猪传染性胃肠炎病毒核衣壳(N)蛋白基因的克隆与表达

以猪传染性胃肠炎病毒亚基因组mRNA为模板根据文献设计一对引物，通过RT－PCR技术，扩增其核衣壳（N）蛋白基因的cDNA；将其按正确的阅读框架定向克隆到表达载体pProEXHTb中特异酶切位点；将重组质粒PHN转化进大肠杆菌TG1株，在浓度为1.0mM IPTG和37℃条件下诱导，PHN基因融合蛋白获得了表达；经SDS－PAGE，Western-blot试验，确定其表达的融合蛋白产物大小为预期的47kD。试验结果证明，在大肠杆菌中表达的TGEV N基因的融合蛋白产物的确具有天然蛋白的抗原性。

Cloning and Expression of Nucleoprotein Gene of Transmissible Gastroenteritis Virus

The viral subgenome mRNA of porcine transmissible gastroenteristis virus(TGEV)was amplified by RT PCR using primer pairs Li01 and Li02 which designed according to the reported reference.A DNA fragment was amplified which contains completely ORF gene and the size is about 1174bp and then the DNA fragement was cloned site specially into pProEXHTb expression vector between the NspV and XhoI cleavage site.The recombinant plasmid(PHN)was verified by restriction endonuclease analysis and nucleotide sequencing.Then it was transformed into E.coli strain TG1 for N gene expression.The expression product was identified by SDS PAGE and Western blot test,a fusion protein about 47KD as we expected was found.The results shows that the vitro expressed protein of N gene via recombinant plasmid vector in the E.coli maintains antigenicity of TGEV.