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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。
Objective To clone and express the functional fragment of apoptosis-inducing factor (SjAIF) of Schistosoma japonicum, and to analyze its biological characteristics. Methods The functional fragment of apoptosis-inducing factor of Schistosoma japonicum was amplified by PCR and the prokaryotic recombinant plasmid was constructed and induced to express. The transcription level of Schistosoma japonicum and adult worms at different developmental stages was analyzed by real-time PCR. Western-blot and indirect The antigenicity and immunogenicity of the recombinant protein were analyzed by ELISA, and the distribution of the protein in the parasite was analyzed by immunohistochemistry. Results The SjAIF functional region fragment was cloned and its size was 831bp. The prokaryotic recombinant plasmid pET-28a (+) - SjAIF was successfully constructed and expressed in E. coli. The recombinant protein has a molecular weight of 35 kD. Quantitative real-time PCR analysis showed that the SjAIF gene transcribed at all developmental stages of the sperm and adult worms. The expression levels of SjAIF gene were lower in the 7d-21d, higher in the 42d and 28d, and higher in the female than in the male. Recombinant protein has good antigenicity and immunogenicity, and induced a higher level of specific IgG antibody in mice after immunization. The protein is mainly present in Schistosoma japonicum body, a small part of the distribution in the parenchyma. Conclusion The functional region of SjAIF gene was successfully expressed and the biological characteristics of SjAIF gene were preliminarily analyzed, which provided a basis for further study on the biological function and function of this gene.