Rabies virus resistant specific humanized antibody and application thereof

A rabies virus and antibody technology, applied in antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve the problem of no drugs being approved for marketing

Active Publication Date: 2014-12-10
LANZHOU INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although some anti-rabies virus-specific McAbs have been reported to have entered the clinical research stage, no drugs have been approved for marketing so far.

Method used

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  • Rabies virus resistant specific humanized antibody and application thereof
  • Rabies virus resistant specific humanized antibody and application thereof
  • Rabies virus resistant specific humanized antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Construction and rescue of anti-rabies virus ScFv phage antibody library

[0040] The anti-rabies virus ScFv phage antibody library is independently constructed by our laboratory. It is based on 32 high-potency healthy human peripheral blood vaccinated against rabies as raw materials. It is extracted from lymphocytes, total RNA is extracted, cDNA is reverse transcribed, and the heavy chain is obtained by PCR. and the light chain variable region gene, and obtained ScFv by overlapping extension PCR (Splicing overlap extension PCR, SOE-PCR). ScFv was digested and ligated with phagemid pS100, and finally electroporated into competent Ecoli TG1 cells to obtain the primary antibody library. After testing, the library capacity of the primary antibody library is about 9.0×10 8 .

[0041] Inoculate the H+λ primary antibody library bacterial solution and the H+κ primary antibody library bacterial solution into 2YT-AG at a ratio of 50 times the capacity of the primary ...

Embodiment 2

[0042] Example 2. Screening of anti-rabies virus phage antibody library

[0043] Prepare inactivated and purified rabies virus using conventional methods. After inactivation and purification, the protein content of rabies virus is 300 μg / ml, which is used for the screening of phage antibody particles.

[0044] Three rounds of selection were performed using purified rabies virus coated immunotubes. The three rounds of antigen coating concentration were 30, 10 and 5 μg / ml in turn, and the washing times were 5, 10 and 20 rounds in turn. The phage particles obtained after screening were re-infected with Ecoli TG1 and spread on 2YT-AG plates, picked clones and cultured in 96-well plates overnight with 2YT-AG, and cultured with 2YT-AK after adding MK1307 helper phage infection. The supernatant was taken and added to an ELISA plate coated with 2 μg / ml purified rabies virus, the color was developed using horseradish peroxidase-labeled mouse anti-M13 antibody, and the absorbance was ...

Embodiment 3

[0048] Example 3, ELISA analysis and screening of candidate antibody monoclonal

[0049] Pick clones from the plate used for titration of phage antibody after enrichment screening and put them in 100 μL of 2×YT-AG. At the same time, pick pS100 empty vector as a negative control, and 3 culture medium only as a blank control, and culture overnight at 37°C. Transfer 10 μL of the culture to a new 96-well plate containing 90 μL of 2×YT-AG, and incubate at 37°C for 1 h. M13K07 was added to each well (to make the phage titer in the culture solution reach 109 cfu / mL, about 25 μL), the MOI was 10-20, and incubated at 37° C. for 30 minutes. Centrifuge at 4000g for 10min, carefully discard the supernatant, resuspend the cells in 100μL of 2×YT-AK, and culture overnight at 30°C.

[0050] The next day, centrifuge at 4000g for 10 min, and use the supernatant for ELISA detection. Coat a 96-well plate with purified rabies virus, 2 μg / mL, 100 μL / well, overnight at 4°C. Wash 3 times with PBST...

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Abstract

The invention aims at providing a rabies virus resistant neutralizing antibody, and particularly provides a humanized or completely humanized monoclonal antibody to meet the requirement for clinically diagnosing and/or treating rabies. A phage antibody library is prepared by adopting a phage antibody library technology and taking 32 parts of high-potency healthy human peripheral blood inoculated with rabies vaccines as a raw material; 7 ELISA positive antibodies are obtained through three rounds of screening from the phage antibody library; and furthermore, the neutralizing activity of the 7 ELISA positive antibodies is measured through an RFFIT method, wherein four ELISA positive antibodies, namely R5, R7, R8 and R9, have higher neutralizing activity in all. The rabies virus resistant neutralizing antibody with high affinity, which is provided by the invention, can be used for substituting ERIG and HRIG to carry out active and/or passive immune therapy on rabies virus seriously-exposed persons.

Description

technical field [0001] The present invention relates to the prevention and treatment of viral diseases, especially the prevention and treatment of rabies. Background technique [0002] Rabies is a worldwide zoonotic infectious disease caused by Rabies virus (RV), and the mortality rate of humans and animals is as high as 100%. According to the latest data from the World Health Organization (WHO), about 55,000 people die from rabies every year in the world, mainly in developing countries in Asia, Africa and Latin America. WHO recommends that active and passive immunotherapy should be administered simultaneously for severe exposures to obtain a rapid protective effect. Currently, the preparations used for passive immunotherapy mainly include equine rabies immune globulin (ERIG) and human rabies immune globulin (human rabies inlmune globulin, HRIG). However, the supply of ERIG and HRIG is limited and the price is high, so it is difficult to popularize their application in und...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10A61K39/42A61P31/14
Inventor 毛晓燕陈继军安晨乔玉玲马瑞
Owner LANZHOU INST OF BIOLOGICAL PROD
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