Anti-HIV genetic engineering divalent antibody and preparation method thereof, and anti-HIV genetic engineering medicine

A genetic engineering and antibody technology, applied in the field of biopharmaceuticals, can solve the problems of limited specific binding effect and ineffective prevention and treatment of HIV virus infection, and achieve prevention and treatment of HIV virus infection. The preparation method is simple and the specific binding effect enhanced effect

Inactive Publication Date: 2013-07-03
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide an anti-HIV antibody that can specifically bind to the HIV virus in view of the defects in the prior art that the specific binding effect between the anti-HIV antibody and the HIV virus is limited, and the prevention and treatment effect on HIV virus infection is not obvious. , Anti-HIV genetic engineering bivalent antibody for effective prevention and treatment of HIV infection, preparation method thereof, and anti-HIV genetic engineering drug

Method used

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  • Anti-HIV genetic engineering divalent antibody and preparation method thereof, and anti-HIV genetic engineering medicine
  • Anti-HIV genetic engineering divalent antibody and preparation method thereof, and anti-HIV genetic engineering medicine
  • Anti-HIV genetic engineering divalent antibody and preparation method thereof, and anti-HIV genetic engineering medicine

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preparation example Construction

[0021] The preparation method of the anti-HIV genetic engineering bivalent antibody of the present invention comprises the following steps: A: obtaining the constant region gene, CD4 gene and CCR5 gene of the human antibody respectively; B: constructing the constant region gene, CD4 gene and CCR5 gene comprising the human antibody Gene expression plasmid; C: The expression plasmid formed in step B is transfected into the expression cell line for culturing to stably express the bivalent antibody; D: The supernatant is collected by centrifugation or the cells are purified to obtain the bivalent antibody. The bivalent antibody with both CD4 and CCR5 can be obtained through cell line expression. This method is easy to operate and facilitates the scaled-up production of the above-mentioned bivalent antibody.

[0022] Further, the above step A specifically includes the following steps:

[0023] A1: Fishing of constant region genes of human antibodies:

[0024] Appropriate primers w...

Embodiment 1

[0047] The CD4 gene and CCR5 gene amplified by PCR were cloned into the recombinant plasmid pCDNA3.1 / His C-IgG containing the constant region gene of IgG1 antibody, and the constant region gene containing IgG1 antibody was flexibly connected to the CD4 gene and CCR5 gene The eukaryotic expression plasmids pCDNA3.1 / HisC-CD4-IgG and pCDNA3.1 / HisC-CCR5-IgG. Specifically, the above-mentioned flexible connection is realized through a flexible connecting peptide. For the amino acid sequence of the flexible linker peptide, see the attached sequence listing.

[0048] Human kidney epithelial cell line 293 was used as the expression cell line to extract high-quality and high-concentration eukaryotic expression plasmids pCDNA3.1 / His C-CD4-IgG and pCDNA3.1 / His C-CCR5-IgG, and use liposome transfection The above plasmids were transfected into 293 cells, and the cells were collected 48 hours later, and the protein was subsequently extracted for Western Blot detection.

Embodiment 2

[0050] Design primers, primer sequence 7 is: 5'-CTT AAG CTT ACC ATG GGG GGT TCT-3', from eukaryotic expression plasmids pCDNA3.1 / His C-CD4-IgG and pCDNA3.1 / His C-CCR5-IgG Amplify the CD4-IgG and CCR5-IgG genes of the eukaryotic expression vector pCDNA3.1 / His C with selection tags (6×His tag and XpressEpitop tag), and load these two genes into pCDNA5 / FRT / TO TOPO TA The expression plasmids pCDNA5 / FRT / TOTOPO TA-CD4-IgG and pCDNA5 / FRT / TOTOPO TA-CCR5-IgG were obtained in the eukaryotic vector.

[0051] The human kidney epithelial cell line Flp-In T-Rex 293 was used as the expression cell line to establish a stable and high-expression cell line, and extract high-quality and high-concentration eukaryotic expression plasmids pCDNA5 / FRT / TO TOPO TA-CD4-IgG and pCDNA5 / FRT / TO TOPO TA-CCR5-IgG was transfected into Flp-In T-Rex 293 cells by liposome transfection method. After 24 hours, the antibiotic Blasticidin B was used for resistance screening. After 10 days, the The 96-well plate dil...

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Abstract

The invention relates to an anti-HIV genetic engineering divalent antibody and a preparation method thereof, and an anti-HIV genetic engineering medicine. The anti-HIV genetic engineering divalent antibody comprises a human antibody constant region and CD4 and CCR5 connected with the human antibody constant region. The preparation method of the anti-HIV genetic engineering antibody comprises the steps that: human antibody constant region gene, CD4 gene, and CCR5 gene are obtained; expression plasmids comprising the three genes are constructed; the expression plasmids are transfected into cell lines and are cultured; and the divalent antibody is obtained after separation and purification. The divalent antibody provided by the invention has both CD4 and CCR5 which can be specifically bound with CD4 and CCR5 loci on HIV virus, specific binding effect is substantially improved, and HIV-infected host cell dual inhibition can be realized. The preparation method provided by the invention is simple and feasible. The anti-HIV genetic engineering medicine with the divalent antibody can effectively act upon HIV virus, and assists in effectively preventing and treating HIV virus infections.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to an anti-HIV genetic engineering antibody, more specifically, to an anti-HIV genetic engineering bivalent antibody, a preparation method thereof and an anti-HIV genetic engineering drug. Background technique [0002] During the infection process of HIV virus, the envelope membrane of HIV virus first fuses with the cell membrane of the target cell. The fusion process is mainly mediated by the envelope glycoprotein gp120 and the transmembrane subunit gp41. gp120 binds to CD4 molecules and co-receptors (chemokine receptors CCR5 or CXCR4, etc.) on target cells successively, resulting in a change in the configuration of gp41, forming a core structure of 6-strand α2 helical bundles, and connecting the virus envelope with the target cells The cell membranes of the cells are drawn closer and fused, completing the infection process of the HIV virus entering the host cell. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85A61K39/42A61P31/18
Inventor 周兆平郑永唐易俊波雷明军苏庆宁
Owner SHENZHEN UNIV
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