[CTLA-4在大肠杆菌中的基因克隆与表达]

[Gene cloning and expression of CTLA-4 in E. coli].

作者信息

Wang C J, Li Y

机构信息

Department of Bioengineering, Institute of Medicinal Biotechnology, CAMS, PUMC, Beijing 100050, China.

出版信息

Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2001 Apr;23(2):154-7.

PMID:12905893

Abstract

OBJECTIVE

To express hsCTLA-4 in E. coli.

METHODS

The hsCTLA-4 gene was obtained by PCR amplification from pE plasmid which contains CTLA-4 gene and was inserted into the expression vector pGEX-2T. The recombination strain was induced by IPTG with different concentrations and time.

RESULTS

The sequence of PCR amplified DNA fragments was identical with the reported CTLA-4 gene. SDS-PAGE and Western blot showed that 0.10 mmol/L IPTG can induce higher production of the fusion protein with molecular weight 40,000 after addition of IPTG 4 hours and GST-CTLA4 had immunological activity.

CONCLUSIONS

hsCTLA-4 can be expressed in soluble form high efficiency.

摘要

目的

在大肠杆菌中表达人源可溶性细胞毒性T淋巴细胞相关抗原4（hsCTLA-4）。

方法

从含CTLA-4基因的pE质粒中通过PCR扩增获得hsCTLA-4基因，并将其插入表达载体pGEX-2T。用不同浓度和时间的异丙基-β-D-硫代半乳糖苷（IPTG）诱导重组菌株。

结果

PCR扩增的DNA片段序列与报道的CTLA-4基因一致。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blot）显示，0.10 mmol/L IPTG在加入4小时后可诱导产生更高产量的分子量为40000的融合蛋白，且谷胱甘肽S-转移酶-CTLA4（GST-CTLA4）具有免疫活性。