作　　者： ; ; ; ; ; ; ; ;

机构地区： 广州中医药大学第一附属医院

出　　处： 《广州中医药大学学报》 2006年第6期522-525,共4页

摘　　要： 【目的】克隆人骨形态发生蛋白(BMP4)基因,构建真核表达质粒,并在中国仓鼠卵巢(CHO)细胞中表达,为进一步研究以BMP4进行骨修复的基因治疗奠定基础。【方法】采用逆转录聚合酶链反应(RT-PCR)法,从人的松质骨中扩增BMP4的环形脱氧核糖核酸(cDNA),插入pGEM-Teasy载体,测序证实后,将其亚克隆至质粒PCDNA3.1B(-)中构建BMP4真核表达载体;采用脂质体将重组质粒转染CHO细胞,经G418筛选获得抗性细胞克隆,以RT-PCR和免疫组织化学方法鉴定转染细胞中BMP4基因的表达。【结果】经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒,RT-PCR和免疫组化方法证明转基因CHO细胞克隆中存在人BMP4基因。【结论】成功获得了人BMP4的全长基因,且序列与已知序列高度一致。 [Objective] To clone human bone morphogenetic protein 4 （BMP4） gene, construct BMP4 eukaryotic expression vector and observe its expression in Chinese hamster ovarian （CHO） cells, thus to provide evidence for the further study of gene therapy for bone repair. [ Methods ] The amplified cDNA of human BMP4 from cancellous bone was obtained by reverse transcription polymerase chain reaction （RT-PCR） and then was inserted into pGEM-Teasy cloning vector. After the sequence of pGEM-Teasy vector including amplified human BMP4 cDNA was confirmed, BMP4 eukaryotic expression vector was constructed from its subclone with PCDNA3.1B （ - ） . Then the recombinant plasmid of PCDNA3.1B （ - ） BMP4 was transfected into CHO cells through lipofectamine method and positive cellular clones were screened with G418. Human BMP4 gene expression in the transfected cells was confirmed with RT-PCR and immunocytochemistry. [ Results] Restriction enzyme digestion and DNA sequencing analysis showed that the target gene was inserted into the recombinant vector. The results of RT-PCR and immunocytochemistry showed the expression of human BPM4 gene in transfected CHO cells. [ Conclusion] The lull-length of human BMP4 is obtained successfully and its sequence is highly consistent with the known sequence.

关 键 词： 骨形态发生蛋白 基因疗法 骨疾病

领　　域： [生物学]