Artificial protein immunogen tci containing multiple ctl epitopes of the major antigens of hiv-1, artificial gene tci encoding polyepitopic protein immunogen tci

 

The invention relates to molecular biology and genetic engineering, specifically to the creation of synthetic polyepitope vaccines against HIV-1. Artificial protein immunogen TCI contains CTL-epitopes as many as possible of the major antigens of HIV-1 to use as a safe and effective DNA vaccines for HIV-1. The length of the artificial protein is a 392 amino acid residue (Fig.2). Designed protein contains about 80 epitopes of proteins Env, Gag, Pol, Nef of HIV-1. The artificial gene synthesis TCI (Fig.3), make use of a PCR method for the amplification of fragments of the genome of HIV, continuous coding regions of protein TCI. Genetically engineered design, coding complete set of epitopes, cloned in the composition expression vector plasmids in E. coli cells. Production of recombinant protein TCI amounted to 10-20% of the total cellular protein. The presence of protein fragments of HIV-1 in the structure of the target immunogen confirmed using ELISA using a panel of HIV-1 positive sera and MAT to protein P24 (Fig.2 and 3 presented in the description). 2 C. p. F.-ly, 4 Il., table 4.

The invention relates to molecular biology and genetic engineering, specifically to the creation of various the implement various forms of viral antigen, including inactivated virus, the modified virus, live attenuated virus, native proteins, genetically engineered peptides and recombinant bacteria, viruses and plasmid DNA [1]. Each of these strategies was assessed, however, the problems of anti-HIV-1 vaccine is still not solved.

One of the promising approaches to the creation of a new generation of safe and effective vaccines based on the identification of viral proteins of T - and b-cell epitopes and the creation of synthetic polyepitope vaccines [2, 3]. Such vaccines should be free from many of the defects which are peculiar to the vaccine, created on the basis of live attenuated and inactivated whole virus or on the basis of native viral proteins. In particular, such a vaccine should not contain unwanted epitopes that induce immunopathology or inhibit protective immunity. Therefore, genetically engineered structures have the potential to improve the immune response against HIV in relation to the response, which is induced by the natural HIV infection.

The proposed approach was used to design the four--spiral artificial protein TBI (T and In Cell Epitopes Containing Immunogen), Copa and five b-cell neutralizing epitopes from proteins of HIV-1 Env and Gag. Mouse immunized with a synthetic protein TBI, showed both humoral and cellular (proliferative responses to HIV-1. Anti-TBI-antibodies had HIV-neutralizing activity.

The results indicate the viability of this approach. Together with this proposed design has some limitations. First, it has a relatively limited antigenic potential (only 9 epitopes). Secondly, it is not shown that this design has a virus-neutralizing activity against primary isolates. Indeed, it was shown that in the case of HIV infection creating vaccines that induce neutralizing antibodies, is a very complex task, because such vaccines were effective only against laboratory HIV strains and were not able to neutralize primary (field) viral isolates [4]. While the escape of the virus from neutralization was not due to the emergence of escape mutants. The explanation of this phenomenon was made possible thanks to the crystal structure analysis of the protein gp120, which revealed a number of mechanisms by which the virus prevents effective induction of antibodies [5].

Therefore, in recent years the focus of many vaccine design is casatella, the responses of cytotoxic T-lymphocytes (CD8+CTL) associated with HIV infection, are important mediators of antiviral immunity and, consequently, HIV-specific CTL can be an important component of an effective vaccine against HIV-1 [6, 7]. It is shown by evidence that in the plasma of HIV-infected individuals find the significant inverse correlation between the frequency of HIV-specific CTL and the load of viral RNA [8]. It is assumed that CTL can protect against HIV infection, as CTL kill HIV-infected cells before they produce new virions [9], and, in addition, CTL release chemokines that inhibit HIV infection [10, 11]. Therefore, we focus our efforts on the development of vaccines that induce CTL responses.

