【作者】 胡森；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 猪传染性胃肠炎病毒（Transmissible gastroenteritis virus,TGEV）是冠状病毒科、冠状病毒属成员，引起猪的传染性胃肠炎（Transmissible gastroenteritis TGE），死亡率很高，呈世界范围分布，严重危害养猪业的发展。TGEV与猪流行性腹泻病毒（PEDV）、猪呼吸道冠状病毒（PRCV）在临床症状上很相似，基因组结构上与TGEV具有较高的同源性，传统的方法很难鉴别。 为了鉴别诊断TGEV与PRCV及对TGEV进行流行病学调查，本研究采用原核表达系统（GST融合表达系统）表达TGEV纤突蛋白（S蛋白）中含有B和C抗原位点的多肽，并且制备了非放射性地高辛标记的核酸探针，通过斑点杂交（Dot-Blot）检测TGEV核酸RNA。 TGEV S蛋白可诱导产生中和抗体，是主要的保护性抗原，其N端抗原位点B和C在PRCV中缺失。根据GenBank TH-98株序列设计第1对引物，应用RT-PCR方法体外扩增TGEV TH-98株S基因5’端含有B、C抗原位点的片段（ts），大小为357bp，并将ts连接到pMD18-T载体上。序列分析结果为，该片段的核苷酸序列与TGEV其它分离株FS772／70、Miller、Purdue15、Pur46-Mad、133的同源性分别为96.92％、97.76％、98.88％、99.72％、99.44％；氨基酸同源性分别为94.92％、95.76％、97.46％、99.07％、98.31％，表明该片段在TGEV不同分离株中较为保守。将ts片段亚克隆到原核表达载体pGEX-6P-1中，将鉴定为阳性的重组质粒转化到感受态细胞BL21（DE₃）pLysS中。挑选含重组质粒pGEX-6P-ts的菌落，经IPTG（终浓度为0.2mmol／L）诱导后，SDS-PAGE分析目的蛋白与谷胱苷肽硫转移酶（GST大小约为26ku）融合后大小约为40ku，目的蛋白分子量约为13.2ku，与理论值相符。优化表达条件后表达量达37.9％。 TGEV S基因5’端285bp片段在TGEV分离株中较为保守，与其它冠状病毒同源性较低。根据GenBank TH-98株序列设计第2对引物，应用RT-PCR方法体外扩增该片段（命名为tp），琼脂糖凝胶纯化后测定其浓度和纯度，进行非放射性地高辛标记。对标记效率测定后，以带正电的尼龙膜为介质进行特异性和灵敏性检测，经非放射性自显影来显示靶核酸。本研究中探针tp检测到TGEV cDNA最低量为120pg。 本研究高效表达了TGEV含有B和C抗原位点的多肽片段（TS）。制备了TGEV非放射性标记的核酸探针，并初步应用于检测TGEV。二者为TGEV鉴别诊断、血清学调查奠定了基础。更多还原

【Abstract】 Transmissible gastroenteritis ( TGE ) was an enteric disease of pigs caused by transmissible gastroenteritis virus (TGEV), a member of coronaviridae family. TGEV was characterized with high mortality and had spread all over the world. The genome structure of TGEV, PEDV and PRCV had high homology and it was difficult to diagnose TGEV by traditional means.Two studies were carried out to differentiate the TGEV from PRCV and investigate the epidemiology of TGE. One was cloning, sequence analysis and expression of the fragment containing the B and C antigenic sites locating at the 5’ terminus in spike gene of TGEV in prokaryotic expression system (fused with GST), the other was preparation of non-radioactive probe labeled by digoxigenin for detecting the RNA extracted from TGEV by assay of Dot-Blot.Spike protein was the main protective antigen inducing the neutralization antibody. The fragment containing antigenic sites B and C locating at the 5’ terminus in spike gene of TGEV was deleted in PRCV. The 1st pair of primer was designed according to strain TH-98.The fragment (named ts) was amplified by RT-PCR and inserted into pMD18-T vector. Sequence analysis showed that homology of nucleotide and amino acid between ts and other TGEV strains Fs772/70, Miller, Purdue15, Pur46-Mad, 133 were 96.92%, 97.76%, 98.88%, 99.72%, 99.44% and 94.92%, 95.76%, 97.46%, 99.07%, 98.31% respectively. Fragment ts was subcloned into expression plasmid pGEX-6P-1, then was transformed into competent cell of BL21 (DE3) pLysS. After screening the positive colony containing recombinant plasmid pGEX-6P-ts, the E.coli was induced by 0.2mmol/L IPTG and was analyzed by SDS-PAGE. The overexpressed protein (TS) was 13.2 ku and showed 40ku in gel fused with GST (26ku) . The amount of TS expressed was 37.9% under optimized condition.The 285 bp fragment (named tp) , locating at 5’terminus of spike gene, had high conservation in TGEV isolations. The 2nd pair of primers was designed according to the sequence of strain TH-98 collected in GenBank. Concentration of tp was analyzed after amplification invitro by RT-PCR, purified by low melt agarose and labeled by digoxigenin. After the estimation of labeling efficiency tp was applicated to hybridize the RNA of TGEV, then was subjected to self-development of X-ray exposed by CSPD to show the target nucleic acid. The minimal amount of TGEV cDNA can be detected was about 120 pg in this research.In the research TS containing antigenic sites B and C was expressed with high efficiency. Nonradioactive nucleic probe to TGEV was prepared and was applicated to detect TGEV atfirst stage, which established foundational work for differentiating TGEV from PRCV and investigation on epidemiology of TGEV.Candidate: Hu Sen Major: Preventive Veterinary ScienceSupervisor: Prof. Wang Junwei更多还原