Recombinant gene structure for inducing immune response, recombinant polypeptide and vaccine

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to obtaining genetically engineered vaccines and can be used in medicine. A recombinant gene structure containing a series of human SLC gene, antigen gene and gene Fc-fragment of IgG 1 is obtained.

EFFECT: invention considerably increases efficiency of the immune response of the body to the introduced antigen compared to existing antigen structures.

10 cl, 15 dwg, 1 tbl, 12 ex

 

The SCOPE of the INVENTION

This invention relates to new recombinant gene sequence of the hybrid protein expressed from this gene, and vaccine derived from this gene, as well as various genetic vaccines obtained by replacing the gene of the antigen in the recombinant sequence, or the use of products expression as vaccines hybrid peptides.

The LEVEL of TECHNOLOGY

Since the opening of secondary lymphoid of the human chemokine (SLC). secondary lymphoid tissue chemokine) he was paid a lot of attention. SLC regulates the secretion of cytokines, changes the immunosuppressive status of the tumor microenvironment and promotes the development of Th1. Thus, SLC useful in antitumor immunity. Further, the SLC has an impact on attracting lymphocytes and dendritic cells (DC, dendritic cells). Intratumoral injection induces local and systemic antitumor immune responses. SLC can also control the suppression of angiogenesis in tumors. Thus, the SLC is very useful in immunotherapy of cancer. However, the application of protein SLC itself or transgenic method did not give satisfactory results in relation to antitumor immunity. It was assumed (Cancer Research 61, 197-205, January 1, 2001)that the antitumor immune response can be enhanced by immunization of mice dendritic CL is DAMI, which transliterowany recombinant retroviral vector carrying the genes encoding the Fc fragment of IgG and antigen of the tumor. The mechanism is that a hybrid protein is expressed and of greater DC, re-captured by dendritic cells via the Fc receptor (designated hereinafter as FcR), and then the epitope hybrid protein prezentuetsya T-helper cells (Th) through molecules MHC class II and cross-prezentuetsya cytotoxic T lymphocytes (CTL) through molecules MHC class I, resulting in induced systemic antitumor humoral and cellular immunity. It was reported (the Journal of Immunology, 1998, 161: 6059-6067)that the ability of DC no capture antigens using FcR-mediated endocytosis 10,000 times higher than through pinocytosis. However, the isolation, amplification and gene transfection in DC increases the complexity of manufacture of the vaccine. In this case, dendritic cells must be prepared individually, which makes quality control and industrial production.

DISCLOSURE of INVENTIONS

To overcome the shortcomings of the prototype, the aim of the invention is the creation of a new recombinant gene construct for the induction of an immune response.

Another purpose of this invention is to provide a hybrid (merged) protein, xpressimage this recombinant gene construct.

Another is the spruce of this invention is to provide a hybrid protein vaccine, encoded by a recombinant gene construct according to the invention, and vaccines recombinant tumor-chemotactic antigen.

To achieve the objectives of the invention proposed the following technical solutions.

The invention relates to a recombinant gene construct for the induction of the immune response containing the gene SLC human antigen gene and gene Fc fragment of IgG1, where gene SLC is left of the antigen gene and gene Fc fragment of IgG1 is to the right of the gene of the antigen. The specified antigen gene contains Her2/neu, P53, PSA, PAP, PSM, MAGE1, MAGE2, MAGE3, BAGE, GAGE1, GAGE2, CAG3, RAGE, NY-ESO-1, Tyrosinase, CEA, Ig-idiotype, gp100, melan A, gp75, TRP-1, TRP-2, CDK4, CASP-8, ras, bcr/abl, MUC-1, and genes encoding proteins related to hepatitis C, HIV and pathogenic microorganisms

In recombinant gene construct according to the invention the sites of the restriction enzyme EcoRI and three genes glycinol are between SLC gene and genome antigen, and the sites of the restriction enzyme EcoRV and five genes glycinol are between antigen gene and gene Fc fragment of IgG1.

Recombinant gene construct according to the invention includes the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:12.

The invention also relates to amino acid sequences encoded by the recombinant gene construct according to the invention. This amino acid sequence contains the sequence of SEQ ID NO:2.

From reenie also relates to amino acid sequences, encoded by SEQ ID NO:12. This amino acid sequence represents SEQ ID NO:13

The invention also relates to a genetic vaccine containing the recombinant gene sequence according to the invention.

In addition, the invention also relates to a vaccine. Preferably the vaccine contains SEQ ID NO:1, SEQ ID NO:12 and the corresponding amino acid sequence

In other words, the invention relates to a recombinant gene construct containing a gene SLC human antigen gene and gene Fc fragment of IgG1, where gene SLC is left of the antigen gene and gene Fc fragment of IgG1 is to the right of the gene of the antigen. The specified antigen gene contains Her2/neu, P53, PSA, PAP, PSM, MAGE1, MAGE2, MAGE3, BAGE, GAGE1, GAGE2, CAG3, RAGE, NY-ESO-1, Tyrosinase, CEA, Ig-idiotype, gp100, melanA, gp75, TRP-1, TRP-2, CDK4, CASP-8, ras, bcr/abl, MUC-1 and other genes encoding proteins related to hepatitis C, HIV and other pathogenic microorganisms. The sites of the restriction enzyme EcoRI and three genes glycinol are between SLC gene and genome antigen, and the sites of the restriction enzyme EcoRV and five genes glycinol are between antigen gene and gene Fc fragment of IgG1.

