摘要
将含完整阅读框架的人t PA (tissue typeplasminogenactivator,t PA)cDNA克隆至昆虫细胞表达转移载体pBacPAK8中 ,获得重组质粒pBac tPA .应用脂质体共转染法 ,将pBac tPA和线性化杆状病毒BacPAK6DNA共转染Tn 5B 1昆虫细胞 .经空斑筛选获得 11株重组病毒 .经PCR鉴定与生物活性测定 ,获得一株高效表达t PA的重组病毒BactPA3.t PA表达活性在感染 (MOI≈ 10 )后 72h左右达到最高 ,为 3 0 4× 10 3IU/mL .经SDS PAGE纤维蛋白自显影分析 ,相对分子质量为 680 0 0左右 .
The t\|PA cDNA containing whole reading frame was inserted into the transfer vector pBacPAK8. The constructed pBac\|tPA contransfected Tn\|5B1\|4 insect cells with the linear BacPAK6 virus DNA by lipofectin\|mediated transfection method.Eleven pure recombinant viruses were isolated by plaque assay, and one of them, nominated BactPA3, was selected by using PCR identification and biological activity comparison.The activity of expressed t\|PA reached the highest level at 72 hours post\|infection.The activity was 3 04×10\+3 IU/mL.SDS\|PAGE fibrin autography showed that the molecular weight of the expressed t\|PA was ahout 68 000.
出处
《中山大学学报（自然科学版）》
CAS
CSCD
北大核心
2000年第6期77-80,共4页
Acta Scientiarum Naturalium Universitatis Sunyatseni
基金
华南生物科学与技术研究中心科技资助项目
