本文選題：豬輪狀病毒 + NSP3�。� 參考：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：輪狀病毒(Rotavirus,RV)屬于呼腸病毒科(Reoviridae)輪狀病毒屬(Rotavirus)成員,是重要的病原體,可以引起多種宿主生物的病毒性腹瀉,如哺乳動(dòng)物和鳥類。豬輪狀病毒(Porcine Rotavirus,Po RV)是引起仔豬腹瀉、嘔吐、脫水等癥狀的主要病原,致死率高,為豬的主要傳染病之一,使我國(guó)的養(yǎng)豬業(yè)遭受了沉重的打擊。RV為雙鏈的RNA病毒,RV基因組是由11段每段均含有一個(gè)開放閱讀框、不連續(xù)的雙股RNA所構(gòu)成,并編碼6種結(jié)構(gòu)蛋白以及6種非結(jié)構(gòu)蛋白。NSP3是RV的非結(jié)構(gòu)蛋白,是一種非糖基化蛋白,該蛋白與病毒粒子感染細(xì)胞有關(guān),能夠關(guān)閉宿主細(xì)胞mRNA的翻譯。非結(jié)構(gòu)蛋白NSP5,主要功能是糖基化和磷酸化的修飾作用,多在RV翻譯后期進(jìn)行參與。其C端富含保守的絲氨酸和蘇氨酸多聚化結(jié)構(gòu)域,參與翻譯后期的磷酸化和糖基化修飾,NSP5的存在可使病毒的復(fù)制轉(zhuǎn)錄等過程順利進(jìn)行,在病毒的復(fù)制周期中起著不可或缺的作用。本研究旨在獲得抗Po RV(G9)血清型NMTL毒株純度高特異性強(qiáng)的單克隆抗體(MAb),加深對(duì)NSP3、NSP5蛋白結(jié)構(gòu)和功能的了解,并對(duì)它們的免疫學(xué)特性進(jìn)行了更深一步的研究。本研究采用RT-PCR方法擴(kuò)增了G9血清型NSP3和NSP5基因,經(jīng)酶切后分別連接至p GEX-6P-1載體中構(gòu)建重組表達(dá)質(zhì)粒p GEX-NSP3和p GEX-NSP5。酶切鑒定后進(jìn)行測(cè)序,獲得測(cè)序正確的陽(yáng)性質(zhì)粒。將其分別轉(zhuǎn)化至E.coli DH5α感受態(tài)細(xì)胞,經(jīng)IPTG進(jìn)行NSP3重組蛋白(r NSP3)和NSP5重組蛋白(r NSP5)的誘導(dǎo)表達(dá),并設(shè)置空載體為對(duì)照。將r NSP3和r NSP5經(jīng)SDS-PAGE分析后進(jìn)行純化,結(jié)果顯示:r NSP3和r NSP5大小分別為60Ku和50Ku。將rNSP3和r NSP5分別與抗RV的陽(yáng)性血清進(jìn)行western blot試驗(yàn),均發(fā)生反應(yīng),結(jié)果表明其具有良好的免疫源性。將含有r NSP3和r NSP5條帶的SDS-PAGE膠搗碎后,采用皮下注射的方法免疫BALB/c小鼠,免疫3次后斷尾取血,間接ELISA檢測(cè)抗體效價(jià)后,進(jìn)行加強(qiáng)免疫于融合的前3d。取加強(qiáng)免疫后小鼠的脾細(xì)胞與生長(zhǎng)狀態(tài)較好的骨髓瘤細(xì)胞(SP2/0)進(jìn)行融合,利用間接ELISA方法進(jìn)行篩選,得到1株能穩(wěn)定分泌抗Po RV(G9)NSP3的雜交瘤細(xì)胞株5B8和1株能穩(wěn)定分泌抗NSP5的雜交瘤細(xì)胞株5E11,均為Ig G1亞類。Western blot及間接免疫熒光(IFA)結(jié)果表明,所獲得的MAbs特異性良好。為確定獲得的MAb所針對(duì)的抗原表位的區(qū)域序列,采用Western blot試驗(yàn)分別對(duì)截短表達(dá)后的NSP3和NSP5蛋白進(jìn)行一系列的鑒定。結(jié)果表明,5B8識(shí)別的抗原表位序列為290QDYDRTFL 297,5E11識(shí)別的抗原表位序列為61 GPSDSA 66。本研究分別成功制備了一株抗NSP3蛋白和一株抗NSP5蛋白的MAb,并對(duì)它們的線性抗原表位進(jìn)行了鑒定。為RV的抗病毒免疫研究、抗體檢測(cè)等均提供了信息及材料,同時(shí)也為進(jìn)一步對(duì)NSP3和NSP5蛋白的結(jié)構(gòu)和功能的研究奠定了良好的基礎(chǔ)。
[Abstract]:Rotavirus (Rotavirus RV) is a member of the Rotavirus family (Reoviridae), which is an important pathogen and can cause viral diarrhea in many host organisms, such as mammals and birds. Porcine RotavirusPo RV is a major cause of diarrhea, vomiting, dehydration and other symptoms in piglets. The RV genome of the double-stranded RNA virus RV is composed of 11 segments containing an open reading frame and discontinuous double-stranded RNA. It also encodes six structural proteins and six nonstructural proteins. NSP3 is a non-glycosylated protein of RV, which is related to viral particle infection and can shut down the translation of host cell mRNA. Nonstructural protein NSP5, whose main function is glycosylation and phosphorylation, is mainly involved in the late stage of RV