采用狂犬病毒G基因原核表达的间接ELISA方法

Construction of indirect ELISA method using rabies virus G gene expressed in prokaryotic system

为获得狂犬病毒G基因并在大肠杆菌中进行表达,进而初步建立用于评价狂犬疫苗免疫效果的方法,根据狂犬病病毒RV(rabies virus)ERA株G基因序列设计引物,从病毒中提取出RNA,经逆转录并扩增得到RVG基因;将该基因片段定向克隆到原核表达载体pET28a(+)中,构建重组质粒PET-RVG并转化到大肠杆菌BL21(DE3)gold中,经IPTG诱导表达并确定表达的最佳条件;Ni亲和层析柱纯化获得重组G蛋白,并以G蛋白为抗原建立检测狂犬病毒抗体的间接ELISA方法.检测结果表明,经灰度分析纯化后纯度可达96%.

In order to acquire rabies virus G gene and express it in E. coli and establish the evaluation method for rabies virus immune,according to Rabies virus ERA G gene sequence to design primer sequences,RNA was extracted from rabies virus,the first strand cDNA was synthesized by reverse transcription. G gene fragments were amplified by PCR. Then the gene fragment was cloned to the prokaryotic expression vector pET28a（ ＋） to construct PET-RVG. The positive recombinant plasmids were transfected into E. coli BL21（ DE3） gold,with IPTG induced expression and determined the best expression condition.Using Niaffinity chromatography purification with G protein,and the indirect ELISA assay for the detection of rabies virus antibodies in serum was established based on the G protein. The results showed that the purity degree was 96%.