| 摘要: |
| 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。
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| 关键词: 鳜, 生长激素, 基因克隆, 原核表达 |
| DOI：10.11693/hyhz201003011011 |
| 分类号: |
| 基金项目:湖南省高等学校科学研究项目(重点项目), 08A007 号; 湖南省高等学校科学研究项目(优秀青年项目), 09B011 号; 长沙市科技局重点项目, K070728—31 号; 湖南省大学生研究性学习和创新性实验计划项目, 284 号 |
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| CLONING AND PROKARYOTIC EXPRESSION OF GROWTH HORMONE GENE IN SINIPERCA CHUATSI |
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LIU Zhen,LUO Xiao-Hua,LU Shuang-Qing,XIE Di-Zhi,FANG Zhi-Jia,XIAO Ke-Yu
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1.Department of Biotechnology＆ Environment Science, Changsha University;2.College of Animal Science and Technology, Hunan Agricultural University
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| Abstract: |
| 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.
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| Key words: Siniperca chuatsi, Growth hormone, Gene clone, Prokaryotic expression |
