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Engineering cell strains capable of stably and efficiently expressing HCV NS3 protein antibody and application thereof

A high-efficiency expression, cell line technology, applied in genetically modified cells, cells modified by the introduction of foreign genetic material, applications, etc., to achieve the effect of stable expression level, high sensitivity and specificity

Active Publication Date: 2019-04-23
山东莱博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the search found that: at present, there is no report of using Chinese hamster ovary cells (CHO) as the host cell to transfect the expression plasmid containing the HCV NS3 protein antibody gene to screen for engineering cell lines that can stably and efficiently express the HCV NS3 protein antibody. Application report of using CHO engineered cells for the preparation of HCV NS3 protein antibody

Method used

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  • Engineering cell strains capable of stably and efficiently expressing HCV NS3 protein antibody and application thereof
  • Engineering cell strains capable of stably and efficiently expressing HCV NS3 protein antibody and application thereof
  • Engineering cell strains capable of stably and efficiently expressing HCV NS3 protein antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Acquisition of engineered cell lines stably and efficiently expressing HCV NS3 protein antibody

[0037] A pair of monoclonal antibodies 3A10B5 and 7E3B6 secreted by two hybridoma cells CCTCC NO.C2018227 and CCTCC NO.C2018228 obtained through experimental screening entrusted a sequencing company (Shanghai Boshang Biotechnology Co., Ltd.) to perform nucleotide sequencing of the antibodies.

[0038] Determine the nucleotide sequence of the light chain of the monoclonal antibody 3A10B5 produced in CCTCC NO.C2018227 hybridoma cell line as shown in SEQ ID NO.5, and the nucleotide sequence of its heavy chain as shown in SEQ ID NO.6, while It is also assumed to be a nucleic acid molecule encoding the genetically engineered antibody NS3-GC1, the nucleotide sequence of its light chain is also shown in SEQ ID NO.5, and the nucleotide sequence of its heavy chain is also shown in SEQ ID NO.6.

[0039] Determine the nucleotide sequence of the light chain of the monoclonal...

Embodiment 2

[0077] Preparation and purification of embodiment 2 genetic engineering antibody

[0078] 2.1 Batch culture of cell lines CHO / NS3-GC1 and CHO / NS3-GC2

[0079] The cell lines CHO / NS3-GC2 and CHO / NS3-GC2 were divided into 0.4×10 6 The density of cell / mL was inoculated into the SF250ml cell culture Erlenmeyer flask, the culture system was 50mL, and the medium used was a special medium (CHO-Med-01) that could significantly increase the antibody production, and its basic formula was: CHO-S-SFM II Dry powder medium 10g / L, NaHCO 3 2.45g / L, 8mM glutamine, 2mM ferric citrate, final pH 7.1. After inoculation, hope blue staining and counting were carried out, and the culture bottle was placed in a cell shaker incubator at 37° C., relative humidity 70-80%, and 5% carbon dioxide, and the rotation speed was 125 rpm.

[0080] Observe the cell growth every day, trypan blue staining and counting, first take 20 μL, add 20 μL of 0.4% trypan blue staining solution to a centrifuge tube and mix...

Embodiment 3

[0089] Example 3 Antibody Identification

[0090] 3.1 Antibody titer identification

[0091] Weigh 5 mg of HRP and dissolve in 1 mL of distilled water; add 0.5 mL of freshly prepared 0.06M NaIO to the above solution 4 Solution, let stand at 4°C for 30min; add 0.5mL of 0.16M polyethylene glycol to the above solution, and let stand at room temperature for 30min; add 5mg of purified antibody (NS3-GC1 or 3A10B5; NS3-GC2 or 7E3B6), mix well and transfer into a dialysis bag, and dialyze against 0.05M pH9.5 carbonate buffer overnight; add 0.2ml of 5mg / mL NaBH the next day 4 solution, after mixing, let stand at 4°C for 2 hours; slowly add an equal volume of saturated (NH4) 2 SO 4 Solution, stand at 4°C for 30 minutes, then centrifuge to remove the supernatant, dissolve the precipitate with a little 0.02M pH7.4 PBS solution, and transfer it to a dialysis bag, dialyze with the same liquid at 4°C overnight to desalt; take it out and centrifuge the next day, and take the supernatant ,...

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Abstract

The invention discloses engineering cell strains capable of stably and efficiently expressing HCV NS3 protein antibody, wherein one strain is Chinese hamster ovary cell strain CHO / NS3-GC1 capable of generating antibody NS3-GC1, the other strain is Chinese hamster ovary cell strain CHO / NS3-GC2 capable of generating antibody NS3-GC2, the cell strains are preserved in China Center for Type Culture Collection on December 12, 2018, and the preservation numbers are CCTCC NO. C2018186 and NO. C2018187 in sequence. The amount of the antibody prepared by using the two strains of cells is about 2g / L, the yield is increased by 80 times compared with that of the previous hybridoma cells, the sensitivity of the antibody is consistent with that of the previous monoclonal antibody, the component antibodypair is specifically combined with HCV NS3 antigen, and ELISA detection can be carried out.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pair of engineering cell lines stably and efficiently expressing HCV NS3 protein antibody and application thereof. Background technique [0002] Hepatitis C is one of the pathogenic factors of chronic liver disease, and hepatitis C is mainly caused by hepatitis C virus (Hepatitis Cvirus, HCV) infection, and nearly 180 million people in the world are infected with hepatitis C virus. Chronic infection of HCV can lead to chronic inflammation, necrosis and fibrosis of the liver, and some patients may develop liver cirrhosis or even liver cancer, seriously endangering human health. The mortality rate associated with HCV infection will continue to increase in the next 20 years, so all effective measures should be taken to avoid infection. [0003] At present, there is no vaccine available to prevent HCV infection, and early detection is the main method for timely treatment a...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/13C07K16/10G01N33/576C12R1/91
CPCG01N33/5767C12N15/85C07K16/109C07K2317/33C07K2317/35C12N2510/00
Inventor 朱之炜解光宁陈振任素平欧兰香王佳颖武建伟张日丽瞿丽丽
Owner 山东莱博生物科技有限公司
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