摘要
【目的】旨在通过构建秦川牛FABP4基因的重组腺病毒载体,为在细胞水平研究FABP4基因的功能和作用机制做准备。【方法】根据GenBank收录的牛FABP4基因mRNA序列设计引物,PCR扩增并克隆测序。将目的基因连接到穿梭载体pAdTrack-CMV上并用PmeⅠ线性化后,转化含有腺病毒骨架载体pAdEasy-1的E.coli BJ5183感受态细胞进行同源重组,得到重组质粒pAd-FABP4,并用PacⅠ酶切鉴定。将经过PacⅠ线性化的pAd-FABP4转染HEK 293A细胞进行病毒包装和扩增,TCID50法测定病毒滴度。【结果】经测序鉴定本次克隆的FABP4序列与GenBank收录的一致。酶切鉴定、绿色荧光观察、PCR及测序检测均证明,重组腺病毒载体构建成功并获得重组腺病毒AD-FABP4,病毒滴度为1.58×109PFU.mL-1。【结论】成功构建了秦川牛FABP4基因重组腺病毒载体并获得高滴度的重组腺病毒。
[ Objective ] The aim of this study was to construct the recombinant adenovirus vector with FABP4 gene of Chinese Qinchuan cattle, so as to provide a basis for studying FABP4 gene functions and mechanisms at cell level. [Method] The primers were designed according to the FABP4 mRNA sequence in GenBank, and the gene was cloned by RT-PCR and then sequenced. The bovine gene fragments containing both FABP4 gene and restriction enzyme were inserted into a shuttle vector pAdTrack-CMV to construct the recombinant shuttle vector pAdTrack-CMV-FABP4. After identifying by digestion and sequencing, pAdTrack-CMV-FABP4 plasmid was linearized by Pine I, and then it was transformed into E. coli BJ5183 competent cells containing backbone vector pAdeasy-1 to obtain recombinant vector by homologous recombination. Then the positive plasmid was linearized by Pac I, and transfected into HEK 293A cells for virus packing, amplification and titer testing by TCID50. [Result] The results of enzyme digestion, sequencing and regular PCR detection showed that the recombinant overexpression vector containing FABP4 CDS region was successfully constructed. The infectious titer of the virus AD-FABP4 was 1.58x 10^9 PFU-mL^-l. [ Conclusion] In this experiment, the recombinant adenovirus vector carrying FABP4 gene was constructed successfully and high titer adenovirus was acauired.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第7期1434-1440,共7页
Scientia Agricultura Sinica
基金
国家"863"计划项目(2011AA100307-02
2013AA102505)
国家自然科学基金项目(31272411
31000998)
国家"十二五"科技支撑计划(2011BAD28B04-03)
国家转基因生物新品种培育重大专项(2011ZX08007-002)
国家肉牛牦牛产业技术体系(CARS-38)
长江学者和创新团队项目(IRT0940)
陕西省科技统筹创新工程计划(2011KTCL02-07)
