汉坦病毒核蛋白重组克隆构建及在梭菌栽体中的表达

发布时间：2018-03-13 23:02

本文选题：肾综合征出血热　切入点：汉坦病毒　出处：《青岛大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的构建含汉坦病毒截短核蛋白基因的重组质粒,并在原核表达系统产气荚膜梭菌中表达,为汉坦病毒鼠用口服疫苗的研制做好前期工作。方法以重组质粒pGEX-4T-2/SA为模板,PCR扩增汉坦病毒截短核蛋白基因片段。Eco81I、BspT104 I双酶切PCR产物和质粒pJRC200,胶回收酶切产物,T_4连接酶连接,构建重组质粒pJRC200-SA,转化E.coli DH5α,菌液PCR、酶切、测序鉴定。将pJRC200-SA电穿孔转化产气荚膜梭菌,先行厌氧增菌培养,后产芽孢培养诱导汉坦病毒截短核蛋白表达。超声裂解产气荚膜梭菌芽孢,对裂解产物进行SDS-PAGE、Western blot鉴定。结果PCR扩增获得约580bp大小基因片段,与目的片段大小相符。菌液PCR得到约580bp大小基因片段,证明插入目的片段大小正确;Eco81I、SalI双酶切得到8290bp和530bp两基因片段,证明插入目的片段连接方向正确;Ec081I、BspT104I双酶切得到8240bp和580bp两基因片段,证明目的片段插入位置正确;测序证明插入目的片段碱基序列正确,无碱基缺失和突变。SDS-PAGE显示截短核蛋白有效表达,大小约27KDa。Western blot分析显示该截短核蛋白能与肾综合征出血热病人阳性血清特异性结合。结论汉坦病毒截短核蛋白重组质粒pJRC200-SA构建成功。截短核蛋白能在原核表达系统产气荚膜梭菌中有效表达,且具有良好的抗原性和特异性。为鼠用汉坦病毒口服疫苗的研制奠定了基础。
[Abstract]:Objective to construct a recombinant plasmid containing Hantavirus truncated nucleoprotein gene and express it in the prokaryotic expression system of Clostridium perfringens. Methods Recombinant plasmid pGEX-4T-2/SA was used to amplify Hantavirus truncated ribosomal protein gene fragment. Eco81IbspT104I double enzyme digested PCR product and plasmid pJRC200. the recombinant plasmid pJRC200-SAwas constructed and ligated to construct recombinant plasmid pJRC200-SA.The recombinant plasmid pJRC200-SAwas transformed into E. coli DH5 伪. Sequencing. PJRC200-SA electroporation was transformed into Clostridium perfringens, then anaerobic culture was used to induce the expression of truncated nucleoprotein of Hantavirus and ultrasonic lysis of Clostridium perfringens spores was carried out by SDS-PAGEG blot. Results about 580bp gene fragment was obtained by PCR amplification, which was consistent with the size of the target gene fragment, and about 580bp gene fragment was obtained by PCR, which proved that the inserted target fragment size was correct and 8290bp and 530bp gene fragments were obtained by double enzyme digestion. The two gene fragments 8240 BP and 580 BP were obtained by double enzyme digestion of Ec081It104I, which proved that the insertion position of the target fragment was correct, the sequence of the inserted target fragment was correct, and the sequence of the inserted target fragment was correct. No base deletion and mutation. SDS-PAGE showed that truncated nucleoprotein was effectively expressed. Western blot analysis of about 27KDa.Western blot showed that the truncated nucleoprotein could specifically bind to the positive serum of patients with hemorrhagic fever with renal syndrome. Conclusion the recombinant plasmid pJRC200-SA of Hantavirus truncated riboprotein was successfully constructed, and the truncated nucleoprotein could be effectively expressed in Clostridium perfringens, a prokaryotic expression system. It has good antigenicity and specificity, which lays a foundation for the development of oral vaccine of Hantavirus in mice.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373.32

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