马冠状病毒核衣壳蛋白基因片段的克隆及其表达  

CLONING AND EXPRESSION OF NUCLEOCAPSID PROTEIN GENE FRAGMENT OF EQUINE CORONAVIRUS

作　　者：顾炳泉[1] 彭志生[2] 张敬友[3] 张鹤晓[4] 秦爱建 李建[1] 高逢结[1] 仇钰 金文杰 邵红霞 孙谦 

机构地区：[1]南通出入境检验检疫局,江苏南通226005 [2]国家质检总局,北京100088 [3]江苏出入境检验检设局,江苏南京210001 [4]北京出入境检验检疫局,北京100026 [5]扬州大学,江苏扬州225009

出　　处：《检验检疫科学》2007年第3期3-6,共4页Inspection and Quarantine Science

摘　　要：根据NCBI发表ECV核衣壳基因序列,用PCR方法扩增出ECV核衣壳基因片段,插入表达型载体pGEX-6p-1中,构建重组质粒pGEX-ECV-N。以IPTG诱导表达,并用SDS-PAGE凝胶电泳分析,结果成功表达了42KD GST-ECV-N融合蛋白。将ECV-N从pGEX-ECV-N载体亚克隆到的杆状病毒转移载体pFastBac1,筛选出重组转移载体pFastBac-ECV-N,在DH1O细菌的转座子作用下,构建重组穿梭质粒Bacmid-ECV-N。提取正确重组的穿梭质粒DNA转染Sf9昆虫细胞,结果获得了重组杆状病毒rBac-ECV-N。PCR和间接免疫荧光试验结果证明,ECV-N在Sf9细胞中得到了很好的表达。该结果对我国马群中冠状病毒感染的流行病学调查及深入研究有指导意义。Equine coronavirus nucleocapsid protein gene fragments were amplified by PCR method based on the gene sequence of Equine coronavirus from NCBI. ECV-N gene fragment was inserted into expression vector pGEX-6p- 1 to establish pGEX- ECV-N. Inducted the BL21 containing pGEX-ECV-N by IPTG, the expressed fusion protein GST-ECV-N was 42KD and identified by SDS-PAGE. Through purified ECV-N fragments, the ECV-N gene from pGEX-ECV-N was subcloned into transfer vector pFastBac 1 to establish pFastBac-ECV-N. Recombinant Bacmid-ECV-N was generated by transposing in the DH10 Bac E.coli ,then transfected into the Sf9 insect cells mediated to produce the recombinant baculovirus rBac-ECV-N. The results of PCR and IFA showed that the sf9 cells expressed ECV-N gene fragment protein. The results are of great significance to epidemiological survey and further research on the horses suffering from Equine coronavirus in China.

关 键 词：马冠状病毒 核衣壳基因 克隆和表达 

分 类 号：S852.65[农业科学—基础兽医学]