昆虫杆状病毒表达系统中人骨形成蛋白3的表达及鉴定

Expression and characterization of human bone morphogenetic protein 3 in baculovirus expression system

目的 探索人骨形成蛋白 3(h BMP3)在昆虫杆状病毒系统中的表达规律 .方法 将 h BMP3全长 c DNA克隆入转移载体 Bac PK8中 ,酶切鉴定后 ,将此载体与病毒 DNA经脂质体包裹转染昆虫细胞 ,重组病毒经蓝白筛选后 ,提取其DNA进行 PCR反应 ,鉴定目的基因 .收集重组病毒感染 5 d的细胞进行免疫荧光及电子显微镜观察 .结果 克隆了目的基因的重组载体经内切酶消化后 ,琼脂糖电泳示有相应大小的目的基因片段切下 .PCR电泳结果见有目的基因片段的扩增 .免疫荧光检测见阳性颗粒分布在昆虫细胞的胞质及胞膜 ,电镜下见重组病毒无多角体颗粒 ,与野生型杆状病毒完全不同 .结论 初步证实 h

AIM To study the expression of human bone morphogenetic protein (hBMP3) in the baculovirus system. METHODS cDNA encoding human bone morphogenetic protein 3 was cloned into the transfer vector BacPK8. The recombinant vector harboring hBMP3 cDNA was identified by restriction enzymes. The linearized virus DNA and the recombinant vector were mixed with lipofectin then transfected into Sf9 cells. The recombinant virus was identified by LacZ blue white selection, PCR analysis, immunofluorescence and electromicroscopic analysis were also made. RESULTS The target DNA fragment in agarose electrophoresis was seen when recombinant transfer vector was incubated with restriction enzyme. The amplified fragment of target gene from recombinant virus DNA by PCR was observed. The positive immunofluorescence granules were observed in cytoplasm and cytomembrane. Unlike wild virus, no polyhedrosis body formation was seen in the recombinant virus under electromicroscopy. CONCLUSION hBMP3 has been expressed in this baculovirus expression system.