Immunogenic composition comprising the chimeric polypeptide of hiv, the polypeptide and the test-system for detection of antibodies to hiv

 

The invention relates to the field of medicine and biotechnology and relates to immunogenic compositions containing the chimeric polypeptide of HIV, method of induction of an immune response to VIC and VIC/2, chimeric polypeptide VIC and VIC/2, a DNA molecule that encodes a chimeric polypeptide, the plasmid vector for expression of a chimeric polypeptide, polyclonal antibodies to VIC or VIC/2 and diagnostic test systems for the detection of antibodies to VIC and VIC. The invention includes an immunogenic composition based on a chimeric polypeptides WHICH or WHICH/2, representing the full-size protein P24, merged with the immunodominant fragment of gp41 plot 575-616 or selected from him (rec 24-41), or merged with immunodominant fragment gp36 the same area or selected from him (rec 24-36). This composition is able to induce a strong immune response to VIC or VIC/2. The invention also includes a gene construct capable of expression of these chimeric polypeptides in cells of E. coli, as well as diagnostic test-system for detection of antibodies to VIC and VIC. The advantage of the invention lies in the fact that the developed gene construct, which allows to Express the chimeric recombinant protein containing part of the n protein fragment shell VIC or VIC, moreover, these recombinant proteins are able to induce a strong immune response to VIC or VIC/2, which allows the use of these chimeric polypeptides not only to create a diagnostic test systems, but also to encourage them to create vaccines. 7 C. and 11 C.p. f-crystals, 3 tab., 5 Il.

The invention relates to medicine and biotechnology and relates to immunogenic compositions containing the chimeric polypeptide of HIV, method of induction of an immune response to VIC or VIC/2, chimeric polypeptide VIC and VIC/2, obtained by recombinant method, a DNA molecule that encodes a chimeric polypeptide, the plasmid vector for expression of a chimeric polypeptide, polyclonal antibodies to VIC or VIC/2 and test systems for the detection of antibodies to VIC and VIC.

It is known that antibodies to proteins encoded by GAG gene, are early markers of HIV infection, antibodies to the protein P24 first appear in the serum in the process of seroconversion [1], and its level correlates with the status of an infected person (drops when symptoms of the disease AIDS) [2, 3] and therefore is a prognostic marker of HIV infection. On the other hand, it is GAG-proteins rather conservative and sledi interest in the development of vaccine and diagnostic products.

Antigens encoded by the gene ENV-gp41 VIC and gp36 WHICH are viral transmembrane glycoproteins. For the proteins encoded by the ENV gene, known short immunodominant sequence of about 40 amino acids) capable of recognizing more than 90% of HIV-positive samples (containing antibodies to HIV). On the contrary, oligopeptides, plays a short sequence of the proteins encoded by GAG gene, and learn no more than 50% of positive sera [4] . Acceptable immunoreactivity are only extended proteins [5].

The genetic engineering techniques allow to obtain industrial quantities of these proteins. However, proteins expressed in the system of E. coli, do not undergo post-translational modification, which is a characteristic of HIV proteins produced in the course of infection, and thus, immunological characterization of recombinant proteins may differ from the properties of viral proteins.

Known test systems based on recombinant proteins, in which one test system uses a mixture of antigens, which includes how long antigens fused with protein carrier (e.g., beta-galactosidase, glutationtransferase and so on [6]), and short fragments, fused with protein-NAPA expression and purification and leads to higher prices of these products. Even more importantly, the difference in physico-chemical properties of antigens leads to uneven adsorption on a solid phase from a mixture of proteins, which causes a decrease in the sensitivity and specificity of the test. The above disadvantages can be overcome by using chimeric structures, which includes the set of relevant antigenic determinants.