The invention also relates to the use of recombinant gene construct according to the invention in the preparation of vaccines. Namely, the vaccine containing a recombinant gene construct according to the invention, in particular the sequence is of nucleotide SEQ ID NO:1, SEQ ID NO:12 and the corresponding amino acid sequence of SEQ ID NO:2 and SEQ ID NO:13.

In other words, the invention relates to a recombinant gene construct containing human SLC gene, the gene of the antigen and gene Fc fragment of IgG1 (see Fig. 13). Gene SLC placed to antigen gene, and inserted the sites of the restriction enzyme EcoRI and three genes glycinol. Gene Fc fragment of IgG1 placed after antigen gene, and inserted the sites of the restriction enzyme EcoRV and five genes glycinol.

According to the invention, the replacement gene of the antigen in these genes at the sites of the restriction enzyme can cause a variety of hybrid genes chemotactic antigen. In particular, this invention relates to a recombinant gene containing the nucleic acid sequence SEQ ID NO:1. The nucleic acid sequence SEQ ID NO:1 is an artificially legirovannoi sequence SLC-Her2/P53-Fc, where SLC gene is located from position 1 to position 402 nucleic acids; ligiously Her2 gene is located from position 418 to position 711 nucleic acid. The nucleotides from position 418 to position 444 in recombinant gene correspond to the nucleotides from position 1105 to position 1131 in the open reading frame (ORF) of the gene Her2/neu. The nucleotides from position 445 to position 546 in recombinant gene correspond to the nucleotides from position 244 to position 345 in the open who nd reading frame (ORF) of the gene Her2/neu, which includes a mutation from G to C in position 250. The nucleotides from positions 547 to position 711 in recombinant gene correspond to the nucleotides from position 1333 to position 1479 in the LFS gene Her2/neu. In recombinant gene part of the P53 gene is located from position 712 to position 1113 corresponding to the nucleotides from positions 475 to position 876 ORS p53. Gene IgG Fc (including introns) is from 1147 to position 2057 in recombinant gene, and two introns are from 1189 to position 1309 and with the provisions of 1640 to position 1739 respectively. The sequence of the nucleic acid with the provisions 1147 to position 1189 is a sequence. hinge-region. The sequence of the nucleic acid with the provisions 1310 to position 1369 is a sequence for CH2, the sequence of the nucleic acid with the provisions of 1740 to position 2057 is a sequence for CH3. Three genes glycinol and EcoRI sites of the restriction enzyme inserted before the fragment Neg/P53, and five genes glycinol and EcoRV sites of endonuclease restriction inserted after Her2/P53. According to this invention, to make a normal biological function expressed factors, 3~5 genes glycinol inserted before and after (right and left) of the gene of the antigen. Gene effect of glycine is to prevent the interference of the recombinant Antiga is and the three-dimensional structure.

This invention also relates to amino acid sequences (612 A.K., Srila) SEQ ID NO:2, encoded by the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:1 is an artificially recombinant SLC-Her2/P53-Fc gene. In the specified amino acid sequence of a fragment of the SLC is from position 1 to position 134, three glycine are from position 135 to 137 provisions, and part of the peptide Neg is from position 140 to position 237. Amino acids from position 140 to position 148 in hybrid protein correspond to the amino acids from position to position 369 377 in Her2/neu. Amino acid position 149 to position 182 in hybrid protein correspond to the amino acids from position 82 to position 115 in Her2/neu, which has a mutation from V to L at position 84. Amino acid position 183 to the position 238 in hybrid protein correspond to the amino acids from position 445 to position 499 in Her2/neu. Part of the P53 peptide is from 238 to position 371 in hybrid protein (corresponding to amino acid position 159 to position 292 in a natural form of P53). Five glycinol are from 374 to position 378. IgG Fc is from position to position 383 612, where the sequence from position to position 383 396 is a loop-region, the sequence from position 397 to position 506 is a sequence for CH2, and consistently is e s position 507 to position 612 is a sequence for CH3.

This invention also relates to another recombinant gene for vaccine against prostate cancer, the sequence of which is shown in SEQ ID NO: 12. The nucleotide sequence of SEQ ID NO: 12 is a sequence artificially very SLC-PSM-mPAP-PSA-Fc (SLC-3P-Fc) gene, which was introduced only a partial sequence of PSM (human Prostate Specific Membrane Antigen), mPAP (mouse Prostatic Acid Phosphatase and PSA (human Prostate Specific Antigen). In a hybrid gene SLC gene is located from position 1 to position 402; part of the PSM gene is located from position 418 to position 603, the corresponding sequence with the provisions of the 1987 to position 2172 in the open reading frame (ORF) PSM; gene mPAP is from position 604 to position 759, corresponding to the sequence from position 328 to position 484 in mPAP ORS; PSA gene is located from the provisions of 760 to position 981, corresponding to the sequence from position 151 to position 372 in PSA ORS; gene IgG Fc (including introns) is from 1003 to the position of 1925, moreover, these two intron are from 1057 to position 1177 and provisions 1508 to position 1607, respectively. Three genes glycinol and EcoRI sites of the restriction enzyme inserted before (left) 3P, and five genes glycinol and EcoRV sites of the restriction enzyme is inserted after (right) 3P.

