摘要
目的:构建炭疽毒素保护性抗原第四结构域PA4基因的重组Sem lik i森林病毒(Sem lik i forest virus,SFV)复制子载体,并观察PA4抗原在重组SFV复制子载体中的表达。方法:将PA4基因克隆到基于RNA和DNA的SFV复制子表达载体中,获得的重组SFV复制子载体直接转染BHK21细胞,通过间接免疫荧光和W estern印迹试验检测PA4在细胞中的表达;与辅助病毒载体共转染制备重组病毒颗粒,通过间接免疫荧光和W estern印迹检测PA4在重组病毒颗粒感染细胞中的表达。结果与结论:成功地构建了基于RNA和DNA的PA4重组SFV复制子载体,并且在体外基于RNA和DNA的重组SFV复制子表达载体能够在细胞中有效地表达非分泌型和分泌型的PA4抗原,制备了具有感染能力并能表达PA4抗原的重组病毒颗粒,为进一步以SFV复制子作疫苗载体观察炭疽新型复制子疫苗的免疫原性奠定了基础。
Objective: To construct recombinant Semliki forest virus(SFV) replieon vectors containing domain 4 of the protective antigen of anthrax ( PA4 ), and observe its expression in mammalian cells. Methods: PA4 Gene from anthrax toxin was cloned into RNA and DNA-based SFV replieon expression vectors, and these recombinant replicon expression vectors were named pSMPA4E, pSMSPA4E, pSCARPA4E, and pSCARSPA4E, respectively. Expression of antigen PA4 was confirmed by indirect (IF) and Western blot after transfecting BHK21 cells with these replieon expression vectors. Recombinant virus particles (RVP) were prepared by eotranfecting these expression vectors with helper vectors and expression of antigen PA4 was also confirmed by indirect immunofluoreseenee (IF) and Western blot in cells which were infected with these recombinant virus particles (RVP). Results and Conclusion: Recombinant SFV replieon vectors containing PA4 gene which could effectively express nonsecreting and secreting antigen PA4 in mammalian cells were successfully constructed. This work laid a foundation for the study of the immunogenieity of SFV replicon vaccines and provided an alternative way to develop new type vaccines against anthrax.
出处
《军事医学科学院院刊》
CSCD
北大核心
2006年第6期521-525,共5页
Bulletin of the Academy of Military Medical Sciences