【摘要】 目的:克隆EB病毒立即早期蛋白ZtaN和衣壳蛋白p23,构建原核表达载体,并在大肠杆菌中表达融合蛋白,为后续利用蛋白进行疾病诊断奠定基础。方法:采用逆转录聚合酶链反应(RT-PCR)技术从B95-8细胞中分别扩增出目的基因BZLF1N和BLRF2,利用融合PCR法将两个基因进行连接,构建重组质粒pGEX-4T-1-BZLF1N-BLRF2,并转化大肠杆菌BL21(DE3)。加入IPTG诱导表达融合蛋白ZtaN-p23,通过SDS-PAGE和Western blot确定蛋白表达量及活性鉴定。结果:重组质粒pGEX-4T-1-BZLF1N-BLRF2经双酶切鉴定和序列分析正确,证实已成功构建原核表达载体;SDS-PAGE显示诱导的蛋白Mr约46 000,与预期值一致;对融合蛋白表达条件进行优化后,发现其主要以包涵体的形式存在于细胞中;亲和层析结果显示纯化后的ZtaN-p23纯度>95%;Westernblot显示ZtaN-p23具有良好的生物活性。结论:成功构建原核表达载体pGEX-4T-1-BZLF1N-BLRF2,并在大肠杆菌中进行了表达纯化,融合蛋白显示出良好的活性。更多还原

【Abstract】 AIM: To construct the prokaryotic expression plasmid pGEX-4T-1-BZLF1N-BLRF2,and express it in Escherichia coli.METHODS: The EB virus BZLF1N gene and BLRF2 gene were amplified by RT-PCR respectively.Then,the two genes were linked by splicing overlap extension PCR method and inserted into the vector pGEX-4T-1,and the recombinant plasmid pGEX-4T-1-BZLF1N-BLRF2 was transformed into E.coli BL21(DE3) strain.The expression protein ZtaN-p23 was analysed by SDS-PAGE and immunoreactivity was proved by Western blotting.RESULTS: Restriction enzyme digestion and DNA sequencing showed recombinant plasmid constructed successfully.The expression product ZtaN-p23 with the molecular weight 46000 was located in the cytoplasm and insoluble.The ZtaN-p23 up to 95% purity was obtained after purified using affinity chromatography.Western blotting showed fusion protein possessed a well bioactivity and specificity.CONCLUSION: The fusion gene BZLF1N-BLRF2 is successfully constructed and effectively expressed in E.coli,which lay the foundation for further research on its biological properties and functions.更多还原