The first design of carrier protein encoding fragments of R24 and immunodominant site gp41, have been described in U.S. patents 5204259 and 5470720 and presents hypernym recombinant polypeptide, soluble in aqueous solutions. On the basis of different variants of these structures are described diagnostic system for ELISA. In the first patent describes a DNA segment that encodes a recombinant HIV P24 protein and HIV p24-gp41 protein and the DNA molecule is able to Express both proteins. Also described cells transformed with recombinant DNA, and a method of obtaining fused protein and the diagnostic system using fused protein. In the description presents the amino acid sequence encoded by this DNA segment, while protein is used to detect antibodies against P24 or against gp41 WHICH, mainly in ELISA. In the second patent describes 4 different hgu C-terminal part of the 7 amino acids, and between these parts is inserted immunoreactive fragment of gp41 and/or gp36 in length from 15 to 20 amino acids, but not more than 35. The circuit structure described chimeric protein can be represented as follows: 1) (P24 fragment - fragment of gp41 - P24 fragment), 2) (P24 fragment - fragment gp36 - fragment R24), 3) (P24 fragment - fragment of gp41 fragment gp36 - fragment of gp41 - fragment - gp41 - P24 fragment), 4) (P24 fragment - fragment of gp41 fragment gp36 - fragment R24). The patent also shows the structure of a DNA segment encoding chimeric polypeptides, and recombinant DNA molecule that is able to Express the chimeric polypeptide in the cells of E. coli. Also described ELISA diagnostic test-system based on chimeric polypeptides, adsorbed on a solid substrate.

In the patent EP 0307149 described enzyme-linked immunosorbent assay for the detection of antibodies against P24 and antibodies against gp41 WHICH based on the use of a chimeric protein consisting of the amino acid sequence of the GAG 121-356 and ENV sequences corresponding to amino acid residues 542-674 (numbering according to [7]). Chimeric protein consists of 379 amino acids, the process of expression of the recombinant protein includes the transformation of the host cells, preferably E. coli, the vector is OK cultivation of cells and secretion of the protein. However, none of these patents chimeric proteins was not used and was not discussed for use in vaccine preparations.

Described [5] diagnostic system based on recombinant antigens, copying VIC-specific sequence of gp41 (amino acids 584-615, numbering authors) and R24 (amino acids 78-437, numbering authors) and VIC-specific sequence gp36. However, the protein described by the authors as recombinant P24, is actually a long fragment predecessor nuclear (core) protein VIC-P55 comprising a fragment of R17, full R24 and fragment R, and is used in a mixture with recombinant gp41, and not in the form of a chimeric protein, and the sequence gp36 not shown.

Describes the work related fused proteins P24 with gp41, which are effectively used in diagnostic test systems. The use of fused proteins as vaccines are not described.

This paper describes a new immunogen WHICH representing recombinant soluble chimeric polypeptide containing fragments of structural proteins VIC. Gene construct encodes the entire internal protein P24 and immunogenic fragment of gp41. Expressive ELISA [8, 9]. The authors suggest the use of this protein as a candidate vaccine. However, these materials do not contain the structure of the chimeric polypeptide.

In connection with the foregoing, the purpose of this invention is to provide such chimeric proteins p24-gp41 and p24-gp36, which can be successfully used not only for diagnosis but also as a vaccine preparation.

The objective of the invention is to develop a gene construct expressing in prokaryotes chimeric recombinant protein containing part of one polypeptide chain of a full-sized polypeptide conformation close to the viral protein P24, and the immunodominant protein fragment shell VIC or VIC, and represents a fused protein containing the N-end full-R24, and-the end - immunodominant fragment of gp41 or gp36, and creation on the basis of the obtained proteins immunogenic composition capable of inducing a strong immune response in mammals, and highly effective diagnostic test system for the detection of antibodies against VIC and/or WIC.

The essence of the present invention includes: an immunogenic composition comprising the chimeric polypeptide of HIV, the method induction of an immune response to Violetto prokaryotes Hypernova polypeptide VIC or HIV 1/2, polyclonal antibodies to VIC or HIV-1/2 test-system for detection of antibodies to VIC and VIC.

The invention includes a group of inventions, United by a common inventive concept.

Immunogenic composition comprising the chimeric polypeptide of HIV, characterized by the fact that she is a full-sized protein P24, merged with the immunodominant fragment of gp41 containing amino acids plot 575-616 (hereinafter numbering [7] and [10]), or selected from him (rec 24-41) (Fig. 1A), or merged with immunodominant fragment gp36 containing amino acids plot 575-616 or selected from him (rec 24-36) (Fig.1B), and a pharmacologically suitable carrier and/or diluent.

