骨形态发生蛋白基因真核表达质粒的构建及其在中国仓鼠卵巢细胞中的表达

Construction of Eukaryotic Expression Vector of Bone Morphogenetic Protein Gene and Its Expression in Chinese Hamster Ovarian Cells

【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.