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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NCTC11637hp0525(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36000,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。
OBJECTIVE: As reported in the literature, Helicobacter pylori is one of the pathogens causing gastric ulcer and gastric cancer. An important factor is the type 4 secretion system encoded by cag pathogenicity island. Hp0525 is an important component of Cag pathogenicity island, is an intimal protein ATPase. However, the expression of Cag protein in Helicobacter pylori and the function of the respective proteins encoded by Cag PAI have not been studied. To further study the pathogenesis of Helicobacter pylori and to develop the diagnostic kit and vaccine for Helicobacter pylori, the clone of Helicobacter pylori NCTC11637hp0525 (caga) gene was cloned and sequenced. The prokaryotic recombinant plasmids were constructed and the HP0525 protein was expressed to study the effect of caga gene on the proliferation of SGC-7901 cells. Methods: The hp0525 gene fragment was amplified by PCR from genomic DNA of H.pylori, cloned into pMD18-T vector, and then inserted into pET-30a vector. The positive clones were screened by double enzyme digestion and analyzed by DNA Instrument for sequencing. After sequencing analysis, the recombinant protein was induced by IPTG. The expressed protein was purified by Ni2 + -NTA column and identified by Western blot and MALDI-TOF. The desalted protein was dialyzed and purified by immunizing New Zealand white rabbits with antibody titer. Of the protein in SGC-7901 cells, using MTT assay protein impact on cell proliferation. Results: The cloned hp0525 gene was 993bp in length and encoded 330 amino acids. The nucleotide sequence of hp0525 shared 97% -99% identity with other H.pylori strains in GenBank. After induced by engineering bacteria, SDS-PAGE showed that the expressed protein band was newborn, with a relative molecular mass of 36000, which was consistent with the expectation. Purified by Ni2 + -NTA column, 98% recombinant protein was obtained. After treated with SGC-7901 cells, the results showed a certain amount of time and dose-dependent. It is an ATPase that has some activity through its assay. Activity was 4.40IU / ml Conclusion: hp0525 gene was successfully cloned and expressed in E.coli BL21, after purification by nickel column to obtain high purity protein, MTT assay to detect recombinant protein inhibition of cell proliferation; at the same time have a certain enzyme activity , Laid the foundation for further study of its biological function.