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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。
OBJECTIVE: To clone Epstein-Barr virus immediate early protein ZtaN and capsid protein p23, construct prokaryotic expression vector and express the fusion protein in Escherichia coli, which lays the foundation for the subsequent diagnosis of disease by using protein. Methods: The target genes BZLF1N and BLRF2 were amplified respectively from B95-8 cells by reverse transcription-polymerase chain reaction (RT-PCR), and the two genes were ligated by fusion PCR to construct recombinant plasmid pGEX-4T-1 -BZLF1N-BLRF2 and transformed into E. coli BL21 (DE3). The expression of the fusion protein ZtaN-p23 was induced by adding IPTG, and the protein expression and activity identification were confirmed by SDS-PAGE and Western blot. Results: The recombinant plasmid pGEX-4T-1-BZLF1N-BLRF2 was confirmed by double enzyme digestion and sequence analysis. The prokaryotic expression vector was successfully constructed. SDS-PAGE showed that the induced protein Mr was about 46,000, which was consistent with the expected value. The protein expression conditions were optimized and found to exist mainly in the form of inclusion bodies; affinity chromatography showed that the purified ZtaN-p23 purity> 95%; Westernblot showed that ZtaN-p23 has good biological activity. CONCLUSION: The prokaryotic expression vector pGEX-4T-1-BZLF1N-BLRF2 was successfully constructed and expressed in Escherichia coli. The fusion protein showed good activity.