Yet another aspect of the invention is a method of inducing an immune response to VIC or HIV 1/2 in mammals, characterized in that the mammal is administered the immunogenic composition according to p. 1, while for the induction of an immune response to WHICH mammal is administered immunogenic composition, representing the full-size protein P24, merged with the immunodominant fragment of gp41 containing amino acids plot 575-616, or selected from him (rec 24-41), and for the induction of immune responses to HIV 1/2 mammal is administered immunogenic composition, n is the notes section 575-616, or selected from him (rec 24-36).

To create immunogenic compositions designed chimeric polypeptide of HIV, characterized in that in the case WHICH he is a full-sized protein P24, merged with the immunodominant fragment of gp41 containing amino acids plot 575-616, or selected from him (rec 24-41) (Fig.1a), and in the case WHICH/2 it is a full-sized protein P24, merged with the immunodominant fragment gp36 containing amino acids plot 575-616 or selected from him (rec 24-36) (Fig.1B).

This immunodominant fragment of gp41 has the following amino acid sequence: SEQ ID NO:1 575-616 L Q A R V T N I E K Y L Q D Q A R L N S W G C A F R Q V C H T T V P W P N D T L K P SEQ ID NO:2 578-609 R V T N I E K Y L Q D Q A R L N S W G C A F R Q V C H T T V P W SEQ ID NO:3 595-607 W G C A F R Q V C H T T V and immunodominant fragment gp36 has the following amino acid sequence: SEQ ID NO:4 575-616 L Q A R V T N I E K Y L Q D Q A R L N S W G C A F R Q V C H T T V P W P N D T L K P
SEQ ID NO:5 578-609
R V T N I E K Y L Q D Q A R L N S W G C A F R Q V C H T T V P W
SEQ ID NO:6 595-607
W G C A F R Q V C H T T V
A DNA molecule encoding a chimeric polypeptide of HIV, is a sequence of bases
SEQ ID NO:7 for chimeric polypeptide WHICH (rec 24-41) and
SEQ ID NO:8 for chimeric polypeptide VIC/2 (rec 24-36).

The plasmid vector used for expressively DNA SEQ ID NO:7 for chimeric polypeptide WHICH (rec 24-41) or SEQ ID NO:8 for chimeric polypeptide VIC/2 (rec 24-36).

In addition, a plasmid vector for expression of a chimeric polypeptide WHICH (rec 24-41), which is a plasmid 15b into which is inserted at the restriction site Bam HI DNA fragment SEQ ID NO:7, and the restriction sites Hind III-Eco R1 inserted par locus.

In addition, a plasmid vector for expression of a chimeric polypeptide WHICH/2 (rec 24-36) is a plasmid 15b into which is inserted at the restriction site Bam HI DNA fragment SEQ ID NO: 8, and the restriction sites Hind III-Eco R1 inserted par locus.

The invention also relates to polyclonal antibodies to VIC or VIC/2, which are immunoglobulins of the IgG class 2a, obtained by immunization mammal a chimeric polypeptide, with a titer of at least 30 000 to the fragment of gp41 and gp36 and at least 200 000 to R24 in indirect ELISA.

The invention also includes a test-system for detection of antibodies to HIV, containing sorbed on a solid substrate, the HIV antigens presented chimeric polypeptides, and detecting reagent, and the antigen WHICH is a full-sized protein P24, merged with the immunodominant fragment of gp41 containing amino acids plot 575-616 or selected from him (rec 24-41), and antigen HIV-1/2 is a full-sized protein P24, merged with IMM is mentioned test system as the detection reagent may contain conjugate antivitamin antibodies to horseradish peroxidase.

The test system may also as a detecting reagent to contain conjugate antivitamin antibodies with alkaline phosphatase.

The test system may also contain as the detecting reagent is a conjugate And protein St. aureus with horseradish peroxidase.

Another option may be the test system, as the detecting reagent containing conjugate antivitamin antibodies with colloidal carbon.

The test system may also contain as the detecting reagent conjugate And protein St.aureus with colloidal carbon.

Another version of the test system may contain as detecting reagent chimeric polypeptides conjugated to horseradish peroxidase.

The test system can also be used as the detecting reagent chimeric polypeptides conjugated with colloidal carbon.

