranged in such a way that the fragments > PST 6, etc., N. inside of each card is aligned. On both maps the sequence of the internal repeat and terminal repeats were indicated by dashed boxes. The arrows under the replays show the orientation of these sequences.

In A specified the c-1 fragments, used for mapping gE deletions and get a physical card> shown in B. the Arrows under the inserts of clones p728, p737 and p754 indicate the regions that had the order to determine the recombination point.

Abbreviations:

A = AliI, E = EcoRI, P = > PST, H = HindIII, r is the recombination point, IR = internal repeat, TR = terminal repeat.

Fig. 12

The determination of the exact recombination point in Usthe region Difivac-1

To determine the exact boundaries E deletions identified in strain Difivac-1, was determined sequence of clone p754 and all clones p728 and p737. The inserts of these clones were shown in Fig. 11. The procedure used sequences have been described in the explanation of Fig. 3.

On A was shown a sequence of mostly AluI-PatI fragment. This sequence begins in the promoter region of the gE gene. Estimated TATA box is underlined. At point r / = recombination point/ this promoter region fused with the sequence that is also found on the opposite site Usregion, and is called the inverted repeat. Accurate recombination point was determined by comparison of repeat identified in the region gE promoter, with a copy of the repeat, vyavleno /under I/ r marking. A similar comparison was made with sequence gE promoter identified in Difivac-1, and gE promoter identified in strain Lam wild type. The point where these sequences diverge, was shown in the B /II/ and also marked "r". Identified point recombination the same.

Fig. 13

Analysis of a partial sequence of the gE gene of BHV-1

Using > PST clone strain of BHV-1 in 1.8 so SC, which falls in the gI, and gE gene of BHV-1 (see Fig. 11/, was determined sequence 284 nucleotides within the coding region of the BHV-1 gI. The procedure used sequences have been described in the explanation for Fig. 3. The translated sequence was based on a universal code of version 1.03 of the computer program PC/gene /November 1987/.

The amino acid sequence of the encoded second frame readout is given in single-letter code below the nucleotide sequence. This sequence of amino acids homologous codereuse region other gI homologues of herpes virus /Cm. Fig. 14/.

Fig. 14

Comparison of the partial amino acid sequence of amino acids of the proposed gI gene of BHV-1 with the corresponding parts of the coding regions of the gI gene of herpes simplex virus, etc 63 gene VI is HSVI sequence starts at AK 80 and sequence of the vzv starts at AK 76 their respective coding regions. Used sequence was published in the documents referred to in the explanation for Fig. 4. The comparison was conducted using Miltalen computer program. Asterisks indicate identical amino acids, and colons indicate similar amino acids.

Fig. 15

Build MSV neo GI plasmids for eukaryotic expression of the gI gene of BHV-1

Based on the comparison of partial amino acid sequences gI gene of BHV-1 was calculated estimated position of the gI gene of BHV-1. Based on this calculation, it was assumed that the SmaI fragment of 1.7, etc., ad must contain the full coding region of the gE gene of BHV-1. The position of this SmaI fragment was specified in A. To blunt the ends of this SmaI fragment of 1.7 T. p. N. were legirovanyh BamHI linkers using standard procedures. The resulting product was digested BamHI and Legerova in the MSV vector-neo eukaryotic expression. The MSV vector neo has a unique BamHI site for the MSV-LTR, which has a strong activity of the promoter. This vector was described in Rijsewijk and others 1987 EMBO y 6, 127-131.

Fig. 16

Construction of fragment double deletions gI/gE BHV-1

The position of the glycoprotein gE-gene and the estimated position of the glycoprotein gI gene in Usthe region of BHV-1 is depicted on the diagram A. Sastry what's significant sites of restriction enzyme relative to the positions of both genes. To build the fragment deletions gI/gE, clone pl.r-SmaI/o containing SmaI fragment, 1.7, etc., ad, which covers the gI gene is digested > PST. Plot > PST remaining insert SmaI- > PST 350 will be made with blunt ends using standard molecular biological procedures. EcoNI-SmaI fragment /Cm. Fig. 6V/ isolated from the fragment HindIII-EcoRI at 4.1 T. p. N., described in Fig. 6A, will also be made with blunt ends and Legerova to the modified PI site. This is illustrated in figure C and D. From the obtained clone pAIE fragment SmaI-DraI 1.4, etc., ad can be isolated for recombination with the DNA of BHV-1 wild-type.

Abbreviations:

E = EcoRI, H = HindIII, S = SmaI, P = > PST, ENI = EcoNI, D = DraI, kb = one thousand pairs of nucleotides /etc., ad/ and Us= unique short.

Fig. 17

The average amount of virus released from the nose calves after vaccination

. = = vaccinated Difivac-1, 0 = not vaccinated control calves.

Fig. 18

Average daily clinical manifestations in calves after control of infection with virulent strain of BHV-1, key designations as in Fig. 17.

Fig. 19

The average rectal temperature of calves, with the control of infection with virulent strain of BHV-1, the key designation as Chewie designations as shown in Fig. 17.

Fig. 21

The average amount of virus released from the nose calves after control of infection with virulent strain of BHV-1, key designations as in Fig. 17.

Fig. 22

The average rectal temperature of calves after control of infection with virulent strain of BHV-1

. = vaccinated Lam gE-, 0 = vaccinated Lam gE-/TK-,

x = non-vaccinated control calves.

Fig. 23

The average growth of calves after control of infection with virulent strain of BHV-1, key designations as in Fig. 22.

Fig. 24

Average daily clinical manifestations in calves after controlling charging of a virulent strain, the key designations as in Fig. 22.

1. The strain of bovine herpesvirus type 1 (BHV-I) CNCM 1-1213 having a deletion in the gene for the glycoprotein de used to prepare the vaccine.

2. The vaccine for the vaccination of cattle against bovine herpes virus type 1 (BHV-I), including strain under item 1.

3. Method of vaccination of cattle against bovine herpes virus type 1 (BHV-I), involving the introduction of a vaccine for p. 2.

4. Diagnostic serological reagent comprising picopsu, vaccinated vaccine for p. 2, from cattle infected with BHV-I wild-type.

5. The way serological differences between cattle vaccinated with a vaccine for p. 2, from cattle infected with BHV-I wild type, including the selection of blood serum from animals vaccinated with the vaccine under item 2 and infected with virulent strain of IOWA, analysis of sera on the interaction of the produced antibodies serological diagnostic reagent under item 4.

 

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