【作者】 沈敏； 王新华； 刘守仁； 钟发刚； 杨华； 连宏军； 陈晓军； 甘尚权；

【Author】 SHEN Min, WANG Xin-hua, LIU Shou-ren, ZHONG Fa-gang, YANG Hua, LIAN Hong-jun, CHEN Xiao-jun, GAN Shang-quan(Xinjiang Research Academy of Farms and Land Reclamation, Shihezi, Xinjiang 832000, China)

【机构】 新疆农垦科学院畜牧兽医研究所；

【摘要】 利用反转录—聚合酶链式反应(RT-PCR)方法,从新疆绵羊脑垂体总RNA扩增出编码绵羊生长激素(oGH)基因序列,T-A克隆入载体质粒pT-Adv。测序结果表明所克隆的oGH cDNA在重组质粒的阅读框架是正确的,共含有654个碱基,其核苷酸序列与已知序列相比有4个碱基的差异,但翻译后的氨基酸序列完全一致,说明从新疆阿勒泰×中国美利奴杂交绵羊中获得的羊生长激素存在有不同品系间的基因多态性变化。将重组质粒pT-Adv/oGH cDNA定向克隆入真核表达载体质粒pcDNA3.1/myc-HisA,构建成重组oGH基因的真核表达载体pcDNA3.1A/oGH。利用脂质体转染法,将重组质粒导入到小鼠骨髓瘤(SP2/0)细胞中,经间接ELISA检测,证明在转染细胞的上清中存在有目的基因产物的外泌表达。更多还原

【Abstract】 Total RNA of pituitary was extracted from sheep in Xinjiang. The cDNA encoding growth hormone (GH) gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) method using isolated total RNA as template. The product of RT-PCR was inserted to plasmid pT-Adv by T-A cloning. The result of nucleotide sequencing showed that the cloned cDNA had positive reading frame, which consisted of 654 bp. Its nucleotide sequence was compared with the published oGH cDNA sequences and the results showed that there were differences between 4 bases. But their amino acid sequences were consistent and this suggested that the ovine growth hormone gene from Altay x Chinese merino containing polymorphisms between different breeds. The oGH cDNA was subcloned into the eukaryote expression vector plasmid pcDNA3.1A and constructed the recombinant eukaryote expression plasmid pcDNA3.1A/oGH cDNA. By liposome transfection, the recombinant plasmid were transferred into SP2/0 cells. The result of indirect ELISA suggested that there be recombinant oGH protein in the cultural supernate of the transfect cells.更多还原