The invention is illustrated by the following example.

Chimeric design that meets a protein gp24 and the fragment of gp41, is collected in the intermediate plasmid, such as pBS. Synthesize primers corresponding 131-136 and 365-370 amino acid sequence of the full-size cDNA plasmids run for R24 and accordingly 584-589 and 610-615 for a fragment of gp41. PCR fragments alternately clone in pBS. In edah, for example in E. coli. This vector can serve red or any other suitable for this purpose, for example pQE31 (Qiagen). The result of the expression is a chimeric protein rec 24-41. Cells treated with 8M urea and ultrasound. The clarified lysate sufficieut and purified affinity column with Ni2+ -agarose, and then on a column of Q-separate (Pharmacia). Product purity is checked by electrophoresis in PAAG. Proteins used for immunization, conjugation with an enzyme and enzyme-linked immunosorbent assay (ELISA), and so on,

The ability of the protein to induce an immune response (immunogenicity of the drug) test laboratory animals. Rabbits weighing 2 kg subjected to immunization with protein rec 24-41 or rec 24-36. Repeated immunization spend 4 weeks after the first. Blood is collected from the ear vein and get the serum in the normal way. Mice subjected to immunization intraperitoneally protein rec 24-41 or rec 24-36. The second immunization spend 2 months after the first. Blood is collected from the cervical artery 7 days after immunization and get the serum as usual.

In immunogenic compositions as physiologically suitable carrier used aluminium hydroxide, as well as pharmacologically suitable diluent any suitable solutions for parenteralnomu way. Chimeric polypeptides rec 24-41 and rec 24-36 adsorb onto the solid phase, for example in the wells of polystyrene tablet add samples of blood sera of infected and HIV uninfected people in an acceptable breeding and conduct enzyme-linked immunosorbent assay in the usual way, using one of the detecting reagent and colorimetric indicator.

Examples of specific embodiment of the invention.

Example 1. Obtaining plasmids pET15par.

pet 15b hydrolyzing the restriction enzyme Hind III, completed the sticky ends of the DNA polymerase, T4, plasmids are set Quiagen and are ligated with par-locus obtained in [11]. Received ligase mixture transform cells of E. coli strain Nova-Blue (Novagen) according to the usual technique [12]. Transformed cells are plated on plates with LB agar containing 25 μg/ml kanamycin. Grown colonies screened for recombinant plasmids. For this purpose, the bacterial cells are lysed and release of plasmid DNA. The selected plasmid DNA treated with restrictase Pst I and Hind III and the hydrolysate explore electrophoresis in 1% agarose. The hydrolysate plasmid DNA RET-par has electrophoregram band DNA size 2270 base pairs.

Example 2. Obtaining plasmids retri(24-41).

Oligonucleotides to receive the ri 95oWith 30 min and annealed to the 15oC for 2 hours in thermostat, add DNA ligase and incubated overnight at 15oC. the Target DNA fragment goes gp41 allocate using the kit for DNA extraction company Qiagen.

The structure of the oligonucleotides to obtain a synthetic fragment of gp41.

5' GAT CTG GGT ACC GAC GAC GAC GAC AAG AAG GCC ATG GCG ATA TCG GAT CTG CAG CGT GTT CTG GCT GTT GAA CGT TAC CTG AAA GAC CAG CAG CTG CTG GGT ATC TGG GGT TGC TCT GGT AAA CTG ATC TGC ACC ACC GCT GTT CCG TGG TAA TAA G-3'
5' GA TCC TTA TTA CCA CGG AAC AGC GGT GGT GCA GAT CAG TTT ACC AGA GCA ACC CCA GAT ACC CAG CAG CTG CTG GTC TTT CAG GTA ACG TTC AAC AGC CAG AAC ACG CTG CAG ATC CGA TAT CGC CAT GGC CTT CTT GTC GTC GTC GTC GGT ACC CA 3'
To obtain the fragment rec R24 plasmid run-10, containing the full genome VIC [7], amplified using the primers N1 and N2. The DNA fragment allocate using the kit for DNA extraction company Qiagen, hydrolyzing the restriction enzyme BamH1 and purified by electrophoresis in 1% agarose.

The structure of the oligonucleotides to obtain a PCR fragment R24 and PCR-fragment 24-41.

