重组日本血吸虫26ku谷胱甘肽-硫-转移酶的表达、纯化及其免疫特性分析用于急性血吸虫病免疫诊断

Immunodiagnosis of acute schistosomiasis using 26 ku glutathione-S-transferase recombinant protein of Schistosoma japonicum

目的将日本血吸虫(中国大陆株)26ku谷胱甘肽硫转移酶(26kuSjGST)基因克隆入pET28a(+)并诱导表达26kurSjGST蛋白,纯化并用于急性血吸虫病免疫诊断。方法构建重组质粒pET28aSjGST,转化到E.coliBL21,经IPTG诱导表达并进行SDS变性蛋白质电泳,以小鼠抗GST单克隆抗体为一抗,进行Westernblot分析。用His·BandPurificationKit亲和层析纯化重组蛋白,以此重组蛋白作为抗原,用ELISA法检测急性血吸虫患者血清和非流行区正常人血清。结果成功地构建了重组质粒pET28aSjGST,SDS变性蛋白质电泳显示可见一与预期分子量大小相符的特异蛋白条带的表达。Westernblot分析表明,该重组蛋白能被小鼠抗GST单克隆抗体识别。ELISA结果表明,rSjGST用于急性血吸虫患者血清中抗体检测的阳性率为92.3%。结论rSjGST得到表达和纯化,该重组蛋白用于急性血吸虫患者血清中抗体的检测具有良好的免疫反应性,为进一步探讨其在血吸虫病诊断中的应用奠定了基础。

Objective To express and purify 26 ku recombinant glutathione-S-transferase of Schistosoma japonicum （26ku rSjGST） and use it to detect acute schistosomiasis. Methods Recombinant plasmid pET28a-SjGST was transformed into E. coli BL21 and induced by IPTG for its expression. The expressed protein was purified using the affinity chromatography, and analyzed by SDS-PAGE and Western blotting with mouse anti-GST monoclonal antibody. The 26 ku rSjGST was applied to ELISA for detecting circulating antibody in serum of patients with acute schistosomiasis. Results There was a special protein band observed between 20. 1 ku to 31 ku in SDS-PAGE gel, and this protein was able to react with human positive sera by ELISA. Twenty-six serum samples from patients with acute schistosomiasis were parallelly detected by ELISA, and the positive rates was 92.3%. Conclusion The study shows that the 26ku rSjGST can be expressed and applied as a diagnostic antigen for acute schistosomiasis.