小反刍兽疫病毒甘肃株F基因的克

发布时间：2018-03-07 11:36

本文选题：小反刍兽疫病毒(PPRV)　切入点：F基因　出处：《农业生物技术学报》2017年03期 　论文类型：期刊论文

【摘要】：小反刍兽疫(peste des petits ruminants,PPR)是由副黏病毒科(Paramyxoviridae)麻疹病毒属(Morbillivirus)的小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起绵羊(Ovis aries)和山羊(Capra hircus)等小反刍兽的一种急性和高度传染性病毒性疾病,由于其高死亡率常造成绵羊和山羊养殖业的重大经济损失。为研究小反刍兽疫病毒甘肃(GS)株融合蛋白(fusion protein)基因序列特征及其免疫原性,参照Gen Bank中PPRV Nigeria 75/1株F基因序列(Gen Bank登录号:HQ197753)设计引物,应用RT-PCR扩增PPRV GS株F基因,在测序及序列分析的基础上,设计引物扩增去信号肽和跨膜区的F基因片段Fa并将其亚克隆至原核表达载体p ET-32a(+)。重组质粒p ET-PPRV-Fa转化大肠杆菌表达菌(Escherichia coli)Transetta(DE3)后经异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达,表达产物纯化后免疫新西兰兔(Oryctolagus cuniculus)制备多克隆抗体,进而应用间接酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)和Western blot对重组蛋白免疫原性进行分析。结果表明,PPRV GS株F基因(Gen Bank登录号:KX822738)完整CDS全长1 641 bp,编码546个氨基酸。去信号肽和跨膜区的F基因片段Fa在大肠杆菌中成功获得表达,重组蛋白大小约为59 k D,且主要以包涵体形式存在;间接ELISA和Western blot结果表明,纯化产物免疫原性良好。研究结果为F蛋白的相关功能研究及小反刍兽疫的快速诊断方法的建立提供了理论依据。
[Abstract]:Peste des petits ruminants (PPRP) is an acute and highly infectious virus of small ruminants, such as Ovis ariesus of sheep and Capra hircus of goats, which is caused by the measles virus of the genus Morbillivirus (Paramyxoviridaeae). In order to study the sequence characteristics and immunogenicity of fusion protein protein fusion gene of small ruminant zoonotic disease virus Gansu GSH strain, the high mortality rate often causes great economic losses in sheep and goat breeding. According to the F gene sequence of PPRV Nigeria 75/1 strain in Gen Bank, the F gene of PPRV GS strain was amplified by RT-PCR, and the F gene of PPRV GS strain was amplified by RT-PCR. On the basis of sequencing and sequence analysis, the F gene of PPRV GS strain was amplified by RT-PCR. Primers were designed to amplify the signal free peptide and the transmembrane F gene fragment Fa and subcloned it into prokaryotic expression vector pET-32a. the recombinant plasmid p ET-PPRV-Fa was transformed into Escherichia coli coli TransettaDE3 and was induced to express by isopropyl 尾 -D-thiogalactopyranoside IPTGG. The purified product was immunized with Oryctolagus cuniculus to prepare polyclonal antibody. Then the immunogenicity of the recombinant protein was analyzed by indirect enzyme-linked immunosorbent assaysa (Elisa) and Western blot. The results showed that the complete CDS was 1 641 BP, encoding 546 amino acids, and the F gene gene-gene-gene-gene-gene-Gen-KX822738 of PPRV GS strain was cloned from Genin Bank accession number: KX822738. The total length of CDS was 1 641 BP, encoding 546 amino acids. And the F gene fragment Fa of the transmembrane region was successfully expressed in Escherichia coli. The result of indirect ELISA and Western blot showed that the recombinant protein was about 59kD and existed mainly in the form of inclusion body. The immunogenicity of the purified product was good. The results provided a theoretical basis for the study of the related function of F protein and the establishment of a rapid diagnostic method for the small ruminant disease.
【作者单位】：甘肃农业大学动物医学院;中国农业科学院兰州兽医研究所;
【基金】：国家自然科学基金(No.31360620) 甘肃省高等学校基本科研业务费项目
【分类号】：S852.65

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