translation. Its C-terminal is rich in conserved serine and threonine domains. The presence of phosphorylation and glycosylation of NSP5 in late translation can make the replication and transcription of the virus go on smoothly. It plays an indispensable role in the replication cycle of virus. The aim of this study was to obtain monoclonal antibody (MAB) against Po RV (G9) serotype NMTL strain with high purity and strong specificity, to understand the structure and function of NSP3NSP5 protein, and to further study their immunological properties. In this study, G9 serotype NSP3 and NSP5 genes were amplified by RT-PCR and ligated into pGEX-6P-1 vector to construct recombinant expression plasmids pGEX-NSP3 and pGEX-NSP5, respectively. The positive plasmids were sequenced and identified by enzyme digestion. They were transformed into E. coli DH5 偽 competent cells, and were induced to express NSP3 recombinant protein (r NSP3) and NSP5 recombinant protein (r NSP5) by IPTG. RNSP3 and rNSP5 were purified by SDS-PAGE. The results showed that the sizes of rNSP3 and rNSP5 were 60Ku and 50Kurespectively. The western blot test of rNSP3 and rNSP5 with anti-RV positive serum showed that rNSP3 and rNSP5 had good immunogenicity. The SDS-PAGE gel containing the bands of rNSP3 and rNSP5 was mashed. BALB / c mice were immunized by subcutaneous injection. After 3 times of immunization, blood was taken from the tail of BALB / c mice. The titer of antibody was detected by indirect Elisa, and then immunized 3 days before fusion. Spleen cells of mice were fused with myeloma cells (SP2 / 0), and indirect Elisa was used to screen the spleen cells. A hybridoma cell line 5B8 and a hybridoma cell line 5E11 secreting anti-Po RV (G9) NSP3 stably were obtained. They were Ig G1 subclass. Western blot and indirect immunofluorescence (IFA). In order to determine the domain sequence of the epitopes targeted by MAb, the truncated NSP3 and NSP5 proteins were identified by Western blot assay. The results showed that the epitope sequence recognized by h5B8 was 290QDYDRTFL 297E11 and the epitope sequence was 61 GPSDSA66. In this study, a strain of MAbs resistant to NSP3 protein and NSP5 protein were successfully prepared, and their linear epitopes were identified. It provides information and materials for the study of antiviral immunity and antibody detection of RV, and also lays a good foundation for further study on the structure and function of NSP3 and NSP5 proteins.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.4

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相關(guān)期刊論文 前1條

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本文編號(hào)：2117595