猪轮状病毒NSP3和NSP5蛋白单抗的制备及抗原表位鉴定

发布时间：2018-07-12 15:22

本文选题：猪轮状病毒 + NSP3　；参考：《东北农业大学》2015年硕士论文

【摘要】：轮状病毒(Rotavirus,RV)属于呼肠病毒科(Reoviridae)轮状病毒属(Rotavirus)成员,是重要的病原体,可以引起多种宿主生物的病毒性腹泻,如哺乳动物和鸟类。猪轮状病毒(Porcine Rotavirus,Po RV)是引起仔猪腹泻、呕吐、脱水等症状的主要病原,致死率高,为猪的主要传染病之一,使我国的养猪业遭受了沉重的打击。RV为双链的RNA病毒,RV基因组是由11段每段均含有一个开放阅读框、不连续的双股RNA所构成,并编码6种结构蛋白以及6种非结构蛋白。NSP3是RV的非结构蛋白,是一种非糖基化蛋白,该蛋白与病毒粒子感染细胞有关,能够关闭宿主细胞mRNA的翻译。非结构蛋白NSP5,主要功能是糖基化和磷酸化的修饰作用,多在RV翻译后期进行参与。其C端富含保守的丝氨酸和苏氨酸多聚化结构域,参与翻译后期的磷酸化和糖基化修饰,NSP5的存在可使病毒的复制转录等过程顺利进行,在病毒的复制周期中起着不可或缺的作用。本研究旨在获得抗Po RV(G9)血清型NMTL毒株纯度高特异性强的单克隆抗体(MAb),加深对NSP3、NSP5蛋白结构和功能的了解,并对它们的免疫学特性进行了更深一步的研究。本研究采用RT-PCR方法扩增了G9血清型NSP3和NSP5基因,经酶切后分别连接至p GEX-6P-1载体中构建重组表达质粒p GEX-NSP3和p GEX-NSP5。酶切鉴定后进行测序,获得测序正确的阳性质粒。将其分别转化至E.coli DH5α感受态细胞,经IPTG进行NSP3重组蛋白(r NSP3)和NSP5重组蛋白(r NSP5)的诱导表达,并设置空载体为对照。将r NSP3和r NSP5经SDS-PAGE分析后进行纯化,结果显示:r NSP3和r NSP5大小分别为60Ku和50Ku。将rNSP3和r NSP5分别与抗RV的阳性血清进行western blot试验,均发生反应,结果表明其具有良好的免疫源性。将含有r NSP3和r NSP5条带的SDS-PAGE胶捣碎后,采用皮下注射的方法免疫BALB/c小鼠,免疫3次后断尾取血,间接ELISA检测抗体效价后,进行加强免疫于融合的前3d。取加强免疫后小鼠的脾细胞与生长状态较好的骨髓瘤细胞(SP2/0)进行融合,利用间接ELISA方法进行筛选,得到1株能稳定分泌抗Po RV(G9)NSP3的杂交瘤细胞株5B8和1株能稳定分泌抗NSP5的杂交瘤细胞株5E11,均为Ig G1亚类。Western blot及间接免疫荧光(IFA)结果表明,所获得的MAbs特异性良好。为确定获得的MAb所针对的抗原表位的区域序列,采用Western blot试验分别对截短表达后的NSP3和NSP5蛋白进行一系列的鉴定。结果表明,5B8识别的抗原表位序列为290QDYDRTFL 297,5E11识别的抗原表位序列为61 GPSDSA 66。本研究分别成功制备了一株抗NSP3蛋白和一株抗NSP5蛋白的MAb,并对它们的线性抗原表位进行了鉴定。为RV的抗病毒免疫研究、抗体检测等均提供了信息及材料,同时也为进一步对NSP3和NSP5蛋白的结构和功能的研究奠定了良好的基础。
[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 伪 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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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