版纳微型猪近交系生长激素基因克隆及原核表达研究  

Cloning and Prokaryotic Expression of Growth Hormone Gene in Banna Mini-pig Inbred Line

作　　者：王淑燕[1] 霍金龙[1] 王配[1] 潘伟荣[1] 曾养志[1] 

机构地区：[1]云南农业大学动物科学技术学院云南省版纳微型猪近交系重点实验室,昆明650201

出　　处：《畜牧兽医学报》2012年第10期1669-1676,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31160439);神农大学生科技创新行动基金

摘　　要：本研究旨在获得版纳微型猪近交系(BMI)生长激素基因编码区序列,揭示其原核表达规律。采用RT-PCR方法从版纳微型猪近交系(BMI)脑垂体中克隆生长激素(GH)基因,并将其连接到pMD 18-T载体进行测序和生物信息学分析。克隆得到的GH基因cDNA序列长690bp,其中CDS长651bp,编码216个氨基酸,前26个氨基酸为信号肽序列。多猪种GH氨基酸序列比对表明其在各猪种中高度保守,版纳微型猪近交系的GH氨基酸序列与五指山猪和宁香猪的相似性均为100%,与藏猪、香猪、大乌猪、太湖猪、成华猪、内江猪、荣昌猪、长白猪、约克夏的相似性均为99%,与雅南猪、杜洛克的相似性均为98%。扩增GH成熟肽区编码序列,定向克隆至表达载体pET-32a(+),转化入大肠杆菌Rosseta(DE3)感受态细胞中,用IPTG诱导表达蛋白。SDS-PAGE和Westernblotting分析表明,重组质粒在大肠杆菌中获得了高效表达,融合蛋白以包涵体形式存在,分子质量约为40.6ku。以上结果为进一步探究GH基因对BMI矮小性状的影响奠定了基础。This experiment was conducted to obtain the coding sequence of Banna Mini-pig Inbred Line （BMI） growth hormone （GH） gene and to study its prokaryotic expression. BMI GH gene sequence was cloned from pituitary by RT-PCR and inserted into pMD 18-T vector for sequencing and bioinformatics analysis. The length of GH cDNA sequence was 690 bp, which contained a CDS of 651 bp. GH CDS encoded 216 amino acids with 26 amino acids of signal peptide. It was found that theGH amino acids sequence shared 100%, 100%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 98% and 98% identity with those of Wuzhishan pig, Ningxiang pig, Tibetan pig, Xiang pig, Dawu pig, Taihu pig, Chenghua pig, Neijiang pig, Rongchang pig, Landrace, Yorkshire, Yanan pig and Duroc pig, respectively. Subcloned the GH encoding mature peptide sequence into the prokaryotic expressing vector pET-32a （＋） directionally to constitute the recombinant plasmid pET-32a-GH, then it was transformed into the E. cell Rosseta bacteria for expression under induction of IPTG. SDS-PAGE and Western blotting analysis showed that recombinant plasmid oET-32a-GH was successfullv exoressed in high level The fusinn nrntein expressed in form of inclusion body and the molecular weight was approximate 40.6 ku. These results will lay the foundation for further studies of the effects of GH on the dwarfism of Banna Mini-pig Inbred Line.

关 键 词：版纳微型猪近交系 生长激素 矮小性状 原核表达 

分 类 号：S828[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]