摘要
构建以伪狂犬病病毒(PRV)为载体表达狂犬病病毒糖蛋白的重组活载体疫苗。采用AseⅠ/MluⅠ切下EG-FP-rgp的表达盒,将其克隆入p8-AA载体的酶切位点处,经酶切鉴定为正向连接,阳性重组子命名为p8-EGFP/rgp。将该质粒与PRV TK-/gI-基因组在质脂体介导下共转染PK-15细胞,获得PRV-EGFP/rgp重组病毒;通过噬斑克隆对其进行筛选、纯化,并通过RT-PCR、Southern-blot、Western-blot和间接免疫荧光对其进行鉴定,并对重组病毒的滴度、外源基因的遗传稳定性及其与亲本病毒株生长特性的关系进行了测定。结果表明:重组病毒的滴度(TCID50)为108.125/mL;外源基因在细胞内得到了有效的表达,并具有良好的免疫反应性和遗传稳定性;PRV-EGFP/rgp与亲本病毒PRV TK-/gI-生长趋势差异不显著。通过构建表达狂犬病病毒糖蛋白正向重组PRV的活载体疫苗的研制,为狂犬病疫苗的研制提供了新的思路和方法。
In order to construct recombinant pseudorabies virus norientation expressing glycoprotein of rabies virs, we used the Ase I/Mlu I restriction enzymes digesting the EGFP-rgp expression cassette, and then cloned into Sph I/Nde I sites of pS-AA. By enzyme identification for the plasmid,we knew that the plasmid was cloned into p8-AA by norientation way,designated p8-EGFP/rgp. By transfecting the PK-15 cells with pS-EGFP/rgp and the genome of PRV TK^-/gI^- through lipofectamine 2000TM, and gained the recombinant virus （PRV-EGFP/rgp）. To screen identify the recombinant virus,we use plaque clone,RT-PCR,Southern-blot,Western-blot and inderect immunofluorescence techniques. We also titered the recombinant virus, and detected the genetic stability, and determined the growth relationship of recombinant virus with parental virus. From the results, we could see that the titer （TCID50）of recombinant viru was 10^8.125/mL, and the foreign gene was expressed well, possessed genetic stability and immune response. The growth tendency was not significant for PRV-EGFP/rgp and PRV TK^-/gI^-. Thus provides a novel strategy and methods by constructing recombinant pseudorabies virus expressing glycoprotein of rabies virus.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2008年第12期1398-1403,共6页
Chinese Journal of Veterinary Science
基金
国家“863”计划资助项目(2007AA02Z401)
