摘要
目的构建双表达骨形态发生蛋白(Bone morphogenic protein,BMP)2、9腺病毒重组体并进行鉴定。方法自单一表达的BMP2或BMP9 AdEasy质粒上扩增BMP2和BMP9片段,先后亚克隆至穿梭质粒pASG2,获得双表达穿梭质粒pASG2-BMP2、9。酶切及PCR鉴定确认、测序正确后同源重组获得双表达BMP2、BMP9腺病毒质粒,转染至HEK-293细胞中包装和扩增得到高滴度双表达BMP2、BMP9腺病毒,体外感染C3H10细胞,RT-PCR鉴定并观察其早期诱导成骨情况。结果成功构建双表达BMP2、BMP9的腺病毒,滴度约为1010IU/mL,RT-PCR证实双表达腺病毒在C3H10细胞中表达,其感染的C3H10细胞早期碱性磷酸酶含量较单一表达的BMP2或BMP9腺病毒组增加。结论成功构建双表达BMP2、9的重组腺病毒载体,为进一步研究BMP2和BMP9的协同成骨作用和制备高效的组织工程人工骨提供了有利的工具。
Objective To reconstruct and identify the recombinant adenovirus co-expressing BMP2 and BMP9.Method The genes of BMP2 and BMP9 were amplified from AdEasy vector by PCR and sub-cloned into pASG2 vector.The co-expression shuttle plasmid pASG2-BMP2,9 was confirmed by restriction endonuclease digestion,PCR and gene sequencing,then pASG2-BMP2,9 was electro-transducted into competent AdEasier cells to acquire recombinant adenovirus plasmid.Then,the recombinant vector was transfected into HEK293 cells and high-titer recombinant adenovirus（AdBMP2,9） was gained after rounds of amplification.The expression and bone induction capacity of AdBMP2,9 was observed in C3H10 cells.Result AdBMP2,9 was constructed successfully and the virus titer was 1010 IU/mL after amplification.AdBMP2,9 could express and induce alkaline phosphatase activity in C3H10 cells.Conclusion The recombinant adenovirus co-expressing BMP2 and BMP9 was constructed successfully,which provides a useful tool for bone tissue engineering.
出处
《中国微生态学杂志》
CAS
CSCD
2011年第7期593-596,共4页
Chinese Journal of Microecology
基金
国家高技术研究发展(863)计划(2007AA02Z4B5)
国家自然科学基金(31000434)
