【作者】 刘文生； 尹立路； 李梦溪； 姜丽娟；

【Author】 Liu Wensheng, Yin Lilu, Li Mengxi, Jang Lijuan (College of Animal Science, South China Agricultural University, Guangzhou 510642, China)

【机构】 华南农业大学动物科学学院；

【摘要】 生长激素(Growth hormone,GH)是由脑垂体嗜酸性细胞分泌的单链多肽类激素,一般由173-191个氨基酸组成。本论文对花鳗鲡GH基因首次进行了克隆、测序,对其在10种组织中的表达情况进行了定量分析,利用克隆得到的花鳗鲡GH基因重组得到能稳定表达的质粒并可成功诱导得到花鳗鲡的GH融合蛋白。主要研究结果:1.利用PCR技术扩增得到花鳗鲡GH基因的完整开放阅读框ORF,以花鳗鲡基因组DNA为模板成功获得花鳗鲡GH基因完整内含子序列。2.花鳗鲡GH蛋白质的一级结构是由209个氨基酸组成,分子量约为23.57KD,等电点(pI)值为6.52,酸性氨基酸(Asp+Glu)含量为11.9%,碱性氨基酸(Arg+Lys+His)含量为13.8%,疏水性氨基酸含量为45.8%,亲水性氨基酸含量为41.8%。在整个氨基酸序列中亮氨酸(Leu)含量最高,为12.4%,其次为色氨酸(Ser),为9.0%。不稳定系数为35.87,表明该蛋白质稳定性较高。3.成功将花鳗鲡GH基因的CDs序列连接进pET-32a质粒,并将重组质粒转化进大肠杆菌DE3(也称BL21),经SDS-PAGE检测证明重组质粒能稳定表达融合蛋白。在IPTG浓度为0.1mg/L,诱导时间为4h,温度37℃时表达量最高。更多还原

【Abstract】 Growth hormone (GH) is a single peptide hormone consists of 173 to 191 amino acids, secreted by theanterior pituitary cell.In this study,the sequence of GH gene was firstly cloned andanalysed in Marbled eel(Anguilla marmorata). Realtime-PCR was used to study the expression profile of GH mRNA in ten tissues. Add the CDS to PET32a ,we got a stable expression plasmid,and it could be induced to get GH of Marbled eel .The results are as follows: 1.According to the GH gene of European eel,special primers were designed to amplify the whole open reading frame(ORF) by PCR.The whole length of introns were amplified from genomic DNA abstracted from Marbled eel blood by PCR technique..2.The GH had 209 amino acid and its moleculer was about 23.57KD, isoelectric point (pI) was 6.52.The percent of acidic amino acid (Asp + Glu)was11.9%,the basic amino acid(Arg + Lys+His)was 13.8%.Hydrophobic amino acid was 45.8%,Hydrophilic amino acids was 41.8%. In the amino acid sequence,the highest content was Leucine(Leu), percent of 12.4%,the following was Tryptophan(Ser),percent of 9.0%.And the instability index was 35.87,which could show that this protein has high stability. 3.In this study,the GH CDs was cloned successfully into the pET-32a expression vector and the recombinant plasmid was estabilished.The expression of the fusion protein expressed successfully by using SDS-PAGE.The maximal expression appeared in 4 hours using 0.1mg/L IPTG at 37℃.更多还原