Currently, there are several synthetic vaccine constructs containing multiple CTL epitopes of HIV-1. In particular, described polyepitope design, containing seven adjacent minimum HL-A2-restrictively CTL-epitopes [12]. HLA-A2-transgenic mice immunized with recombinant vaccinia virus coding for CTL-polyepitope, generate CTL-specific responses to each enabled epitope. In addition, the epitope-specific the products. These data illustrate the suitability of the proposed approach for the design of HIV vaccines.

More complex vaccine design, containing 20 overlapping CTL epitopes person from proteins of HIV-1 Gag, Pol, Env and Nef, restrictively twelve HLA class I molecules was proposed by Hanke et al. [13 prototype]. In this design were also included one mouse and three monkey epitope for testing immunogenicity, and choose the optimal doses and vaccination regimens in experimental animals. For delivery and expression obtained vaccine constructs were used DNA plasmid and modified virus Ankara (MVA). Received vaccine candidates induced CD8+CTL and production of IFN-y after a single immunization of mice [13, 14]. In addition, it was shown that a combined regimen of vaccination, in which mice were first premirovany DNA vaccine, and then busterboy MVA, was the most optimal (efficient) Protocol for the induction of both IFN-and CD8+CTL [15]. A combined regimen of vaccination (DNA-MVA) was also effectivnes the immunization of monkeys, rhesus Macaque, which showed a high frequency of circulating CTL comparable to that which was observed in SIV-infec PITOMNIK DNA and MVA vaccines it may be effectivnes for the induction of high levels of T-cell immunity in humans.

Together with this, an effective vaccine in addition to the induction of high-level responses of cytotoxic T-lymphocytes for selected epitopes must also provide an immune response against a fairly wide range of CTL epitopes. The above prototype poly-TL-epitope DNA vaccine contains 20 CTL-epitopes of HIV-1 that is not more than 10% of all identified currently CTL-epitopes (see Los Alamos HIV Molecular Immunology Database), which cannot guarantee the high efficiency of this vaccine.

Accordingly an object of the invention is the creation (design and construction) of the new artificial poly CTL-epitope T-cell immunogen containing CTL epitopes as many as possible of the major antigens of HIV-1, for use as safe and effective DNA vaccines against HIV-1.

The problem is solved by developing a selection strategy epitopes involved in the induction of HIV-1-specific CTL responses in infected individuals.

It is known that viral infection induces CD8+CTL responses by presenting viral antigen in conjunction with MHC molecules of class I (Major Hisrocopatibi not as full-sized proteins, and as short peptides (8-12 A. K.), associated with specific alleles of MHC class I [18, 19]. These short antigenic epitopes appear from viral cytoplasmic proteins in the proteasome-mediated processing [20, 21]. Because the distribution of HLA alleles varies for populations of different geographical regions, an effective vaccine against AIDS must contain epitopes, restrictionenzyme different HLA alleles to cover the genetic diversity of the molecules of the major histocompatibility complex (MHC) class I for the population of the geographic region.

Effective polyepitope CTL vaccines, except for certain HLA specificity must also contain a large number of conservative epitopes involved in the induction of both CD8+CTL and CD4+Th-lymphocytes. Now identifitsirovana enough of these epitopes. Their amino acid sequences are summarized in Los Alamos HI V Molecular Immunology Database. To construct a CTL immunogen, candidate DNA vaccine were selected those that met the following criteria:

1. Were selected epitopes presented in the Los Alamos HIV Molecular Immunology Database, which induce both CD8+CTL and CD4+Th and yavlyaetsya.

2. Epitopes were selected from the major viral protein antigens Env, Gag, Pol and Nef, which induce CTL responses, which are important components of HIV prevention.

3. In those cases, when it was known, in consideration taken epitopes that induce CTL that recognize the corresponding naturally processed epitopes.

