日本血吸虫组织蛋白酶B基因的克

发布时间：2018-01-28 19:39

本文关键词： 日本血吸虫组织蛋白酶B 基因克隆原核表达　出处：《福建医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:构建表达日本血吸虫(Schistosoma japonicum)组织蛋白酶B(Cathepsin B,CB/ Sj31)基因的原核表达系统,优化表达条件,以期获得重组组织蛋白酶B的高效表达,为进一步研究组织蛋白酶B的生化特性和生物学功能奠定基础。方法: 1.基因克隆:提取日本血吸虫总RNA。Genebank中获得Sj31基因全长,通过RT-PCR扩增Sj31基因的cDNA序列(含768个碱基),基因克隆技术构建pET30a- Sj31重组表达载体。采用氯化钙共沉淀法将重组子转化至E.coli DH5α和BL21(DE3)。PCR、酶切、测序方法筛选阳性重组子。 2.蛋白表达:①抗血清的制备:取感染日本血吸虫的大白兔心脏血,分离取血清。经大肠杆菌裂解液吸附法处理。②IPTG诱导BL21(DE3)细菌重组Sj31蛋白表达,经SDS-PAGE电泳及用5×His单抗(小鼠源)抗原鉴定融合蛋白。用兔抗日本血吸虫多克隆抗体,检测蛋白的抗原性。优化诱导表达的时间和IPTG的诱导浓度,诱导重组蛋白大量表达。结果: 1.提取了日本血吸虫成虫的总RNA。两步法RT-PCR扩增出Sj31基因的cDNA序列。基因克隆技术成功构建pET30a- Sj31重组表达载体。 2.重组子可被IPTG诱导表达,诱导表达的重组蛋白可被5×His单抗(小鼠源)抗原识别;并且重组蛋白可被兔抗日本血吸虫多克隆抗体识别。当诱导表达时间为4h、IPTG终浓度为1.0mmol/L时,表达量最大。结论:成功构建Sj31基因的原核表达系统,获得高效表达;重组蛋白肽段具有良好的抗原性。为进一步研究其功能打下了必要的基础。
[Abstract]:Objective: to construct a cathepsin B (Cathepsin B) expressing Schistosoma japonicum (Schistosoma japonicum) from Schistosoma japonicum. The prokaryotic expression system of CBSj31) gene was optimized to obtain the high expression of recombinant cathepsin B. It will lay a foundation for the further study of the biochemical characteristics and biological functions of cathepsin B. Methods: 1. Gene cloning: the full length of Sj31 gene was obtained from total RNA.Genebank of Schistosoma japonicum. The cDNA sequence of Sj31 gene (including 768 bases) was amplified by RT-PCR. The recombinant expression vector pET30a- Sj31 was constructed by gene cloning. The recombinant plasmid was transformed into E. coli DH5 伪 and BL21(DE3).PCR by calcium chloride coprecipitation. The positive recombinant was screened by enzyme digestion and sequencing. 2. Preparation of antiserum against protein expression of 1: 1: blood from heart of rabbits infected with Schistosoma japonicum. The recombinant Sj31 protein expression of BL21DE3 was induced by E. coli lytic solution adsorption. The fusion protein was identified by SDS-PAGE electrophoresis and 5 脳 His monoclonal antibody (mouse origin). Rabbit polyclonal antibody against Schistosoma japonicum was used. To determine the antigenicity of the protein, to optimize the time of induction and the concentration of IPTG, and to induce the expression of recombinant protein in large quantities. Results: 1. The total RNA of adult Schistosoma japonicum was extracted. The cDNA sequence of Sj31 gene was amplified by two-step RT-PCR. The gene cloning technique successfully constructed pET30a-. Sj31 recombinant expression vector. 2.Recombinant can be induced to express by IPTG, and the recombinant protein can be recognized by 5 脳 His monoclonal antibody (mouse) antigen. The recombinant protein could be recognized by rabbit polyclonal antibody against Schistosoma japonicum, and when the induced expression time was 4 h, the final concentration of IPTG was 1.0 mmol / L, the expression level was the highest. Conclusion: the prokaryotic expression system of Sj31 gene was successfully constructed, and the recombinant protein peptide had good antigenicity, which laid a necessary foundation for further study of its function.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383.2

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