Fab segment of human-source human immunodeficiency virus (HIV) antibody and coded gene and application thereof

A technology that encodes genes and antibodies, used in applications, antibodies, gene therapy, etc.

Inactive Publication Date: 2011-10-12
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-source human immunodeficiency virus (HIV) antibody and coded gene and application thereof
  • Fab segment of human-source human immunodeficiency virus (HIV) antibody and coded gene and application thereof
  • Fab segment of human-source human immunodeficiency virus (HIV) antibody and coded gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 is the screening and preparation method of HY498; Example 2 is the neutralizing activity of HY498 against HIV; Examples 3-5 are the reactivity of HY498; Example 6 is the screening and epitope analysis of HY498 epitope mimetic peptides.

[0044] Example 1, Preparation of Fab Fragment of Human Anti-HIV Antibody

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

[0046] 1. Construction of phage antibody library

[0047]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, preparation of phage expression vector, etc. Firstly, the PCR amplification of the human IgG Fab segment gene was carried out. The steps are: use lymphocyte separation fluid to is...

Embodiment 2

[0055] Example 2, Expression and Purification of Fab Fragment of Human Anti-HIV Antibody

[0056] Transform the recombinant vector pComb3XSS-Fab containing the coding gene of the Fd fragment of HY498 shown in Sequence 2 and the light chain coding gene of HY498 shown in Sequence 1 obtained from the above screening into Escherichia coli TOP10F' competent cells, and pick a single colony Add to 500ml containing 20mM MgCl 2 and 50 μg / ml ampicillin in SB medium at 37°C at 250rpm 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 10000 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 Fab fragment (HY498) of the purified human anti-HIV antibody was obtained.

Embodiment 3

[0057] Example 3, HY498 neutralizing activity detection of HIV

[0058] The antiviral activity of Fab fragments of human anti-HIV antibodies was evaluated by HIV pseudovirus infection system. The specific steps are, the plasmid pcDNA3.1-ENV expressing the ENV protein of HIV strain SF162 or HXB2 and the backbone plasmid pSG3 ΔENV (Express all proteins in the HIV genome except ENV), transfect 293T cells at a mass ratio of 1:2, and set pSG3ΔENV control at the same time, that is, only transfect the same amount of pSG3 ΔENV . At 37°C, 5% CO 2After incubating in the cell incubator for 6 hrs, the plasmid entered the cells, then changed the medium, continued to incubate in the cell incubator for 48 hr, and the pseudovirus was secreted into the supernatant. Use a pipette to suck out the supernatant in the cell culture flask or plate as much as possible, filter through a 0.45 μm filter or centrifuge at 1000g for 10 minutes to get the supernatant, add FBS to it to make the final conce...

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Abstract

The invention discloses an Fab segment of a human-source human immunodeficiency virus (HIV) antibody, a coded gene thereof and application thereof. The Fab segment of the antibody consists of a variable region (VH) and a constant region subunit CH1 of a heavy chain of the antibody and a light chain of the antibody, wherein the light chain consists of a light chain variable region (VL) and a lightchain constant region (CL); both of the VH and the VL consist of complementary determining regions (CDRs) and framework regions (Framework Region, FRs); the complementary determining region consists of a CDR1, a CDR2 and a CDR3; the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the VL are respectively shown as the 27th to the 32nd positions of the sequence 2, the 50th to the 52nd positions of the sequence 2 and the 89th to 98th positions of the sequence 2; and the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the VH are respectively shown as the 26th to the 33rd positions of the sequence 3, the 51st to the 58th positions of the sequence 3 and the 97th to 116th positions of the sequence 3. The Fab segment and the coded gene thereof are used for preparing genetic engineering antibodies in different forms and medicaments, vaccines and diagnostic reagents for treating, preventing and diagnosing HIV infection and acquired immune deficiency syndrome.

Description

technical field [0001] The invention relates to a Fab fragment of a human HIV antibody, its coding gene and application. 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 the discovery of AIDS in 1981, more than 60 million people have been infected worldwide, and about 25 million of them have died. The number of new infections and deaths is increasing every year. According to WHO estimates, there are now 16,000 new infections every day; it is estimated that about 100 million people will be infected in ten years, which will undoubtedly be a catastrophe for human society. my country's HIV infection has entered a period of rapid growth from the spread period, and there are about 700,000 infected people, including more than 80,000 AIDS patients. In Yunnan, Henan, Xinjiang and other prov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/63C12N5/10C12N1/21C12N7/01G01N33/569C12Q1/70C12Q1/68A61K39/42A61K48/00A61P31/18C12R1/93C12R1/92
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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