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采用聚合酶链反应 (PCR)技术对鲤和鲑生长激素cDNA的 5’端和 3’端进行定向改造。将改造后的5种基因分别克隆到大肠杆菌表达质粒 pBV2 2 0进行原核表达 ,以研究鱼生长激素基因在大肠杆菌中的表达水平。实验结果表明 :(1)核糖核蛋白体结合位点 (SD)与起始密码子AUG之间的距离对鱼生长激素的表达水平有影响 ;(2 )鲑生长激素基因的终止密码子UAG可能不会有效终止该基因在大肠杆菌中翻译的进行而造成部分通读 ,加入大肠杆菌强终止码UAA可避免通读和产生超长蛋白 ;(3)对鲤和鲑生长激素cDNA的 5’端和 3’端进行相同的修饰 ,但两者生长激素的表达量却有很大差异 ,说明基因的密码子偏性对表达水平有影响 ;(4 )提高 5’端RNA二级结构自由能和密码子的适配指数有利于外源基因的高效表达
The 5 ’and 3’ ends of carp and salmon growth hormone cDNA were engineered by polymerase chain reaction (PCR). The modified five genes were cloned into E. coli expression plasmid pBV220 prokaryotic expression in order to study the expression of fish growth hormone gene in Escherichia coli. The experimental results showed that: (1) the distance between the ribosome binding site (SD) and the start codon AUG had an influence on the expression of fish growth hormone; (2) the stop codon UAG of the salmon growth hormone gene might Will not effectively stop the translation of the gene in E. coli resulting in partial read-through, adding Escherichia coli strong stop codon UAA to avoid read-through and produce the long protein; (3) on the carp and salmon growth hormone cDNA 5 ’end and 3 ’End of the same modification, but both the expression of growth hormone are very different, indicating that gene codon bias on expression levels have an impact; (4) to improve the 5’ end RNA secondary structure free energy and codon The fit index is conducive to the high expression of foreign genes