Recombination human rabies viruses resisting antibody

A rabies virus and antibody technology, applied in the field of recombinant monoclonal antibodies, can solve the problem that the comprehensive effect of antiserum cannot be completely replaced, and achieve the effect of small storage capacity and great flexibility

Inactive Publication Date: 2008-08-06
吉林圣元科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some people have expressed human anti-rabies virus antibody fragments Fab and scFv in Escherichia coli, but these small molecule antibodies cannot completely replace the comprehensive effects of antiserum in vivo, including antibody-dependent cytotoxicity and antibody-mediated cellular cleavage, etc., so the production of intact human anti-rabies immunoglobulins became the target of further research

Method used

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  • Recombination human rabies viruses resisting antibody
  • Recombination human rabies viruses resisting antibody
  • Recombination human rabies viruses resisting antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Construction of pPICZα / Fc expression vector

[0068] 1.1 PCR amplification of human antibody Fc fragment

[0069] Take 100ng of plasmid pPICZα-H (containing the full-length sequence of human antibody heavy chain) and amplify the Fc upstream primer:

[0070] 5'-TAAGGGCCCCTGAGCCCAAATCTTGTG-3' (SEQ ID NO: 1)

[0071] Fc amplification downstream primer: 5'-CCGTCTAGATCATTTACCCGGAGACAGGG-3' (SEQ ID NO: 2) Use these two amplification Fc primers to establish the following reaction system in a 0.2ml EP tube:

[0072] 20 μl of the above cDNA reaction solution

[0073] MgCl 2 6μl

[0074] 10×LA PCR buffer 8μl

[0075] TaKaPa LATaq 1 μl

[0076] Amplify Fc upstream primer 1μl

[0077] Amplify Fc downstream primer 1μl

[0078] Sterile distilled water Final volume 100μl

[0079] Cyclic amplification was performed on a PCR machine. The amplification procedure is:

[0080] ①94℃2min

[0081] ② 94°C for 30s, 58°C for 30s, 72°C for 1.5min, 28 ...

Embodiment 2

[0128] Example 2: Construction of anti-rabies virus scFv-Fc small molecule antibody library expression plasmid

[0129] Blood donation volunteers were immunized with the French PM strain Vero cell purified vaccine (deltoid muscle injection in the upper arm) and the antibody titers reached the corresponding protection level. 20ml of blood was collected (1ml±separated serum was used for antibody detection), and put into sterile heparin anticoagulant tubes respectively. Lymphocytes were separated and extracted with lymphocyte separation medium, and then total RNA was extracted by Trizol method.

[0130] 2.1 Extraction of total RNA

[0131] 2.1.1 Isolation of lymphocytes

[0132] 1) Add an equal volume of balanced salt solution to dilute the peripheral blood, and mix slowly.

[0133] 2) Add lymphocyte separation liquid into the centrifuge tube (add 3-5ml separation liquid to 10ml of blood), tilt at 45°C, and slowly add diluted blood along the tube wall at a distance of 1cm from...

Embodiment 3

[0217] Example 3: Expression of scFv-Fc small molecule antibody in Pichia pastoris

[0218] 3.1 Preparation of linearized recombinant plasmid

[0219] Transformation of super competent cells XL with scFv-Fc recombinant plasmid pPICZα / scFv-Fc 1 -blue, the bacterial liquid obtained after shaking overnight at 37°C, was extracted according to the instructions of the plasmid extraction kit.

[0220] 1) Use a 1.5ml EP tube to collect 3-5ml of bacterial liquid, centrifuge at 12000g for 30s, and dry the supernatant;

[0221] 2) Add 150 μl of solution B (50 mM Tris-Cl, 10 mM EDTA), 10 μl of solution A (RNase A), shake and suspend fully, and place at room temperature for 5 minutes;

[0222] 3) Add 300 μl of solution C (1% SDS, 0.2M NaOH), gently invert and mix 4 to 6 times, and place at room temperature for 5 minutes;

[0223] 4) Add 450 μl solution D (4M guanidine hydrochloride), and mix by inversion repeatedly 4-6 times;

[0224] 5) Centrifuge at 12000g for 5min to remove the prec...

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Abstract

The invention relates to a production method of recombination human antibody, in particular to a production method of recombination human anti rabies virus antibody, which is characterized in that the recombination antibody is neutralizing antibody specially combined with rabies virus, which is determined by the specific gene sequence in hypervariable region (CDRs) of antibody heavy chain and light chain variable region and can be expressed in procaryotic cell and eukaryotic cell. The CDR area, part or all gene of the antibody can generate the antibody in any expression system of procaryotic cell and eukaryotic cell, to prevent and treat rabies clinically.

Description

field of invention [0001] The present invention relates to a recombinant monoclonal antibody, in particular to the preparation method and application of a universal vector carrying the Fc segment of the heavy chain constant region of a human antibody used for screening and expressing a human antibody scFv-Fc small molecule antibody in an antibody expression library. Expression of recombinant human anti-rabies virus antibodies in Saccharomyces pasturii. Background of the invention [0002] The outstanding feature of antibody molecules is the duality of their functions and molecular structures: recognizing different antigens requires a huge amount of structural diversity; interacting with in vivo effector systems requires structural constancy. This feature makes antibody molecules the most complex molecules in the body and has long been a research hotspot. [0003] Since the end of the 19th century, the method of obtaining antiserum by immunizing animals with antigens has bee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N1/19A61K39/395A61P31/12C12R1/84
Inventor 颜炜群王丁丁
Owner 吉林圣元科技有限责任公司
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