【摘要】：目的:漢坦病毒(Hantavirus,HV)是腎綜合征出血熱(Hemorrhagic Fever with Renal Syndrome,HFRS)的病原體,人感染后可導(dǎo)致HFRS和漢坦病毒肺綜合征(Hantavirus Pulmonary Syndrome,HPS)兩種嚴(yán)重的疾病。HV的包膜糖蛋白(Glycoprotein,GP)可刺激機(jī)體產(chǎn)生中和抗體,GP包括G1和G2兩種蛋白。本文將HV編碼G1的M1基因在巴氏畢赤酵母(Pichia pastoris,P.pastoris)表達(dá)系統(tǒng)中進(jìn)行了克隆和表達(dá),并對(duì)表達(dá)產(chǎn)物進(jìn)行鑒定。 內(nèi)容和方法:使用Vero-E6細(xì)胞系分別增殖HV漢灘型(HTN)Z10株和漢城型(SEO)L99株。待HV增殖到一定程度,即免疫熒光法檢測(cè)細(xì)胞中HV感染量達(dá)+++～++++時(shí),提取總RNA,RT-PCR擴(kuò)增編碼G1蛋白的M1基因片段,連接入pMD18-T simple載體,測(cè)序驗(yàn)證目的片段。使用限制性內(nèi)切酶Xba Ⅰ、Kpn Ⅰ對(duì)重組質(zhì)粒進(jìn)行雙酶切以獲得目的片段,經(jīng)純化后,連接入經(jīng)同樣內(nèi)切酶雙酶切的畢赤酵母分泌表達(dá)載體pPICZαA,接頭測(cè)序驗(yàn)證其讀碼框架。將重組表達(dá)載體經(jīng)BstX Ⅰ酶切線性化后電轉(zhuǎn)化入處于感受態(tài)的P.pastoris,重組菌株經(jīng)Zeocin篩選,將陽性重組子傳代4次以穩(wěn)定其性狀。提取重組菌株染色體DNA,PCR檢測(cè)目的片段是否整合入P.pastoris染色體。使用甲醇誘導(dǎo)法對(duì)重組菌株進(jìn)行目的蛋白的誘導(dǎo)表達(dá),并應(yīng)用Westem Blot和Dot Blot對(duì)表達(dá)產(chǎn)物進(jìn)行鑒定。 結(jié)果:M1基因RT-PCR產(chǎn)物連接入pMD18-T simple載體后,獲得重組質(zhì)粒pMD18T-Z10M1、pMD18T-L99M1。經(jīng)測(cè)序后與GenBank登錄的核苷酸序列進(jìn)行比較,與Z10株RNA序列(AF 276987)相比,目的片段有9個(gè)堿基突變:與L99株mRNA序列(AF 288298)相比,目的片段有5個(gè)堿基突變。經(jīng)限制性內(nèi)切酶KpnⅠ、XbaⅠ對(duì)其進(jìn)行雙酶切并定向克隆入經(jīng)同樣內(nèi)切酶雙酶切的質(zhì)粒載體pPICZαA中,重組質(zhì)粒分別命名為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，

本文編號(hào)：2213239