汉坦病毒M1基因克隆及其在毕赤酵母系统中的表达
发布时间：2018-08-30 13:39
【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,人感染后可导致HFRS和汉坦病毒肺综合征(Hantavirus Pulmonary Syndrome,HPS)两种严重的疾病。HV的包膜糖蛋白(Glycoprotein,GP)可刺激机体产生中和抗体,GP包括G1和G2两种蛋白。本文将HV编码G1的M1基因在巴氏毕赤酵母(Pichia pastoris,P.pastoris)表达系统中进行了克隆和表达,并对表达产物进行鉴定。 内容和方法:使用Vero-E6细胞系分别增殖HV汉滩型(HTN)Z10株和汉城型(SEO)L99株。待HV增殖到一定程度,即免疫荧光法检测细胞中HV感染量达+++～++++时,提取总RNA,RT-PCR扩增编码G1蛋白的M1基因片段,连接入pMD18-T simple载体,测序验证目的片段。使用限制性内切酶Xba Ⅰ、Kpn Ⅰ对重组质粒进行双酶切以获得目的片段,经纯化后,连接入经同样内切酶双酶切的毕赤酵母分泌表达载体pPICZαA,接头测序验证其读码框架。将重组表达载体经BstX Ⅰ酶切线性化后电转化入处于感受态的P.pastoris,重组菌株经Zeocin筛选,将阳性重组子传代4次以稳定其性状。提取重组菌株染色体DNA,PCR检测目的片段是否整合入P.pastoris染色体。使用甲醇诱导法对重组菌株进行目的蛋白的诱导表达,并应用Westem Blot和Dot Blot对表达产物进行鉴定。 结果:M1基因RT-PCR产物连接入pMD18-T simple载体后,获得重组质粒pMD18T-Z10M1、pMD18T-L99M1。经测序后与GenBank登录的核苷酸序列进行比较,与Z10株RNA序列(AF 276987)相比,目的片段有9个碱基突变:与L99株mRNA序列(AF 288298)相比,目的片段有5个碱基突变。经限制性内切酶KpnⅠ、XbaⅠ对其进行双酶切并定向克隆入经同样内切酶双酶切的质粒载体pPICZαA中,重组质粒分别命名为pPICZαA-Z10M1、pPICZαA-L99M1。
[Abstract]:Objective: Hantavirus (Hantavirus,HV) is the pathogen of hemorrhagic fever with renal syndrome (Hemorrhagic Fever with Renal Syndrome,HFRS). Human infection can lead to HFRS and Hantavirus pulmonary syndrome (Hantavirus Pulmonary Syndrome,HPS) two serious diseases. HV envelope glycoprotein (Glycoprotein,GP) can stimulate the production of neutralizing antibody GP including G1 and G2 proteins. The M1 gene encoded by HV was cloned and expressed in Pichia pastoris (Pichia pastoris,P.pastoris) expression system, and the expressed product was identified. Content and methods: Vero-E6 cell line was used to proliferate HV Hantaan (HTN) Z10 strain and Seoul (SEO) L99 strain respectively. When HV proliferates to a certain extent, that is, when the amount of HV infection is detected by immunofluorescence, the M1 gene fragment encoding G1 protein is amplified by total RNA,RT-PCR and ligated into pMD18-T simple vector, and the target fragment is verified by sequencing. The recombinant plasmid was digested with restriction enzyme Xba 鈪，
本文编号：2213239
[Abstract]:Objective: Hantavirus (Hantavirus,HV) is the pathogen of hemorrhagic fever with renal syndrome (Hemorrhagic Fever with Renal Syndrome,HFRS). Human infection can lead to HFRS and Hantavirus pulmonary syndrome (Hantavirus Pulmonary Syndrome,HPS) two serious diseases. HV envelope glycoprotein (Glycoprotein,GP) can stimulate the production of neutralizing antibody GP including G1 and G2 proteins. The M1 gene encoded by HV was cloned and expressed in Pichia pastoris (Pichia pastoris,P.pastoris) expression system, and the expressed product was identified. Content and methods: Vero-E6 cell line was used to proliferate HV Hantaan (HTN) Z10 strain and Seoul (SEO) L99 strain respectively. When HV proliferates to a certain extent, that is, when the amount of HV infection is detected by immunofluorescence, the M1 gene fragment encoding G1 protein is amplified by total RNA,RT-PCR and ligated into pMD18-T simple vector, and the target fragment is verified by sequencing. The recombinant plasmid was digested with restriction enzyme Xba 鈪，
本文编号：2213239
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