摘要
采用聚乙二醇（PEG）沉淀法纯化克隆刚地弓形虫（Toxplasmagondii）p30基因的转移载体质粒pSXIVVI+X3－p30DNA；将pSXIVVI＋X3－p30DNA与粉纹夜蛾核型多角体缺陷型病毒TnNPV－SVI－GDNA共转染草地夜蛾（Sfg）细胞，构建出既能形成多角体又能表达外源基因的重组病毒TnNPV－p30。经空斑纯化挑选出6株单克隆重组病毒，感染细胞裂解液在相对分子质量约为34000～35000位置出现一条表达带；其含量占细胞总蛋白的5.13％～5．62％，株间差异不明显；用抗弓形虫多克隆抗血清进行Western－Blot呈一特异反应条带。感染后72h蛋白表达量最高，可达7．18％；96小时次之，为5．91％。
The transfer plasmid pSXIVVI+X3-p30 DNA cloned with the p30 gene of Toxoplasma gondii was purified with PEG. A recombinant virus TnNPV-p30 was constructed by cotransfecting the Spodoptera frugiperda (Sf) cells with the parental virus. It could express foreign gene and form polyhedrin in the meantime. We selected six strains of monoclonal re combinant virus by plaque assay. The infected Sf cells were fractionated and analyzed by SDS-PAGE. A specific expression band in the position of 34 to 35 kDa was found,and its content was 5. 13~ 5. 62% of the total cell protein. There was no marked difference between differe strains,and the protein was recognized by a polyclonal antiserum against ToxoPlasma gondii in Western immunoblot 72 h post-infection cells achieved the maximun expression (7. 18% ),and the expression of 96 h post infection cells were 5. 91%.
出处
《第一军医大学学报》
CSCD
1998年第2期91-95,共5页
Journal of First Military Medical University
基金
国家自然科学基金!39400115
关键词
P30基因
弓形体
真核系统
基因表达
Toxoplasma gondii
expression
p30 gene
baculovirus expression vector
