犬冠状病毒TN449株M基因的克隆与转移载体的构建

用蔗糖密度梯度离心纯化犬冠状病毒TN449株病毒液,提取总RNA并反转录,同时设计1对5′端加有BamHⅠ和HindⅢ内切酶位点的引物,对病毒cDNA进行了PCR扩增,回收PCR产物将其连接入pGEM-TEasy载体并转化大肠埃希氏菌。并对阳性重组质粒菌测序和序列分析。结果,犬冠状病毒与牛冠状病毒、猫冠状病毒、猫传染性腹膜炎病毒、人冠状病毒、鸡传染性支气管炎病毒、鼠传染性肝炎病毒、猪呼吸道冠状病毒、人重症急性呼吸道综合征病毒和猪胃肠炎病毒的同源性分别是39.39%、85.41%、83.98%、36.80%、26.64%、37.23%、93.51%、29.00%和94.81%;犬冠状病毒M蛋白有3个疏水性结构域。同时构建了pET28a-CCV-M表达质粒。

Cloning and construction of transfer vector of Mgene of canine coronavirus TN449 strain

Canine coronavirus(CCV) TN449 virus liquid was collected and purified by sucrose density gradient centrifugation. The total RNA was extracted and reversely transferred. The virus membrane protein gene was amplified from virus cDNA by a pair of primers added with BamH and Hind enzyme site in 5 end. The PCR product was purified, linked with pGEM-T Easy vector and transfected into (E.coli.) (Analysis) of amino acid sequence deduced from nucleotide acid sequence indicated homogeneity of amino acid sequence of CCV membrane protein with that of bovine coronavirus, feline coronavirus, feline infectious peritonitis virus, human respiratory coronavirus, avian infectious bronchitis virus, mouse hepatitis virus, porcine respiratory coronavirus, serious actute symptom respiratory virus and porcine transmissible gas- (troenteritis) virus was 39.39%,85.41%,83.98%,36.80%,26.64%,37.23%,93.51%,29.00% and (94.81%) respectively. Analysis of amino acid sequence of CCV membrane protein by bioinformation indicated CCV membrane protein owned three hydrophobe structure domains which were presumed as its transmembrane structure. The recombined pET28a-CCV-M was constructed, which laid foundation to study the function of CCV membrane protein gene.