This invention also relates to the amino acid sequence of SEQ ID NO: 13, encoded by the NUS is eotide sequence of SEQ ID NO: 12. In particular, the invention relates to amino acid sequences (568 A.K.)corresponding to artificially recombinant gene SLC-3P-Fc. In this hybrid amino acid sequence SLC is from position 1 to position 134, three glycine are from position 135 to position 137, PSM is from position 140 to position 201, the portion of the peptide mPAP is from position 202 to position 253, part of PSA peptide is from 254 to position 327, five glycinol are from 374 to position 378, and IgG1-Fc is to position 335 to position 568.

This invention also relates to the use of the recombinant gene sequence according to the invention, including SEQ ID NO:1, in the production of vaccines.

As can be seen from the above, the strategy for chemotactic antigen according to the invention is as follows: attach the chemokine SLC to the left of the antigen and joining Fc immunoglobulin IgG to the right of the antigen; the introduction of a hybrid gene consisting of three parts, directly in healthy or tumor tissue for the expression and secretion; education consisting of two parts United to the body through the hinge region of the Fc. The indicated recombinant protein actively attracts DC and T cells due to the chemotactic activity of SLC in vivo. DC grabs a hybrid protein with high efficiency using pinii the oz, especially with the help of Fc receptor and SLC receptor CCR7. While DC is antigen into the cell. After processing and processing of the antigen appears to be Th and CTL using molecules MHC-II and MHC-I., And finally induced antitumor specific humoral and cellular immune response. In addition, the DC can be activated by the combination of the fragment Fc Fc receptor DC. SLC can actively remove DC and T lymphocytes, as well as to promote the secretion of cytokines, such as IL-12, interferon-γ and interferon-inducible protein (IP-10), to promote the development of Th1, to suppress the production of TGF-β and VEGF. Thus, the SLC is very useful for antitumor immunity. For the reasons described above, this combination has a synergistic effect and can induce a stronger antitumor immune response (see Figure 1).

In this invention used in a variety of recombinant genes, but this invention is not limited. The study revealed that in the form of plasmids, and in the form of a hybrid protein recombinant chemokinesis antigen can be obtained industrially. Stages of preparation for DC in this application was omitted. But DC can be with high efficiency targeting in vivo. Moreover, introduction sites of the restriction enzyme from two sides of the gene of the antigen makes it possible to substitute antigen for polucheniya different chemokine against antigens of neoplastic and non-neoplastic diseases. For example, a new vaccine chemokine antigen can be obtained by replacing antigen HP SLC-HP-Fc antigen is related to prostate cancer.

Recombinant gene can be constructed in eukaryotic vectors using direct injection of plasmids into the body or tumor. Other techniques, such as gene gun or electrical impulse, can increase the degree of transfection and effectiveness of expression. Constructed recombinant virus such as retrovirus, adenovirus, adeno-associated virus, vaccinia virus, herpes simplex virus, and the like, can also be used as vaccines for injection in vivo. Eukaryotic cells (e.g. Cho) can be transliterowany plasmids and cultured in vitro for expression and secretion. Thanks to the Fc fragment of the hybrid protein may be easily and efficiently isolated and purified by the methods of affinity chromatography and used as a protein vaccine.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows a scheme of the mechanism of gene vaccines chemotactic antigen according to the invention.

Figure 2 shows the construction of plasmids. HP indicates Her2/neu, p53 hybrid gene; sig denotes a signal peptide.

Figure 3 shows the recombination SLC-Her2/neu-Fc according to the invention.

Figure 4 shows the definition of Western blotting of cellular expression and secrets the hybrid protein in the cells, transfected gene vaccine pSLC-HP-Fc. And indicates the lysates of cells transfected with pSLC-HP-Fc; means In the culture supernatant of cells transfected with pSLC-HP-Fc; C indicates the culture supernatant of cells transfected with pcDNA.

Figure 5 shows the effect on the inhibition of tumors in vivo by pre-vaccination gene vaccine tumor-chemotactic antigen (pSLC-HP-Fc).

6 shows the survival of mice that entered the tumor, using pre-vaccination gene vaccine tumor-chemotactic antigen (pSLC-HP-Fc).

Fig.7 shows the effect on the inhibition of tumors in vivo when exposed to a genetic vaccine tumor-chemotactic antigen (pSLC-HP-Fc).

Fig shows the time of survival when exposed to a genetic vaccine tumor-chemotactic antigen (pSLC-HP-Fc).

Fig.9 shows CTL activity after immunization gene vaccine tumor-chemotactic antigen (pSLC-HP-Fc).

Figure 10 shows the number of specific antibodies to HP in serum after 2 and 4 weeks after vaccination. The serum of mice was diluted in ratio 1:100. Used the ELISA analysis.

11 shows that the vaccine pSLC-HP-Fc can induce specific CTL against Her2/neu and p53 in vitro.

Fig shows the construction of plasmids pSLC-3P-Fc.

Fig shows the effect on the inhibition of tumors in vivo after exposure to the root of the nd vaccine tumor-chemotactic antigen (pSLC-3P-Fc).

Fig shows the time of survival after exposure to genetic vaccine tumor-chemotactic antigen (pSLC-3P-Fc).

Fig shows a fundamental structure of the recombinant gene according to the invention.

