摘要
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 recombinant adenovirus vector carrying the human bone morphogenetic protein12 （BMP12） gene and to assess the protein expressions of BMP12 in infected adipose-derived stem cells. Methods BMP12-cDNA was inserted into adenovirus shuttle plasmid pAdtrack-CMV to generate a recombinant plasmid pAdtrack-CMV-BMP12, then the Pme Ⅰ linearized plasmid pAdtrack-CMV-BMP12 and adenovirus backbone plasmid pAdEasy-1 were electroporated into BJ5183 cells. The DNA containing the identified recombinant plasmid was digested with Pac Ⅰ and transfected into 293A cells to package the adenovirus, followed by identification of the recombinant adenovirus by means of observation of green fluorescence protein expression under fluorescent microscopy. After amplified in 293A cells, the adenovirus was purified. Then the Ad-BMP12 was used to transduce adipose-derived stem cells. The BMP12 expression was assessed by Elisa. Results Recombinant adenoviral vector carrying BMP12 was constructed successfully, which was confirmed by restriction enzyme digestion and GFP expression. The protein expressions of BMP12 were detected after Ad-BMP12 infected adipose-derived stem cells. Conclusion The recombinant adenoviral vector carrying BMP12 can be successfully constructed, and provides the basis of BMP12 gene therapy.
出处
《中国运动医学杂志》
CAS
CSCD
北大核心
2008年第2期198-201,共4页
Chinese Journal of Sports Medicine
