Method for constructing CHO cell strain for stably and efficiently expressing human serum albumin and interleukin II fusion protein

A human serum albumin, fusion protein technology, applied in the field of biopharmaceuticals, can solve problems such as degradation, excessive glycosylation, production and application limitations, etc.

Inactive Publication Date: 2015-07-22
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Guan Bo et al. successfully expressed the fusion protein IL-2-HSA with biological activity in Pichia pastoris through albumin fusion technology, but when the fusion protein was expressed in Pichia pastoris, the product was heterogeneous, hyperglycosylated and Degradation and other issues are greatly restricted in production applications

Method used

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  • Method for constructing CHO cell strain for stably and efficiently expressing human serum albumin and interleukin II fusion protein
  • Method for constructing CHO cell strain for stably and efficiently expressing human serum albumin and interleukin II fusion protein
  • Method for constructing CHO cell strain for stably and efficiently expressing human serum albumin and interleukin II fusion protein

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Embodiment Construction

[0029] The present invention will be specifically described below in conjunction with the accompanying drawings.

[0030] 1. Experimental materials:

[0031] Chinese hamster ovary cells (CHO-S), geneticin G418 (sigma), expression vector pMH 3 , adherent medium D001, suspension medium B001, and fed-batch medium F001 were all from Hangzhou Anruipu; rabbit anti-human IL-2 and mouse anti-human HAS were from HRP; mouse anti-human HAS was from abcam; goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were from Beyontian; other reagents used were of analytical grade.

[0032] 2. Experimental equipment

[0033] Electrotransfer instrument; cell incubator; cell shaker; conventional disposable consumables.

[0034] 3. Experimental steps

[0035] 3.1. Plasmid extraction

[0036] Solution preparation: Solution I: 50mM glucose; 25mM Tris-cl (pH 8.0); 10mM EDTA (pH 8.0), sterilized and placed at 4°C after preparation. Solution II: 0.2mol / L NaoH; 1% (m / L) SDS, re...

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Abstract

The invention discloses a method for constructing a CHO cell strain for stably and efficiently expressing a human serum albumin and an interleukin II fusion protein. The CHO monoclonal cell strain for stably and efficiently expressing the human-derived recombinant protein can be obtained by electrically transferring a plasmid with the human serum albumin and the interleukin II fusion gene into the CHO cell. The monoclonal cell strain obtained by the invention is capable of secretory expression of the human serum albumin and the interleukin II fusion protein, the protein expression quantity is high, and the fusion protein can be obtained through the separation and purification; the in vitro biological activities of two fusion proteins are higher than the mol monomer 1L-2 activity. The CHO cell strain can be widely applied to the medicines for treating various diseases such as tumor, hepatitis, pneumonia and immunodeficiency disease.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to designing a CHO cell strain expressing recombinant proteins HSA-IL-2 and IL-2-HSA respectively by means of genetic engineering. Background technique [0002] Interleukin-2 (Interleukin-2, IL-2), also known as T cell growth factor, is a factor produced when Morgan et al stimulate T lymphocytes with mitogen and concanavalin. IL-2 plays an important role in the body's immune response and is clinically used for the treatment of tumors, hepatitis, and immunodeficiency diseases. Among them, hepatitis B is a global infectious disease, especially in my country. Since cytokines play an important role in regulating immune responses, the use of cytokines to enhance the immune response induced by immunization has attracted great attention and promising experimental results have been obtained. However, the use of cytokines to To enhance the immune response and use it clinically, no substan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C07K19/00A61K38/20A61K47/48A61P35/00A61P1/16A61P11/00A61P37/04
Inventor 金坚李华钟卢慧惠彭林陈蕴蔡燕飞段作营
Owner JIANGNAN UNIV
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