DETAILED disclosure of the INVENTION

The invention will be described hereinafter in detail and illustrated. You must understand that the purpose of the illustrated embodiments is only to illustrate, but not limit the invention.

Example 1. Cloning of the human gene SLC

mRNA was extracted from lymph nodes of a patient suffering from cancer. Using methods well known in the art, the Kpnl site of the restriction enzyme was added to direct the SLC primer (5'-cggtaccacagacatggctcagtcac-3') and a sequence encoding a three glycine and the EcoRI site of the restriction enzyme was added to the reverse primer SLC (5'-taaattctcctcctcctggccctttagg-3'). Gene SLC was obtained FROM RT-PCR amplification. The resulting fragment was purified and inserted into the vector pUCmT for sequencing (Fig-SLC). The results show that the sequence of the received fragment corresponds to a scheduled sequence (the underlined part).

In this invention sequencing of PCR products was carried out using a 377 DNA sequencing machine (firm ABI) using four-color fluorescent termination sequencing, the basis of the tion on the method of Sanger termination of dideoxynucleotides. The sequence from position 36 to position 464 is a sequence of the cloned fragment according to the invention (with reference to SEQ ID NO: 3).

Example 2. Preparation and connection of the gene Her-2/neu

First, two fragments of the gene Her-2/neu cloned by PCR. The first fragment was received as follows: use the direct primer 5'-agaattcaagatctttgggagcctggcatttctgggctacctgctcatcgctcac-3', carrying the EcoRI site of the restriction enzyme, and used the reverse primer 5'-gatgcccagcccttgca gggccagggcatagttgtc-3'. The plasmids containing the extracellular segment of the neu gene, was taken as template and amplified fragment corresponding to the amino acid sequence from position 82 to position 115, where the amino acid V at position 103 was replaced by the amino acid L. the Second fragment was obtained in the following way: use direct primer 5'-ctgcaagggctgggcatc-3' and reverse primer carrying the NcoI site of the restriction enzyme 5'-tccatggcccggttggcagtgtggag-3'. Was amplified fragment corresponding to the amino acid sequence from position 445 to position 499.

Two of PCR product were collected and combined in a PCR system. Next, the resulting fragment was used as a matrix, and using the direct primer of the first fragment and the reverse primer of the second fragment was amplified hybrid gene size 294 BP Then the resulting hybrid gene would is inserted into the vector pUCmT (pUC-Her2P) and sequenced. The results show that the sequence of the resulting hybrid gene corresponds to the scheduled sequence (underlined sequence).

The result is shown in SEQ ID NO: 4, where the sequence from position 121 to the position 465 is a sequence of the cloned fragment according to the invention.

Example 3. Cloning a segment of the gene p53

The gene fragment 402 BP, encoding amino acids from position 156 to position 289 of p53 protein, was cloned from a plasmid containing a full-sized gene p53. Used the following primers: forward primer with a single NcoI site of the restriction enzyme was represented by 5'-ACC atg gcc atc tac aag cag tca cag CAC atg ac-3'; reverse primer with a single EcoRV site of the restriction enzyme was represented by 5'-tga tat ctt tct tgc gga gat tct ctt c-3'. The obtained fragment was inserted into the vector pUCmT (pUC-p53P) for sequencing. The results show that the sequence of the received fragment corresponding to the expected sequence (the underlined segment).

The result is shown in SEQ ID NO: 5, where the sequence from position 39 to position 445 is a sequence of fragment according to the invention.

Example 4. The connection of fragments of Her2/neu and p53

As shown in Figure 3, the smaller fragment from pUC-Her2P, cut with EcoRI/NcoI, and a smaller fragment from pUC-p53P, cut with NcoI/EcRV, were legirovanyh with a large fragment of pcDNA3z, cut with EcoRI/EcoRV to obtain plasmid pcDNA-Her2/p53, also referred to as pcDNA-HP. HP means a gene legirovannye of Her2/neu and p53. Artificially legirovannye fragment of Her2/neu-p53 contains genes encoding the peptide linking molecule HLA-I and HLA-II.

Example 5. Construction of PcDNA-Fc

First chemically synthesized polylinker was used to replace polylinker pcDNA3. 1.

Was used following the method of construction.

Chemically synthesized two of the oligonucleotide.

Direct primer:

5'-GCTAGCGAAGCTTTGGTACCGTAGGATCCACGAATTCAGTCCA-GGATATCGGCGGTGG-3'

Reverse primer:

5'-GGTTTAAACGTTAACCCCGGGCCCTCGAGCTCTAGAGCCTCCT-CCACCGCCGATATC-3'

The last 15 bases on the 3' ends of each primer complementary to each other. The PCR product was received in a system with a mixture of two primers in the ratio of 1:1, DNA Taq polymerase and dNTP at 45°C for renaturation and elongation. The purified product was inserted into the vector pUCmT for sequencing. The results show that the sequence of the resulting product is the desired sequence. The underlined sequence shows the synthesized website polychloropene. The final site polychloropene represented: "Nhel-HindIII-Kpn1-BamH1-EcoR1-EcoRV - glycine × 5-Xbal - Xho1 - Apa1-Sma1 - Hpa1-Pme1". In this sequence a small fragment of the gene (GGCGGTGGAGGAGGC), Cody the existing five glycinol, was inserted method sites EcoRV and Xbal. Cut the cloned plasmid Nhel/Pmel and received a fragment of about 100 BP Then this fragment is ligated with the large fragment of pcDNA3. 1, cut with Nhel/Pmel, to obtain a new vector, called pcDNAf.

