日本血吸虫凋亡相关基因SjTNFR的初步研究

发布时间：2018-04-04 16:05

本文选题：日本血吸虫　切入点：SjTNFR　出处：《上海师范大学》2017年硕士论文

【摘要】：血吸虫病是一种全球分布、影响较为严重的重要人兽共患病。目前可用治疗药物存在潜在抗性,同时缺乏有效的疫苗,使得新药的开发显得尤为重要。细胞凋亡是细胞程序性死亡的一种主要方式。日本血吸虫细胞凋亡的深入研究可为筛选日本血吸虫新药靶提供新思路。本实验通过对日本血吸虫凋亡相关基因SjTNFR的克隆、原核和真核表达、及该基因生物学功能的初步探讨,为深入阐述日本血吸虫的凋亡机制积累了知识,为评估SjTNFR作为血吸虫病治疗药物靶标的潜力提供了基础。1.日本血吸虫凋亡相关基因SjTNFR的克隆表达及生物学特性分析利用PCR技术扩增了日本血吸虫SjTNFR的全长序列,构建了重组质粒。序列分析表明该基因ORF含603bp,编码200个氨基酸,理论分子量为22.3Da,理论等电点为7.6。生物信息学分析表明该基因不含有信号肽以及跨膜区序列。结构域分析表明该基因编码蛋白属于TNFR超家族成员。构建了重组表达质粒pET28a(+)-SjTNFR,并在大肠杆菌中成功表达。Real-time PCR结果显示该基因在大、小鼠来源14d、23d、32d、和42d四个不同发育阶段虫体均有表达,其中大鼠来源虫体SjTNFR的表达水平在14d后都明显高于小鼠来源虫体,在感染后32d和42d表达差异更明显(P0.05)。提示该基因的差异表达可能是影响血吸虫在大、小鼠不同适宜性宿主体内生长发育的因素之一。2.SjTNFR的真核表达及功能初步分析通过PCR技术扩增到SjTNFR功能区片段,成功构建了真核重组表达质粒pxj-40-SjTNFR,利用脂质体转染法把重组质粒转入293T细胞。细胞间接免疫荧光实验显示,转染pxj-40-SjTNFR质粒的293T细胞表达SjTNFR蛋白。Western blotting分析结果进一步证明了重组质粒在293T细胞中成功表达,SjTNFR重组蛋白分子量大小约为26 kDa。流式细胞术实验结果表明,重组质粒转染细胞的细胞早期凋亡比例为25.3%,而对照组为2.73%,转染组的细胞晚期凋亡比例为11.2%,对照组为2.73%。转染组的早期凋亡和晚期凋亡比例均明显高于对照组。综合分析表明日本血吸虫凋亡相关基因SjTNFR具有一定的促凋亡作用。本文为深入开展SjTNFR的生物学功能分析,及筛选针对SjTNFR的药物靶标提供了基础。
[Abstract]:Schistosomiasis is a global distribution, which has a serious impact on zoonosis.The potential resistance of available therapeutic drugs and the lack of effective vaccines make the development of new drugs particularly important.Apoptosis is a major way of programmed cell death.Further study on apoptosis of Schistosoma japonicum cells may provide a new idea for screening new drug targets of Schistosoma japonicum.Through the cloning, prokaryotic and eukaryotic expression of the apoptosis-related gene SjTNFR of Schistosoma japonicum, and a preliminary study of the biological function of the gene, we have accumulated knowledge for the further elaboration of the apoptotic mechanism of Schistosoma japonicum.This provides a basis for assessing the potential of SjTNFR as a therapeutic target for schistosomiasis.Cloning, expression and Biological characteristics of SjTNFR, a apoptosis related Gene of Schistosoma japonicum, the full-length SjTNFR sequence of Schistosoma japonicum was amplified by PCR, and the recombinant plasmid was constructed.Sequence analysis showed that the gene ORF contains 603bpand encodes 200 amino acids with a theoretical molecular weight of 22.3 Daa and a theoretical isoelectric point of 7.6.Bioinformatics analysis showed that the gene did not contain signal peptide and transmembrane region sequence.Domain analysis showed that the gene encoded protein belonged to TNFR superfamily.The recombinant expression plasmid pET28a-SjTNFR was constructed, and the expression of .Real-time PCR in E. coli showed that the gene was expressed in four different developmental stages, E. coli.The expression level of SjTNFR in the rat body was significantly higher than that in the mouse after 14 days, and the difference was more obvious at 32 days and 42 days after infection.These results suggest that the differential expression of this gene may be one of the factors affecting the growth and development of Schistosoma japonicum in different suitable hosts. 2. The eukaryotic expression and function of SjTNFR were amplified into the functional region of SjTNFR by PCR technique.The eukaryotic recombinant expression plasmid pxj-40-SjTNFR was successfully constructed, and the recombinant plasmid was transfected into 293T cells by liposome transfection.Cell indirect immunofluorescence assay showed that 293T cells transfected with pxj-40-SjTNFR plasmid expressed SjTNFR protein. Western blotting analysis further demonstrated that the molecular weight of recombinant SjTNFR protein expressed in 293T cells was about 26kDa.The results of flow cytometry showed that the proportion of early apoptosis was 25.3in the transfected cells with recombinant plasmids, 2.73in the control group, 11.2in the transfection group and 2.73in the control group.The proportion of early and late apoptosis in transfection group was significantly higher than that in control group.Comprehensive analysis showed that the apoptosis-related gene SjTNFR of Schistosoma japonicum could promote apoptosis to some extent.This paper provides a basis for further analysis of biological function of SjTNFR and screening of drug targets for SjTNFR.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.735

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