中文摘要：

目的：构建携带小鼠BMPRⅠb基因的慢病毒载体,并转染神经干细胞,为研究BM-PRⅠb在BMPs调控神经干细胞分化中作用奠定基础。方法：利用RT-PCR从小鼠脑组织中获得BMPRⅠb基因,然后定向插入慢病毒表达质粒,进行双酶切及测序鉴定。将鉴定成功的重组慢病毒质粒和另外两种辅助质粒共转染包装细胞,收集、浓缩慢病毒并检测其滴度。利用获得的慢病毒载体转染NSCs,并观察目的基因表达情况。结果：RT-PCR产物经电泳及测序证实克隆成功BM-PRⅠb基因,酶切及测序鉴定成功构建重组慢病毒质粒,共转染293T细胞72h后大部分细胞表达绿色荧光,病毒滴度为5×108 TU/ml。慢病毒感染后的NSCs表达绿色荧光且BMPRⅠb mRNA表达上升。结论：成功构建BMPRⅠb基因慢病毒载体,并成功转染NSCs。

英文摘要：

Objective： To construct lentivirus vectors carring mouse Type Ⅰb bone morphogenetic protein receptor gene and transfect neural stem cells.Method： The gene fragment of BMPRⅠb was obtained by using rt-pcr method with total RNA extracted from mouse brain tissue.Gene recombinant technology was employed to chlone BMPR1b gene to lentivirus vector to construct a recombinant lentivirus plasmid.The plasmid was transfected respectively with two other plasmids into packaging cells.The lentiviral vector lentiCMV-BMPRⅠb/CMVeGFP were collected and tittered.The lentiviral vector lentiCMV-BMPRⅠb/CMVeGFP was used to infect NSCs.After infection,the expression of BMPR Ⅰb was indentified by using rt-pcr.Results： A 1600bp band was seen by rt-pcr amplification.Result： The gene sequencing result confirmed that BMPRⅠb gene was successfully cloned.The recombinant lentivirus plasmid was identified by double-digested with BamHI/XhoI.After transfection,a large number of 293T cells expressed green fluorescence.The concentration of virus titer was 5×108TU/ml.After infection,the expression of BMPR Ⅰb mRNA increased.Conclusion： mouse BMPR Ⅰb gene expression lentiviral vectors are successfully constructed and can transfect neural stem cells.