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

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

Inactive Publication Date: 2013-03-27
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 against 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 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 separat...

Embodiment 2

[0058] Example 2, HY1397 neutralizing activity detection of HIV

[0059] 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 HIV strain SF162ENV protein 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 pSG3 ΔENV Control, i.e. only transfected with the same amount of pSG3 ΔENV . At 37°C, 5% CO 2 After 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 concentration 20%, and tran...

Embodiment 3

[0060] Embodiment 3, the reactivity of HY1397 and HIV envelope protein

[0061] The cross-reactivity between HY1397 and 15 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 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. Develop with TMB for 30 min, 2M H 2 SO 4 Termination, the microplate reader detects the absorbance A value. It was found that HY1397 could react with envelope pro...

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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 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 Patents(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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