中文摘要：

目的：利用SUMO标签构建人TNFα原核表达载体,通过表达及纯化获得重组蛋白,为深入研究和利用人TNFα奠定基础。方法：利用PCR技术,从质粒pET32a-hTNFα中扩增出人TNFα成熟肽编码序列,并在其上游添加SUMO标签,与原核表达载体pET28a连接,构建表达质粒pET28a-SUMO-hTNFα。在BL21（DE3）工程菌中表达融合蛋白,经Ni-NTA纯化体系纯化,切除SUMO标签,纯化获得hTNFα成熟蛋白。CCK-8法检测TNFα对L929细胞的细胞毒性,以测定TNFα的生物学活性。结果：成功构建pET28a-SUMO-hTNFα原核表达质粒,酶切鉴定和测序分析与预期结果完全一致。在BL21（DE3）工程菌中实现了融合蛋白的可溶性表达。经纯化、水解酶切除标签及再次纯化获得hTNFα成熟肽。CCK-8法检测得所制备的TNFα蛋白ED50约为12.8μg/ml。结论：成功构建原核表达载体pET28a-SUMO-hTNFα,经表达、纯化、酶切及再纯化,获得有生物活性的hTNFα蛋白,为深入研究和利用hTNFα奠定基础。

英文摘要：

Objective： To construct a prokaryotic expression plasmid of human TNFα with SUMO,obtain recombinant protein by expression and purification,which is expected to provide basis for the further research and utilization of TNFα. Methods： The gene encoding mature TNFα protein was amplified from plasmid pET32ahTNFα by PCR and cloned into pET28 a with SUMO tag on the upstream. The fusion protein SUMO-hTNFα was expressed in BL21（ DE3） engineering bacteria,then was purified by Ni-NTA Resin purification system. The purified production was digested by SUMO protease. One more purification in order to obtain mature TNFαprotein. The biological activity was measured in a cytotoxicity assay using L929 cells by CCK-8 test. Results： The prokaryotic expression plasmid pET28a-SUMO-hTNFα was successfully constructed. The results identified by enzyme digestion and nucleotide sequencing corresponded exactly well as expected. The fusion protein SUMOhTNFα expressed in a soluble form in BL21（ DE3）. Fusion protein was purified by Ni-NTA Resin purification system. Mature hTNFα protein was obtain after digestion by Sumo protease and purification. ED50 of recombinant hTNFα is 12. 8μg /ml. Conclusion： The prokaryotic expression plasmid pET28a-SUMO-hTNFα was successfully constructed. TNFα protein was expressed and obtained by digestion and purification. These established a foundation of further research and utilization of hTNFα.