4. Was chosen, if possible, the so-called optimal epitopes that studies titration overlapping truncated peptides defined as the minimal epitopes that cause the most effective sensitization CTLs that are specific to certain molecules MHC class I.

5. Considered CD8+CTL epitopes, which together restrictively ten different optimally matched alleles of HLA class I. As you know, this is enough to cover the genetic diversity of antigens on MHC class I in the population virtually any geographic area [13, 22, 23]. It was chosen epitopes specific to HLA alleles, which are most common in the population of Russia. These include those antigens HLA class I and II, which are found in 20% of cases, namely A1, A2, A3, A9, a10, Aw19, B7, B12, V, Cw3, Cw4, DR1, DR2, DR3, DR4, DR5, DR7 (see tab. 1).

In the result of the analysis for COSM above criteria. Such epitopes are listed in table.2 and 3. It should be noted that some of the listed in the table.2 epitopes able to contact additional HLA molecules, which were not originally taken into consideration, because their alleles have low frequency of occurrence in the population. It can be assumed that such epitopes recognized by several HLA alleles may increase the potential generated on the basis of the vaccine.

As a strategy for inclusion (Union) set selected from different epitopes of viral proteins in a single polypeptide chain was chosen approach, based on the aggregation of epitopes as overlapping peptides. In this case, overlapping epitopes are located relative to each other as well as in native proteins. This overlapping epitopes allows to minimize the total length of the immunogen. It turned out that in the present case, most of the selected epitopes (table. 2) is mapped to the sequence of native viral proteins as partially or fully overlapping peptides and localized in multiple contiguous regions (table. 3). Such antigen-active regions containing overlapping epitopes were identified and used to design a poly-what about the immunogen, candidate for use as DNA vaccines against HIV-1. Further to refer to this immunogen will be used abbreviation TCI (T Cell Imunogen). The overall design TCI-immunogen is shown in Fig 1. The length of the artificial protein is a 392 amino acid residue (sequence shown in Fig.2). Designed protein contains about eighty epitopes (as CD8+CTL and CD4+Th), many of which overlap and together restrictively ten different HLA alleles. To explore the CTL responses induced by DNA vaccines in experimental animals, the composition of the target immunogen includes additional epitopes presented by MHC molecules of class I mice and monkeys rhesus Macaque.

The problem is solved by constructing an artificial gene encoding TCI immunogen.

Note that the sequence of a gene encoding a protein TCI (392 A. K.) is relatively long and is 1176 nucleotides (nucleotide sequence shown in Fig.3). Therefore, the total chemical synthesis of a gene TCI is rather time-consuming task. An alternative and more efficient approach to the synthesis of the gene TCI may be based on the use of the method concern is poly. Indeed, as already noted, CTL immunogen contains fragments that are sufficiently long and continuous amino acid sequences of native viral proteins. That is why the idea of synthesis of the target gene TCI PCR is quite attractive, as it allows for the amplification of fragments of the target gene native sequence of the genome of HIV-1. The most suitable candidate for this purpose was the genome sequence of strain BH-10 HIV-1 [24], since the corresponding amino acid sequence of the viral proteins and TCI coincide. A match is found can be used for amplification of fragments of the target gene TCI wherever possible, the sequence of the genomic cDNA of HIV-1 (strain BH-10).

In accordance with the overall design TCI immunogen (Fig.1) the sequence of a gene encoding a protein TCI, was divided into three blocks (Fig.4). Block 1 contains fragments encoding CTL-epitopes gag gene, unit 2 - epitopes of the env gene and block 3 - epitope genes pol and nef. They developed a unique block amplification of the gene TCI, according to which fragments of the target gene are synthesized using a set of overlapping primers for basmati is trice - genomic cDNA and thermophilic polymerases. The required primers were designed synthesized (table. 4), after which the fragments (blocks 1, 2 and 3) were obtained according to the proposed scheme (Fig.4).