N1 GGGG GAT CCT CAA AAT TAC CCT ATA GTG
N2 GGGG GG ATS CAA TAG TTG GCT CAT TGC TTC
N3 GGGGG GAT CCT TAT TAC CAC GCA AC
T7 AAT ACG ACT CAC TAT AG
To obtain the full-length DNA fragment encoding a protein rec 24-41, in a test tube 0.5 ml mix both DNA fragment (P24 and gp41) and performed PCR using primers N1 and N3. The target fragment is allocated, as obeschannoi ligase mixture transform cells of E. coli strain Nova-Blue (Novagen) according to the usual technique [12]. Transformed cells are plated on plates with LB agar containing 25 μg/ml kanamycin. Grown colonies screened for recombinant plasmids. For this bacterial clones grow and produce plasmid DNA. The selected plasmid DNA analyze PCR using primers N3 and T7. The structure of the DNA fragment confirmed by sequencing according to the method of Sanger using primers T7 and N3 [12]. The structure of the DNA fragment shown in Fig.1A, scheme design expressing vector shown in Fig.2A.

Example 3. Obtaining plasmids retri(24-36).

The structure of the oligonucleotides to obtain a synthetic fragment gp36.

5' GAT CTG GGT ACC GAC GAC GAC GAC AAG AAG GCC ATG GCG ATA TCG GAT CTG CAG GAG CGT GTT ACC GCT ATC GAA AAA TAC CTG CAG GAC CAG GCT CGT CTG AAC AGC TGG GGT TGC GCT TTC CGT CAG GTT TGC CAC ACC ACT GTT CCG TGG TAA TAA G-3'
5' A TCC TTA TTA CCA CGG AAC AGT GGT GTG GCA AAC CTG ACG GAA AGC GCA ACC CCA GCT GTT CAG ACG AGC CTG GCC CTG CAG GTA TTT TTC GAT AGC GGT AAC ACG CTC CTG CAG ATC CGA TAT CGC CAT GGC CTT CTT GTC GTC GTC GTC GGT ACC CA 3'
The DNA fragment that encodes a protein rec 24-36, get in the same way as described in example 1. The structure of the DNA fragment shown in Fig. 1B, circuit design expressing vector shown in Fig. 2B.

Example 4. Expression, isolation and purification of rec 24-41 VIC.

The strain E. coli BL21 (DE3) containing plasmid retri(24-41), grown in 1 lit is iglucruise) to a concentration of 1 mm, incubated for another 4 hours and collect cells by centrifugation at 4000g. Cells are suspended in 50 ml of 6 M urea, treated with ultrasound, it clarified the lysate add tetrathionate potassium and sodium sulfite to a concentration of 25 mm and 100 mm, respectively, incubated for 16 h and the mixture chromatographic on affinity column with Ni2+ - agarose (Qiagen) according to the recipe of the manufacturer. Rechromatography carried out on a column of Q-separate in 6 M urea and a linear gradient of NaCl (0-0,2 M). Product purity is assessed by electrophoresis in PAAG (Fig.3).

For holding refolding the protein solution add cysteine to a final concentration of 10 mm and incubated for 72 h atoAnd cialiswhat speed vs 6 M-1 M urea, and then against 10 mm NaHCO3and protein lyophilized. Amino acid sequence of the hybrid polypeptide determined on the basis of the nucleotide sequence shown in Fig.1A. The structure is confirmed by mass spectrum of MALDI-TOF and results tripticase hydrolysis.

Example 5. Expression, isolation and purification of rec 24-36 VIC.

To obtain protein using E. coli strain BL21 (DE3) containing plasmid retri (24-36). Expression, isolation and purification of the protein were carried out as described in example 4. Amino acid Posen in Fig. 1B.

Proteins used for immunization, conjugation with an enzyme and colloidal carbon enzyme-linked immunosorbent assay (ELISA) and dot-analysis.

Example 6. Indirect enzyme-linked immunosorbent assay (ELISA).

