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Aids recombination gland virus vaccine

A technology of recombinant adenovirus and adenovirus, applied in the direction of antiviral agent, virus/bacteriophage, biochemical equipment and method, etc., can solve the problems of HIV immune escape, poor effect, immune protection and resistance failure, etc.

Inactive Publication Date: 2005-03-09
杭州康泰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Phase III clinical trial completed by VaxGen in 2003 used a DNA vaccine expressing HIV-1 BE subtype gp120 protein. Unfortunately, its therapeutic and protective effects were disappointing; in addition, many companies (including the famous pharmaceutical giant Merck , Roche, etc.) AIDS vaccines are still undergoing clinical phase I-III clinical trials. According to some preliminary results that have been published so far, although some vaccines can induce HIV-specific CD4+, CD8+ T cell responses, control the virus Replication, reducing the viral load, but its effective rate is still not high, and HIV immune escape occurred in the later stage of the test, resulting in the failure of vaccine-induced HIV-specific immune protection and resistance
[0007] The development and application of AIDS vaccines have shown that in terms of inducing effective immune protection against HIV in humans, adenovirus-based vaccines have a better effect, while naked DNA immunization, although performing well in mouse models, is not effective in primates. Less effective in animals and humans

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1. Obtaining of secreted human IL-15 gene

[0091] Human peripheral blood leukocytes were collected, total RNA was extracted by one-step guanidine isothiocyanate method, the first strand was synthesized by AMV reverse transcriptase, and the wild-type human IL-15 cDNA containing 10 N-terminal ATGs was amplified by PCR respectively using it as a template sequence:

[0092] Primers for wild-type human IL-15:

[0093] Upstream primer 5'CAG CCA GGA CTC GAT GGA GAA TCC 3' (SEQ ID NO: 17)

[0094] Downstream primer 5'G ATC GGA TCC TGT CTA AGC AGC AGA GTG ATG 3' (SEQ ID NO: 18)

[0095]The PCR reaction volume was 50 μl, the primer concentration was 0.4 μM, and the amplification parameters were 94° C. for 30 seconds, 51° C. for 45 seconds, and 72° C. for 50 seconds. After 30 cycles, the products were analyzed by 2% agarose gel electrophoresis. The size of the PCR product of wild-type human IL-15 is about 790bp, and the PCR product is directly cloned into the pMD18T (T...

Embodiment 2

[0101] Embodiment 2. Synthesis of HIV-1 polyepitope peptide coding gene

[0102] For the design of HIV-1 multi-epitope combinations, twelve optimal epitopes capable of generating specific CTL responses effective against HIV-1 virus infection in HIV-1 infected individuals were selected. These CTL epitopes are all HLA-A2 restricted, and the sequences are shown in Table 1. The designed multi-epitope peptide combination is formed by linking corresponding flanking sequences and connecting amino acids (GAAAG) according to the sequence shown in the table, and consists of 181 amino acids in total. According to the 181 amino acid sequence, the coding gene sequence of the corresponding multi-epitope peptide was synthesized, with a total length of 543 bp (SEQ ID NO: 13).

[0103] knsavSLLNA TDIAVgaaag RGPGRAFVTI gaaagRIRQG LERAgaaagY 50

[0104] VDRFYKTLga aagSLYNTVA TLgaaagPLT FGWCYKLgaa agVLEWRFDS 100

[0105] RLgaaagAII RILQQLgaaa gFLGKIWPSW KgaaagALVE ICTEMgaaag 150

[0106] VIYQ...

Embodiment 3

[0124] Example 3. Acquisition of the gene encoding the fusion protein of HSP65 and HIV-1 multi-epitope peptide

[0125] Mycobacterium bovis was obtained from the Chinese Type Culture Collection (ATCC) of Wuhan University. Extract the total DNA of the bacterium as a template for PCR to amplify the coding gene sequence of the Mycobacterium bovis HSP65 protein, and the primers are:

[0126] Upstream primer 5'TAG AAT TCA TGG CCA AGA CAA TTG CGT AC 3' (SEQ ID NO: 21)

[0127] Downstream primer 5'TA GGTACC CAA ATC TTT AGA AGG GTC3' (SEQ ID NO: 22)

[0128] The PCR reaction volume was 50 μl, the primer concentration was 0.4 μM, and the amplification parameters were 94° C. for 30 seconds, 52° C. for 45 seconds, and 72° C. for 100 seconds. After 30 cycles, the products were analyzed by 2% agarose gel electrophoresis. The PCR product size of HSP65 coding gene was 1665bp. The PCR product was digested with EcoRI, recovered and purified from the gel, and ligated with the human IL-2 sign...

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Abstract

A recombinant gene vaccine AdHIV-Vac able to express the polyepitope gene HSP 65-HIV and human gene IL-15 for preventing and treating AIDS, its preparing process, its relative application, and the composite medicine containing it are disclosed.

Description

technical field [0001] The present invention relates to the field of gene therapy. More specifically, the present invention relates to a new recombinant adenovirus co-expressing HSP65-HIV epitope peptide fusion gene and cytokine (human interleukin 15) gene, the preparation method and application of the recombinant adenovirus, and the recombinant adenovirus containing the Pharmaceutical composition of recombinant adenovirus. Background technique [0002] Since the first discovery of AIDS (Acquired immunodeficiency syndrome, AIDS) in 1981, its spread has spread all over the world. At present, the cumulative number of people infected with AIDS virus in the world has reached 47 million, causing nearly 14 million deaths. The spread and spread of AIDS has seriously affected human health, social life and production, and has become one of the most serious social problems facing mankind. my country's AIDS prevention and control is in a critical period. The total number of AIDS vir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/235A61P31/18C12N7/01C12N15/33
Inventor 王建莉周向阳夏大静
Owner 杭州康泰生物技术有限公司
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