To clone blocks 1-3 gene TCI in E. coli cells was used previously developed vector plasmid pFH123 [25]. This plasmid carries out specific polylinker containing sites for SII-endonuclease restriction, allowing to obtain the cloned DNA fragments with the planned unique sticky ends. Cloning of block 1 in plasmid pFH123 was carried out at the sites Wamn and XhoI/SalI, a units 2 and 3 sites BamHI, SalI. The use of plasmids pFH123 for cloning fragments of the target immunogen provides an unambiguous Assembly full gene TCI. To this end, the blocks of the target gene were isolated from recombinant plasmids pFH123 (unit 1 through the restriction sites BamHI and FokI, block 2, FokI restriction enzymes cut sites and block 3 on the websites of the FokI restriction and SalI). To provide a clear “cut” cloned blocks, primers (table. 4) were laid synonymous nucleotide change leading to the disappearance of the restriction sites of AMN, and SalI I and FokI. After allocating blocks 1-3 and SalI were simultaneously cloned into the vector plasmid pFH123 sections retirada blocks and complete sequence of a target gene, were selected standard procedures screening using PCR and restriction analysis. The structure of all cloned sequences were confirmed by sequencing. Fix mutations introduced Taq polymerase was carried out on the basis of stepwise amplification of the gene sequence TCI using the correcting oligonucleotide primers. Thus, the proposed scheme allows us to synthesize the target gene corresponding to a given structure that directs the synthesis of synthetic recombinant immunogen TCI.

Note that the technology block Assembly has a number of advantages:

1) independent synthesis and cloning of three different fragments of the gene TCI actually leads to three different CTL-immunogenum, immunogenic and protective properties which can be studied individually;

2) combinations of blocks allow fast enough to synthesize a whole new set of genes (including target gene TCI);

3) introduction to one of the source sequences are of special sites allows you to get new designs genes by adding sequences coding for new epitopes.

To prove that artificial protein TCI is immunoactive in HIV-1-positice, encoding a full set of epitopes, was cloned in E. coli cells in the series of vector expression plasmids. Production of recombinant protein TCI amounted to 10-20% of the total cellular protein. The presence of protein fragments of HIV-1 in the structure of the target immunogen confirmed using ELISA using a panel of HIV-1-positive sera and monoclonal antibodies to the protein P24.

Thus, the obtained data confirm that the immunochemical properties of the product of gene expression TCI matches the target immunogen TCI.

The list of graphical materials

Fig.1. The overall design TCI-immunogen, a candidate vaccine against HIV-1. The rectangles labeled sequences of proteins of HIV-1 containing the selected epitopes. The position of the individual epitopes are indicated by lines. Restrictions epitopes on antigens of the major histocompatibility complex of humans, mice and monkeys marked by letters (HLA-A, B, Cw - human, H-2a, b, d, f, k, p, u, q - mouse, Mamu - A*01 - Macaca mulatta). Th - helper epitopes.

Fig.2. Amino acid sequence synthetic protein immunogen TCI.

Fig.3. The nucleotide sequence of the synthetic gene TCI.

- restriction site NheI;-the Kozak sequence;- initiating codon;stop-codon.

Fig.4. The scheme of synthesis and Assembly of a gene TCI.

Stages PCR (vertical arrows) are numbered in Arabic numerals. Oligonucleotides primers (horizontal arrows) are numbered in Roman numerals. Alternating black and gray rectangles within blocks of the gene TCI correspond to the sequences of the genome of HIV-1, encoding the continuous regions of the protein TCI. In oligonucleotides 1, 5, 7, 10, 15, 22 laid the sites of hydrolysis of restricts for cloning in the vector pFH, and oligonucleotides 2, 3, 5 introduced the “silent” mutations, leading to the disappearance of the FokI site. In addition, oligonucleotide 7 entered the initiating codon ATG, and the oligonucleotide 5 is a stop codon.

For a better understanding of the essence of the present invention the following are examples of its implementation.

