鸡传染性支气管炎病毒分离株核衣壳基因克隆、序列分析和真核表达

根据GenBank公开序列自行设计一对引物，采用RT—PCR扩增出鸡传染性支气管炎病毒（Infectious bronchitis virus，IBV）W和C9001分离株完整的核衣壳（N）基因，并将其克隆至pMD18-T载体进行核苷酸序列测定和分析。结果表明，扩增的2个IBV分离株核衣壳基因片段长度均为1230bp，编码409个氨基酸，彼此间核苷酸和氨基酸同源性分别为88．0％和89．5％，与GenBank中有代表性的参考毒株相应基因核苷酸和氨基酸序列比较显示，w株核苷酸序列与GenBank中的广东分离株（AY646283）同源性最高，为94．1％，氨基酸序列同源性为94．6％；与国内部分毒株核苷酸序列同源性在86．1％～88．0％之间，氨基酸序列同源性在88．0％～90．7％之间；C9001株与国内部分毒株核苷酸序列同源性在86．4％～99．8％之间，氨基酸序列同源性在88．0％～99．8％之间。从核衣壳基因编码的氨基酸序列的系统进化树可见，W株与C9001株处于不同的进化分枝，亲缘关系较远。同时将核衣壳基因构建于真核表达质粒pVAX1中，用脂质体法将重组质粒转染入COS-7细胞中，间接免疫荧光检测出核衣壳蛋白的体外表达。研究结果为进一步研究IBV核衣壳蛋白的结构与功能以及基因工程疫苗的研制奠定了基础。

Eukaryotic expression of nucleocapsid gene of infectious bronchitis virus isolates

The complete nucleocapsid genes of infectious bronchitis virus(IBV) strain W and C9001 were amplified by RT-PCR and cloned into the pMD18-T vector.Positive clones were identified by the restriction endonuclease analysis and PCR.The gene were sequenced and compared with those of other IBV strains from China in GenBank.The results showed that the nucleocapsid genes of strain W and C9001 shared 88.0 % nucleotide identity and 89.5 % amino acid homology.The strain W and strain Guangdong shared 94.1 % nucleotide homology and 94.6 % amino acids.Strain C9001 shared 86.4 %-99.8 % nucleotide homology and 88.0 %-99.8 % amino acid homology with other strains.The nucleocapsid genes from the pMD18-T vector were cloned into eukaryotic expressing vector pVAX1.The recombinant plasmids were transfected into COS-7 cells with liposome.Expression of nucleocapsid proteins was successfully detected by indirect Immunofluorescence assay.The results provided foundation for further studies on structure and function of nucleocapsid gene and development of the IBV genetic engineered vaccine.