Antigens adsorb on the surface of the holes tablets ELISA plate (Greiner, 756071). For this purpose, solutions containing a mixture of antigens, which are the subject of the present invention is VIC rec 24-41 and VIC/2 rec 24-36 with concentrations of 8 ág/ml each in 0.05 M carbonate-bicarbonate buffer, pH 9.5 to contribute to the wells of tablets and incubated for 40 hours at room temperature. Then the solutions were removed and the tablets are dried for 2 hours at room temperature. For setting ELISA the wells contribute serum of HIV-infected patients or normal serum non-HIV-infected people, diluted in PBSAT (phosphate saline buffer with albumin and tween-20), and vyderjat for 1 hour at 37oC. Then the wells are washed 4 times with PBST (phosphate saline buffer with tween-20). Then in the wells made a conjugate of horseradish peroxidase to antibodies against human IgG (Sigma, no A 0170) working dilution of PBSAT and incubated for one hour at 37oC. Then the wells are washed 4 times with PBST. Later in the wells add the peroxidase substrate (o-phenylenediamine dehydrator in the dark for 15 minutes The enzymatic reaction stopped by the addition of 1N sulfuric acid and the optical density of the solution in the wells was determined by spectrophotometer reader MCC 340 at 492 nm. The results are shown in table. 1.

Thus, the indirect variant of ELISA based on recombinant proteins, which are the subject of this invention, provides recognition of 100% of HIV-positive sera (477 of 477) specificity 100% (1032 negative result on 1032 negative sample or a single positive result at 1032 negative sample).

This way is the production of enzyme immunoassay using as a developing reagent of alkaline phosphatase conjugated with antivirovym antibodies, but in this case, from solutions for sample dilution and conjugate and wash solution are eliminated phosphates, instead of using Tris, and applies a substrate for a phosphatase, such as paranitrophenylphosphate.

Enzyme-linked immunosorbent assay also performed as described in example 6, with the difference that as the detecting reagent is used conjugate And protein St. aureus with horseradish peroxidase.

Example 7. Enzyme-linked immunosorbent assay using labeled antasia mixture of purified antigens, which is the subject of the present invention is VIC rec 24-41 and VIC/2 rec 24-36 - at a concentration of 8 µg/ml each in 0.05 M carbonate-bicarbonate buffer, pH 9.5 to contribute to the wells of tablets and incubated for 40 hours at room temperature. Then the solutions were removed and the tablets are dried for 2 hours at room temperature. To obtain the indicator reagent is prepared ([13]) conjugates with horseradish peroxidase antigens that are the subject of the present invention is VIC rec 24-41 and VIC/2 rec 24-36. For setting ELISA the wells contribute serum of HIV-infected patients or normal serum non-HIV-infected people, diluted in PBSAT, and incubated for 1 hour at 37oC. Then the wells are washed 4 times with PBST. Then in the hole made by the conjugate of horseradish peroxidase with antigens that are the subject of the present invention, in a working dilution of PBSAT and incubated for one hour at 37oC. Then the wells are washed 4 times with PBST. Later in the wells add the peroxidase substrate (o-phenylenediamine of the dihydrochloride of 1.6 mg/ml in citrate-phosphate buffer, pH 5.5, with 0,006% H2O2) and incubated at room temperature in the dark for 30 minutes Enzymatic reaction stopped by the addition of 1N sulfuric acid, and DMS are shown in the table.2.

Thus, immunodeficiency option ELISA based on recombinant proteins, which are the subject of this invention, provides recognition 36 out of 36 (100%) WHICH positive sera specificity 100% (420 negative results on 420 negative sample or a single positive result at 420 HIV-negative samples).

Example 8. The dot-analysis (indirect variant, recombinant proteins, which are the subject of this invention, sorbed on the solid phase - nitrocellulose membrane, the indicator reagent is a protein St. aureus, conjugated with colloidal carbon).

For sorption prepare a solution containing a mixture of recombinant proteins, which are the subject of the present invention is VIC rec 24-41 and HIV 1/2 rec 24-36 with a final concentration of 200 μg/ml each (i.e. a total of 400 μg/ml) in carbonate-bicarbonate buffer 0.05 M, pH of 9.6. This solution was applied to the nitrocellulose membrane (Millipore, 5 μm), pre-marked into squares of side 1 cm, 1 ál (i.e., 0.4 μg of a mixture of antigens) on the point at the centre of each square, and dried in air at room temperature for 1 hour. The membrane is soaked in a blocking solution (2% BSA at a FSB of 0.05% Tween-20) for 2-20 hours, then extracted the corresponding reagent is used conjugate And protein with colloidal carbon. The result is read visually. A positive result appears as black spots (Fig.4).

