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目的:构建及制备人骨形态发生蛋白12(BMP12)基因重组腺病毒,证明其可感染脂肪组织来源干细胞并表达蛋白。方法:将包含有BMP12cDNA全长序列的BMP12-pED6质粒用限制性内切酶EcoRI进行酶切,得到一个1260bp大小的含有BMP12cDNA的目的基因片段。将目的基因片段插入质粒pcDNA3.1后,用KpnI和PmeI进行双酶切,插入pAdtrack-CMV。插入目的基因片段的穿梭质粒pAdtrack-CMV-BMP12用PmeI进行酶切线性化后与腺病毒骨架载体pAdEasy-1一起电转化入感受态BJ5183菌株。用PCR及多种酶切方法鉴定重组体。最终将线性化的重组质粒利用脂质体转入293A细胞中进行病毒包装。BMP12基因随着重组腺病毒在感染的293A细胞中扩增而得到复制,并通过CsCl梯度离心法得以纯化。Elisa法检测Ad-BMP12感染脂肪组织来源干细胞后BMP12的蛋白表达。结果:pAdtrack-CMV-BMP12经酶切证实有1260bp的插入片段。酶切及PCR鉴定证实BMP12基因重组腺病毒载体构建成功。GFP(green fluorescent protein)表明重组腺病毒扩增成功并制备出高滴度重组病毒。该重组病毒可成功感染脂肪来源组织干细胞并表达BMP12蛋白。结论:BMP12基因能够重组于腺病毒载体,可以进行有效扩增,产生高浓度的重组腺病毒,从而用于BMP12基因治疗的研究。
OBJECTIVE: To construct and prepare human recombinant adenovirus carrying human bone morphogenetic protein 12 (BMP12) gene and to prove that it can infect adipose tissue-derived stem cells and express proteins. Methods: BMP12-pED6 plasmid containing the full-length BMP12 cDNA sequence was digested with restriction endonuclease EcoRI to obtain a 1260bp BMP12 cDNA containing the target gene fragment. The target gene fragment was inserted into the plasmid pcDNA3.1, double digested with KpnI and PmeI, inserted into pAdtrack-CMV. The shuttle plasmid pAdtrack-CMV-BMP12 inserted into the target gene fragment was linearized by restriction enzyme digestion with PmeI and electrotransformed into the competent BJ5183 strain together with the adenoviral backbone vector pAdEasy-1. Recombinants were identified by PCR and multiple digestion methods. Finally, the linearized recombinant plasmids were transformed into 293A cells by liposomes for viral packaging. The BMP12 gene is replicated as the recombinant adenovirus is amplified in infected 293A cells and purified by CsCl gradient centrifugation. Elisa method was used to detect the protein expression of BMP12 after Ad-BMP12 infection of adipose tissue-derived stem cells. Results: pAdtrack-CMV-BMP12 confirmed by digestion 1260bp insert. Digestion and PCR identification confirmed BMP12 gene recombinant adenovirus vector was successfully constructed. GFP (green fluorescent protein) indicates that the recombinant adenovirus has been successfully amplified and prepared with high titers of recombinant virus. The recombinant virus can successfully infect adipose-derived tissue stem cells and express BMP12 protein. Conclusion: BMP12 gene can be recombined into adenovirus vector and can be effectively amplified to produce high concentration of recombinant adenovirus for BMP12 gene therapy.