【摘要】 为了在哺乳动物细胞中表达A型肉毒毒素Hc抗原,构建了含A型肉毒毒素受体结合区Hc基因的基于RNA和DNA的重组Semliki森林病毒(Semliki forest virus,SFV)复制子表达栽体。RNA和DNA复制子栽体转染BHK21细胞后,经间接免疫荧光、Western印迹和ELISA检测,结果表明非分泌型和分泌型的Hc抗原在细胞中都得到了有效地表达;而且复制子表达载体与辅助病毒载体共转染均可制备高滴度的重组病毒颗粒,该重组病毒颗粒感染细胞后,也都能表达Hc抗原。以上结果表明,基于RNA和DNA的重组SFV复制子表达载体在细胞中均可有效地表达Hc抗原和制备具有感染能力并能表达Hc抗原的重组病毒颗粒。基于RNA和DNA的重组SFV复制子表达载体的构建和含A型肉毒毒素受体结合区Hc基因的重组病毒颗粒的获得,为进一步观察SFV复制子疫苗的免疫原性奠定了基础,从而为A型肉毒毒素新型疫苗的研制提供了新途经。更多还原

【Abstract】 To produce the C-terminal fragment of heavy-chain receptor(Hc)of the Clostridium botulinum neurotoxin serotype A (BoNT/A)in mammalian cells,we cloned the Hc gene from BoNT/A into the RNA and DNA-based Semliki Forest virus(SFV)rep- licon expression vectors,resulting in recombinant replicon expression vectors pSMHc,pSMSHc,pSCARHc,and pSCARSHc.Effec- tive expression of non-secreted and secreted antigen Hc were confirmed by indirect imumofluorescence(IF),Western blot and ELISA after transfecting BHK21 cells with these replicon expression vectors based on both DNA and RNA.Recombinant virus par- ticles(RVP)were prepared by cotranfecting these expression vectors with helper vectors(pSFV-helper2 or pSHCAR)and the ex- pression of antigen Hc was confirmed by indirect imumofluorescence(IF)and Western blot in cells(BHK21)that were infected with these recombinant virus particles(RVP).RNA and DNA-based recombinant SFV replicon expression vectors and recombinant virus particles(RVP)containing Hc gene all effectively expressed antigen fragment Hc in cells.This work laid a foundation for the study of the immunogenicity of SFV replicon vaccines and provided an alternative to develop new type vaccines against botulinum neuro- toxin serotype A.更多还原