【作者】 林涛；

【导师】 颜其贵； 郭万柱；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 本研究选用齐口裂腹鱼(Schizothorax prenanti)作为实验材料,从齐口裂腹鱼脑垂体中提取总RNA。设计合成了一对齐口裂腹鱼生长激素基因的特异性引物,上游引物加入EcoRⅠ酶切位点;下游引物加入XholⅠ酶切位点。应用逆转录-聚合酶链式反应(RT-PCR)技术克隆得到编码齐口裂腹鱼生长激素(SGH)基因的cDNA,并定向克隆到pMD18-T载体上。正反向测序结果分析表明:克隆的齐口裂腹鱼生长激素基因cDNA全长633bp,包含完整的开放阅读框(ORF),编码210个氨基酸。生物信息学分析结果表明其分子量为23.55KD,等电点为5.46;疏水氨基酸占36.7%,亲水氨基酸占28.57%,碱性氨基酸占10.47%,酸性氨基酸占12.38%。将得到的序列在GenBank和EMBL数据库中进行了同源比较,结果显示:本研究克隆到的齐口裂腹鱼GH基因与GenBank中登记的其他鲤科鱼类的生长激素基因的同源性在92%-97%之间,影响蛋白质高级结构的保守二硫键为2个。将齐口裂腹鱼生长激素成熟肽(mSGH)基因片段定向插入融合表达载体pET-32a(+)质粒中,转化大肠杆菌BL21,用PCR方法筛选阳性克隆;以XholⅠ和EcoRⅠ酶切鉴定重组质粒。以IPTG诱导融合蛋白表达,SDS-PAGE分析表明mSGH以融合蛋白的形式得到了表达,分子量约为41KD,与预期的大小一致。融合蛋白表达量占菌体总蛋白量的42.6%。证明所构建的齐口裂腹鱼GH的重组质粒能高效表达,命名为pET-mSGH。通过投喂齐口裂腹鱼幼鱼实验结果表明:实验组幼鱼食量明显增强,体重和体长的增长率分别比对照组高20.82%和17.35%,证明该蛋白具有生物活性。为进一步开展齐口裂腹鱼生长激素的应用、真核表达研究及研制新型调节鱼类生长的基因制剂的应用奠定了的基础。更多还原

【Abstract】 The cDNA encoding growth hormone(GH) peptide was amplified by reverse transcription polymerase chain reaction(RT-PCR),based on the total RNA isolated from the pituitary of Schizothorax prenanti.The amplified cDNA was inserted into plasmid pMD18-T.The sequencing result showed that the length of SGH cDNA fragment is 633bp,and the ORF included of 633bp,encoding 210 amino acids with molecular mass of 23.55 Kda and pI of 5.46. The SGH peptide included 10.7%basic amino acids,12.38%acidic amino acids,28.57% hydrophilic amino acids,36.7%hydrophobicity amino acids.By blasting the homologous sequences in GenBank database,the sequence of Schizothoraxprenanti GH from pituitary is 92%-97%homologous compared with the previously cloned GH of other fish in the same family.The SGH cDNA fragment was inserted into pET-32a(+) to construct the expression plasmid, and the recombinant plasmid was identified by PCR and digested with EcoRⅠand XhoⅠ,and the positive recombinant plasmid was named as pET-mSGH.Then the pET-mSGH was transformed into E.coli BL21 competent cells.The mRNA and expressed protein were assayed by SDS-PAGE.The results showed that a new protein band was found in SDS-PAGE with molecular mass of about 41 KDa.These indicated that the Schizothorax prenanti GH gene was correctly transcribed and translated in the recombinant bacteria.Throught animal experiment,it is demonstrated that the test fishes was higer in growth rate of body weight and stem length,respectively 20.82%and 17.35%higer than control group, additionally,the appetition of test fishes was enhanced obviously,and the protein was proved to be biological active consequently.Conclusively,this study has established substantial bases for further use of GH,eukaryotic expression of GH,and developing new genetic praeparatum regulating the growth of fish.更多还原