GFP标记的重组新城疫病毒HN基因乳酸菌表达载体的构建及原核表达

为构建具有示踪特性的新城疫病毒(NDV)HN基因乳酸菌穿梭表达载体,采用PCR方法扩增NDV HN基因与绿色荧光蛋白(GFP)基因,将两基因克隆至乳酸菌表达载体pW425et中,转化入表达宿主菌BL21(DE3)后利用IPTG诱导表达。用荧光显微镜检查重组菌的绿色荧光效应,采用SDS-PAGE和Western-blotting检测其在大肠杆菌中的表达情况。结果显示,NDV HN和GFP基因与表达载体pW425et正确重组。荧光显微镜下可见阳性重组菌落的绿色荧光,且该重组菌传代30代次以上,荧光效应依然稳定。SDS-PAGE显示表达出约63ku的条带,与已知表达的HN蛋白大小相符。Western-blotting进一步证实该蛋白能被NDV阳性血清所识别,具有反应原性。构建了GFP标记的重组NDV HN基因乳酸菌穿梭表达载体且能在大肠杆菌中表达。

Construction and prokaryotic expression of recombinant Lactobacillus expression vector labelling with GFP gene of HN gene of Newcastle disease virus

To express a recombinant GFP-labelled Newcastle disease virus(NDV) HN with Lactobacillus shuttle expression vector pW425et,HN and GFP genes were amplified by PCR and cloned into the pW425et vector,respectively.The recombinant was transformed into Escherichia coli BL21(DE3) and induced with IPTG.Result showed that the fluorescence of recombinant strain could be observed under fluorescence microscope for at least 30 generations.SDS-PAGE and Western-blotting results showed that NDV HN with the molecular weight of 63 ku had reactogenicity with NDV positive serum.In conclusion,the recombinant NDV HN Lactobacillus expression vector labelling with GFP was successfully constructed and expressed in E.coli.