肝癌相关抗原kinectin基因片断的重组表达

RECOMBINATION AND PURIFICATION OF HEPATOCELLULAR CARCINOMA-ASSOCIATED ANTIGEN kinectin

目的:研究肝癌相关抗原kinectin基因片断的体外重组表达方法,为该抗原的生产奠定基础。方法:用PCR法扩增肝癌相关抗原kinectin的基因片断,将目的基因插入表达载体pMAL-C2上麦芽糖结合蛋白(MBP)基因下游的多克隆位点,转化DH5α菌。通过蓝白斑筛选、PCR鉴定、内切酶鉴定及DNA测序筛出正确的重组子,并用其转化TB1菌。用IPTG对重组的TB1工程菌进行诱导表达,SDS-PAGE电泳鉴定。结果:kinectin基因片断正确插入pMAL-C2载体的多克隆位点,用IPTG对工程菌进行诱导,可见目的蛋白条带出现。结论:pMAL-C2载体可用于对肝癌相关抗原kinectin的体外基因重组表达。

Objective：To study the method to recombine and express the gene segment of HCC-associated antigen-kinectin in vitro, which will provide a basis for the production of this antigen. Methods： The gene segment of kinectin was amplified by PCR, and then inserted into the vector pMAL-C2 at the downstream of the gene encoding the MBP protein. The recombinant vector was transfected into E. coli DH5α. The blue-white selection assay, PCR, incision enzyme assay and DNA sequencing were performed to find out the right recombinant clone. The right clone was transfected into the expression host （E. coli TB1）, then the engineering bacteria was induced by IPTG. The induction products were analyzed through the SDSPAGE electrophoresis. Result： The gene segment of kinectin have been successfully inserted into the vector pMAL-C2 at the multiple clone site, and the interest protein was expressed when the engineering bacteria was induced by IPTG. Conclusion： The expression vector pMAL-C2 can be used to recombine and express the gene segment of kinectin in vitro.