Example 1. The design (synthesis) artificial gene TCI

A list of all oligonucleotide primers for Assembly blocks gene TCI presented in table.4. Previously prepared solutions of all oligonucleotide primers at a concentration of 10 pmol/µl, i.e., a 10 mm solution of mixed four deoxynucleotides (dNTP) at a concentration of 5 mm each is literatu with an activity of 10 units act./ál (hereinafter polymerase and buffer for Tte DNA polymerase X10 (the buffer). At all stages used distilled deionized sterile water (the water).

1.1. The receiving unit 1 gene TCI

Were prepared reaction mixture for PCR the following composition:

Water - 70 µl; buffer - 10 ál; cDNA 1 μl; dNTP - 16 μl; primer 7 - 1 μl; primer 8 - 1 ál; Taq polymerase - 1 mm. Mixture to obtain a fragment A.

Water - 70 µl; buffer - 10 ál; cDNA 1 μl; dNTP - 16 μl; primer 9 - 1 μl; primer 10 - 1 mm; polymerase - 1 mm. Mixture to obtain a fragment Century

Conditions for the reactions of:

92C - 0.8 min; 48S - 0,6 min; 70C - 0.5 min 2 cycles;

92S - 0,6 min; 52S - 0,6 min; 70C - min 0,2 - 13 cycles;

92With - 1,2 min; 56With - 1,2 min; 70With - 1,2 min - 1 cycle.

Upon completion of the reaction in each reaction mixture were added 100 μl of chloroform and 100 μl of phenol, was intensively shaken and centrifuged in a centrifuge type Eppendorf (12000 g) for 1 min the Aqueous phase was transferred to another test tube, add 10 μl of potassium acetate 3 M and 250 μl of ethanol were mixed, videris what canola, centrifuged and dried at room temperature for 20 minutes DNA fragments were separated in a 6% polyacrylamide gel (PAG), the strips of the gel containing the PCR products of the estimated length, washed in water at room temperature for 1 hour, then carefully crushed, was added 500 μl of water and kept at 37C for 16 hours, the Suspension of the gel was centrifuged at 12000 g for 3 min and the upper phase containing the PCR fragments and carefully transferred to another tube.

To obtain a PCR fragment was prepared With the reaction mixture of the following composition:

water - 57 μl; buffer to 10 μl; dNTP - 16 μl; fragment - 5 μl; primer 10 - 6 μl; primer 11 - 1 μl; primer 12 - 1 μl; primer 13 - 1 μl; primer 14 - 2 ál; Taq polymerase - 1 µl.

The conditions of the reaction of:

92With - 1,2 min; 45S - 0,6 min; 70C - 0.5 min 2 cycles;

92S - 0,6 min; 50S - 0,6 min; 70With - 0,3 min - 22 cycle;

92With - 1,2 min; 52With - 1,2 min; 70With - 1,2 min - 1 cycle.

The PCR fragment was isolated from the reaction mixture according to the scheme described above for the PCR-fragment A.

For PCR srisa - 1 µl.

The conditions of the reaction of:

92With - 1,2 min; 45C - 1.5 min; 50C - 1.5 min; 70C - 1.5 min - 1 cycle; then the reaction mixture was added primer 7 and primer 10 to 1 μl and PCR was performed under the following conditions:

92C - 0.8 min; 50C - 0.8 min; 70S - 0,6 min - 19 cycles;

92With - 1,3 min; 52With - 1,2 min; 70C - 1.5 min - 1 cycle.

The target PCR fragment was treated with a mixture of phenol-chloroform, besieged and dried, as described above, is then hydrolyzed in restrictase BamHI and XhoI in 100 μl of reaction mixture at 37C for 16 h, besieged and were separated by electrophoresis in 4% SDS page the same as the above. After elution of DNA fragments from a gel material besieged, washed and dried as described above. DNA was dissolved in 20 μl of TE buffer to a final concentration of 0.1 pmol/μl. Kept at -20C.

