本文選題：腎綜合征出血熱　切入點(diǎn)：漢坦病毒　出處：《青島大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的構(gòu)建含漢坦病毒截短核蛋白基因的重組質(zhì)粒,并在原核表達(dá)系統(tǒng)產(chǎn)氣莢膜梭菌中表達(dá),為漢坦病毒鼠用口服疫苗的研制做好前期工作。 方法以重組質(zhì)粒pGEX-4T-2/SA為模板,PCR擴(kuò)增漢坦病毒截短核蛋白基因片段。Eco81I、BspT104 I雙酶切PCR產(chǎn)物和質(zhì)粒pJRC200,膠回收酶切產(chǎn)物,T_4連接酶連接,構(gòu)建重組質(zhì)粒pJRC200-SA,轉(zhuǎn)化E.coli DH5α,菌液PCR、酶切、測(cè)序鑒定。將pJRC200-SA電穿孔轉(zhuǎn)化產(chǎn)氣莢膜梭菌,先行厭氧增菌培養(yǎng),后產(chǎn)芽孢培養(yǎng)誘導(dǎo)漢坦病毒截短核蛋白表達(dá)。超聲裂解產(chǎn)氣莢膜梭菌芽孢,對(duì)裂解產(chǎn)物進(jìn)行SDS-PAGE、Western blot鑒定。 結(jié)果PCR擴(kuò)增獲得約580bp大小基因片段,與目的片段大小相符。菌液PCR得到約580bp大小基因片段,證明插入目的片段大小正確;Eco81I、SalI雙酶切得到8290bp和530bp兩基因片段,證明插入目的片段連接方向正確;Ec081I、BspT104I雙酶切得到8240bp和580bp兩基因片段,證明目的片段插入位置正確;測(cè)序證明插入目的片段堿基序列正確,無堿基缺失和突變。SDS-PAGE顯示截短核蛋白有效表達(dá),大小約27KDa。Western blot分析顯示該截短核蛋白能與腎綜合征出血熱病人陽性血清特異性結(jié)合。 結(jié)論漢坦病毒截短核蛋白重組質(zhì)粒pJRC200-SA構(gòu)建成功。截短核蛋白能在原核表達(dá)系統(tǒng)產(chǎn)氣莢膜梭菌中有效表達(dá),且具有良好的抗原性和特異性。為鼠用漢坦病毒口服疫苗的研制奠定了基礎(chǔ)。
[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 偽. 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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373.32

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