人骨形态发生蛋白2基因重组腺病毒的构建与鉴定

Construction and identification of recombinant adenovirus vectors encoding human bone morphogenetic protein 2

背景:骨形态发生蛋白2是诱导成骨关键物质,参与调节从细胞增殖、决定种系分化方向到细胞死亡等一系列的生物过程。目的:利用AdMax系统构建人骨形态发生蛋白2基因的重组腺病毒载体并鉴定。方法:以人cDNA为模板PCR扩增人骨形态发生蛋白2基因,转化E.coli感受态细胞,经菌落PCR鉴定阳性转化子、阳性克隆测序无误后,扩增、抽提。将带有目的基因的腺病毒表达载体和携带有腺病毒大部分基因的辅助包装质粒共转染293细胞进行病毒包装扩增,PCR检测目的基因、Western Blot检测目的蛋白及终点稀释法检测病毒滴度。结果与结论:PCR获得长度为1223bp的人骨形态发生蛋白2目的基因片段,同源重组表达载体经阳性克隆PCR及测序鉴定,结果正确。293细胞内包装、扩增,经Westernblot及PCR鉴定无误后,获得病毒滴度为5×1013pfu/L的重组腺病毒。实验成功构建携带有人骨形态发生蛋白2基因的重组腺病毒载体。

BACKGROUND：Bone morphogenetic protein 2 plays a key role in inducing osteogenesis.It involves in a series of bioprocess,including cell proliferation,determining the differentiation direction of germ line and cell death.OBJECTIVE：To construct and identify the recombinant adenovirus vectors encoding human bone morphogenetic protein 2 gene by using AdMax system.METHODS：First,human bone morphogenetic protein 2 gene sequencing was amplified by PCR from human cDNA template and then cloned.Second,the recombinant shuttle plasmid was constructed and transformed into Escherichia coli competent cells DH5α.After the positive colonies were identified by colonies PCR and sequencing,the expression vectors were amplified and extracted.Next,the adenovirus expression vectors with target gene and the helper packaging plasmid carrying a majority of adenovirus genes were co-transfeced into 293 cells for virus packaging and amplification.Finally,target genes were detected by PCR,and target protein was detected by Western blot method,as well as infectious titer of the recombinant adenovirus was detected by end point dilution method.RESULTS AND CONCLUSION：Gene fragment of a length of 1 223 bp human bone morphogenetic protein 2 was obtained by PCR.The expression vectors constructed by homologous recombination techniques were identified by PCR cloning and sequencing;the results were correct.After virus packaging and amplification in 293 cells were identified by Western blot and PCR methods,the virus titer of recombinant adenovirus was 5×1013 pfu/L.These results suggest that the recombinant adenovirus vectors carrying human bone morphogenetic protein 2 gene have been constructed successfully.