幽门螺杆菌Ⅳ型分泌系统HP0527-C基因的克隆表达及其生物学功能的初步研究

Cloning,Expressing and Analysis of Biological Function of HP0527-C of Type IV Secretion System in Helicobacter Pylori

为克隆表达幽门螺杆菌(Helicobacter pylori,H.pylori)NCTC 11637 hp0527-c基因,探讨其生物学功能。设计引物扩增,T-A克隆,测序正确后构建表达载体pET-28a-hp0527-c,转化大肠杆菌BL21,经IPTG诱导表达后以Ni2+-NTA柱获得纯度为98%重组蛋白。经Western blot鉴定正确后,免疫新西兰大白兔获得了较高滴度的多克隆抗体;将重组蛋白作用于BGC-823细胞,RT-PCR检测细胞IL-8mRNA表达增高;同时用MTT法来检测重组蛋白对细胞增殖的影响,结果表明其活性呈现一定量的时间和剂量依赖性。已成功克隆了HP0527-C的重组蛋白,初步探讨了其与细胞之间的相互作用,为进一步阐明H.pylori致病机制奠定了基础。

Objective ：To obtain recombinant soluble HP0527-C protein of Helicobacter pylori （H. pylori） NCTC 11637 , with biological activity . Methods ： The hp0527-c coding region was amplified from the Helicobacter pylori NCTC11637 by PCR then was subcloned into expression vector pET-28a for sequence analysis. Recombination plasmid was used to transform E. coli BL21 cells and fusion protein HP0527-C expression was induced by IPTG. The products were purified by Ni-NTA chromatography column and identified by SDS-PAGE and Western blot. After the protein was co-cultured with BGC-823 cells, the expression of IL-8 mRNA in cells can be determined by reverse transcriptase polymerase chain reaction （RT-PCR）. Rime time PCR was also applied to determine the mRNA expression level of IL-8. The proliferation inhibition activity of HP0527-C was detected by MTT method. Results：The target protein expressed in E. coli BL21 （DE3） had the same Mr as that of expected and could be detected by Western blot. The expressed product reached a purity of 98% after Ni^2＋ -NTA column chromatography. The protein after dialyzed and annealed , was co-cultured with BGC-823cells, the expression of IL-8 mRNA in BGC-823 cells could be determined. A real-time PCR analysis on IL-8 at different time showed that, the expression level of IL-8 peaked in 24h. The protein of different concentration co-cultured with BGC-823 cells for different times, It was found that the protein in low concentration promotes the proliferation of cells, while achieving 100μg/ml concentration, it inhibits proliferation of cells along with multiplication of the concentration of the protein; Conclusion： A 1494 base pairs long hp0527-c gene, which encodes a product of 497 amino acid, was obtained using PCR method and was subcloned into expression vector pET-28a for sequence analysis. It is indicated that the hp0527-c gene was amplified and expressed in E. coli BL21 （DE3）. High purified protein was purified by Ni^2＋ -NTA column chromatography. Recombinantthe HP0527-C protein with biological activity was examined . This proepective research shed light on further research on its biological activity and function.