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Human HIV broad spectrum neutralization antibody A16, associated biological material and applications thereof

A technology of biological materials and antibodies, applied in the fields of application, antibodies, analysis materials, etc., can solve problems such as the separation of HIV-infected persons that have not yet been seen

Inactive Publication Date: 2013-07-31
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 antigenic epitopes mediating serum neutralizing activity have yet to be identified (9)
A variety of HIV subtypes are prevalent in my country, especially the B' subtype (Thailand B), B / C and A / E recombinant types have become the main epidemic strains accounting for more than 90%, but unfortunately, There is no report on the isolation of broad-spectrum neutralizing antibodies from HIV-infected patients in my country

Method used

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  • Human HIV broad spectrum neutralization antibody A16, associated biological material and applications thereof
  • Human HIV broad spectrum neutralization antibody A16, associated biological material and applications thereof
  • Human HIV broad spectrum neutralization antibody A16, associated biological material and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 is the screening and preparation method of A16; Example 2 is the expression and purification of A16; Example 3 is the reactivity of A16 with HIV envelope protein antigen; Example 4 is the detection of the neutralizing activity of A16 to HIV ; Example 5 is to determine whether A16 acts on the conformational epitope of gp120; Example 6 is to determine whether A16 can inhibit the combination of gp120 and CD4.

[0061] Example 1, Preparation of Fab Fragment of Human Anti-HIV Antibody

[0062] 1. Obtaining the gene sequence and amino acid sequence of the Fab fragment of the human anti-HIV antibody

[0063] 1. Construction of phage antibody library

[0064] The database construction technology mainly refers to the method 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 and PCR amplification of antibody gene, prep...

Embodiment 2

[0073] Example 2, Expression and Purification of Human Fab Antibody A16

[0074] Transform the recombinant vector pComb3XSS-Fab containing the A16 coding gene obtained through the above screening into Escherichia coli TOP10F' competent cells, pick a single colony and add it to 500ml containing 20mM MgCl 2 and 50 μg / ml ampicillin in SB medium at 37°C at 250 rpm for 5-8 hours (OD 600 =1), add 1ml 0.5M IPTG, shake at 37°C 250rpm for 16 hours, freeze-thaw bacteria at -80°C, add 25μl polymyxin B solution, shake well on ice with 0.5μl nuclease for 1 hour, centrifuge at 10,000 rpm for 30 Minutes, the supernatant was purified with Ni-NTA Superflow N column or protein G column (according to the method provided in the manual). The purified anti-HIV antibody human Fab antibody A16 was obtained.

Embodiment 3、A16

[0075] Embodiment 3, the reactivity of A16 and HIV envelope protein

[0076] The cross-reactivity of A16 with 11 envelope protein recombinant antigens (gp120 or gp140) from different subtypes of HIV, including HIV subtypes A, B and C, was detected by ELISA. The procedure is to mix each protein with 100 μl 0.1M NaHCO at a concentration of 1 μg / ml 3 (pH8.6) solution were coated respectively, overnight at 4°C. The next day, block with 200ml 3% BSA at 37°C for 1 hour, discard the blocking solution, wash once with 0.05% PBS-T, add 100 μl of a small amount of induced bacterial supernatant, incubate at 37°C for 1 hour, and wash three times with 0.05% PBS-T . 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. Develop with TMB for 30 min, 2M H 2 SO 4 Termination, the microplate reader detects the absorbance A value. It was found that A16 could react with envelope protein recombinant anti...

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Abstract

The invention discloses human HIV broad spectrum neutralization antibody A16, an associated biological material and applications thereof. The A16 comprises an antibody heavy chain Fd fragment and an antibody light chain, wherein the heavy chain Fd fragment comprises a variable region VH and a constant region subunit CH1, the VH and the VL comprise determinant complementarity regions and framework regions, the determinant complementarity region comprises CDR1, CDR2 and CDR3, amino acid sequences of the CDR1, the CDR2 and the CDR3 of the VL are respectively represented by sites 26-31, sites 49-51 and sites 88-98 of a sequence 3, and amino acid sequences of the CDR1, the CDR2 and the CDR3 of the VH are respectively represented by sites 26-33, sites 51-58 and sites 97-115 of a sequence 4. The A16 can be adopted to prepare drugs, vaccines and diagnostic reagents for treatments, preventions and diagnosis of HIV infection and AIDS.

Description

technical field [0001] The present invention relates to a human HIV-specific Fab antibody and related biological materials and applications thereof, in particular to human HIV neutralizing antibody A16 and related biological materials and applications. Background technique [0002] Acquired immunodeficiency syndrome (AIDS), referred to as AIDS, is a syndrome mainly caused by T cell immune dysfunction caused by human immunodeficiency virus (human immunodeficiency virus, HIV) infection. Since AIDS was discovered in 1981, more than 60 million people worldwide have been infected with HIV, and about 25 million of them have died. According to WHO statistics, the number of new infections and deaths is increasing year by year, and now there are about 16,000 new infections every day. The first AIDS patient was discovered in my country in 1985. So far, 434,000 HIV-infected people have been reported, of which 88,000 died. In Yunnan, Henan, Xinjiang and other regions, HIV infection ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12Q1/70C12Q1/68G01N33/571A61K39/42A61K48/00A61P31/18
Inventor 何玉先满来孟佳子
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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