| 摘要: |
| 【目的】构建秦川牛ADIG基因的重组腺病毒载体，包装并扩繁病毒，为下一步研究该基因在脂肪细胞分化过程中的分子作用机制和相关信号通路奠定基础。【方法】根据NCBI收录的家牛ADIG基因(NM_001113720.1)mRNA序列设计引物，以秦川牛组织样提取的总RNA反转录而成的cDNA为模板，通过RT-PCR和PCR扩增获得目的基因，将其连接到pMD-19-T Simple载体并测序。质粒提取纯化后通过BglⅡ与SalⅠ双酶切，然后胶回收酶切产物，将其连接到穿梭载体pAdTrack/CMV上，构建重组穿梭质粒pAdTrack/CMV-ADIG。将pAdTrack/CMV-ADIG质粒用PmeⅠ酶切线性化，转化含有腺病毒骨架载体pAdEasy-1的大肠杆菌 BJ5183感受态细胞中进行同源重组，构建重组腺病毒质粒pAD-ADIG，并用PacⅠ酶切鉴定，提取质粒后转化大肠杆菌Top10进行扩繁。用PacⅠ酶切线性化pAD-ADIG载体并回收质粒大片段，转染293A细胞进行病毒包装，然后完成病毒扩繁和病毒滴度的测定。【结果】PCR扩增获得了399 bp ADIG基因CDS区；成功构建了重组穿梭质粒pAdTrack/CMV-ADIG和重组腺病毒质粒pAD-ADIG。经检测，ADIG重组腺病毒包装成功，扩繁后得到了高滴度的腺病毒（1×108 PFU/mL）。【结论】成功构建了秦川牛ADIG基因的重组腺病毒表达载体pAD-ADIG，完成了病毒包装和扩繁。 |
| 关键词: ADIG 腺病毒 同源重组 秦川牛 |
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| 基金项目:国家转基因重大专项（2013ZX08007-002）；“十二五”国家“863”计划项目(2013AA102505)；国家现代农业（肉牛牦牛）产业体系专项（CARS-38）；国家自然科学基金项目（31272411）；陕西省科技统筹创新工程计划项目（2014KTZB02-02） |
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| Construction of recombinant adenovirus vector and viral packaging of ADIG gene in Qinchuan cattle |
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ZHANG Qiong,JIANG Bi-jie,CHENG Gong,et al
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| Abstract: |
| 【Objective】The aim of this project was to construct recombinant adenovirus vector of ADIG gene,then package and amplify the corresponding recombinant adenovirus,which would improve further study on its molecular mechanism and related signaling pathway on adipocyte differentiation.【Method】The ADIG gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR),and the template was mixed cDNA from different tissues of Qinchuan cattle.Then PCR product was inserted to pMD-19-T Simple vector and confirmed by sequencing.The plasmids were prepared and then digested with BglⅡ and SalⅠ before gel extraction.The product was then inserted into the adenoviral shuttle plasmid pAdTrack/CMV to obtain pAdTrack/CMV-ADIG.The linearized plasmid pAdTrack/CMV-ADIG was co-transformed into competent cells BJ5183 containing adenoviral backbone plasmid pAdEasy-1 to obtain recombinant adenovirus vector pAD ADIG.The recombinant adenovirus vector pAD-ADIG was verified by enzyme cleavage with PacⅠ.The confirmed recombinant adenovirus plasmid pAD-ADIG was transformed into E.coli Top10 competent cells for further amplification.Plasmid of pAD-ADIG was then prepared and digested with PacⅠ to be totally linearized before being transfected into 293A cell line for packaging and amplification of the recombinant adenovirus.【Result】PCR product with length of 399 bp was obtained and the shuttle vector pAdTrack/CMV-ADIG together with the recombinant adenovirus vector pAD-ADIG were obtained and verified by sequencing.ADIG recombinant adenovirus was packaged successfully and high concentration (1×108 PFU/mL)of ADIG adenovirus was obtained.【Conclusion】A recombinant adenovirus vector containing ADIG gene was successfully constructed to package and amplify the recombinant adenovirus. |
| Key words: ADIG adenovirus vector homologous recombination Qinchuan cattle |
