【摘要】 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Gen Bank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Bam HⅠ/KpnⅠ双酶切后定向克隆到p Fast Bac-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r Bacmid-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-Trap HP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。更多还原

【Abstract】 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 Gen Bank, we designed a pair of specific primers for PCR amplification, c DNA worked as the PCR template. The sequence of G protein gene was amplified by specific primers designed according to the CVS-11 strain from Gen Bank. The PCR-amplified RVG gene was cloned into the p Fast BacGP67 B plasmids with digestion by restriction enzyme Bam H I and Kpn I. Positive clone was transformed into E.coli DH10 Bac competent cells and then the recombinant r Bacmid-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 wayfor producing efficacious subunit vaccine and screen neutralizing antibodies in future research.更多还原