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Anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and preparation and application thereof

A technology of genetically engineered antibodies and rabies virus, applied in antiviral immunoglobulin, genetic engineering, antiviral agents, etc., can solve problems such as difficult large-scale production, difficult antibodies, limited applications, etc., achieve efficient expression and overcome technical bottlenecks Effect

Inactive Publication Date: 2011-10-19
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the most reported genetically engineered antibodies are scFv, scFv-Fc and Fab, etc. scFv has made great contributions to the diagnosis of various diseases, but due to the instability of its spatial structure, its application in clinical treatment is greatly limited. Applications
scFv-Fc and Fab have large molecules and contain multiple glycosylation sites. It is difficult to complete the maturation of antibodies in prokaryotic systems, and often require expression and preparation in eukaryotic systems. However, due to the limitations of current fermentation technology in eukaryotic cells making it difficult to form large-scale production

Method used

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  • Anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and preparation and application thereof
  • Anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and preparation and application thereof
  • Anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Construction of ribosome display single-chain antibody gene library (VH / k)

[0053] The construction of the antibody gene library is divided into four major steps:

[0054] 1-1) Extraction of total cell RNA and cDNA synthesis

[0055] Extraction of total cell RNA The rabies virus Vero vaccine (France·Lyon Pasteurieux Serum Vaccine Company) was used for basic immunization of three volunteers. Two weeks later, another booster immunization was performed. Take 40 ml of peripheral blood from three immunized volunteers, and add 5 U heparin per ml of blood. Dilute the peripheral blood with an equal amount of PBS, gently add it from the tube wall to the centrifuge tube containing the lymphocyte separation solution (Institute of Blood, Peking Union Medical College), set a low-speed tabletop centrifuge at 2500r / min, and centrifuge for 20min. Use a capillary pipette to gently suck the lymphocyte layer and place it in another 15ml centrifuge tube. Dilute the same amount with...

Embodiment 2

[0075] Example 2: Construction of ribosome display single chain antibody gene library

[0076] The construction of the single-chain antibody gene for ribosome display was completed by assembly PCR. The purified VH / K antibody gene was used as a template, and two-step PCR was performed with RDTb / Ck / for and RDT7 / Ck / for as primers. The first Add a prokaryotic ribosome binding site (SD sequence) to the N-terminus. The second step is to construct T7 promoter, 5'-stem-loop structure and 3'-stem-loop structure at both ends of the gene. TaqDNA polymerase 5U, total volume 50μl, 94℃60s, 50℃80s, 72℃120s, after 30 cycles of amplification, the constructed gene fragment is about 1000bp, see figure 2 , The constructed ribosome display antibody gene library has a capacity of 6.7×10 12 .

[0077] The required primer sequences are as follows:

[0078] Ck / for 5’ccg cac acc agt aag gtg tgc ggt gct cta gaa cac tct cc 3’

[0079] RDTb 5’aga cca caa cgg ttt ccc tct aga aat aat ttt gtt taa ctt taa

[0080] ...

Embodiment 3

[0082] Example 3: In vitro transcription and translation

[0083] In vitro transcription and translation were performed using invitrogen's expresswayTM Plus ExpressionSystem, according to the operating manual. After terminating the reaction, 1 / 5 volume of pre-cooled WBT containing 10% (w / v) BSA was added to the supernatant. After the reaction mixture was centrifuged at 1400g for 5 minutes at 4°C, the supernatant was transferred to a new ice-precooled EP tube.

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Abstract

The invention relates to an anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and a preparation and an application thereof. The antibody is VH-connecting peptide-VK of a three-structural single-stranded antibody comprising a variable region of heavy chain and a variable region of light chain by using 12 sequences of amino acid of the 1 (CH1) 5' end of a constant region of a heavy chain as the connecting peptide AKTTAPSVYPL, and the antibody realizes efficient expression in a prokaryotic system. The biological characteristic studies show that the RD9 is ananthropogenic genetic engineering antibody which has high appetency and better stability and can specially centralize rabies virus as well as genes and gene products of the antibody can be used for preparing clinical drugs for preventing and treating the rabies; the preparation method of the antibody is easy for massive industrial production. The invention solves the problem in the other technology of the application of genes of the variable region of the heavy chain and the variable region of a light chain of the antibody and polypeptide coded by the genes in the drugs for preventing and treating the rabies.

Description

(1) Technical field: [0001] The invention belongs to the field of biotechnology and relates to an anti-rabies virus antibody and its preparation and application, in particular to a human anti-rabies virus glycoprotein neutralizing genetic engineering antibody RD9 and its preparation and application. (2) Background technology: [0002] Rabies is an infectious disease with the highest mortality rate, with a case fatality rate of 100%. Rabies is widely distributed around the world, and more than 60,000 people die from rabies every year. my country is also a country with a high incidence of rabies. At present, another peak of rabies epidemic is taking shape in our country! The prevention and treatment of rabies has become one of the most important public health problems in our country. It is almost impossible for humans to eliminate rabies from the earth, because rabies is a symbiotic disease of humans and animals, and humans still cannot control the occurrence of animal rabies. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/63G01N33/569C12Q1/68A61K39/42A61K48/00A61P31/14
Inventor 赵小玲杨志华弓景波
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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