Recombinant vector vaccine expressing HIV antigen

A recombinant vector and vaccine technology, applied in the field of recombinant vector vaccine and its construction, can solve problems such as unsuitability for application

Inactive Publication Date: 2016-03-30
CSPC ZHONGQI PHARM TECH (SHIJIAZHUANG) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] At present, most of the HIV vaccines used in foreign preclinical research are based on the epidemic strains in Europe, America and Africa, w

Method used

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  • Recombinant vector vaccine expressing HIV antigen
  • Recombinant vector vaccine expressing HIV antigen
  • Recombinant vector vaccine expressing HIV antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Construction of non-transmissible Sendai virus vector (SeV / dF) with deletion of F gene

[0040] The present invention obtains the Sendai virus vector with F gene deletion through a series of PCR, enzyme digestion and connection means, and the specific construction process is as follows:

[0041] 1. Clone the full base sequence of the Z strain in Sendai virus to obtain the plasmid pSeV with the full-length cDNA of SeV.

[0042] 2. Digest the plasmid pSeV with restriction endonucleases SphI and KpnI at the same time, recover a 3500bp fragment, and name it pSeV△SK plasmid, and name the excised 14670bp fragment 1, most of which are the genome sequence of Sendai virus.

[0043] 3. Carry out insertion mutation by PCR: insert 18 bp sequence with NotI site at the 119th position of Sendai virus genome sequence, obtain plasmid and pSeV18.

[0044] The primer sequences and methods used are as follows, and the NotI restriction site is underlined.

[0045] OP1,5'-TCTGACACATGCAGCTC...

Embodiment 2

[0060] HIV gag Gene Cloning, Modification and Introduction of Sendai Virus Vector with F Gene Deletion

[0061] 1. Collect venous blood from HIV-positive patients in AIDS-endemic areas in Henan;

[0062] 2. Using Qiagen's QiaAmpBlood kit to extract total cellular DNA from the above venous blood;

[0063] 3. According to the consensus sequence of the gag gene, design primers, use the total DNA in step 2 as a template, amplify the gag gene and connect it to the pMD18-Teasy vector (purchased from Promega) to obtain the plasmid pMD18-T-gag, and transform into DH5α competent Cells (Promega), smeared on a plate, cultured, picked a single clone and cultured, extracted plasmids for enzyme digestion identification;

[0064] 4. Sequencing the correct plasmid identified by enzyme digestion in step 3, and comparing the sequencing results with the gag gene of the B subtype standard strain, the results show that the gene has the sequence characteristics of the B subtype;

[0065] 5. Accordi...

Embodiment 3

[0068] Construction of Particles Expressing Sendai Virus Helper Protein

[0069] 3.1 Construction of plasmid pGEM-NP expressing Sendai virus NP protein:

[0070] 1. Digest pSeV-TDK (from DNADEC) and pGEM11Zf(+) (purchased from Promega) with restriction endonucleases NotI / XhoI respectively, and connect the nucleotide sequence containing the NP protein to pGEM11Zf after digestion (+) on;

[0071] 2. Design the following primers to amplify the nucleotide sequence encoding the NP protein using the product obtained in step 1 as a template:

[0072] forward primer, 5'-CCGGAATTCAACAAATGGCCGGGTTGTTGAGCACCTTCGA-3';

[0073] reverse primer, 5'-CCGGAATTCCTAGATTCCTCCTATCCCAGCTACTGCTGCTCG-3';

[0074] 3. Digest the PCR product with EcoRI and connect it to the vector pGEM11Zf(+) to obtain the pGEM-NP plasmid.

[0075] 3.2 Construction of plasmid pGEM-P expressing Sendai virus P protein:

[0076] 1. Use SeVcDNA as a template to amplify the gene encoding the P protein, and the primer seq...

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Abstract

The invention provides a recombinant sendai vector vaccine expressing HIVGag, construction method and application thereof. The vaccine is constructed based on an F gene defective sendai virus vector. The polynucleotide expressing HIVGag is properly modified and reconstructed so as to be able to realize high expression in eukaryotic cells without depending on regulatory protein Rev. The test result shows that the recombinant vector vaccine can express HIVGag correctly and can effectively induce in-vivo and in-vitro cellular immunity.

Description

technical field [0001] The invention relates to the field of antiviral immunology, in particular, the invention relates to a recombinant vector vaccine expressing HIV antigen and its construction method and application. Background technique [0002] Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (human immunodeficiency virus, HIV) after invading the human body through CD4 molecules and certain chemokine receptors on the cell surface to invade host cells, destroy the immune function of the body, and cause Secondary immunodeficiency syndrome characterized by malignant disease due to severe opportunistic infections. According to statistics, as of 2012, the total number of people infected with AIDS in the world has exceeded 35 million, more than half of the total population of France. In 2012, there were 2.3 million new infections and about 1.6 million deaths due to AIDS. The number of AIDS infections and morbidity in China has also ente...

Claims

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Application Information

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IPC IPC(8): C12N15/86A61K48/00A61K39/21A61P31/18
Inventor 张锋杨延新王明晓
Owner CSPC ZHONGQI PHARM TECH (SHIJIAZHUANG) CO LTD
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