【作者】 颜文卿；

【导师】 吴德峰； 颜江华；

【作者基本信息】 福建农林大学 ， 临床兽医学， 2006， 硕士

【摘要】 犬细小病毒病是由犬细小病毒（Canine parvovirus，CPV）感染引起的犬的一种急性传染病，以剧烈的呕吐、出血性肠炎和白细胞显著减少为主要特征，并可引起犬急性心肌炎。该病发病急，病程短，传染性强，死亡率高，是危害我国养犬业最为严重的传染病之一，可造成严重的经济损失。 根据GeneBank已发表的犬细小病毒VP₂基因序列，设计一对特异引物，采用SDS-蛋白酶K-酚氯仿抽提法，从犬细小病毒／犬瘟热二联苗中提取犬细小病毒的基因组DNA，并以此为模板进行PCR扩增，获得了犬细小病毒VP₂基因，将其克隆到质粒pET22b（+）的相应位点上。重组质粒经酶切鉴定和核苷酸序列分析，结果表明所插入的阅读框正确，序列无误，从而成功地构建了pET22b／VP₂原核表达载体。 将测序正确的重组质粒pET22b／VP₂分别转化E．coli BL21（DE3）和E．coli Rosetta^TM（DE3）两种不同宿主菌，IPTG诱导表达，SDS-PAGE电泳结果表明：重组质粒pET22b／VP₂在E．coli BL21（DE3）中未见明显的目的蛋白表达带；而在E．coli Rosetta^TM（DE3）中则获得了高效表达，在相应位置上（68kd处）可见明显的目的蛋白表达带；重组蛋白主要以包涵体的形式表达，且表达量占菌体总蛋白的30％以上；Ni²⁺亲和层柱纯化收集蛋白，纯化后的蛋白经复性处理后，能与犬细小病毒阳性血清发生特异反应。 本试验成功地克隆了犬细小病毒VP₂基因完整片段，并实现了其在原核表达系统中的高效表达，为犬细小病毒基因工程疫苗以及新型诊断试剂的研制奠定了基础。更多还原

【Abstract】 The disease of Canine parvovirus is a kind of acute infectious disease, which is caused by Canine parvovirus（CPV）. The characteristic clinical symptoms of CPV are vomiting、 bloody enteritis and serious lucopenia, which also cause acute cardiac muscle inflammation. The course of the disease is short, the mortality is high. The disease is one of the most infectious diseases in our country, which not only endanger the dogs but also make a lot of loss.A pair of primers was designed according to the sequence of VP₂ gene from Genebank, the genome DNA of CPV was extracted from CPV/CDV vaccine, then it was used as templates for polymerase chain reaction （PCR） to amplify the VP₂ gene. The VP₂ gene then was cloned into the restriction of plasmid pET22b（+）. The recombinant plasmid was identified by restriction enzymes analysis and nucleotide sequence analysis, the results showed that the VP₂ gene has been correctly cloned into plasmid pET22b（+）, the prokaryotic expression vector of pET22b/VP₂ was successfully constructed.The corrcet recombinant pET22b/VP₂ was transformed into Escherichia coli BL21（DE3）and Escherichia coli Rosetta^TM（DE3） with IPTG inducing. The expressed protein was measured by SDS-PAGE, the results showed that there was not obvious expression strip in Escherichia coli BL21 （DE3） ;While in Escherichia coli Rosetta^TM（DE3） , it can get high expression, the expressed protein was morn than 30% of total bacterial protein and existed in the form of inclusion bodies. Target proteins was purified by Ni²⁺ affinity chromatography. After renaturation, the protein can be recognized by the positive serum of CPV.The trail has successfully cloned the full segment of VP., gene, and made it got high expression in the prokaryotic expression system. All these work will laid foundation for the research and development of genetic engineering vaccine and new diagnose reagent.更多还原