论文部分内容阅读
目的克隆、表达日本血吸虫线粒体外膜转运酶34基因(SjTOM34),并对其生物特性进行初步研究。方法应用RT-PCR方法从42d日本血吸虫成虫cDNA中扩增SjTOM34基因,并进行生物信息学分析。应用实时定量RT-PCR方法分析了SjTOM34基因在日本血吸虫不同发育阶段虫体中的表达状况。构建了SjTOM34基因重组原核表达质粒,并在大肠杆菌中进行表达,Western blotting分析了重组抗原的免疫原性及该蛋白在不同发育阶段虫体中的表达情况,利用免疫组化方法观察了该蛋白在虫体内的分布情况。结果获得了SjTOM34的基因序列,其开放阅读框(ORF)为1 083bp。该基因在所检测的不同发育阶段童虫和成虫中均有表达,其中在35d虫体中的表达量略高于其他时期的虫体。成功构建了重组的原核表达质粒并且在大肠杆菌中获得表达,重组蛋白在上清和沉淀中都有存在,并且具有较好的免疫原性,在所检测的各时期虫体中均可检测到该蛋白的存在,并且主要分布在虫体的实质组织中。结论获得了日本血吸虫SjTOM34基因,初步明确了该基因在日本血吸虫童虫和成虫中的表达情况,及在成虫中的分布。
Objective To clone and express the Schistosoma japonicum Mitochondrial transmembrane transporter 34 gene (SjTOM34) and study its biological characteristics. Methods The SjTOM34 gene was amplified from cDNA of 42 days adult Schistosoma japonicum by RT-PCR and analyzed by bioinformatics. The real-time quantitative RT-PCR method was used to analyze the expression of SjTOM34 gene in different developmental stages of Schistosoma japonicum. The recombinant prokaryotic expression plasmid of SjTOM34 gene was constructed and expressed in E. coli. The immunogenicity of the recombinant antigen and the expression of the recombinant protein in different developmental stages were analyzed by Western blotting. The protein was observed by immunohistochemistry In the distribution of parasites. Results The gene sequence of SjTOM34 was obtained, with an open reading frame (ORF) of 1,083 bp. The gene was expressed in all stages of adult worms and adults tested. The expression level of the gene in 35 days was slightly higher than that in other stages. The recombinant prokaryotic expression plasmid was successfully constructed and expressed in E.coli. The recombinant protein was found in both supernatant and sediment, and had good immunogenicity. The recombinant protein could be detected in all the tested pests The presence of protein, and the main distribution in the parenchyma of parasites. Conclusion The SjTOM34 gene of Schistosoma japonicum was obtained, and the expression of SjTOM34 gene in Schistosoma japonicum and its adult worms and its distribution in adults were preliminarily determined.