摘要
根据已克隆的半滑舌鳎(Cynoglossus semilaevis)生长激素(GH)基因和生长激素受体Ⅰ(GHR-Ⅰ)基因的cDNA序列信息设计引物,用于扩增半滑舌鳎GH、GHR-Ⅰ基因序列。随后,分别利用表达载体pET-32a和pGEX-6P-1成功构建了包含半滑舌鳎GH、GHR-Ⅰ基因ORF全序列及不同ORF片段的6个重组质粒,并将获得的重组质粒在表达宿主菌大肠杆菌(Escherichia coli)BL21(DE3)中,用IPTG诱导表达。SDS-PAGE电泳结果显示:重组GH的pET-32a-CSGH质粒在40 kD处有特异性的蛋白条带,重组GHR-Ⅰ胞内区的pGEX-6P-1-CSGHR1-2质粒在70 kD处发现诱导的蛋白条带,其表达量分别占细胞蛋白总量的37.6%和20.2%;而构建的pGEX-6P-1-CSGH重组质粒和含有GHR-Ⅰ基因跨膜区的重组质粒(pET-32a-CSGHR1-1、pET-32a-CSGHR1-2和pGEX-6P-1-CSGHR1-1)均未获得表达产物。由此可见,半滑舌鳎GHR-Ⅰ基因的跨膜区可能影响其在大肠杆菌中的表达。研究结果为实现半滑舌鳎GH、GHR-Ⅰ制剂的开发及应用于养殖生产实践奠定基础。
In this study,the GH and GHR-I sequences of the tongue sole（Cynoglossus semilaevis） were sub-cloned into the expression vectors pET-32a and pGEX-6P-1,respectively.Subsequently,these recombinant plasmids were expressed in Escherichia coli BL21（DE3） cells.The total bacterial proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）.As a result,a new specific GH band expressed by using the pET-32a vector（pET-32a-CSGH） was found with molecular mass of about 40 kD,and a new specific GHR-I band expressed by using the pGEX-6P-1 vector（pGEX-6P-1-CSGHR1-2） was found with molecular mass of about 70 kD.The GH and GHR-I recombinant proteins were expressed and composed of 37.6% and 20.2% of the total bacterial proteins,respectively.However,pGEX-6P-1-CSGH and the other three recombinant plasmids（pET-32a-CSGHR1-1,pET-32a-CSGHR1-2 and pGEX-6P-1-CSGHR1-1） containing the sequences of GHR-I trans-membrane domain were not successfully expressed.The relevant results showed that the trans-membrane domain of the GHR-I might have an influence on its prokaryotic expression from C.semilaevis.These findings may lay a theoretic foundation for applying the growth-promoting effects of GH and GHR-I to the aquaculture industry.
出处
《中国水产科学》
CAS
CSCD
北大核心
2012年第6期956-962,共7页
Journal of Fishery Sciences of China
基金
国家自然科学基金资助项目(30871913,31201981)
山东省泰山学者工程专项(2009.03-2014.02)
山东省自主创新成果转化重大专项(2009ZHZX1A1201)
关键词
半滑舌鳎
生长激素基因
生长激素受体Ⅰ基因
原核表达
Cynoglossus semilaevis
growth hormone gene
growth hormone receptor I gene
prokaryotic expres-sion
