弓形虫三磷酸核苷水解酶-II的克隆、表达及初步应用

目的探讨弓形虫RH株速殖子NTPase-II重组蛋白的免疫反应性及其在抗体检测中的应用。方法通过PCR获得NTPase-II基因,克隆入pGEM-T Easy载体,经酶切与测序鉴定后亚克隆至表达质粒pBAD-HisB,构建原核表达重组质粒,在大肠杆菌中以包涵体形式获得高效表达。SDS-PAGE和Western-blot分析蛋白表达产物。用纯化的表达产物作为诊断抗原,通过对反应条件的优化,初步建立检测抗NTPase-II抗体的间接ELISA方法。结果PCR扩增得到特异的弓形虫NT-Pase-II基因序列,经测序鉴定无基因突变。重组质粒诱导表达产物相对分子量约70kD,与理论值相符。经Western-blot证实,重组蛋白可刺激机体产生相应的抗体。具有良好的抗原性和免疫原性。用表达的重组NTPase-II蛋白初步建立了用于NTPase-II抗体检测的间接ELISA方法。结论重组NTPase-II蛋白具有较强免疫原性,可作为诊断抗原用于急性弓形虫感染诊断试剂的研发。

Cloning and expression of NTPase gene of Toxoplasma gondii and application of the recombinant protein

To investigate the immunoreactivity of the recombinant proteins encoded by the nucleoside triphosphate hydrolase(NTPase)-II gene of the tachyzoite form of Toxoplasma gondii RH strain and explore the significance of proteins in detection for anti-NTPase-II antibodies,genes encoding NTPase-II protein of T.gondii were obtained by PCR and cloned into vector pGEM-T Easy.Positive clones were screened and identified by digestion and sequenced.The target gene was then subcloned into the expression vector pBAD-HisB.It was found that the recombinant plasmids were over-expressed in E.coli as inclusion body with molecular weight of 70 kD.The indirect ELISA for the detection of anti-NTPase-II antibodies in mice sera was established after management of the optional working conditions,and the results of Western-blo and ELISA indicated the purified recombinant proteins have highly antigenicity and immunogenicity.The study suggests the fact of the advantage of NTPase-II as the antigen for serodiagnosis.