幽门螺杆菌Cag-PAI hp0525基因的克隆表达及其重组蛋白HP0525对SGC-7901细胞增殖的影响

目的：由于文献报道幽门螺杆菌（Helicobacter pylori）是产生胃溃疡和胃癌的致病菌之一，一个重要的影响因素是由cag致病岛编码的四型分泌系统。Hp0525是Cag致病岛中重要成分，是一种内膜蛋白ATPase。而幽门螺杆菌中Cag蛋白的表达以及Cag PAI编码的各自蛋白功能研究得还很少，为进一步研究幽门螺杆菌的致病机制和研发幽门螺杆菌诊断试剂盒及疫苗，特克隆幽门螺杆菌NCTC 11637hp0525 (caga) 基因，并对其进行测序，构建原核重组质粒，表达HP0525蛋白，初步研究其对SGC-7901细胞增殖的影响。方法：应用PCR技术从H.pylori基因组DNA中扩增hp0525编码基因片段，克隆至pMD18-T载体后，再将其定向插入pET-30a载体中，双酶切鉴定筛选阳性克隆，以DNA自动分析仪进行序列测定。测序分析正确后，经IPTG诱导表达，表达蛋白以Ni2＋-NTA柱进行纯化，并经Western blot和MALDI-TOF鉴定，透析除盐后的蛋白，通过免疫新西兰大白兔和抗体效价的测定，纯化的蛋白作用于SGC-7901细胞，用MTT法检测蛋白对细胞增殖的影响。结果：成功克隆hp0525基因，全长993bp，编码330个氨基酸，与GenBank 公布的其他H.pylori菌株基因序列的核苷酸同源性为97%~99%。工程菌诱导后SDS-PAGE显示新生表达蛋白带，相对分子质量为36 000，与预期一致，经Ni2＋-NTA柱纯化后可获得纯度为98％重组蛋白。蛋白作用于SGC-7901细胞后，结果呈现一定量的时间和剂量依赖性。它是一种ATPase，通过测定具有一定的活性。活性为4.40IU/ml结论：成功克隆hp0525基因，并在大肠杆菌BL21中表达，经过镍柱纯化后得到纯度较高的蛋白, MTT法来检测出重组蛋白抑制细胞增殖；同时具有一定的酶活力，为进一步研究其生物学功能奠定了基础。

Cloning,Expressing and Analysis of Helicobacter pylori NCTC 11637 hp0525 Gene and Study the Influence of Proliferation of SGC-7901 Cell

Objective : To construct recombinant plasmid containing hp0525 gene of Helicobacter pylori ( H. pylori ) NCTC 11637 , and analysis of sequence nucleic acid, express it in E.coliBL21 and study the influence of the HP0525 protein on proliferation of SGC-7901 cells . Methods H. pylor hp0525 gene was amplified from the genome DNA by PCR,then operated T-A cloning and sequenced. The hp0525gene fragment was inserted directionally into vector pMD18-T to construct recombinant clones of hp0525 and was sequenced. The recombinant plasmid was transformed into E.coli BL21for expressing under induction of IPTG. Purify the expressed protein by Ni2＋-NTA column chromatography. Expressed product was analyzed by Western blot and MALDI-TOF. Added the purified protein into SGC-7901 cells, the effect of cell proliferation in SGC-7901 cells induced by the recombinant protein was observed by MTT. Results A 993 base pairs long hp0525 gene, which encodes a product of 330 amino acid, was obtained using PCR method and was cloned into pMD18-Tvector successfully. The sequence analysis for hp0525 showed that it shares 97%-99% homology with other strains in Gene bank. SDS-PAGE showed a protein band with a relative molecular weight of 36 000, which was consistent with the expectation. The expressed product reached a purity of 97% after Ni2＋-NTA column chromatography. The protein after dialyzed and annealed , was co-cultured with SGC-7901 cells, The protien of different concentration co-cultured with SGC-7901 cells for different times, found that the protein in low concentration stimulates proliferation of cells, to achieve some concentration, it inhibits proliferation of cells along with multiplication of the concentration of the protein; The protein inhibits proliferation of cells relay on the extension of time and concentration. Conclusion It is indicated that we have obtained the correct hp0525 gene and expressed in E.coli BL21. We obtained high purified protein by Ni2＋-NTA column chromatography, immunized serum was tested by indirect ELISA and the titers were up to 1：8000. the protein can inhibits proliferation of SGC-7901cells , and T-ATPase activity which posed a basis for further researching on its biological function.