狂犬病毒N基因的原核表达及间接ELISA方法的建立

目的用表达的狂犬病核蛋白建立检测狂犬病抗体的间接ELISA方法。方法根据GenBank发表的狂犬病病毒(Rabies Virus,RV)ERA株N基因序列设计引物,利用PCR的方法从重组质粒pMD-N中扩增RV N基因,将该基因片段定向克隆到原核表达载体pET28a(+)中,构建原核表达载体pET-N。阳性重组质粒转化原核表达宿主菌BL21(DE3),用IPTG诱导表达并确定表达N基因的最佳诱导时间。通过凝胶薄层扫描分析和Western-blot分析确定表达蛋白的表达量和特异性。用纯化的表达产物作为诊断抗原,通过对各反应条件的优化,初步建立检测RV抗体的间接ELISA方法。结果扩增了RV N基因,构建了原核表达载体pET-N和原核表达菌,表达了N基因且目的蛋白以包涵体形式存在表达菌中,最佳诱导时间为4 h。重组蛋白表达量占菌体总蛋白的56.8%,并能与RV阳性血清发生特异性反应。用表达的狂犬病重组N蛋白建立了用于RV抗体检测的间接ELISA方法。结论成功表达了狂犬病核蛋白,用表达的狂犬病核蛋白建立的间接ELISA方法可以用于RV抗体的检测。

Establishment of a putative indirect ELISA assay with the prokaryotically expressed gene product of N gene from rabies virus

In this study,the complete length of N gene from rabies virus was amplified by PCR using a pair of specific primers designed according to the relevant sequences from GenBank,and the PCR product was cloned into prokaryotic expression vector pET28a( ) to obtain the prokaryotically expressed plasmid pET-N.The target gene was then expressed in the E.coli BL21(DE3) cells by inducted with IPTG and the expression was optimized with a proper induction time of 4 hours. The highest expression of the target protein was up to 56.8% of total bacterial proteins through the analysis of gel scanning,and the good immunoreactivity to rabies virus antibodies was proved by Western blot analysis.By using this prokaryotically expressed gene product of N gene from rabies virus,the indirect ELISA assay for the detection of rabies virus antibodies in canine serum was established after management of the optional working conditions.