In this way perform setting of the dot-analysis using as a detecting reagent conjugate antivitamin antibodies with colloidal carbon.

Similarly spend the dot-analysis in immunometric case, where the chimeric polypeptides that are the subject of this invention, sorbed on the solid phase - nitrocellulose membrane, and as the detecting reagent used chimeric polypeptides conjugated with kolloidnyi carbon.

Example 9. Immunogenic properties of soluble recombinant chimeric protein WHICH rec 24-41.

The immunogenicity of the drug test on laboratory animals. Rabbits breed "Chinchilla" weighing 2 kg subjected to immunization is the subject of this invention protein VIC rec 24-41 in physiological solution, 1 mg per animal. Repeated immunization spend 4 weeks after the first. Blood is collected from the ear vein and get the serum in the normal way. Of mice (CBA x S B1/6) F1 females, weighing 16-18 g subjected to immunization intraperitoneally is the subject of this invention protein VIC rec 24-41, 100 µg in 0.5 ml of physiological age is the cut 7 days after immunization and get the serum as usual.

The immune response was determined by ELISA. For carrying out ELISA antigens adsorb on polystyrene tablets Greiner 756071. The antibody titer is determined in indirect ELISA and sandwich version. In indirect ELISA developing reagent in the analysis of the sera of mice is the conjugate of horseradish peroxidase to antibodies goat against mouse IgG (Sigma, A 3673), and the analysis of the serum of the rabbit - conjugate And protein St. aureus with peroxidase ([13]). For the sandwich options are preparing conjugates of recombinant antigens with peroxidase ([13]). Substrate for the colorimetric reaction is (o-phenylenediamine of the dihydrochloride of 1.6 mg/ml in citrate-phosphate buffer, pH 5.5, with 0,006% H2O2). The results recorded at 492 nm using a spectrophotometer MCC 340. For each dilution of serum perform three simultaneous determination and calculate the average value And492For titer take this serum dilution at which25% exceeds the level of the slice. Because the drug is a chimeric polypeptide containing a full-sized protein P24 VIC and merged with him a fragment of gp41 WHICH, investigate the immune response to both of these fragments. Table 3 shows the results of the study with anantnag proteins: VIC gp41, with only one HIV-specific fragment - part of gp41, WHICH R24, containing only one HIV-specific fragment of a full - sized R24, and WHICH rec24-41 containing both HIV-specific fragment - part of gp41 and full-R24, i.e. the same antigen that was used for immunization.

From table 3 it is seen that in response to the introduction of the chimeric polypeptide is developing a strong immune response as gp41, and P24. The difference in the titles of certain indirect and sandwich ELISA, due to different conditions of presentation of antigen on the solid phase and the related different accessibility determinants.

Example 10. The functional activity of antibodies against chimeric soluble recombinant antigen VIC rec 24-41.

Evaluation of the affinity of chimeric recombinant antigen VIC rec 24-41 viral prototype and the ability of antibodies raised by animals (mice and rabbits, example 9) when the specified immunization with antigen, to learn proteins culture of the virus is performed using a Western blot test. To do this, use strips of nitrocellulose from commercial set LavBlot (Sanofi Pasteur, France), which, after separation in SDS-PAAG, transferred proteins cultural VIC. Strips treated divorced in PBSAT siemenia, then washed 3 times with PBST and treated with indicator reagents. As indicator reagents are peroxidase conjugate with antibodies goat against mouse IgG (Sigma, A 3673) (when the detection specificity of murine antibodies) and peroxidase conjugate with a protein (detection specificity rabbit antibodies). Antibodies induced in animals in response to immunization with the recombinant protein, learn gp41 and P24 culture of the virus, as well as the precursor protein, P24-P55 (Fig.5). Therefore, the antibodies induced by recombinant protein that specifically recognize the natural viral protein, which indicates that the functional activity of antibodies and the similarity of the structure recombining protein and viral prototype.

