重组宁乡猪生长激素真核表达质粒(pCI-GH-EGFP)的构建及其在Vero细胞中的表达  

Construction of recombinant eukaryotic expression plasmid of growth hormone in Ningxiang pigs(pCI-GH-EGFP) and its expression in Vero cells

作　　者：王文策[1,2] 翟双双 耿梅梅[2] 褚武英[3] 魏永伟[4] 于涟[4] 杨琳[1] 

机构地区：[1]华南农业大学动物科学学院/国家生猪种业工程技术研究中心,广州510642 [2]中国科学院亚热带农业生态研究所,长沙410125 [3]长沙大学生物工程与环境科学系,长沙410003 [4]浙江大学动物预防医学研究所,杭州310027

出　　处：《黑龙江畜牧兽医》2016年第7期86-89,277,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学青年基金项目(31501959);国家现代农业产业技术体系水禽产业技术体系项目(nycytx-45-09);华南农业大学校长基金项目(4300-k12226)

摘　　要：为了构建宁乡猪生长激素(GH)真核表达系统,并转染非洲绿猴肾细胞系(Vero)观察其表达情况,试验选用宁乡猪脑垂体组织总RNA反转录获得的c DNA为模板,克隆宁乡猪生长激素(nxGH)基因序列,将其定向插入p CI载体构建重组质粒p CI-GH,并将增强型绿色荧光蛋白(EGFP)基因酶切后连入质粒p CI-GH中,构建真核表达载体p CI-GH-EGFP。将该重组质粒在脂质体作用下转染Vero细胞,倒置荧光显微镜下观察到GH-EGFP融合蛋白可稳定表达。结果表明:宁乡猪生长激素基因编码区序列含有1个由651个核苷酸组成的开放阅读框(ORF),编码216个氨基酸残基。To construct an eukaryotic expression system of growth hormone （GH） in Ningxiang pigs and transfect African green monkey kidney cells （Vero） to observe its expression, a eDNA obtained from total RNA of pituitary tissues in Ningxiang pigs by reverse transcription was selected as a template. Ningxiang porcine growth hormone （nxGH） gene sequence was cloned, and then directionally inserted into pCI vector to construct a recombinant plasmid pCI -GH. The enhanced green fluorescence protein （EGFP） gene was ligated into the plasmid pCI -GH to construct a eukaryotie expression vector pCI - GH - EGFP. Vero cells were transfected with pCI - GH - EGFP under the action of liposomes, and it was found that GH - EGFP fusion protein could stably express, which was observed under an inverted fluorescence microscope. The results showed that the coding region sequence of nxGH gene contained an open reading frame of 651 bp, encoding for a protein of 216 amino acid residues.

关 键 词：宁乡猪 生长激素(GH)基因 增强型绿色荧光蛋白(EGFP) 克隆 真核表达 

分 类 号：Q575.11[生物学—生物化学]