猪传染性胃肠炎病毒S基因片段的表达及鉴定

发布时间：2018-04-22 05:04

本文选题：猪传染性胃肠炎病毒 + 杆状病毒载体表达系统　；参考：《南阳师范学院》2016年硕士论文

【摘要】：猪传染性胃肠炎(Porcine transmissible gastroenteritis, TGE)是由猪传染性胃肠炎病毒(Porcine transmissible gastroenteritis virus, TGEV)引起的猪的一种以腹泻、呕吐、脱水等为主要症状的高度接触性消化道传染病,对两周龄以内的仔猪致死率可达100%,给全球养猪业造成了巨大危害。在TGEV基因组中,ORF2编码的S蛋白携带有主要的B细胞抗原识别决定簇,经研究证实S蛋白N端的543个氨基酸残基内含有A、B、C、D四个抗原位点,是唯一能诱导机体产生中和抗体的主要结构蛋白。因此,S蛋白的表达对TGEV基因工程疫苗和疾病诊断试剂的研制具有重要意义。本研究根据已发表的TGEVS基因cDNA序列,设计并合成了3对特异性引物,通过PCR扩增得到4个含有不同抗原位点的S基因片段：含D位点的S2基因,含A位点的S3基因,含A和D位点的S(2+3)基因以及含有A、B、C和D位点的Sfull基因。将S基因分别克隆到pMD19-Tsimple载体并进行测序鉴定。用DNA　star软件将测序结果中不同S基因与Genebank中收录的TGEV　S基因参考序列相比较,其核苷酸同源性为100%。将测序验证正确的S基因的不同片段分别克隆到杆状病毒转移载体pFBDMY-IM中,随后将获得的阳性重组转移载体通过Tn7转座重组至杆状病毒Bacmid中,然后将重组Bacmid转染Sf9昆虫细胞获得重组病毒。将重组病毒感染Sf9昆虫细胞,72小时后用荧光显微镜进行观察,发现Sf9细胞出现明显的细胞病理变化和红色荧光,说明重组杆状病毒已经成功构建。提取重组杆状病毒基因组DNA并用TGEV S基因的特异性引物进行PCR鉴定,结果证实TGEV S基因已经成功重组至杆状病毒基因组中。用SDS-PAGE和Western Blot分析蛋白表达产物,结果表明：S2、S3、Sfull基因在杆状病毒表达系统中成功得到表达,表达的重组蛋白大小分别为25.9 KDa、27.5KDa和85.1KDa；S(2+3)基因在杆状病毒表达系统中没有表达。因此,本研究将含不同抗原位点的S基因片段在杆状病毒表达系统中进行表达,为以后利用S蛋白开发TGEV基因工程疫苗和疾病诊断试剂奠定了基础。
[Abstract]:Porcine transmissible enteritisis (TGEV) is a highly contagious infectious disease of the digestive tract caused by porcine transmissible gastroenteritis virus (TGEV), which is characterized by diarrhea, vomiting, dehydration, etc. The fatality rate of piglets within two weeks of age can reach 100, which has caused great harm to the global pig industry. In the TGEV genome, the S protein encoded by ORF2 carries the main B cell antigen recognition determinant. The 543 amino acid residues at the N-terminal of S protein contain four antigenic sites of Agna Bu CfU D. It is the only major structural protein that can induce the production of neutralizing antibodies. Therefore, the expression of S protein plays an important role in the development of TGEV gene engineering vaccine and disease diagnosis reagent. According to the published cDNA sequence of TGEVS gene, three pairs of specific primers were designed and synthesized. Four S gene fragments containing different antigenic sites were amplified by PCR: S2 gene containing D locus and S3 gene containing A locus. The Szc23) gene with A and D loci and the Sfull gene with Agna C and D loci. S gene was cloned into pMD19-Tsimple vector and sequenced. DNA star software was used to compare the different S genes in the sequencing results with the reference sequences of TGEV S gene included in Genebank. The nucleotide homology was 100%. The different fragments of the correct S gene were cloned into the baculovirus transfer vector pFBDMY-IM, and the obtained positive recombinant transfer vector was recombined into the baculovirus Bacmid by Tn7 transposition. Then the recombinant Bacmid was transfected into Sf9 insect cells to obtain the recombinant virus. After 72 hours of infection with recombinant virus, Sf9 insect cells were observed by fluorescence microscope. It was found that there were obvious cellular pathological changes and red fluorescence in Sf9 cells, which indicated that the recombinant baculovirus had been successfully constructed. The genomic DNA of recombinant baculovirus was extracted and identified by PCR with specific primers of TGEV S gene. The results showed that the TGEV S gene had been successfully recombined into the genome of baculovirus. SDS-PAGE and Western Blot were used to analyze the protein expression products. The results showed that the cell S2S3Sfull gene was successfully expressed in the baculovirus expression system, and the expressed recombinant protein was 25.9 KDa-27.5KDa and 85.1 KDA-Sf23) gene, which was not expressed in the baculovirus expression system. Therefore, the S gene fragments with different antigenic sites were expressed in baculovirus expression system, which laid a foundation for the development of TGEV gene engineering vaccine and disease diagnosis reagent by using S protein.
【学位授予单位】：南阳师范学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.651

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