猪传染性胃肠炎病毒SC-Y株N基因的克隆及原核表达

Cloning and Prokaryotic Expression of Nucleocapsid Protein Gene of Transmissible Gastroenteritis Virus SC-Y Strain

通过PCR方法扩增猪传染性胃肠炎病毒SC-Y株核衣壳蛋白(N)基因,然后将重组到pMD18-T载体中的约1174 bp的基因片段亚克隆到PET-32a(+)表达载体上,通过酶切及PCR鉴定阳性的重组质粒命名为PET-N,核苷酸及推导的氨基酸序列分析表明,该基因与其他猪传染性胃肠炎病毒相应基因具有很高的同源性,将阳性重组质粒转化BL21(DE3),经IPTG诱导可表达分子量约66 kD的融合蛋白,N基因的表达可为传染性胃肠炎病毒的诊断提供良好的物质材料。

The nucleocapsid protein gene of transmissible gastroenteritis virus SC- Y strain was amplified by reverse transcription-polymerase chain reaction （RT- PCR）. The recombinated fragment in pMD18 - T was subcloned into the prokaryotic expression vector PET- 32a（ ＋ ）. The recombinant expression vector that contained the target fragment was identified by using restriction endonudeases and named as PET- N. The nucleotide and deduced amino acid sequences of the cloned N gene from ransmissible gastroenteritis virus SC- Y strain were analyzed. The results show that the gene has a relatively high homology with the corresponding gene from other transmissible gastroenteritis virus strains. The recombinant plasmid is transformed into BL21 and the recombinant bacteria is induced by IPTG. SDS- PAGE are performed to detect the N fusion protein. Result indicates the molecular weight of N fusion protein is 66 kD. The expression of N protein is to provide a basis for the research on diagnosis of TGEV. biological functions. The constucton of recombinant N gene.