汉坦病毒S基因在大肠埃希菌中的克隆和表达

发布时间：2017-12-31 13:19

本文关键词：汉坦病毒S基因在大肠埃希菌中的克隆和表达　出处：《天津医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的:汉坦病毒(Hantavirus,HV)是肾综合征出血热(Hemorrhagic Fever with Renal Syndrome,HFRS)的病原体,可导致人类两种疾病:HFRS和汉坦病毒肺综合症(Hantavirus Pulmonary Syndrom,HPS)。HV核蛋白(Nucleocapsid Protein,NP)是其主要的结构蛋白之一,本文旨在对编码NP蛋白的S基因在大肠埃希菌(Escherichia coli,E.coli)BL21(DE3)中进行克隆和表达,并对表达产物进行鉴定。方法:以pGEM-T-L99S和pGEM-T-Z10S为模板,分别经PCR扩增L99S和Z10S的ORF,前者经EcoRI及XhoI双酶切,后者经SacI及XhoI双酶切,纯化后将二者与经相同双酶切所得的pET32a和pET28a载体大片段相连接,转入E.coli Top10感受态细胞中;在抗生素(pET32a为氨苄青霉素抗性,pET28a为卡那霉素抗性)筛选下,挑取阳性重组子。经微量碱裂解法提取重组质粒,以PCR法、酶切法和序列分析法进行鉴定。继而将正确定向克隆的重组质粒转入表达宿主Ecoli.BL21(DE3)感受态中,通过IPTG对重组菌进行NP蛋白的诱导表达,并使用HFRS患者阳性血清进行Western Blot鉴定。同时,摸索不同浓度的IPTG和不同诱导时间对重组菌中目的蛋白产量的影响。结果:PCR扩增获得的L99 S基因和Z10 S基因定向克隆入pET32a和pET28a后,挑取抗性筛选的阳性重组子,分别择其一命名为:pET32a-L99S、pET32a-Z10S、pET28a-L99S和pET28a-Z10S。PCR法、酶切法和序列分析对上述重组质粒鉴定结果显示,L99 S基因和Z10 S基因已经正确连接入pET32a和pET28a载体,且未改变读码框架。将上述重组质粒转化入Ecoli.BL21(DE3)感受态中,挑取阳性重组菌分别命名为E.coli BL21(DE3)/pET32a-L99S、E.coli BL21(DE3)/pET32a-Z10S、E.coli BL21(DE3)/pET28a-L99S和E.coli BL21(DE3)/pET28a-Z10S。IPTG诱导重组菌E.coli BL21(DE3)/pET32a-L99S和E.coli BL21(DE3)/pET32a-Z10S,结果显示重组蛋白表达量达菌体总蛋白的40%;
[Abstract]:Objective : Hantavirus ( HV ) is a pathogen of hemorrhagic fever with renal syndrome and hemorrhagic fever with renal syndrome . Nucleocapsid Protein ( NP ) is one of its main structural proteins . The purpose of this study is to clone and express the S gene encoding NP protein in E . coli BL21 ( DE3 ) and to identify the expression product . Methods : The ORFs of L99S and Z10 were amplified by PCR . The recombinant plasmids were digested with the same dienzyme and then transferred into E . coli Top10 competent cells . The recombinant plasmids were transformed into E . coli Top10 competent cells . The recombinant plasmids were transformed into E . coli Top10 competent cells . The recombinant plasmids were transformed into the expression host Ecoli BL21 ( DE3 ) competent cells . coli BL21 ( DE3 ) / pET32a - L99S , E . coli BL21 ( DE3 ) / pET32a - L99S and E .

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

【参考文献】