The sequencing chemically synthesized polylinker shown in SEQ ID NO: 6, where the sequence from position 3 to position 100 is a sequence of fragment according to the invention.

The method of constructing the plasmids PcDNA-Fc

The Fc fragment was cut from the plasmid containing IgG1 Fc, using the XbaI/ApaI restriction endonucleases and inserted into the vector pcDNAf, which has been treated with the same endonucleases. The resulting construct was named PcDNA-Fc. The sequence of the IgG1 Fc was confirmed by sequencing of the resulting plasmid.

Sequencing of the fragment Fc

The Fc fragment with a size of 900 BP was amplified from a plasmid containing the gene IgG1 Fc, using PCR and inserted into the vector pUCmT for sequencing. Were used for amplification the following primers: forward primer: 5'-gaattcggagttaacgagcccaaatcttg-3'; reverse primer: 5'-gggccctcatttacccggagac-3'. The sequencing results indicate that the plasmid contains the hinge region and the region CH2 and CH3 from IgG1. This means that the plasmid contains Fc (see underlined sequence). The result is shown in SEQ ID NO:7, which follow etelnost from position 78 to the position 991 is a sequence according to the invention.

Example 6. The connection of the gene SLC-Her2/neu-Fc

As shown in Figure 3, a larger fragment from pcDNA-HP, cut Cloned/EcoRI, was attached to a smaller fragment from pUC-SLC, cut Cloned/EcoRI, to obtain pcDNA-SLC-HP. Then a smaller fragment from pcDNA-SLC-HP, cut Cloned/EcoRV, was attached to a larger fragment of pcDNA-Fc, cut Cloned/EcoRV, to obtain the construct pcDNA-SLC-HP-Fc (also called pSLC-HP-Fc).

The design of the control plasmids shown in figure 2

Example 7. The definition of Western blotting of cellular and secreted recombinant protein in cells transfected with the gene vaccine

Recombinant plasmid pSLC-HP-Fc were transliterowany cells B16-F10 method of liposomes. Positive clones were selected using G418. Cells were cultured in serum-free 1640 medium for 24 hours, and then collected and concentrated supernatant. Method Western blotting was used to determine the expression of the hybrid protein. Used primary antibody was a polyclonal rabbit antibody to human p53, the secondary antibody was conjugated to a horseradish peroxidase goat anti-rabbit antibody. The blots visualize using the kit for chemiluminescent determination of ECL-Plus (Amersham Pharmacia Biotech). In conclusion, the film is subjected to the action of x-rays. The results show that Express what I hybrid protein can be determined in the supernatant and cell lysate B16-P10 cells, transfected vector pSLC-HP-Fc. Molecular weight as expected (see Figure 4). It was confirmed that the sequence of the hybrid protein represents SEQ ID NO: 2.

Example 8. Cloning of human prostate-specific membrane antigen PSM (Prostate Specific Membrane Antigen)

Total RNA was extracted from cell lines of human prostate cancer LNCaP. RT-PCR was carried out using the direct primer (5'-ACTCGAGAT GAAGACATACAGTGTATC-3') and reverse primer (5'-TGATATCTTAGGCTAC TTCACTCAAAG-3'). After the electrophoresis was carved band hPSM size 390 BP Fragment was purified and extracted by the method of Glass Milk. The purified fragment was Legerova in vector pUCm-T with getting pUCm-hPSM. A positive clone were collected and sequenced. The results are what you expected. If you look at the SEQ ID NO: 8, the obtained gene fragment corresponds to the sequence with the provisions of 1864 to position 2253 LFS human PSM. The sequence from position 229 to position 618 in the received sequence is a sequence according to the invention.

Example 9. Cloning of the gene fragment of human prostate-specific antigen PSA (Prostate Specific Antigen)

Total RNA was extracted from cell lines of human prostate cancer LNCaP. RT-PCR was carried out using the direct primer (5'-TCTCGAGGGC GGTGTTCTGGTGCA-3') and operatoroperator (5'-AGATATCATGTCC AGCGTCCAGCAC-3'). After the electrophoresis was carved band of about 600 BP Fragment was purified and extracted by the method of Glass Milk. The obtained fragment was inserted into the vector pUCm-T with getting pUCm-PSA. A positive clone were collected and sequenced. The results shown in SEQ ID NO: 9. The obtained gene fragment corresponds to the sequence from position 151 to position 609 LFS human PSA. The sequence from position 45 to position 503 in the received sequence is a sequence according to the invention.

Example 10. Cloning a fragment of the gene of mouse PAP (Prostatic Acid Phosphatase)

Total RNA was extracted from tissues of the mouse prostate. RT-PCR was carried out using the direct primer (5'-TCTAGATGAGAGCTGTTCCTCTG-3') and reverse primer (5'-GGGCCCTTAATTCCGTCCTTGGTG-3'). After the electrophoresis was cut strip size 1146 BP Fragment was purified and extracted by the method of Glass Milk. The obtained fragment was inserted into the vector pUCm-T with getting pUCm-mPAP. A positive clone were collected and sequenced. The results shown in SEQ ID NO: 10. The sequence from position 48 to position 1193 in the received sequence is a sequence according to the invention.

