【作者】 巫雪艳；

【导师】 刘世贵； 高荣；

【作者基本信息】 四川大学 ， 遗传学， 2003， 硕士

【摘要】 本研究选用草鱼(Ctenspharynogodom idellus)作为实验材料，从草鱼脑垂体中提取总RNA。设计合成了一对草鱼生长激素基因的特异性引物，上游引物加入BamHI酶切位点；下游引物加入EcoRI酶切位点。应用逆转录-聚合酶链式反应(RT-PCR)技术克隆得到编码草鱼生长激素(CGH)的基因cDNA，并定向克隆到pUC18载体上。正反向测序结果分析表明：克隆的草鱼生长激素基因cDNA全长669bp，开放阅读框(ORF)包含633个碱基，编码210个氨基酸。分子量为23.6KD，等电点为6.28；疏水氨基酸占43.3％，亲水氨基酸占32.4％，碱性氨基酸占12.4％，酸性氨基酸占11.9％。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：本研究克隆到的草鱼GH基因与GenBank中登记的X60474草鱼GH基因有12个碱基的差异，编码的氨基酸有3个氨基酸残基的差异，同源性为98％，影响蛋白质高级结构的保守二硫键为2个。证明本实验成功克隆到了草鱼生长激素基因的编码序列。 采用DNA重组技术将已克隆到的草鱼生长激素(CGH)cDNA片段定向插入融合表达载体pGEX-4T-1质粒中，转化大肠杆菌BL21，用菌落PCR和质粒PCR方法筛选阳性克隆；以BamHⅠ和EcoRⅠ酶切鉴定重组质粒。以IPTG诱导融合蛋白表达，并作RT-PCR及SDS-PAGE分析。RT-PCR分析确证重组质粒在大肠杆菌中能正确转录；SDS-PAGE分析证明诱导重组质粒表达了融合蛋白，分子量约为50KD，与预期的26 KD的GST带和23.6KD的草鱼生长激素基因编码蛋白质构成的融合蛋白大小一致。融合蛋白表达量占总蛋白量的20％。实验证明得到高效表达草鱼GH的重组质粒，命名为pGCGH。 本研究克隆到草鱼生长激素基因cDNA序列，并首次选用融合表达载体作为鱼类生长激素基因的原核表达工具，成功构建了重组CGH原核表达质粒，实现了CGH的高效表达。为进一步开展草鱼生长激素的应用和生物活四川大学硕士学位论文草鱼生长激素墓因的克隆及原核表达研究性检测分析、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坚实的基础。更多还原

【Abstract】 Total RNA was isolated from grass carp (Ctenspharynogodom idellus) pituitary. The cDNA encoding growth hormone (GH) peptide was amplified by reverse transcription polymerase chain reaction (RT-PCR) method using isolated total RNA as template. The amplified cDNA fragment was inserted to plasmid pUC 18. The sequencing result shows that length of CGH cDN A fragment is 669bp. ORF include of 633bp, encoding 210 amino acids with molecular 23.6 KDa weight and pI is 6.28. CGH peptide include 12.4% Basic Amino Acids, 11.9% Acidic Amino Acids, 32.4% Hydrophilic Amino Acids, 43.3% Hydrophonic Amino Acids. By blasting the homologous sequences in GenBank databases, the sequence of grass carp GH cDNA from pituitary is 98% homologous compared with the previously cloned GH cDNA of grass carp .The CGH cDNA fragment was inserted into pGEX-4T-l to construct the expression plasmid. The recombinant plasmid was digested by BamH I and EcoR Ito identify whether the CGH cDNA fragment was inserted into the plasmid, the pGCGH was transformed into E.coli BL21 competent cells. The mRNA and protein expression were assayed by RT-PCR and SDS-PAGE. The results were found that the specific 670bp DNA bases of CGH was detected by RT-PCR in the recombinant bacteria and a new protein band was found in SDS-PAGE with molecular mass of about 50 KDa which is consisted of a 23.6 KDa protein deduced from the CGH gene sequence and GST (26 KDa). These indicated that the grass carp GH gene was correctly transcribed and translated in the recombinant bacteria, and could be employed to further develop a novel growth-regulating reagents to promote the health development offish.更多还原