【作者】 孙跃辉；

【导师】 黄金海；

【作者基本信息】 天津大学 ， 食品科学， 2010， 硕士

【摘要】 由山羊关节炎脑炎病毒(CAEV)引发的山羊关节炎脑炎(CAE)是世界养羊业中破坏力最强、经济学意义最重要的病毒性传染病。本研究对CAEV89-GB1026株进行了全基因组克隆和序列测定,分析了CAEV的分子特性;应用环介导等温扩增技术,建立了CAEV快速检测方法;同时通过构建衣壳蛋白P25的原核表达载体,获得高纯度的重组表达蛋白,初步建立了CAEV感染羊抗体监测的ELISA方法。采用PCR的方法,扩增出覆盖CAEV全基因组的5个cDNA片段,分别将其克隆到pGEM-T easy载体,经序列测定、拼接获得了CAEV89-GB1026株的全基因组序列。该毒株基因组全长9065nt,包括一段453nt的5’LTR、3个结构基因(gag、pol和env)、2个调节蛋白编码基因(rev和tat)、1个辅助蛋白编码基因(vif)和一段308nt的3’LTR。序列比较结果显示,该毒株与甘肃分离株相似性为99.6%,与意大利分离毒株的相似性仅为73.0%~73.5%。依据gag基因对国内外分离株同源关系分析表明,该毒株属于小反刍动物慢病毒B亚群B2亚型,Gag蛋白中存在多个与MVV相同的抗原表位。在p25基因的高保守区设计了5条引物,应用LAMP扩增技术建立了CAEV快速检测方法。采用LAMP实时浊度仪对扩增反应进行检测,可在36min内检测数个拷贝的质粒,灵敏度相当常规PCR的100倍。分别应用琼脂糖凝胶扩散实验(AGID)、常规PCR和LAMP对68份临床样品进行了检测,阳性率分别为38.2%, 41.2%和50%。该方法可用于细胞培养物、感病动物全血和外周血淋巴细胞中CAEV原病毒DNA的特异性检测。应用生物信息学方法对衣壳蛋白P25和P25融合蛋白的生化特性进行分析,设计了不同的原核表达载体。将p25基因克隆至原核表达载体pGEX-6P-1,在E.coli BL21细胞中可溶性表达,经GST亲和层析获得重组蛋白GST-P25。将p25基因克隆至原核表达载体pET28a,将重组质粒转化E.coli BL21(DE3)诱导表达,通过包涵体变性、复性的方法获得重组蛋白His-P25。应用免疫印迹两种重组蛋白的抗原性进行分析,重组蛋白可与CAEV阳性血清特异性地结合,表明重组蛋白具有良好的抗原性,并以His-P25为抗原,建立了检测CAEV抗体的间接ELISA。更多还原

【Abstract】 Caprine arthritis and encephalitis (CAE) caused by Caprine arthritis encephalitis virus (CAEV) is one of the most destructive and economically important viral disease of the goat industry worldwide. The disease is commonly associated with polyarthritis, interstitial pneumonia, mastitis, and progressive weight loss in adults, while the newborn and young kids often suffer from CAE-induced encephalitis, accompanied with a high mortality rate.In this study, the whole genome of CAEV89-GB1026 strain was cloned and sequenced, and the molecular characterization was determined. Additionally, a rapid detection assay based on loop-mediate isothermal amplification (LAMP) has been developed for detecting CAEV proviral DNA. At last, the p25 gene was cloned into expression vectors and expressed in E.coli, and the indirect ELISA was developed for detection of antibody in serum to CAEV.Five DNA fragments covering the whole genome of CAEV89-GB1026 strain were amplified from total DNA extracted from CAEV infected GSM cell by employing polymerases chain reaction, and were cloned into pGEM-Teasy vector respectively. Then the whole genome sequence was determined by sequencing and assembling these fragments. CAEV89-GB1026 strain has a genome size of 9065nt, with a 453nt of 5’LTR, followed by three structural gene (gag, pol and env), two regular gene (rev and tat) and an accessory gene (vif), and a 308nt 3’LTR. Sequence comparison showed the complete nucleotide sequence of CAEV89-GB1026 strain shared 99.6% identity with gansu strain (AY900630), but only 73.0%~73.5% identity with Italy isolates (EU293537,GQ381130). The homology tree was generated based on gag gene, showed that CAEV89-GB1026 strain belongs to SRLV subtype B2. The secondary structure and epitopes of structural proteins were analyzed, several function domains were identified, CAEV and MVV shared several epitopes in gag protein.A rapid detection method was developed by employing LAMP assay, a set of five primers was designed against highly conserved sequences located within the p25 gene region. The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and separated PBMCs, had no cross reaction with the negative control. Amplification was monitored with the Loopamp real-time turbidmeter, several copies of CAEV proviral DNA can be detected in 36 min, which is 100 fold more sensitive than PCR. 68 clinical samples were tested using AGID, PCR and LAMP assay and the positive rates were 38.2%, 41.2% and 50%, respectively.The biochemical characteristics of the capsid protein (P25) and presumptive P25 fusion proteins were analyzed by bioinformatics analysis method, and the recombinant P25 was obtained by constructing different expression vector. The P25 protein was expressed as a fusion protein with glutathione-S-transferase (GST) in E.coli BL21 by using prokaryotic expression vector pGEX-6P-1, the GST-P25 was obtained by a single step of affinity chromatography using Glutathinione Sepharose 4B. The pET28-p25 expression vector was constructed using pET28a vector, the recombinant protein (His-P25) with his-tag located in N and C-terminal was expressed in E coli BL21(DE3), and was purified through denaturizing and refold of inclusion bodies. The recombinant proteins were analyzed by Western blot, results showed that the recombinant proteins could specifically react with sera from CAEV infected goats, indicating that the recombinant proteins shared good immunogenicity. An indirect ELISA was developed for detection of antibody in serum to CAEV, using purified His-P25 as coating antigen.更多还原