Example 11. The connection of fragments of genes PSM, mPAP and PSA (3P)

A fragment of the gene hPSM was cloned from the plasmid pUCm-hPSM by PCR with the following primers: forward primer corresponding to posledovatel the surface with the provisions of the 1987 to the provisions of the 2007 LFS hPSM (inserted EcoRI site of the restriction enzyme) was a 5'-AGAATTCATGATGAATGATCAACTCATG-3'; reverse primer consisted of a 5'-AGCACTCATCAAAGTCCTGGCCTTGGAAGGGTCCAC-3' (the underlined part corresponds to the sequence from position 328 to the provisions of ORS 345 mPAP, the rest corresponds to the sequence from position 2155 to position 2172 ORS hPSM). After electrophoresis, the obtained fragment hPSM was cut out and purified by the method of Glass Milk. A fragment of the gene mPAP was cloned from the plasmid pUCm-mPAP using PCR with the following primers: forward primer corresponding to the sequence from position 328 to position 348 OPC mPAP was a 5'-AGGACTTTGATGAGTGCTATG-3'; reverse primer corresponding to the sequence from position to position 463 483 OPC mPAP was a 5'-AGGGCAGTCTCTGAAAGGCAG-3'. After electrophoresis, the obtained fragment mPAP was cut out and purified by the method of Glass Milk. A fragment of the gene hPSA was cloned from the plasmid pUCm-hPSA by PCR with the following primers: forward primer consisted of a 5'-CCTTTCAGAGACTGCCCTGGCGGTGTTC TGGTGCAC-3' (underlined part corresponded to the sequence from position 466 to position 483 OPC mPAP, and the remainder corresponded to the sequence from position 151 to position 168 OPC hPSA); reverse primer consisted of a 5'-AGATATCGAGCAGCATGAGGTCGT-3'. After electrophoresis, the obtained fragment hPSA was cut out and purified by the method of Glass Milk.

Primer 5'-AGAATTCATGATGAATGATCAACTCATG-3' was used for amplification hPSM, and primer 5'-AGGGCAGTCTCTGAAAGGCAG-3' was used in isulan for amplification mPAP individually in 25 ál system 10 cycles, to get the single chain. Then the two systems have been mixed for a further PCR in 50 μl of system 18 cycles. After electrophoresis, the obtained fragment hPSM-mPAP size 352 BP was excised and purified by the method of Glass Milk.

Primer 5'-AGGACTTTGATGAGTGCTATG-3' (corresponding to the sequence from position 328 to position 348 OPC mPAP) was used for amplification mPAP, and primer 5'-AGATATCGAGCAGCATGAGGTCGTG-3' (corresponding to the sequence from position 355 to position 372 OPC PSA) was used for amplification hPSA individually in 25 ál system 10 cycles, to obtain a single chain. Then the two systems have been mixed for a further PCR in 50 μl of system 18 cycles. After electrophoresis, the obtained fragment mPAP-hPSA size 378 BP was excised and purified by the method of Glass Milk.

Primer 5'-AGAATTCATGATGAATGATCAACTCATG-3' was used for amplification hPSM-mPAP, and primer 5'-AGATATCGAGCAGCATGAGGTCGTG-3' was used for amplification mPAP-hPSA individually in 25 ál system 10 cycles, to obtain a single chain. Then the two systems have been mixed for a further PCR in 50 μl of system 18 cycles. After electrophoresis, the obtained fragment hPSM-mPAP-hPSA size 564 BP was excised and purified by the method of Glass Milk. The obtained fragment was inserted into the vector pUCm-TV for sequencing. The results are what you expected. See SEQ ID NO: 11, where the sequence from position 54 to position 629 sequence is what elnett according to the invention.

Example 12. Recombination gene chemokine antigen SLC-3P-MS

After sequencing, the plasmid containing the correct gene sequence 3P (3P denotes a hybrid gene PSM-PAP-PSA size 564 BP that encodes a 188 amino acids) was cut EcoRI/EcoRV restriction endonucleases. Received a smaller fragment 3P was Legerova cut with EcoRI/EcoRV vector pSLC-HP-Fc with obtaining construction pSLC-3P-MS (see Fig). The sequence of the recombinant gene SLC-3P-Fc is shown in SEQ ID NO: 12, which corresponds to the amino acid sequence shown in SEQ ID NO: 13.

Test examples

Test 1. Definition chemotactically activity of the hybrid protein in relation to immune cells in the chamber of Boyden

Removing activity of human SLC in vitro:

A. Cell line melanoma B16F10 was transliterowany purified plasmid pcDNA3z-SLC-HP-Fc and control plasmid. After 48 hours incubation collected culture medium and concentrated using PEG20000 (as a negative control was used supernatant nitrostilbene cells B16-F10).

B. Cells of peripheral blood mononuclear (RVMS, from peripheral blood mononuclear cells) were collected from 5 ml of healthy whole peripheral blood using the environment for the Department of limitato (Lymphocyte Separation Medium), washed twice with saline and resuspendable in serum-free cf is de RPMI-1640. RVMS were counted and diluted to a concentration of 1×106/ml.

