牛冠状病毒N基因的原核表达及初步应用

Prokaryotic expression of the nucleocapsid protein gene in bovine coronavirus and its preliminary application

目的获得牛冠状病毒N基因的全长序列,实现N基因在大肠杆菌中的高效表达,并进行初步应用。方法根据已发表的牛冠状病毒Mebus株的序列设计引物,对BCV-DQ株进行RT-PCR扩增、克隆和测序;将该基因插入到原核表达载体pET-30a(+)上进行原核表达,SDS-PAGE和Western blot检测;用纯化的重组N蛋白作为包被抗原,建立ELISA检测方法,对部分临床样本进行检测。结果BCV-DQ株N基因全长1347bp,与GenBank已发表的6株BCV的N基因相比较,核苷酸同源性98.3%以上。构建的重组质粒pET-N能表达出一条大小约为60ku的蛋白,且能与鼠抗BCV血清发生特异性反应。用建立的ELISA方法对黑龙江不同地区送检的共256份牛血清样品进行检测,结果阳性率65.23%,与病毒中和试验的符合率达95.31%。结论BCV N基因保守性很高,并在原核表达系统中获得了高效表达,其表达产物具有良好的免疫反应原性。临床检测结果表明N蛋白可作为诊断抗原用于牛冠状病毒病的流行病学调查。

To obtain and analyze the sequence of the nucleocapsid gene from bovine coronavirus,and to produce the fusion protein of the N gene in E.coli in order to use this recombinant protein for the study of bovine coronavirus.The N gene of BCV-DQ strain was amplified by RT-PCR,in which the primers were designed on the basis of N gene sequence of BCV-Mebus strain.The PCR products of 1 347 bp in length were cloned and sequenced,and then inserted into the prokaryotic vector pET30a.The recombinant plasmids were then transformed into Escherichia coli BL21 and identified by SDS-PAGE and Western blot assay.ELISA assay was optimized of N protein as the coating antigen to detect the viruses in the clinical samples.In comparison with 6 BCV strains in GenBank,the sequence identity was proved to be more than 98.3%.Result in SDS-PAGE showed that the fusion protein had a molecular weight of 60 ku,and could be specifically recognized by mouse serum against BCV.The indirect ELISA was used to test 256 serum samples collected from Heilongjiang province and 65.23% samples were positive.On testing field samples,an overall agreement of 95.31% was generated between the the neutralization test of viruses（VN）and indirect ELISA.It is apparent that the N gene was highly conservative and is expressed in E.coli in high level,also the prokaryotic expression products of this gene show a fine reactiongenicity in immune responses.It was also suggested that the N protein may be a useful antigen for sero-diagnosis and epidemiological investigation of BCV.