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Expression carrier for high-efficient screening target protein, its preparation method and use

A technology of eukaryotic expression vectors and vectors, applied in the field of genetic engineering vectors, can solve problems such as the application of Neo gene 546 site mutations, and achieve the effects of reducing screening workload, improving screening efficiency, and saving time

Inactive Publication Date: 2006-09-06
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the application of Neo gene 546 site mutation in eukaryotic expression vectors

Method used

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  • Expression carrier for high-efficient screening target protein, its preparation method and use
  • Expression carrier for high-efficient screening target protein, its preparation method and use
  • Expression carrier for high-efficient screening target protein, its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Construction of pCMV-HFI-mNeo vector

[0024] 1. Material

[0025] Escherichia coli strains and CHO cells are international standard strains, kept in this room, PCR primer synthesis and sequence determination are completed by Shanghai Boya Biological Co., Ltd. T4DNA ligase and DMEM medium are products of GibcoBRL company, Vent DNA polymerase, nucleic acid restriction The endonuclease is the product of Biolab, the plasmid extraction kit is the product of Beijing Broadtech Company, and the DNA fragment recovery kit is the product of Dalian Bao Bioengineering Co., Ltd. The goat anti-human IgG is made by our laboratory and is labeled with horseradish peroxidase. Goat anti-human IgG was purchased from Beijing Zhongshan Biotechnology Company. The vector pCI-neo was purchased from Promega, the light and heavy chain variable region genes of the anti-c-erbB-2 antibody (Genbank: A22466; A22467) were provided by Dr. Lei Yu, School of Pharmacy, University of Utah, and the huma...

Embodiment 2

[0029] Example 2 The role of pCMV-HFI-mNeo vector in efficient screening of target protein

[0030] 1. Cell culture, transfection and screening

[0031] Chinese hamster ovary cells (CHO) were cultured in DMEM medium containing 10% fetal bovine serum. The plasmid vector was transfected into CHO cells by the plastoliposome (purchased from Gibco) mediated method, at 37℃ and 5% CO 2 After 8 hours of culture in the incubator, the antibiotic-free and serum-free medium was replaced with DMEM medium containing 10% fetal bovine serum, and the normal medium was replaced with medium containing 300 μg / ml G418 after 48 hours. Then single clones were selected by limiting dilution method. When the cells overgrown the bottom of the culture flask, the supernatant was harvested, and the culture supernatant was used to detect the expression of fusion protein by ELISA. The 96-well plate was coated with goat anti-human IgG, and the bound fusion protein was detected with goat anti-human IgG labeled wit...

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Abstract

An expression carrier for effectively screening target protein is prepared through introducing an internal ribosome to the eukaryotic expression carrier containing CMV promoter and dhfr amplification system, linking the fusion gene of anti-erbB2-seFv-Fc gene and IL-2 to the neomycin-resistant gene, site mutation to Neo gene by PCR, and configuring the expression carrier pCMV-HFI-mNeo.

Description

Technical field [0001] The invention relates to a genetic engineering vector, in particular to an expression vector for efficient screening of target proteins, as well as its preparation method and application. Background technique [0002] The bioengineering of antibodies has significant research and application value, as well as huge market potential. High-efficiency antibody expression has always been the bottleneck technology of genetic engineering antibody preparation. Improving antibody expression yield and reducing its production cost have become an urgent problem to be solved. The efficient expression of genes in mammalian cells depends on many factors, including transcription, translation control elements, RNA metabolism, gene copy number, mRNA stability, and gene location on host cell chromosomes. At present, most eukaryotic expression vectors are randomly integrated vectors, and the sequences on both sides of the gene integration site can greatly affect the expression ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/64C12N15/31C12N15/67C12N15/62C07K16/00C07K19/00
Inventor 陈立勇解志刚郭宁沈倍奋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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