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

A technology of eukaryotic expression vector and vector, applied in the field of genetic engineering vector, can solve the problems such as the application of 546 site mutation of Neo gene, which can reduce the screening workload, improve the screening efficiency, and achieve the effect of efficient and rapid screening.

Inactive Publication Date: 2005-08-24
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
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Effect test

Embodiment 1

[0023]The construction of embodiment one pCMV-HFI-mNeo vector

[0024] 1. Materials

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

Embodiment 2

[0029] Example 2 The effect of pCMV-HFI-mNeo vector in high-efficiency 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 liposome (purchased from Gibco) mediated method, at 37°C and 5% CO 2 After culturing in the incubator for 8 hours, the antibiotic-free and serum-free medium was replaced with DMEM medium containing 10% fetal bovine serum, and the normal medium was replaced with a medium containing G418 at a concentration of 300 μg / ml 48 hours later. Single clones were then picked by limiting dilution. When the cells covered the bottom of the culture flask, the supernatant was harvested, and the expression of the fusion protein was detected by ELISA. Coat the 96-well plate with goat anti-human IgG, detect the bound fusion protein with horseradish peroxidase-labeled goat anti-hu...

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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 carrier, in particular to an expression carrier for efficiently screening target proteins, and also relates to its preparation method and application. Background technique [0002] Antibody bioengineering has great research and application value, and also has huge market potential. The high-efficiency expression of antibodies has always been the bottleneck technology for the preparation of genetically engineered antibodies. Improving the yield of antibody expression and reducing its production cost has become an urgent problem to be solved. High-efficiency gene expression in mammalian cells depends on many factors, including transcriptional and translational control elements, RNA metabolism, gene copy number, mRNA stability, and location of genes on host cell chromosomes. At present, most eukaryotic expression vectors are random integration vectors, and the sequences on both sides of the gene integration...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/62C12N15/64C12N15/67
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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