【作者】 赵焕云；

【导师】 高云英；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2008， 硕士

【摘要】 根据已知狂犬病毒株核蛋白基因(N)和糖蛋白基因(G)序列设计合成了4对检测引物和2对克隆引物,建立了狂犬病RT-PCR及套式PCR诊断技术;自英国引进3株荧光素标记的针对狂犬病毒核蛋白的单克隆抗体,建立了狂犬病直接免疫荧光诊断技术。使用建立的诊断方法对云南省部分地区的56份犬脑组织样品(小脑、脑干、海马角)进行检测,结果检出阳性样品共计12份,阳性率21.4%。病犬脑组织样品RT-PCR和套式PCR检测结果与直接免疫荧光试验结果一致。同时对2006-2007年间云南曲靖(YNQJ07)、昭通(YNZT07)、楚雄(YNMD06)、保山(YNTC06)狂犬病毒株N基因和G基因进行RT-PCR扩增,克隆至pMD18-T载体进行测序,并与已知代表性毒株进行比对及系统发育分析。结果发现:云南狂犬病毒株均属于基因Ⅰ型毒株,YNQJ07、YNZT07、YNMD06以及近年部分广西、浙江、江苏、湖北、安徽、江西省毒株N基因和G基因核酸序列同源性分别介于97.0～99.3%、97.4～99.3%,与其它基因Ⅰ型毒株同源性分别介于86.3～90.2%、82.9～93.0%。YNTC06与泰国和部分广西毒株同源性介于95.0～95.8%,与其它基因Ⅰ型毒株同源性介于82.9～92.9%。研究表明YNQJ07、YNZT07、YNMD06属于亚组群Ⅱ毒株,YNTC06毒株属于亚组群Ⅲ毒株,云南狂犬病毒核蛋白和糖蛋白存在特异性氨基酸变异位点。用EcoRⅠ和XbaⅠ(N基因)或KpnⅠ(G基因)双酶切目的基因PCR产物,纯化回收后定向克隆至pPICZαA载体中,构建了酵母表达质粒pPICZαAN和pPICZαAG。更多还原

【Abstract】 The direct immunofluorescent assay (DFA) was developed by importing 3 fluorescent labelled monoclonal antibodies raised against nucleoprotein of rabies virus from British. 4 pairs of detect primer and 2 clone primer were designed and synthesized based on Nucleoprotein (N or NP)gene and Glycoprotein gene (G) of known rabies viruses, the reverse transcriptase - polymerase chain reaction (RT-PCR) assay and the nested polymerase chain reaction (nested PCR) assay were developed. 56 dog brains of Yunnan province was detected by the diagnostic method, 12 was positive samples, positive rate is 21.4%. The results of the RT-PCR and nested PCR is same as the DFA. Meanwhile, the N and G genes of rabies strains from Qujing (YNQJ07), Zhaotong (YNZT07), Chuxiong (YNMD06) and Baoshan (YNTC06) of Yunnan province during 2006 to 2007, were amplified by reverse transcription– polymerase chain reaction (RT-PCR) and cloned into pMD18-T vectors for sequencing respectively. The sequence data were done alignment and phylogenetic analysis with known representative strains. The results showed Yunnan strains of rabies virus all belonged to genotypeⅠ. The nucleotide homologies of N and G genes between YNQJ07, YNZT07, YNMD06 and recent strains isolated from Guangxi, Zhejiang, Jiangsu, Hubei, Anhui, and Jiangxi provinces etc. were 97.0～99.3% and 97.4～9.3% respectively. The homologies between YNQJ07, YNZT07, YNMD06 and other genotypeⅠs trains were 86.3～90.2%、82.9～93.0% respectively. The homologies among YNTC06, Thailand and some Guangxi strains were 95.0～95.8% and the homologies between YNTC06 and other genotypeⅠstrains were 82.9～92.9%. This result of the nucleotide sequence analysis indicated that YNQJ07, YNZT07, YNMD06 belonged to subgroupⅡstrains and YNTC06 belonged to subgroupⅢstrain. There were some specific mutations of amino acid on nucleoprotein and glycoprotein of Yunnan strains.The purpose gene was enzyme digestion by the EcoRⅠa nd XbaⅠ(N gene)or KpnⅠ(G gene),after purification and recovery oriented cloned into pPICZαA vectors, successful constructed the donator plasmids of Yeast Expression pPICZαAN and pPICZαAG.更多还原