重组沙门菌侵袭蛋白A的原核表达与纯化

Prokaryotic Expression and Purification of Recombination Salmonella Invasion Protein

目的在大肠杆菌中表达重组沙门菌侵袭蛋白A,并对表达产物进行分离和纯化。方法提取沙门菌基因组模板,PCR扩增侵袭因子invA基因目的片段,插入表达质粒载体pET-30c(+)中,构建pET-invA重组子,经双酶切和测序证实后,转化表达宿主大肠杆菌BL21(DE3),异丙基-β-D硫代半乳糖(IPTG)诱导重组蛋白表达,镍琼脂糖凝胶亲和层析纯化重组蛋白,SDS-PAGE和Westernblot检测鉴定表达蛋白。结果成功构建了沙门菌pET-invA大肠杆菌表达重组子,实现了该蛋白在大肠杆菌中的高效表达,分离纯化的表达产物纯度达到电泳纯。结论沙门菌侵袭蛋白A的成功表达和分离纯化,为该蛋白的免疫学性能研究、相应抗体的制备以及沙门菌快速检测方法的建立奠定了基础。

Objective To express recombinant Salmonella invasion protein A（InvA） in E. coli and purify it. Methods The invA gene of Salmonella was amplified by PCR from the Salmonella genome and cloned into expression vector pET-30c （＋） to generate the pET-invA recombinants. They were comfirmed by restriction endonuclease digestion and sequence analysis. The verified recombinant was transformed into E. con BL21 （DE3）. After inducing with isopropyl-~D-thiogalactopyranoside （IPTG）, the recombinant protein was purified via NiNTA affinity chromatography under denature conditions. The recombinant proteins were analyzed with SDSPAGE and Western blot. Results The pET-invA recombinant for Salmonella was successfully constructed and the recombinant InvA protein was expressed in E. coli at a relatively high level. The SDS-PAGE results for the purified recombinant protein demonstrated that the purified protein had reached the electrophoresis purity. Conclusion The successful expression of the Salmonella InvA protein will be very helpful for the further study on its antigenicity, immunological activity, and the development of rapid detection methods for Salmonella strains.