CAJ | 학술논문

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1 347bp,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60 ku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.
목적 획득우관상병독N기인적전장서렬,실현N기인재대장간균중적고효표체,병진행초보응용.방법 근거이발표적우관상병독Mebus주적서렬설계인물,대BCV-DQ주진행RT-PCR확증、극륭화측서;장해기인삽입도원핵표체재체pET-30a (+)상진행원핵표체,SDS-PAGE 화Western blot검측;용순화적중조N단백작위포피항원,건립ELISA검측방법,대부분림상양본진행검측.결과 BCV-DQ주N기인전장1 347bp,여GenBank이발표적6주BCV적N기인상비교,핵감산동원성98.3%이상.구건적중조질립pET-N능표체출일조대소약위60 ku적단백,차능여서항BCV혈청발생특이성반응.용건립적ELISA방법대흑룡강불동지구송검적공256빈우혈청양품진행검측,결과양성솔65.23%,여병독중화시험적부합솔체95.31%.결론 BCV N기인보수성흔고,병재원핵표체계통중획득료고효표체,기표체산물구유량호적면역반응원성.림상검측결과표명N단백가작위진단항원용우우관상병독병적류행병학조사.
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.