摘要
利用反转录-聚合酶链式反应(RT-PCR)方法,从新疆阿勒泰×中国美利奴杂交F1代绵羊脑垂体总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的3条绵羊序列相比共有7个碱基的差异,除第472位和529位的碱基差异能够引起氨基酸序列的变化外,其余均为无义突变,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/oGHcDNA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。
Total RNA of pituitary was extracted from Xinjiang Ahay × Chinese Merino F1 sheep. The eDNA 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 nucleotlde sequencing showed that the cloned c.DNA had positive reading frame and its length was 654 bp. Its nucleotide sequences were compared with three piece of published ovine growth hormone gene （ oGH ） eDNA sequences and the result showed that there were differences between 7 bases. These variation are all silent except the two base changes found at 472 and 529 nt. This suggested that the oGH gene from Ahay x Chinese merino cotains polymorphisms between different breeds. The oGH eDNA 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 ELLSA suggested that there were recombinant oGH protein in the cultural supernate of the transfect cells.
出处
《新疆农业科学》
CAS
CSCD
北大核心
2009年第3期466-473,共8页
Xinjiang Agricultural Sciences
基金
国家发改委高技术产业化示范工程项目(00041296800)
新疆生产建设兵团农业科学研究与技术开发计划项目(NKB01NKYNK13XM)
关键词
绵羊
生长激素
克隆
表达
sheep
growth hormone
cloning
expression
