【作者】 朱伟；

【导师】 刘棋；

【作者基本信息】 广西大学 ， 预防兽医学， 2007， 硕士

【摘要】 本课题首先建立了羊痘与羊口疮二重PCR诊断方法，试验表明该方法特异性强、敏感性高，可检测到4pg的DNA模板，能很好区分症状相似的山羊痘和羊口疮，本试验检测了7份疑似山羊痘病料，检出山羊痘阳性6份，羊口疮阳性1份。对广西山羊痘6份阳性病料和国内现用的山羊痘疫苗株的p32蛋白的全基因进行了PCR扩增、克隆和测序，并将序列上传至Genbank中(序列号分别是EF522176、EF522177、EF522178、EF522179、EF522180、EF522181)，序列分析结果表明，广西分离株之间的核苷酸同源性99.4-100％，推导的氨基酸同源性为98.1-99.7％，山羊痘广西分离株与哈萨克斯坦AY077835、NC004003以及印度的AY382369、AY159333和中国哈尔滨的AY881707分离株的核苷酸同源性为99.6—99.9％，推导的氨基酸同源性为98.8—99.7％；与国内使用的疫苗株的核苷酸同源性为99.3-99.8％，推导的氨基酸同源性为97.8-99.4％；广西分离株、哈萨克斯坦的AY077835、NC004003，印度的AY382369、AY159333和中国哈尔滨的AY881707与现用的疫苗株相比，在34—35位都缺失了2个氨基酸；广西分离株核苷酸651位的碱基全部由T变为C，647-652位核苷酸构成由TGTATA变异为TGTACA，多了1个限制性内切酶Bsp1407Ⅰ位点，而疫苗株和国内及国外的AY077835、NC004003、AY382369、AY159333、AY881707在647-652位核苷酸都没有该限制性内切酶酶切位点，因此用限制性内切酶Bsp1407Ⅰ可以区分疫苗株与广西分离株，本文建立了PCR-RFLP方法，疫苗株可以切成135bp和604bp两个片段，广西分离株可以切成135bp、226bp和378bp三个片段。为了进一步研究P32基因，本研究利用重组PCR技术消除P32基因跨膜区，用DNAstar软件对跨膜区缺失前后的蛋白图谱分析可知，跨膜区缺失前后的蛋白跨膜区位置的折叠构象、螺旋发生了一定变化，但抗原性在缺失前后未见变化，因此构建了一个缺失片段的重组质粒pGEX-S6，把该重组质粒转化到大肠杆菌BL21(DE3)，用IPTG诱导进行原核表达，经SDS-PAGE检测表达产物，结果显示检测到P32基因的表达产物，用BandScan软件对表达产物分析，优化反应条件，最佳诱导时间为5h，IPTG最佳诱导浓度是0.8mmol／l，最佳诱导温度是37℃，优化后表达的蛋白占菌体蛋白的29.4％，表达产物以融合蛋白的形式存在，融合蛋白出现在沉淀中，分子量为58KDa，Western-blot分析结果显示，表达产物可被山羊痘标准阳性血清识别。更多还原

【Abstract】 The multiplex PCR meothod were developmented for differentiation goatpoxvirus from orf virus. The sensitivity of the PCR assay was evaluated using 10-fold dilutions of a culture .The detection limit of the method was 4pg DNA. 7 field samples were detected by this multiplex PCR, and 6 were goatpoxvirus, 1 was orf virus.The P32 protein genes of 6 strains from Guangxi were amplified by PCR and sequenced, the Genbank number for our nucleotide sequences are EF522176, EF522177, EF522178, EF522179, EF522180, and EF522181,respectively.The results of nucleotide sequences analysis shows,the homologies of nucleotide and animo acid sequence among the strains from Guangxi are 99.5-99.8%,and 98.5-99.4% ,respectively.The homologies of nucleotide and animo acid sequence between the Chinese vaccine strain and strians from Guangxi are 96.6-99.9%,and 99.1-100%. And the homologies between reference strians published in genbank and the strain from Guangxi are 99.4-99.6%, and 98.1-98.8%.It shows deletion of 2 amino acid at the position 34 to 35 in those stains isolated from Guanggxi. And substitution T 651 C, and a additional restriction sites of Bsp1407 I are found in strians from Guangxi, while it doesn’t exist in the other reference strains and vaccine strain. Bsp1407 I can digests the PCR products of P32 gene of strains from Guangxi into 3 fragments of 135bp, 226bp and 378bp, however, vaccine strain was digested into 2 fragments of 135bp and 604bp.Furthermore, the protein P32 was expressed in Eco.li using pGEX-6p-lvecor. For P32 protein has transmembrane domain, the expression level of complete P32 protein using the common method is rather low. So a recombine PCR was used to delete the transmembrane domain. Then, P32 protein with the transmembrane domain deletetion or not were analysised by the software DNAstar. Results indicated that the Alpha amphipathic regions, the Beta amphipathic regions, flexible regions were changed, but the antigenicity wasn’t change. The DNA of P32 gene without transmembrane domain was inserted into pGEX-6p-l vector to construct, then recombinant vector pGEX-S6 was transformed into BL21(DE3), and induced by IPTG in different time, concentration and temperature, detected using SDS-PAGE and Western blot, and analysised using BandScan software. The results indicated that protein almost expressed in body of fusion protein with GST protein, molecular weight of expression product was 58KDa, and expression product could be identified by stand serum of goatpox.更多还原