狂犬病病毒糖蛋白在昆虫细胞中的表达及其免疫学特性分析

目的:利用Bac-To-Bac杆状病毒昆虫细胞表达系统对狂犬病病毒糖蛋白(rabies virus glycoprotein,RVG)基因进行克隆?表达?纯化,并对重组 RVG 进行免疫学特性鉴定?方法:参照 GenBank 收录的狂犬病病毒 CVS-11 株RVG基因序列,设计特异性引物,扩增目的基因?RVG 基因经BamHⅠ/KpnⅠ 双酶切后定向克隆到 pFastBac-GP67B 载体上,阳性重组转座质粒进一步转化E.Coli DH10Bac 感受态细胞,经蓝白斑筛选及 PCR 鉴定后获得重组穿梭载体 rBacmid-RVG,将其转染至对数生长期的Sf-9昆虫细胞,进行重组RVG的真核表达?His-TrapHP 纯化目的蛋白,SDS-PAGE 和 Western blot 进行鉴定?重组RVG免疫小鼠,检测其免疫原性和血清中和活性?结果:用Bac-To-Bac杆状病毒昆虫细胞表达系统表达并纯化了重组RVG,分子量约58 000?经重组RVG免疫后的小鼠血清具有中和活性?结论:重组RVG具有天然RVG的活性,为进一步研制亚单位疫苗及制备筛选中和抗体奠定了基础?

Expression of insect cell-based rabies virus glycoprotein and assessment of its immunogenicity efficacy in mice

Objective:Rabies virus glycoprotein (RVG) was cloned using Bac-to-Bac baculovirus expression vector system and expressed in Spodoptera frugiperda (Sf-9) cells. Furthermore, the purified r-RVG protein was used to evaluated the immunological characteristics. Methods: Referring to the sequence of G protein gene of rabies virus CVS-11 strain from GenBank, we designed a pair of specific primers for PCR amplification, cDNA worked as the PCR template. The sequence of G protein gene was amplified by specific primers designed according to the CVS-11 strain from GenBank. The PCR-amplified RVG gene was cloned into the pFastBac-GP67B plasmids with digestion by restriction enzyme BamH I and Kpn I. Positive clone was transformed into E.coli DH10Bac competent cells and then the recombinant rBacmid-RVG was identified by blue /white selection and PCR analysis. The recombinant baculovirus was generated by transfecting Sf-9 cells and the recombiant RVG from eukaryotic expression was purified with His-Trap, characterized by SDS-PAGE and Western-blot analysis. The immunogenicity and serum neutralizing activity of recombiant RVG were assessed using mice model. Results: Recombiant RVG protein was efficiently expressed in eukaryotic expression and purified, with molecular weight of 58 000. The mice serum showed neutralizing activity after immunization by recombiant RVG. Conclusion: The results of the study indicated that recombiant RVG retained the biological activity as the native conformation, thereby paving the way for producing efficacious subunit vaccine and screen neutralizing antibodies in future research.