PEDV-RBD基因在昆虫杆状病毒中的表达与鉴定

Expression and identification of PEDV-RBD gene in baculovirus expression vector system

受体结合域是决定病毒侵入宿主及其嗜性的关键因素。该研究目的在于获得具有天然构象且反应原性良好的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)的受体结合域(receptor-binding domain,RBD)蛋白。根据PEDV S蛋白序列(GenBank登录号:AKN45969.1),设计、优化并合成其RBD部分基因序列,并将其亚克隆至杆状病毒双表达载体pFastBacTMDual上,得到了重组质粒pFBD-PER。将pFBD-PER转化到大肠杆菌DH10BacTM感受态细胞中,进行蓝白斑筛选,获得重组杆状质粒Bacmid-PER,通过脂质体转染至Sf9细胞中进行病毒拯救。转染120 h后,收取上清病毒液,盲传3代后收取Sf9细胞,利用间接免疫荧光法(IFA)及Western blot确定PEDV-RBD蛋白是否表达。结果显示,重组质粒pFBD-PER酶切验证正确,重组杆粒Bacmid-PER成功构建,杆状病毒能够表达PEDV-RBD蛋白,且能与strep tag抗体发生特异性反应。IFA鉴定出现特异性绿色荧光。结果表明,成功表达了PEDV-RBD蛋白且具有反应原性,为深入开展PEDV抗体制备、新型疫苗研发奠定基础。

Receptor binding domain(RBD) is the key factor to determine the host and tropism of virus. The aim of the research was to obtain the porcine epidemic diarrhea virus(PEDV),RBD protein with natural conformation and good reactionogenicity. According to the sequence of PEDV?s protein(GenBank: AKN45969.1),the RBD part was designed, optimized and synthesized, and subcloned into the baculovirus double expression vector pFastBacTMDual to obtain the recombinant plasmid pFBD-PER containing PEDV-RBD gene. The pFBD-PER was transformed into DH10 BacTM competent cells and a blue/white colony screening assay was implemented to obtain the recombinant rod-like plasmid Bacmid-PER that was transfected into Sf9 cells by lipofectamine for viral rescue. 120 hours after transfection, the supernatant of viruses was collected. Sf9 cells were collected after viruses were cultured for three generations. the target protein was to verify whether it was expressed by using indirect immunofluorescence, and a further verification by using Western blot.The results showed that the recombinant plasmid pFBD-PER was proved to be correct.The PEDV-RBD protein expressed in baculovirus reacted specifically to strep tag antibody.The specific green fluorescence was detected by IFA showed that the PEDV-RBD protein was expressed successfully and had the reactionogenicity.This study laid a foundation for the further preparation and the development of a new vaccine for PEDV antibody.