The preceding description and examples are given as illustrations and do not exhaust the possibilities of the present invention (sequence given at the end of the description).

Literature
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Claims

1. Immunogenic composition comprising the chimeric polypeptide of HIV, characterized by the fact that she is a full-sized protein P24, merged with the immunodominant fragment of gp41 containing amino acids plot 575-616 or selected from him (goes 24-41), or merged with immunodominant fragment gp36 containing amino acids plot 575-616 or selected from him (goes 24-36), and a pharmacologically suitable carrier and/or diluent.

2. Method of induction of an immune response to VIC or VIC/2 in mammals, characterized in that the mammal is administered the immunogenic composition according to p. 1, while for the induction of an immune response to WHICH mammal is administered immunogenic composition, representing pobrania from it (goes 24-41), and for the induction of an immune response to VIC/2 mammal is administered immunogenic composition, representing the full-size protein P24, merged with the immunodominant fragment gp36 containing amino acids plot 575-616 or selected from him (goes 24-36).

3. The chimeric polypeptide of HIV, characterized in that in the case WHICH he is a full-sized protein P24, merged with the immunodominant fragment of gp41 containing amino acids plot 575-616 or selected from him (goes 24-41), and in the case WHICH/2 it is a full-sized protein P24, merged with the immunodominant fragment gp36 containing amino acids plot 575-616 or selected from him (goes 24-36).

4. The chimeric polypeptide of HIV-p. 3, characterized in that the immunodominant fragment of gp41 has the following amino acid sequence: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3.

5. The chimeric polypeptide of HIV-p. 3, characterized in that the immunodominant fragment gp36 has the following amino acid sequence: SEQ ID NO:4; SEQ ID NO:5; SEQIDNO:6.

6. A DNA molecule encoding a chimeric polypeptide of HIV-p. 3, representing a sequence of bases of SEQ ID NO:7 for chimeric polypeptide WHICH (goes 24-41) and SEQ ID NO:8 for chimeric polypeptide VIC/2 (goes 24-36).

7. Plasmids is on p. 7, characterized in that for expression of a chimeric polypeptide WHICH (goes 24-41) he is a plasmid 15b into which is inserted at the restriction site Barn H1 DNA fragment SEQ ID NO:7, and the restriction sites Hind III - Eco R1 inserted par locus.

9. Plasmid vector under item 7, characterized in that for expression of a chimeric polypeptide WHICH/2 (goes 24-36) he is a plasmid 15b into which is inserted at the restriction site You H1 DNA fragment SEQ ID NO:8, and the restriction sites Hind III-Eco R1 inserted par locus.

10. Polyclonal antibodies to VIC or VIC/2, representing the immunoglobulins of the IgG class 2a, obtained by immunization with the chimeric polypeptide according to p. 3 with a titer of at least 30,000 to the fragment of gp41 and gp36 and titer not less than 200000 to R24 in indirect ELISA.

11. Test-system for detection of antibodies to VIC and VIC, including HIV antigens absorbed on a solid substrate, and the detecting reagent, characterized in that as antigens it contains a chimeric polypeptides under item 3, the antigen WHICH is a full-sized protein P24, merged with the immunodominant fragment of gp41 containing amino acids plot 575-616 or selected from him (goes 24-41), and antigen VIC/2 is a full-sized protein P24, merged with immunode the EMA on p. 11, characterized in that as the detecting reagent is used conjugate antivitamin antibodies to horseradish peroxidase.

13. The test system on p. 11, characterized in that as the detecting reagent is used conjugate antivitamin antibodies with alkaline phosphatase.

14. The test system on p. 11, characterized in that as the detecting reagent is used conjugate And protein St.aureus with horseradish peroxidase.

15. The test system on p. 11, characterized in that as the detecting reagent is used conjugate antivitamin antibodies with colloidal carbon.

16. The test system on p. 11, characterized in that as the detecting reagent is used conjugate And protein St.aureus with colloidal carbon.

17. The test system on p. 11, characterized in that as the detecting reagent is used chimeric polypeptides under item 3, conjugated to horseradish peroxidase.

18. The test system on p. 11, characterized in that as the detecting reagent is used chimeric polypeptides under item 3, conjugated with colloidal carbon.

 

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