秦川牛SREBP1基因重组腺病毒载体的构建与病毒包装  

Construction of Recombinant Adenovirus Vector Specific to SREBP1 Gene ofQinchuan Cattle and the Packaging and Amplifying of the Corresponding Adenovirus

作　　者：付常振[1] 昝林森[1,2] 王虹[1] 姜碧杰[1] 成功[1,2] 王洪宝[1,2] 朱光星[1] 李耀坤[1] 王洪程[1] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]国家肉牛改良中心,杨凌712100

出　　处：《畜牧兽医学报》2013年第8期1323-1329,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：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);国家自然科学基金(31272411);"十二五"国家863计划(2011AA100307-02);教育部"长江学者和创新团队发展计划"(IRT0940);"十二五"国家科技支撑计划(2011BAD28B04-03)

摘　　要：克隆秦川牛的SREBP1基因并构建重组腺病毒表达载体,包装扩繁获得高滴度病毒,拟为在细胞水平上开展基因功能的研究奠定基础。本试验以秦川牛脂肪组织为试验材料,提取总RNA并反转得到cDNA,以Gen-Bank收录的牛的SREBP1基因mRNA序列设计引物,PCR扩增SREBP1基因与克隆载体pMD19-T Simple连接并测序鉴定。挑选测序正确的SREBP1基因酶切后连接到腺病毒穿梭载体上构建pAdTrack-CMV-SREBP1表达载体,用PmeⅠ限制酶酶切线性化,然后转染到含有骨架载体pAdEasy-1的E.coli BJ5183感受态进行同源重组,得到腺病毒重组载体pAd-SREBP1。用PacⅠ限制酶酶切线性化pAd-SREBP1载体并回收质粒大片段,转染293A细胞包装病毒并扩繁提高病毒滴度,绿色荧光蛋白(GFP)标记法测定腺病毒的滴度。本试验成功克隆了秦川牛的SREBP1基因,测序结果与GenBank收录的牛的基因序列比较有2处位点突变,均已排除扩增酶的保真性不高等外界因素造成的。将SREBP1基因与穿梭载体连接构建了pAdTrack-CMV-SREBP1表达载体,并与骨架载体重组得到重组腺病毒载体pAd-SREBP1,用PacⅠ酶切线性化包装病毒,扩繁得到病毒滴度为1.5×109 GFU·mL-1高滴度病毒。本研究成功克隆秦川牛SREBP1基因并重组成病毒重组子,包装扩繁得到高滴度腺病毒。The objective of this research was to construct recombinant adenovirus vector specific to SREBP1 gene of Qinchuan cattle and further package and amplify recombinant adenovirus carrying SREBP1 gene,aimed at studying SREBP1 gene function at cellular level.A pair of exclusive primers was designed according to the GenBank sequence information of SREBP1 gene to amplify the complete CDS area of SREBP1 gene via polymerase chain reaction（PCR）.The obtained PCR products were then sub-cloned into pMD19-T simple vector.The confirmed fragments containing CDS area of SREBP1 gene were first digested from clone vector and then insert into the shuttle vector to construct the pAd-Track-CMV-SREBP1 plasmid.The resultant plasmid was linearized by digesting with restriction endonuclease PmeⅠ and subsequently transformed into BJ5183 containing pAdEasy-1 to obtain the expression vector pAd-SREBP1.Overall,the confirmed recombinant adenovirus plasmid pAd-SREBP1 was digested with PacⅠ and transfected into 293A cell line to package and amplify the recombinant adenovirus.The viral titer was determined by GFP labeled method.SREBP1 gene was cloned from Qinchuan cattle with two mutations in the DNA fragments.The two mutations were proved to be caused probably by the varietal difference,not by external factors.Recombinant plasmid pAdTrack-CMV-SREBP1 and pAd-SREBP1 were successfully constructed to the package and amplify the recombinant adenovirus with 1.5×109 GFU·mL-1 as its titer.

关 键 词：秦川牛 SREBP1 腺病毒 

分 类 号：S823[农业科学—畜牧学]