摘要
目的构建TAT-HBx-EGFP质粒,转化大肠埃希菌BL21,进行蛋白的表达、鉴定及纯化。方法 PCR扩增目的基因HBx,进行EcolR1单酶切,与经EcolR1单酶切的TAT-EGFP质粒进行连接,转化大肠埃希菌BL21,利用IPTG对其进行诱导。应用West-ern blot方法鉴定表达的融合蛋白,采用Ni2+-金属螯合亲和层析柱法对融合蛋白进行纯化。结果得到构建好的TAT-HBx-EGFP质粒及纯化的目的融合蛋白TAT-HBX-EGFP。结论通过对TAT-HBx-EGFP质粒的构建和TAT-HBX-EGFP蛋白的表达与纯化,为进一步研究HBX蛋白的致肝癌作用奠定了实验基础。
Objective To construct a TAT-HBx-EGFP plasmid transformed into E.coli BL21,then protein expression was identified and purified.Methods PCR was used to amplify gene HBx.HBx and TAT-EGFP plasmid were digested by EcolR1 single restriction enzyme,then joined together.The plasmid was transformed into E.coli BL21,and induced by IPTG.Western blot was used to identify the protein and purified by Ni2 ＋-metal chelate affinity chromatography.Results We got the constructed TAT-HBx-EGFP plasmid and the purified fusion TAT-HBX-EGFP.Conclusion The construction of TAT-HBx-EGFP plasmid,expression and purification of TAT-HBX-EGFP protein have laid a foundation for further study of HBX protein function caused liver cancer.
出处
《胃肠病学和肝病学杂志》
CAS
2011年第1期41-43,共3页
Chinese Journal of Gastroenterology and Hepatology