Century Cooking chamber Boyden: the camera was purchased from Neuro Probe Co. The lower wells of the chamber were filled with 27 μl of conditioned culture medium, and the top 50 ál of cell suspension of 1×106/ml RVMS. The upper and lower wells were separated by a polycarbonate filter (Neuro Probe) hole size 5 microns. The chamber was incubated at 37°C With 5% CO2within 4 hours

, The Filter is unloaded, and the cells were scraped off from the top side of the filter, and then washed, fixed and stained filter. Migrated cells were counted in five randomly selected high-power fields (×200). Chemotactic index Cl (Chemotactic Index) was calculated as follows: Cl = number of cells in five high-power fields in the studied wells / number of cells in five high-power fields in the control wells. Value Cl negative group was 1.

Table 1
Definition chemotactically activity of the hybrid protein
GroupCounting hemotoxicity cellsClThe value P
Negative control 34±10,41
The control vector44,6±11,91,31>0,05*
psig-HP68,6±9,52,02<0,05*
pSLC-HP-Fc157,4±19,24,63<0,01*<0,05**
pSLCof 156.6±20,3br4.61<0,01*<0,05**
* relative to the negative control
** regarding psig-HP

Results: the Level chemotactically activity of the serum-free supernatant of cells transfected with pSLC-HP-Fc significantly higher than the other, which means that after transfection of the plasmid can be expressed, and expressed active protein. Level chemotactically capacity of the cell, transtitional pSLC-HP-Fc similar to that in cells, transtitional pSLC, which means that a hybrid protein does not damage the activity of SLC.

Test 2. Antitumor immune response on gene vaccine human figure is toksicheskogo antigen in mice

Gold particles were loaded plasmids (naked genes) and deposited on the inner surface of the Tefzel tubing, cut into appropriate segments for bullets to deliver 1 µg DNA, according to the instructions BioRad. There are several control groups: control vector, pSLC-Fc psig-HP (plasmid expressing HP, containing the signal peptide SLC), pSLC-HP and psig-HP-Fc.

Test 3. The immune response against cancer

14 days and 7 days prior to injection of tumor mice were vaccinated twice pSLC-HP-Fc or control vectors in the skin of the abdominal. On day 0, each mouse was injected with 5×104tumor cells B16F10 (B16F10 cells were transliterowany genome HP and selected on G418) in 0.2 ml. Tumor size was recorded 2-3 times per week. The tumor size was calculated as length (cm) × width (cm2).

Results: Vaccination twice with pSLC-HP-Fc significantly improved the effect of tumor suppression (see Figure 5, 6), and the average time of survival of mice was significantly extended. Therapeutic effect pSLC-HP-Fc significantly better than the other vectors (p<0,05), indicating a synergistic immune action fragments SLC and Fc.

Test 4. Immunotherapy of cancer

On day 0, each mouse was injected with 5×104tumor B16F10 cells in 0.2 ml. On days 6, 12 and 18 mice were vaccinated using system gene gun (Gene Gun System). Tumor size was recorded 2-3 times per week. the size of the tumor was evaluated, as specified above.

Results: As shown in Fig.7 and 8, the vaccine pSLC-HP-Fc shows the most effective suppression of tumor growth. On day 28 the difference between groups pSLC-HP-Fc and psig-HP-Fc is significant (P<0,05). The average time of survival of mice was prolonged by immunization pSLC-HP-Fc. Mice in group B16 (pSLC-HP-Fc) was injected cells B16 wild type. After three immunizations pSLC-HP-Fc group B16 showed no effect of suppressing the tumor, meaning that the immune response induced pSLC-HP-Fc is specific to HP antigen.

Test 5. CTL activity induced by a genetic vaccine tumor-chemotactic antigen

Two weeks after the triple vaccination system gene gun tumor bearing mice were killed and analyzed for CTL cytotoxicity was performed using spleen cells using LDH Kits (Promega). The ratio of spleen cells and target cells (effector:target e:M) was 40:1, 20:1 and 10:1 respectively.

Results: Lymphocytes derived from mice immunized with vaccine chemokine nucleotide showed a significant cytotoxic effect on cells B16F10-HP (see Fig.9). When relations e:M were 40:1 or 20:1, there was still a significant difference between the study vaccine and control vectors (P<0,05), which confirms that the vaccine is stronger in relation to the induction of CTL actively the tee.

Test 6. Determination of specific antibodies in the serum of immunized mice

The number of HP-specific antibodies in the serum of the group immunized mice was determined using ELISA after 2 and 4 weeks after immunization (pSLC-HP-Fc). Briefly, microtiter plates, coated with recombinant p53 protein, were incubated overnight, blocked with 20% calf serum and incubated with primary antibodies (serially diluted mouse serum) and, in turn, a secondary antibody conjugated with horseradish peroxidase goat artemisinine IgG). After washing and visualization using O-phenylenediamine, the value of OD was measured at 490 nm. Monoclonal antibody against p53 was used as a positive control, and normal mouse serum was used as negative control.

Results: the Level of p53 antibodies in mice immunized with SLC-HP-Fc, is the highest and significantly higher than the level of other vectors (see Figure 10).

