摘要
根据Bergeron等报道的猪细小病毒(PPV)基因组,设计一对包含VP2全基因的PCR引物,上下游均引入一个BamHⅠ位点,扩增得到VP2基因后,将其插入到pUSK载体中,构建了转移载体pUSK VP2。采用脂质体介导的转染方法,将伪狂犬病毒TK-/gG-/LacZ+株的基因组DNA与pUSK VP2共转染PK 15细胞,待细胞病变后收集病毒液,在空斑纯化的同时,利用检测PPVVP2基因和LacZ基因的PCR方法筛选重组病毒TK-/gG-/VP+2株,Southernblotting、SDS PAGE、Westernblotting和电镜观察鉴定重组病毒,并在不同细胞上测定重组病毒的增殖滴度,接种小鼠进行安全性试验。结果发现,外源基因VP2已成功地插入到TK-/gG-/LacZ+亲本株的基因组中,并获得了表达。表达的VP2蛋白可以与猪细小病毒阳性血清反应,而且可以自行装配成病毒样颗粒。同时发现,VP2基因的插入不影响重组病毒的增殖特性,其毒力与亲本株相当。
A pair of primers of VP_2 gene was designed according to PPV genomic sequence reported by Bergeron.BamHⅠ sites were introduced on the upper stream and lower stream of the primers.The VP_2 gene obtained by PCR was inserted into pUSK vector,and the pUSK-VP_2 was then constructed.The genomic DNA of PRV TK~ -/gG~ -/LacZ~ + strain and pUSK-VP_2 were co-transfected into PK-15 cell with lipofectin,and the purified recombinant virus TK~ -/gG~ -/VP~ +_2 was screened out with PCR method by examining VP_2 gene and LacZ gene respectively.The recombinant virus were analyzed through Southern blotting,SDS-PAGE,Western blotting and electron micrograph,which indicated that the VP_2 gene had been inserted into the genome of TK~ -/gG~ -/LacZ~ + strain,the VP_2 protein expressed might react with PPV positive sera,and could form virus-like particles.Test of virus titers and virulent test on Balb/C rat showed that the insertion of VP_2 gene did not influence proliferation of recombinant virus.Its virulence was reduced considerably and equalled with that of parent strain.
出处
《病毒学报》
CAS
CSCD
北大核心
2004年第2期133-137,共5页
Chinese Journal of Virology
基金
国家863高技术研究发展计划(2001AA213051-1)
