摘要
【目的】构建广西巴马小型猪载脂蛋白E基因(ApoE)真核表达载体,为生产转ApoE基因的广西巴马小型猪奠定基础。【方法】采用RT-PCR从广西巴马小型猪肝组织中扩增出ApoE基因编码序列,将该基因连接至pEGFP-C1载体上,利用双酶切和测序对重组质粒pEGFP-C1-ApoE进行鉴定,并将重组质粒pEGFP-C1-ApoE转染PK15细胞。【结果】广西巴马小型猪ApoE基因编码区序列长954bp,编码317个氨基酸,与GenBank已公布的猪ApoE基因cDNA序列(NM_214308)对应区段同源性为100%。构建的重组质粒pEGFP-C1-ApoE转染PK15细胞48h后,在荧光显微镜下转染细胞发出荧光,细胞形态清晰,可见细胞核。【结论】广西巴马小型猪ApoE基因能与pEGFP-C1载体重组构建pEGFP-C1-ApoE真核表达载体,有效表达出ApoE蛋白。
Objective An eukaryotic expression vector of Apolipoprotein E （ApoE） gene in Guangxi Bama mini-pig was constructed to provide references for producing Guangxi Bama mini-pig with ApoE. Method The coding sequence of ApoE gene was amplified from liver of Guangxi Bama mini-pig by RT-PCR, and this sequence was inserted into pEGFP-C1 vector. The recombinant plasmids pEGFP-C1-ApoE were identified by sequence and double digestion, and then the positive plasmids were transfected into PK15 cells. ResultWith the length of 945 bp, the ApoE gene coding sequence of Guangxi Bama mini-pig encoded 317 amino acids and shared 100% homologue to the previously reported cDNA sequence in GenBank（NM_214308）. After 48 hours transfection of the recombinant plasmids pEGFP-C1-ApoE into the PK15 cells, the cells had visible expression of fluorescent and cell nucleus. Conclusion The ApoE gene of Guangxi Bama mini-pig can recombine into pEGFP-C1 vector and effectively express the Apolipoprotein E.
出处
《南方农业学报》
CAS
CSCD
北大核心
2013年第6期1022-1025,共4页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31060293)
广西科技基础条件平台建设项目(09-090-09)
