人类胞内氯离子通道蛋白3在原核细胞及真核细胞内的表达

The expression of human intracellular chloride channel protein 3 in eukaryotic and prokaryotic cells

目的研究人类胞内氯离子通道蛋白3(CLIC3)在真核细胞中的定位和表达,及其GST融合蛋白在原核细胞中的表达。方法以含人CLIC3的全长cDNA序列的质粒为模板,PCR扩增CLIC3片段,构建真核表达载体pcDNA3.1-CLIC3-FLAG,检测其定位及表达;构建原核表达载体pGEX-5X-3-CLIC3,转化到大肠杆菌BL21菌株,IPTG诱导融合蛋白GST-CLIC3表达。结果细胞免疫荧光结果表明CLIC3在COS7细胞质和细胞核中均有分布;Western blot结果显示CLIC3在HEK-293T细胞中能有效表达;考马斯亮蓝染色结果表明融合蛋白GST-CLIC3在BL21菌株中能有效表达。结论人类的CLIC3蛋白COS7、HEK-293T及大肠杆菌BL21菌株均能有效表达,为进一步了解CLIC3的功能奠定了一定的基础。

Objective To investigate the accuracy of PCR for detection of non-EV71 and non-CoxA16 by using u- niversal primers with VP4 gene of HEV. Methods When identified by VP4 gene of EV71 and CoxAI6 unrelated HFMD, two isolates of Sabin-1 were found（ such as Fy-01and Fy-02）. They were identified by co-primers such as EVP4/Q8 and specific primers of VP1 gene. Results Fy-01 isolates were appeared with an unusual frayment and identified as CoxA10 after being sequenced and the selected independent clones were sequenced and blasted in GenBank. It proved that both Sabin-1 and CoxA10 existed. Conclusion Clinical diagnosis of EVT1 and CoxA16 unrelated HFMD,when identifying the type of HFMD pathogen only by using the amplification product of VP4 gene after being sequenced directly is not enough, especially HFMD with mixed infections caused by enterovirus.