鳜(Siniperca chuatsi)生长激素基因克隆和原核表达

CLONING AND PROKARYOTIC EXPRESSION OF GROWTH HORMONE GENE IN SINIPERCA CHUATSI

采用逆转录-聚合酶链式反应(RT-PCR)技术,从鳜脑垂体总RNA中扩增生长激素(GH)成熟肽基因,将成熟生长激素的cDNA定向克隆到表达载体pET-32a(+),并转入大肠杆菌BL21(DE3)中。结果表明,鳜生长激素(GH)基因含开放阅读框(ORF)615个核苷酸,编码204个氨基酸,蛋白分子量为23kDa,等电点为7.07,其中酸性氨基酸占10.78%,碱性氨基酸占12.74%,疏水氨基酸为占44.12%,极性氨基酸占32.35%。在IPTG终浓度1.0mmol/L、温度37℃和培养时间4h的最佳诱导表达条件下,鳜生长激素基因(GH)在大肠杆菌中获得高效表达,重组菌体裂解物经SDS-PAGE可检测到分子量约为43kDa的鳜生长激素与硫氧还蛋白(Thioredoxni,Trx)融合蛋白,其表达量约占菌体总蛋白58%。Western印迹分析也证实含6×His标签的重组融合蛋白能够很好地与抗6×His单抗发生反应。本研究为下一步鳜生长激素基因(GH)的生物学功能及应用奠定了基础。

GH cDNA was amplified and cloned from total RNA isolated from pituitary gland of Siniperca chuatsi by RT-PCR.Sequence analysis showed an open reading frame （ORF） of S.chuatsi growth hormone gene in size of 615bp,encoding 204aa of 23kDa and pI at 7.07,comprising 10.78% acidic,12.74% basic,44.12% hydrophobic,and 32.35% hydrophilic.The cDNA of mature growth hormone was directionally cloned in pET-32a（＋）,and then the recombinant expression vector was transformed into E.coli BL21 （DE3 plys）.After induction by IPTG,an expected 43kDa fusion protein was found by SDS PAGE analysis,accounting for 58% of the total protein.Western blot demonstrated that the recombinant fusion protein can be recognized by anti-6×His monoclonal antibody.