1.2. The receiving unit 2 gene TCI

For PCR reactions with primers II unit mixed:

Water - 70 µl; buffer - 10 ál; cDNA 1 μl; dNTP - 16 μl; primer 15 - 1 μl; primer 16 - 1 mm; polymerase - 1 mm. The mixture for poluchenii. Mixture to obtain a fragment that is

Water - 70 µl; buffer - 10 ál; cDNA 1 μl; dNTP - 16 μl; primer 21 - 1 μl; primer 22 - 1 mm; polymerase - 1 mm. Mixture to obtain a fragment F.

Conditions for the reactions of:

92C - 0.8 min; 48S - 0,6 min; 70C - 0.5 min 2 cycles;

92S - 0,6 min; 52S - 0,6 min; 70C - min 0,2 - 13 cycles;

92With - 1,2 min; 56With - 1,2 min; 70With - 1,2 min - 1 cycle.

Purified fragments D, E, F were obtained similarly to fragment A.

To obtain the fragment G was mixed:

Water - 62 μl; buffer to 10 μl; dNTP - 16 μl; fragment E - 5 ál; fragment F - 5 ál; Taq polymerase - 1 µl.

The conditions of the reaction of:

92With - 1,2 min; 45With - 1,2 min; 50With - 1,2 min; 70With - 1,2 min - 1 cycle; then the reaction mixture was added primer 19 and primer 22 1 μl and PCR was performed under the following conditions:

92C - 0.8 min; 50C - 0.8 min; 70With only 0.4 min - 19 cycles;

92With - 1,2 min; 55With - 1,2 min; 70The conditions of the reaction of:

92With - 1,2 min; 45S - 0,6 min; 70With only 0.4 min - 1 cycle;

92S - 0,6 min; 50S - 0,6 min; 70S - 0,2 min - 18 cycles;

92With - 1,2 min; 52With - 1,2 min; 70C - 0.8 min - 1 cycle.

Receiving target PCR fragment, containing II block gene TCI, were performed similarly to the block Assembly 1, using the appropriate primers block 2.

The selection of PCR-fragment unit II gene TCI was performed according to the scheme described for block 1. For the formation of 5 - protruding sticky ends instead of restrictase XhoI used SalI.

1.3. The receiving unit 3 gene TCI

For PCR reactions with primers unit 3 was mixed:

Water - 70 µl; buffer - 10 ál; cDNA 1 μl; dNTP - 16 μl; primer 1 - 1 μl; primer 2 - 1 ál; Taq polymerase - 1 mm. The mixture to obtain the fragment K.

Water - 70 µl; buffer - 10 ál; cDNA 1 μl; dNTP - 16 μl; primer 3 - 1 μl; primer 4 - 1 ál; Taq polymerase - 1 mm. Mixture to obtain a fragment of L.

Conditions for the reactions of:

92C - 0.8 min; 48S - 0,6 mi is 6 min; 70C - min 0,2 - 13 cycles;

92With - 1,2 min; 56With - 1,2 min; 70C - 0.8 min - 1 cycle.

To the reaction mixture fragment M mixed:

Water - 70 µl; buffer - 10 ál; cDNA 1 μl; dNTP - 16 μl; primer 5 - 1 μl; primer 6 - 1 mm; polymerase - 1 µl.

The conditions of the reaction of:

92With - 1,2 min; 48S - 0,6 min; 70C - 0.8 min 2 cycles;

92S - 0,6 min; 52S - 0,6 min; 70With only 0.4 min - 13 cycles;

92With - 1,2 min; 56With - 1,2 min; 70C - 0.8 min - 1 cycle.

Purified fragments K, L, M singled out like fragments a, b, C, D, E.

Unit 3 was designed using the received fragments K, L and M, which were mixed with the following components:

water - 57 μl; buffer to 10 μl; dNTP - 16 μl; fragment of a K - 5 ál; fragment of L - 5 ál; fragment of M - 5 ál; Taq polymerase - 1 µl.

