人骨形成蛋白-2基因的克隆及其重组腺病毒载体的构建  

Cloning of human bone morphogenetic protein-2 gene and construction of recombinant adenoviral vector

作　　者：张莉[1] 马宁[1] 高文信[1] 张明[2] 

机构地区：[1]吉林大学口腔医学院颌面外科,吉林长春130041 [2]吉林大学基础医学院法医学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2007年第3期435-439,共5页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅资助课题(20030424-03)

摘　　要：目的:克隆人骨形成蛋白-2(BMP-2)基因cDNA,构建含BMP-2基因的重组腺病毒载体。方法:采用RT-PCR方法克隆BMP-2基因cDNA,并插入克隆载体pGEM-T中进行全序列分析。将BMP-2基因cDNA克隆到穿梭载体pShuttle-CMV中,构建pShuttle-BMP2重组质粒,再将其中的表达盒克隆入Adeno-X腺病毒DNA中,获得重组腺病毒DNA-pAd-BMP。结果:成功地克隆长约1200 bp的BMP-2 cDNA,并成功地构建其腺病毒载体,经线性化的pAd-BMP2 DNA转染HEK293细胞,包装、扩增后得到人骨形成蛋白-2重组腺病毒,其滴度约为1×1011nfu.L-1,该滴度可满足进一步的BMP-2成骨作用的研究。结论:成功地构建BMP-2腺病毒载体。Objective To clone the human bone morphogenetic protein-2 （hBMP-2） gene cDNA and construct its replication-deficient type 5 adenoviral vector. Methods Using osteoblastic sarcoma tissue to extract cell total RNA, hBMP-2 cDNA was amplified by RT-PCR. The hBMP-2 cDNA was then inserted into cloning vector pGEM-T. The recombinant plasmid was screened and the recombinant plasmid which was confirmed by restriction enzyme, PCR and sequence analysis was gotten. To construct the recombinant adenoviral vector, the BMP-2 cDNA was subcloned into a shuttle vector and the recombinant pShuttle-BMP2 vector was obtained. It was subcloned into Adeno-X adenoviral genomic DNA and the pAd-BMP2 DNA was obtained. Results A length of 1 200 bp BMP-2 eDNA was cloned, and it was subcloned into adenoviral vector, the recombinant adenovirus was constructed successfully. After measuring the titre of virus （1 × 10^11 nfu ·L^-1）, the target gene was evaluated by PCR. Conclusion The hBMP-2 adenoviral is obtained. The successful construction of pAd-BMP2 may provide us useful target for the further research.

关 键 词：骨形态发生蛋白质类 遗传载体 基因 转染 

分 类 号：Q78[生物学—分子生物学]