日本血吸虫凋亡诱导因子SjAIF功能区片段的克隆及表达分析

Cloning and expression analysis of a functional fragment of apoptosis-inducing factor(AIF) gene in Schistosoma japonicum

目的克隆表达日本血吸虫凋亡诱导因子(SjAIF)功能区片段,并对其生物学特性进行初步分析。方法应用PCR技术扩增日本血吸虫凋亡诱导因子的功能区片段,构建原核重组表达质粒并诱导其表达,实时定量PCR分析其在不同发育时期童虫和成虫的转录水平,通过Western-blot和间接ELISA法分析重组蛋白的抗原性和免疫原性,应用免疫组化分析该蛋白在虫体内的分布情况。结果克隆了SjAIF功能区片段,大小为831bp。成功构建了原核重组表达质粒pET-28a(+)-SjAIF,并在大肠杆菌中获得表达,重组蛋白分子量35kD。实时定量PCR分析表明SjAIF基因在童虫和成虫的各个发育阶段均有转录,其中7d^21d虫体表达量较低,42d和28d虫体表达量较高,雌虫表达量高于雄虫。重组蛋白具有较好的抗原性和免疫原性,免疫小鼠后诱导产生了较高水平的特异性IgG抗体。该蛋白主要存在于日本血吸虫体被,少部分分布于实质组织中。结论成功表达了SjAIF基因的功能区片段,对其生物学特性进行了初步分析,为进一步研究该基因生物学功能和作用提供了基础。

We cloned and expressed a functional fragment of apoptosis-inducing factor（AIF）gene in Schistosoma japonicum and further analyzed its biological characteristics.PCR technique was employed to amplify the functional fragment of SjAIF by employing a cDNA of 14d（day）schistosomula as template.The fragment of AIF was subcloned into a pET28a（＋）vector and the recombinant plasmid was transformed into competent E.coil/BL21 for producing recombinant protein.The expression level of SjAIF was determined at several different development stages of schistosomula and adult worms by using real-time RT-PCR.The recombinant protein was purified and then its antigenicity was accessed by Western blotting and ELISA.The distribution of the protein in Schistosoma japonicum was analyzed by immunolocalization.Real-time PCR analysis revealed that the expression of SjAIF was lower in 7d,14 dand 21dthan that in other stages.Western-blotting showed that the recombinant had good immunogenicity.The vaccinated group showed a good ability to induce IgG as examined by ELISA.Immunolocalization analysis revealed that the SjAIF was mainly distributed in tegument and parenchyma.The fragment of SjAIF was obtained and its molecular characterizations were preliminarily investigated.This study provided an important basis for further investigation of the biological characteristics and mechanism of the protein in Schistosoma japonicum.