Preparing method for recombinant human anti-rabies monoclonal antibodies

A monoclonal antibody and rabies virus technology, applied in the field of genetic engineering, can solve problems such as cumbersome operation, difficulty in industrialization, and poor stability

Active Publication Date: 2008-01-09
NCPC NEW DRUG RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the stability of this method is poor, the operation i

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Construction of recombinant vector pCAdhfr-NM57 expressing NM57 antibody

[0016] The obtained NM57 antibody heavy chain and light chain variable region genes were cloned into the corresponding restriction enzymes EcoRI and NotI sites of the vector pCAdhfr according to conventional methods, and the ligation products were transformed into E. coli according to conventional methods. Recombinant vectors containing the complete NM57 antibody heavy and light chain encoding genes were named pCAdhfr-NM57H and pCAdhfr-NM57L, respectively.

Embodiment 2

[0017] Example 2 Construction of a recombinant CHO cell line expressing NM57 antibody

[0018] Take 20ug of recombinant vector pCAdhfr-NM57H and pCAdhfr-NM57L DNA respectively, mix them and use electrotransfection method to transfect CHO-K1 cell line (purchased from China Type Culture Collection (CCTCC No. GDC018), electrotransfection conditions are in accordance with BIO- RAD recommends the conditions. For specific parameters, please refer to the GenePulser Electroprotocol Survey number 086 program. After adherence culture, transient expression is detected. After confirming the expression of the target product, perform clonal culture and high-expressing cell line screening, and add 0.1-10uM MTX for addition. Pressure screening, the expression level of the obtained cell line under the adherent culture condition is 50ug / 10 6 Cells / day, named CHO-K1-NM57.

Embodiment 3

[0019] Example 3 Engineering cell culture

[0020] The cells recovered from the production seed bank were expanded and cultured. After being qualified, they were inoculated in DMEM medium at a seeding rate of 1:3~1:5 for suspension culture and domestication. The cells were successively subcultured and expanded, harvested, and the above-mentioned cells The suspension was inoculated into a NBS 5L bioreactor, continuously perfused and cultured, and the cell culture solution was collected.

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Abstract

Production of recombinant human rabies virus mono-cloning antibody NM57 is stable and has high purity and excellent specificity. It can be used to prevent rabies and substitute ERIG and HRIG.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a human anti-rabies virus monoclonal antibody gene, a recombinant vector containing the gene, and a host transformed with the vector to prepare a human anti-rabies virus monoclonal antibody and applications thereof. Background technique [0002] Rabies is a natural epidemic or zoonotic acute infectious disease caused by rabies virus. It has a wide epidemic and a fatality rate of nearly 100%, which poses a serious threat to people's lives and health. Human rabies is mainly caused by bites, scratches, or mucosal infections of diseased animals, and can also be transmitted through respiratory aerosols under certain conditions. Infectious animals are mainly dogs (over 90%), followed by cats. [0003] According to data from the China Epidemic Reporting System, the annual increase in rabies epidemics after 1998 has been higher and higher. In 2001, there were 891 cases ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/85C07K16/18C12P21/08
Inventor 贾茜高健惠觅宙魏敬双赵伟陶冉程立均贺建功白冠仁
Owner NCPC NEW DRUG RES & DEV
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