骨形成蛋白9腺病毒表达载体的构建及鉴定

Construction and identification of a adenovirus vector containing BMP9 gene

目的构建骨形态发生蛋白9(BMP9)基因腺病毒表达载体及稳定产毒细胞系,为观察其对T2DM糖、脂代谢异常的干预效应奠定基础。方法质粒pCMV/BMP9双酶切克隆至腺病毒载体Ad5.F,构建重组腺病毒表达载体Ad5.BMP9.F,酶切测序鉴定;转包装细胞,经抗性筛选检测病毒滴度,挑选滴度较高的稳定产毒细胞克隆进行逆转录PCR(RT-PCR)鉴定。结果建立BMP9基因腺病毒表达载体Ad5.BMP9.F经酶切及测序证实目的基因插入位点及读码框架正确;建立稳定产毒细胞克隆最高病毒滴度达7.4×105 CFU/mL,RT-PCR证实BMP9基因整合其中并稳定表达。结论成功构建含BMP9基因的腺病毒表达载体及其稳定产毒细胞系。

Objective To establish a adenovirus vector containing BMP9 gene and a stable toxin-producing cell line,for observing the intervention effect of the glucolipid metabolic on type2 diabetes mellitus.Methods The plasmid pCMV/BMP9 was digested by EcoRI/BamHI,then cloned to adenovirus vector Ad5.F,constructed the recombinant plasmid Ad5.BMP9.F.The plasmid Ad5.BMP9.F was evaluated by enzyme-digestion and sequencing.The plasmid Ad5.BMP9.F was transfect to packaging cell line293.The titer of the recombinant virus was detected.The high-titer cell lines were chosen for culture,and then evaluated by RTPCR.Results The retroviral vector containing BMP9 gene was established successfully,which was confirmed by enzyme-digestion and sequence analysis.The toxin-producing cell lines was established.The highest titer of the recombinant virus was 7.4×10^5CFU/mL.BMP9 gene can express stably in 293 cells.Conclusion A adenovirus vector containing BMP9 gene and a high-titer vector production cell line are successfully constructed.