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Fab segment of human HIV antibody, and coding gene and application thereof

A technology that encodes genes and fragments, and is used in applications, antibodies, genetic engineering, etc.

Inactive Publication Date: 2011-09-21
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, only a small number of HIV broad-spectrum neutralizing antibodies have been found so far, and most of the epitopes that mediate serum neutralizing activity have yet to be identified (Nature, 2009)

Method used

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  • Fab segment of human HIV antibody, and coding gene and application thereof
  • Fab segment of human HIV antibody, and coding gene and application thereof
  • Fab segment of human HIV antibody, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 is the screening and preparation method of HY1397; Example 2 is the neutralizing activity of HY1397 on HIV;

[0043] Examples 3-5 are the reactivity of HY1397; Example 6 is the screening and epitope analysis of HY1397 epitope mimetic peptides.

[0044] Example 1. Preparation of Fab fragments of human anti-HIV antibodies

[0045] 1. Obtaining the gene sequence and amino acid sequence of the Fab fragment of human anti-HIV antibody

[0046] 1. Construction of phage antibody library

[0047] The library construction technology mainly refers to the methods introduced by Barbas et al. (Carbos F. Barbas III, Dennis R. Burton, Jamie K. Scott, Gregg J. Siverman. Phage Display-A Laboratory Manual. Cold Spring Harbor Laboratory Press. New York), Such as primer design of antibody gene and PCR amplification, preparation of phage expression vector, etc. First, PCR amplification of human IgG Fab segment gene is performed. The steps are: use lymphocyte separation solution to separat...

Embodiment 2

[0058] Example 2. Detection of HIV neutralization activity of HY1397

[0059] The HIV pseudovirus infection system was used to evaluate the antiviral activity of the Fab fragments of human anti-HIV antibodies. The specific steps are to combine the plasmid pcDNA3.1-ENV expressing the HIV strain SF162ENV protein and the backbone plasmid pSG3 ΔENV (Express all proteins except ENV in the HIV genome), transfect 293T cells at a mass ratio of 1:2, and set pSG3 ΔENV Control, ie only transfect the same amount of pSG3 ΔENV . At 37℃, 5% CO 2 After 6 hours of incubation in the cell incubator, the plasmid was allowed to enter the cells, then the medium was changed, and the cells were incubated in the cell incubator for 48 hours, and the pseudovirus was secreted into the supernatant. Use a pipette to aspirate as much of the supernatant in the cell culture flask or cell culture plate as possible, filter through a 0.45 μm filter or centrifuge at 1000 g for 10 min to take the supernatant, add ...

Embodiment 3

[0060] Example 3. Reactivity of HY1397 with HIV envelope protein

[0061] ELISA was used to detect the cross-reactivity of HY1397 with 15 recombinant antigens (gp120 or gp140) of envelope proteins from different HIV subtypes, including HIV subtypes A, B and C. The procedure is to use 100μl 0.1M NaHCO for each protein at a concentration of 1μg / ml 3 (pH8.6) The solutions were separately coated, overnight at 4°C. On the next day, block with 200ml 3% BSA at 37°C for 1 hour, discard the blocking solution, wash with 0.05% PBS-T, add 100μl small amount of induced bacterial supernatant, incubate at 37°C for 1 hour, wash with 0.05% PBS-T for three times all over. Add 100 μl enzyme-labeled anti-human Fab secondary antibody diluted 1:30000, incubate at 37°C for 1 hour, and wash three times with 0.05% PBS-T. Use TMB to develop color for 30 minutes, 2M H 2 SO 4 Stop, the microplate reader detects the absorbance A value. It was found that HY1397 can react with recombinant antigens of envelo...

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Abstract

The invention discloses a Fab segment of a human HIV antibody, and a coding gene and application thereof. The Fab segment of the antibody consists of a heavy chain variable region VH and a constant region subunit CH1 of the antibody, and a light chain of the antibody, wherein the light chain consists of a variable region VL and a constant region CL; the VH and VL respectively consist of a complementary-determining region (CDRs) and a framework region (FRs); the complementary-determining region consists of CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1, CDR2 and CDR3 of the VL are shown as the 27th-32nd position, 50th-52nd position and 89th-98th position in a sequence 2; and the amino acid sequences of the CDR1, CDR2 and CDR3 of the VH are shown as the 26th-33rd position, 51st-57th position and 96-109th position in a sequence 3. The Fab segment and the coding gene thereof prepare gene engineering antibodies in different forms so as to prepare medicines, vaccines and diagnostic reagents for treating, preventing and diagnosing HIV infection and human immunodeficiency virus.

Description

Technical field [0001] The invention relates to a Fab fragment of human HIV antibody and its coding gene and application. Background technique [0002] Acquired immunodeficiency syndrome (Acquired immunodeficiency syndrome, AIDS) is abbreviated as AIDS, which is a syndrome mainly caused by human immunodeficiency virus (HIV) infection with T cell immune function deficiency. Since AIDS was discovered in 1981, more than 60 million people have been infected worldwide, of which about 25 million have died. The number of new infections and deaths has increased year by year. According to WHO estimates, there are 16,000 new infections every day; it is estimated that about 100 million people will be infected in ten years. This will undoubtedly be a major disaster for human society. HIV infection in my country has entered a period of rapid growth from the dissemination period. There are currently about 700,000 infected people, including more than 80,000 AIDS patients. In Yunnan, Henan, X...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/63C12N5/10C12N1/21C12N1/19C12N7/01C12Q1/70C12Q1/68G01N33/569A61K39/42A61P31/18C12R1/93
Inventor 何玉先满来万超孙坚萍张超
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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