Test 7. Specific human CTL against Her-2/neu or p53, induced by immunization with pSLC-HP-Fc in vitro

Using centrifugation using the environment to separate the lymphocytes were collected mononuclear cells (MNCs, mononuclear cells from whole peripheral blood of healthy donors HLA-A2+. Half of the MNCs was transliterowany PLA is Midol pSLC-HP-Fc using liposomes and incubated with untreated MNCs in the ratio of 1:1 at 37°C With 5% CO 2. On day 6 were counted viable cells, resuspendable and cultivated with a variety of target cells in different ratios e:M in 96-well microplate. Analysis of LDH (Promega Kits) was used for analysis of cytotoxicity.

Results: After mixing of cultures transfetsirovannyh pSLC-HP-Fc MNC induced specific CTL to kill tumor cells that sverkhekspressiya Her2/neu or p53 MNF-restriction follows (see 11). That is, only specific CTL largely kill HLA-A2+tumor cells that Express Her2/neu or p53, indicating the specificity of the CTL induced gene vaccine chemotactic antigen.

Test 8. Combating prostate cancer gene vaccine chemokine antigen pSLC-3P-MS in vivo

5×104cancer cells B16-3P (transfetsirovannyh pcDNA-3P cancer cells B16, selected on G418) were introduced in the side to C57BL/6 mice (eight mice in each group). Mice were immunized on the third, eighth and thirteenth day after injection of tumor cells. In the control groups were used to control vectors pSLC-Fc and psig-3P (a plasmid containing the gene signal peptide SLC to the left of gene 3P). Watched the tumor size and survival time.

Results: As shown in Fig and 14, gene vaccine chemokine antigen (pSLC-3P-Fc) significantly inhibited tumor growth (compared with the control groups, P< 0.05) and significantly prolongs the survival time (in comparison with the control groups, P<0,05), indicating that the change in antigen gene vaccine chemotactic antigen can give other vaccines against the corresponding antigen.

1. Recombinant gene construct for the induction of the immune response containing the gene SLC human antigen gene and gene Fc fragment of IgG1, where gene SLC is left of the antigen gene and gene Fc fragment of IgG1 is the right of the antigen gene.

2. Recombinant gene construct according to claim 1, where the specified antigen gene includes Her2/neu, P53, PSA, PAP, PSM.

3. Recombinant gene construct according to claim 1 or 2 where the sites of the restriction enzyme EcoRI and three genes glycinol are between SLC gene and genome antigen, and the sites of the restriction enzyme EcoRV and five genes glycinol are between antigen gene and gene Fc fragment of IgG1.

4. Recombinant gene construct according to claim 1, Kotor which has the sequence of SEQ ID NO:1.

5. Recombinant gene construct according to claim 1, which has the sequence of SEQ ID NO:12.

6. Recombinant polypeptide for the induction of the immune response, characterized by the amino acid sequence encoded by the sequence SEQ ID NO:1.

7. The polypeptide according to claim 6, where the amino acid sequence represents SEQ ID NO:2.

8. Recombinant polypeptide for the induction of the immune response, characterized by the amino acid sequence encoded by the sequence SEQ ID NO:12, where the amino acid sequence represents SEQ ID NO:13.

9. The vaccine for the induction of an immune response that contains recombinant genetic construct according to claim 1 or the corresponding recombinant polypeptide.

10. The vaccine according to claim 9, where this gene construct represented by SEQ ID NO:1, SEQ ID NO:12, or the corresponding amino acid sequence.



 

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FIELD: biotechnologies.

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6 cl, 4 dwg, 2 tbl, 10 ex

FIELD: medicine.

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2 cl, 3 dwg, 1 tbl, 8 ex

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FIELD: chemistry; biochemistry.

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11 cl, 13 ex, 4 tbl, 20 dwg

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FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic and protein engineering and can be used in biomedical industry. A genetic make up is proposed, which codes a peptide in which two domains bonding the growth hormone (GH) receptor are bonded into a tandem by a "semirigid" or "rigid" linker, which consists of at least 1-4 copies of the A(EAAAK)A amino acid sequence.

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16 cl, 4 dwg, 1 tbl, 13 ex

FIELD: medicine.

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FIELD: medicine.

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FIELD: medicine.

SUBSTANCE: method is suggested for production of antibody for binding to NK-cells, which crossly interacts with products of gene KIR2DL1 and KIR2DL2/3 and neutralises inhibitor activity of such KIR. Mentioned method includes selection of such antibodies that crossly interact at least with products of gene KIR2DL1 and KIR2DL2/3, are able to restore lysis with NK cells Cw3+ or Cw4+ target cells and are bound with NK cells or polypeptide of KIR primate. Antibodies produced by this method are described, as well as their derivatives, where antibody is linked with toxin, radionuclide, recognisable aggregation, solid carrier or polyethylene glycol.

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7 cl, 13 dwg, 4 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: there is offered application of humanised fused protein for making a medicine used for stimulation of immune response and stabilisation of disease progressing in patients with GD2-positive tumours. The antibody contains antibody H14.18 caught with surface glycosphingolipid GD2 of human cells, and cytokine IL2. There is disclosed method of increase in ADCC and lysis activity of natural killers in cancer patients by introduction of the fused protein. The invention can be applied in GD2-overexpression cancer therapy.

EFFECT: application of the invention provides low-immunogenicity antibody.

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FIELD: chemistry; biochemistry.

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EFFECT: invention enables to lower immunogenicity of the modified anti-CD20 antibody compared to unmodified molecules and increase anti-tumour activity with respect to CD20.

15 cl, 17 dwg, 3 tbl, 8 ex

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