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秦川牛FABP4基因重组腺病毒载体的构建与鉴定
引用本文:魏胜娟,昝林森,王洪宝,成功,季舒涵,王虹,付常振,姜碧杰.秦川牛FABP4基因重组腺病毒载体的构建与鉴定[J].中国农业科学,2013,46(7):1434-1440.
作者姓名:魏胜娟  昝林森  王洪宝  成功  季舒涵  王虹  付常振  姜碧杰
作者单位:1.西北农林科技大学动物科技学院,陕西杨凌 712100
2.国家肉牛改良中心,陕西杨凌 712100
基金项目: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)
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1.58×109 PFU?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。

关 键 词:秦川牛      FABP4基因      pAdTrack-CMV      pAdEasy-1      腺病毒
收稿时间:2012-10-16

Construction of the Recombinant Adenovirus of FABP4 Gene of Qinchuan Cattle
WEI Sheng-Juan,ZAN Lin-Sen,WANG Hong-Bao,CHENG Gong,JI Shu-Han,WANG Hong,FU Chang-Zhen,JIANG Bi-Jie.Construction of the Recombinant Adenovirus of FABP4 Gene of Qinchuan Cattle[J].Scientia Agricultura Sinica,2013,46(7):1434-1440.
Authors:WEI Sheng-Juan  ZAN Lin-Sen  WANG Hong-Bao  CHENG Gong  JI Shu-Han  WANG Hong  FU Chang-Zhen  JIANG Bi-Jie
Institution:1.College of Animal Science and Technology, Northwest A & F University, Yangling 712100, Shaanxi; 2.National Beef Cattle Improvement Center in China, Yangling 712100, Shaanxi
Abstract:【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 PmeⅠ, 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 PacⅠ, 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 1.58×109 PFU•mL-1.【Conclusion】In this experiment, the recombinant adenovirus vector carrying FABP4 gene was constructed successfully and high titer adenovirus was acquired.
Keywords:Qinchuan cattle  FABP4 gene  pAdTrack-CMV  pAdEasy-1  recombinant adenovirus
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