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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 the direction of antiviral immunoglobulin, genetic engineering, antiviral agents, etc. The effect of overcoming technical bottlenecks

Inactive Publication Date: 2009-10-07
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-displayed single-chain antibody gene library (VH / k)

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

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

[0055] Extraction of total cellular RNA: Rabies virus Vero vaccine (Bas de Merieux Serum Vaccine Company, Lyon, France) was used for basic immunization of three volunteers, and a booster immunization was given 2 weeks later. 40ml of peripheral blood was drawn from three immunized volunteers, and 5U of heparin was added to each milliliter of blood. Dilute the peripheral blood with an equal amount of PBS, gently add it from the tube wall to the centrifuge tube filled with lymphocyte separation solution (Hematology Institute, Peking Union Medical College, China), place in a low-speed desktop centrifuge, 2500r / min, and centrifuge for 20min. Use a capillary pipette to gently aspirate the lymphocyte layer and place it in another 15ml...

Embodiment 2

[0074] Example 2: Construction of ribosome-displayed single-chain antibody gene library

[0075] The construction of the single-chain antibody gene for ribosome display was completed by assembly PCR, using the purified VH / K antibody gene as a template, and using RDTb / Ck / for and RDT7 / Ck / for as primers for two-step PCR, the first The first step is to add a prokaryotic ribosome binding site (SD sequence) at the N-terminus. In the second step, the T7 promoter, 5'-stem-loop structure and 3'-stem-loop structure were respectively constructed at both ends of the gene. TaqDNA polymerase 5U, total volume 50μl, 94°C for 60s, 50°C for 80s, 72°C for 120s, after 30 cycles of amplification, the constructed gene fragment is about 1000bp, see figure 2 , the capacity of the constructed ribosome display antibody gene library reached 6.7×10 12 .

[0076] The required primer sequences are as follows:

[0077] Ck / for 5’ccg cac acc agt aag gtg tgc ggt gct cta gaa cac tct cc 3’

[0078] RDTb 5’...

Embodiment 3

[0080] Example 3: In vitro transcription and translation

[0081]The in vitro transcription and translation were carried out by expresswayTM Plus ExpressionSystem of Invitrogen Company according to the operation manual. After terminating the reaction, pre-cooled 1 / 5 volume of WBT containing 10% (w / v) BSA was added to the supernatant. After the reaction mixture was centrifuged at 1400 g for 5 min at 4 °C, the supernatant was transferred to a new ice-cold 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 an anthropogenic 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. (two) background technology: [0002] Rabies is the infectious disease with the highest mortality rate, with a case fatality rate of 100%. Rabies is widely distributed in the world, and more than 60,000 people die of rabies every year in the world. my country is also a country with a high incidence of rabies. At present, another rabies epidemic peak 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 human beings to eradicate rabies from the earth, because rabies is a symbiotic disease between humans and animals, and humans have not yet been able to contr...

Claims

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Application Information

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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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