The conditions of the reaction of:

92With - 1,2 min; 45C - 1.5 min; 50C - 1.5 min; 70With - 1,2 min - 2 cycles; then the reaction mixture was added to primer 1 and primer 5 1 ál and p://img.russianpatents.com/chr/176.gif">S - 0,6 min - 19 cycles;

92With - 1,3 min; 52With - 1,2 min; 70C - 1.5 min - 1 cycle.

Further processing of the PCR fragment in block 3 was similar to the scheme described for block 2.

1.4. Cloning a PCR fragment containing the blocks 1, 2 and 3 gene TCI

10 μg of plasmid pFH123 were digested with restrictase BamHI and SalI in 100 μl of reaction mixture for 6 h at 37C, then the vector portion of the plasmid was isolated according to the scheme described for pre-engineered gene TCI. The concentration of vector brought to 0.1 pmol/μl.

The crosslinking of each of the selected fragment and vector were mixed three ligase mixture of the following composition:

Water - 38 μl; legasy buffer (10) - 5 ál; vector 1 μl; block 1 - 5 ál; T4-DNA-ligase (20 units act./ál) - 1 µl. The mixture was stirred for 16 hours at 5C.

For the second and third blocks of the ligase mixture was obtained according to the scheme described for block 1.

10 μl of ligase mixture was used to transform competent cells of E. coli Xlbluel by selection on medium containing 50 μg/ml ampicillin. Recombinant DNA was isolated by the standard method [26], was then digested with restrictase Wamn and > PST and analyzed by electrophoresis on under what generowanie method Singer. The plasmid containing the target gene sequence TCI corresponding raschetnym, were designated as plasmid pFHblockl, pFHblock2, pFHblock3.

Example 2. Construction of recombinant plasmids pFH-TCI

To obtain plasmids pFH-TCI containing the full target gene TCI, used the vector described in example 1.4, and three fragments of the gene TCI, which were isolated from plasmids pFHblockl, pFHblock2, pFHblock3. This plasmid pFHblockl were digested with restriction enzyme Wamn, and then by the restriction enzyme FokI and allocated fragment block 1/BamHI-FokI similar to that described in example 1.1. Plasmid pFHblock2 were digested with restriction enzyme FokI and allocated fragment block2/FokI-FokI. Plasmid pFHblock3 were digested with restriction enzyme SalI, and then FokI and allocated fragment block3/SalGI-FokI. The concentration of the selected blocks resulted in an amount equal to 1 pmol/μl.

To obtain recombinant plasmids with full genome TCI mixed:

water - 26 μl; legasy buffer (10) - 5 ál; vector 2 µl; block I/BamHI-FokI - 5 ál; block II/FokI-FokI - 5 ál; block III/FokI-SalI - 5 ál; T4-DNA-ligase (20 units act./ál) and 2 ál.

The conditions of the reaction and selection of recombinant clones were similar to those described for the plasmid pFH1block, pFH2block, pFH3block in example 1.4.

The structure of the gene sequence TCI made of 3 blocks in recombinant plasmides, gene expression TCI in E. coli cells

To obtain recombinant plasmids pET-TCI plasmid pet-32A (Novagen) sequentially hydrolyzed by the restriction endonucleases BamHI and SalGI under standard conditions. Vector pET32a/BamHI-SalGI allocated according to the scheme described for vector pFH123/BamHI-SalGI in example 1.4. Bam-Sal fragment of the gene with a 5'-protruding sticky ends was obtained by hydrolysis of plasmid pFH-TCI same restrictases similar to that described in example 2.

