牛冠状病毒核衣壳蛋白原核表达及鉴定

Prokaryotic Expression and Identification of Bovine Coronavirus Nucleocapsid Protein

【目的】诊断致犊牛腹泻冠状病毒。【方法】采集石河子、沙湾、奎屯等10个规模化奶牛场141份腹泻犊牛粪样,采用酶联免疫吸附实验(ELISA)进行冠状病毒检测,PCR方法进行核酸复检。同时根据GenBank登录的牛冠状病毒N基因序列,设计特异性引物,对Mebus分离株N基因进行扩增,克隆到PMD 19-T载体后,将测序正确的基因片段经EcoRⅠ和Hind Ⅲ双酶切后连接到PET-28a和PET-32a载体中,构建重组表达载体PET-28a-N和PET-32a-N,进行测序,亚克隆入BL21(DE3)表达载体,用IPTG进行诱导重组菌,SDS-PAGE和Western blot分析表达产物。【结果】所采集141份腹泻犊牛粪便中,用ELISA检测阳性率70.21%;再用RT-PCR检测出阳性率为61.7%同时,克隆了牛冠状病毒N基因,片段大小为1 400 bp,双酶切鉴定目的条带正确,测序结果与标准序列的进行比对同源性达到98.22%,通过SDS-PAGE分析证实表达的重组蛋白PET-28a-N-DE3相对分子质量为54KD;重组蛋白BCV-32a-N-DE3相对分子质量为70KD,Western blot分析表明该重组蛋白BCV-32a-N-DE3和BCV-28a-N-DE3和可以与牛冠状病毒阳性血清发生特异性反应。【结论】犊牛冠状病毒是导致石河子、沙湾、奎屯等10个规模化奶牛场和部分肉牛场犊牛腹泻的主要病原;ELISA方法和RT-PCR方法结合应用检测犊牛冠状病毒效果具有参考意义。获得的牛冠状病毒N蛋白具有很好的免疫反应性,为牛冠状病毒诊断试剂研发奠定基础。

【Objective 】To preliminarily diagnose calf diarrhea coronavirus. 【Method 】141 calf feces samples from 10 dairy cattle farms in Shihezi,Shawan and Kuitun were collected. The coronavirus was detected by enzyme linked immunosorbent assay（ELISA）,and the PCR method was used to test the nucleic acid.At the same time,specific primers were designed to amplify the N gene of the Mebus isolate based on the sequence of the Gen Bank virus N gene,and the gene fragment was cloned into the PMD 19-T vector. The sequence of the correct gene fragment was connected to the PET-28a and PET-32a carriers after EcoRⅠ and Hind Ⅲ double enzymes,and the recombinant expression carrier PET-28a-N and PET-32a-N were constructed. After sequencing,subclones were inserted into BL21（DE3） expression vector,and IPTG was used to induce recombinant bacteria. SDS-PAGE and Western blot were used to analyze the expression products.【Result】In the feces of 141 diarrhoea calves,the positive rate was 70. 21 by ELISA and 61. 7% by RT-PCR. The bovine coronavirus N gene was cloned successfully,the size of the fragment was 1,400 bp; The target strip was identified correctly by double enzyme cutting,and the sequence results were compared with the standard sequence to reach 98. 22% of homology. The relative molecular mass of recombinant protein PET-28a-N-DE3 expressed by SDS-PAGE analysis was 54 KD,and the relative molecular mass of recombinant protein BCV-32a-N-DE3 was 70 KD,and Western blot analysis showed that the recombinant protein BCV-32a-N-DE3 and BCV-28a-N-DE3 could react specifically to bovine coronavirus positive sera.【Conclusion】Calf coronavirus is the main cause of diarrhea in 10 large-scale dairy cattle farms in Shihezi,Shawan and Kuitun,and the results of the combination of ELISA and RT-PCR methods are of reference significance for the detection of calf coronavirus. The bovine coronavirus N protein has good immunoreactivity,which lays the foundation for the research and development of bovine coronavirus diagnostic reagents.