细粒棘球绦虫pET30a-EgA31-Eg95重组质粒构建及鉴定

发布时间：2019-01-03 17:13

【摘要】： 目的:克隆细粒棘球绦虫Eg95、EgA31抗原基因,构建pET30a-EgA31及pET30a- EgA31-Eg95原核表达载体,并在大肠埃希菌宿主系统中表达EgA31及EgA31-Eg95重组蛋白,对表达产物进行免疫印迹分析。方法:从细粒棘球绦虫原头蚴及成虫组织中提取总RNA,反转录生成cDNA,分别以原头蚴cDNA及成虫cDNA为模板RT-PCR克隆获得Eg95和EgA31抗原基因,将其克隆至pUCm-T载体,测序确定其正确性。利用定向克隆技术将EgA31抗原基因片段克隆至原核表达质粒pET30a上,转化E.coli DH5α,根据选择标记的卡那霉素抗性基因筛选到阳性克隆,通过PCR分析和酶切鉴定筛选出阳性克隆,转化BL2(1DE3),IPTG初步诱导表达pET30a-EgA31重组蛋白,SDS-PAGE电泳检测,凝胶图像分析系统确定目的蛋白表达水平。以测序正确的pET30a-EgA31重组质粒为载体,运用酶切连接技术构建pET30a-EgA31 -Eg95重组质粒,表达pET30a-EgA31-Eg95重组蛋白,SDS-PAGE电泳检测,并经凝胶图像分析确定目的蛋白的表达水平。结果:测序表明选取的pET30a-EgA31及pET30a-EgA31-Eg95阳性克隆均为正确连接目的基因的重组质粒。经IPTG诱导后重组蛋白均得到成功表达,分别在相对分子量约为31 kDa及46 kDa处有表达条带,表达量约占菌体总蛋白质的26%及20%。结论:成功克隆并构建了pET30a-EgA31及pET30a-EgA31-Eg95原核表达质粒,初步诱导表达出EgA31、EgA31-Eg95及Eg95重组蛋白,为进一步研究其免疫特性奠定了基础。
[Abstract]:Aim: to clone the Eg95,EgA31 antigen gene of Echinococcus granulosus, construct the prokaryotic expression vector of pET30a-EgA31 and pET30a- EgA31-Eg95, and express EgA31 and EgA31-Eg95 recombinant proteins in the host system of Escherichia coli. Methods: Eg95 and EgA31 antigenic genes were cloned from Echinococcus granulosus (Echinococcus granulosus) protocaria and adult Echinococcus granulosus (Echinococcus granulosus) tissues by reverse transcription of total RNA, to generate cDNA,. Eg95 and EgA31 antigenic genes were cloned into pUCm-T vector using cDNA and cDNA as templates, respectively. Sequencing confirmed its correctness. The EgA31 antigen gene fragment was cloned into prokaryotic expression plasmid pET30a by directional cloning technique, and transformed into E.coli DH5 伪. The positive clones were screened by selective marker kanamycin resistance gene. The positive clones were screened by PCR analysis and restriction endonuclease analysis. Transforming BL2 (1DE3), IPTG induced expression of pET30a-EgA31 recombinant protein), SDS-PAGE electrophoresis detection, gel image analysis system to determine the expression level of the target protein. The recombinant plasmid of pET30a-EgA31-Eg95 was constructed by restriction endonuclease ligation with the pET30a-EgA31 recombinant plasmid which was sequenced correctly. The recombinant pET30a-EgA31-Eg95 protein was expressed and detected by SDS-PAGE electrophoresis. The expression level of the target protein was determined by gel image analysis. Results: sequencing showed that the selected pET30a-EgA31 and pET30a-EgA31-Eg95 positive clones were recombinant plasmids with correct ligation of the target gene. After induction by IPTG, the recombinant protein was successfully expressed, and the relative molecular weight was about 31 kDa and 46 kDa, respectively. The expression amount of the recombinant protein was about 26% and 20% of the total cell protein, respectively. Conclusion: the prokaryotic expression plasmids of pET30a-EgA31 and pET30a-EgA31-Eg95 were cloned and constructed successfully, and the recombinant proteins of EgA31,EgA31-Eg95 and Eg95 were induced and expressed, which laid a foundation for further study of their immunological characteristics.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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