Was prepared following the ligase mixture of:

vector 1 μl (0.05 pmol); fragment - 5 ál (0.5 pmol); legasy buffer (10) - 2 ál; water - 11 ál; T4DNA ligase (20 units act./mm); total volume of 20 µl. Ligase, the mixture was stirred overnight at 5°C. 10 μl of the mixture was used for transformation Rb-competent cells JM109. Analysis of the recombinant clones was performed by the method of amplification of their DNA using a pair of primers included in the 2nd block of the gene TCI, as well as supporting the restriction of recombinant DNA specified restrictase. 10 ng of the resulting plasmid pET-TCI was used to transform competent cells of E. coli HELL 494 (pLys).

For expression of recombinant gene TCI 5 ml overnight culture of E. coli cells the HELL 494 (pLys), transformed with the plasmid pET-TCI, resuspendable in 50 ml of LB medium with ampi is 0,8-0,9. To the culture was added IPTG up to 1 mm concentration of inducer and cultured for another 4 hours. Cell biomass was collected by centrifugation at 3000 rpm./min for 10 minutes Then cells resuspendable in 5 ml of PBS buffer1 (0.02 M Na-phosphate buffer pH 7.5; 0.15 M NaCl) with a solution of MgCl2benzamidine and lysozyme concentrations up to 5 mm, 0.5 mm and 1 mg/ml, respectively. The mixture was stirred for 15 min at room temperature, and the cells were destroyed by three freeze in nitrogen, followed by rapid thawing in a water bath at 65C. Cell hydrolysate was centrifuged 20 min at 12 thousand rpm, the supernatant was removed, and the residue resuspendable in 3 ml of 7 M urea, 10 mm Tris pH 8, 0.1 M NaCl, 5 mm DTT and gently stirred 5 hours at room temperature. The extract was centrifuged for 30 min at 16 thousand rpm the Supernatant was used for determination of specific immunochemical activity within it the recombinant protein TCI containing the determinants of HIV-1.

Example 4. The definition of specific immunochemical activity of the recombinant protein TCI

The resulting solution containing TCI-immunogen, was adsorbed on 100 µl of the hole in the polystyrene tablet p as a few positive control (K+) were used solutions P24 protein of HIV-1, R envelope protein of HIV-1, and the lysate eukaryotic cells infected with HIV-1, with a final concentration of 2-4 µg/ml as two negative controls ( -) were used solution lysate proteins untransformed cells of E. coli obtained similar fractions of recombinant protein TCI, and the lysate of uninfected eukaryotic cells. Sorption positive and negative controls antigenic material was performed under the conditions described above.

After sorption, the plate was shaken and the wells were added to 150 µl of blocking solution (0.2% casein, 0.1 M sodium phosphate buffer, pH 7.5, 0.05% tween), kept for 1 hour at 25And the tablet is then thoroughly washed 4 times with water. The reaction of immunochemical binding to the immobilized antigens was performed by standard methods ELISA analysis using solutions of liofilizovannyh polyclonal serum P24 protein, envelope protein R, vysokotirazhnoj serum viral proteins of HIV-1 and ascitic fluid of mouse monoclonal obtained (288-309) determinants of protein P24 (instruction test systems “Recombinant anti-HIV 1+2”, the production company “Vector-best”, FS-3847-99). All cultivation of recombinant antitime protein of HIV-1 virus. IgG antibodies two monoclonal sera 29F2 and A steadily worked on the tablet with antigens P24 protein lysate eukaryotic cells infected with HIV-1, as well as with recombinant protein TCI.

ELISA activity of the above negative controls was low and did not exceed background values.

Thus, for the first time successfully carried out the design and construction of artificial protein immunogen TCI containing a set of 80 CTL-epitopes of proteins Env, Gag, Pol, Nef HIV-1, which causes the development of a cytotoxic immune response.

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Claims

1. Artificial protein immunogen TCI containing multiple CTL epitopes of the major antigens of HIV-1 having the amino acid sequence shown in Fig.2.

2. Artificial gene TCI encoding polyepitopic protein immunogen TCI having the nucleotide sequence shown in Fig.3.

 

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