摘要
采用特异性引物自贵州黑羽乌骨鸡胃部组织克隆了Ghrelin的基因,基因全长2330bp,含有4个外显子,3个内含子。采用RT-PCR技术,获得了Ghrelin cDNA片段(563bp),序列分析推测,通过可变剪切可产生两段mRNA,cDNA编码区与基因组中的编码区完全一致。黑羽乌骨鸡Ghrelin基因与已知鸡种比较有13～18个碱基不同,相似性为99.1%～99.3%,但其氨基酸序列完全一样,表明禽类的Ghrelin多肽高度保守。将其中Ghrelin编码区亚克隆到原核表达载体pTYB11中,经序列测定碱基序列正确,获得重组质粒pTYB11/G。在IPTG的诱导下,重组质粒在大肠杆菌ER2566中获得表达。当OD600=0.5,IPTG为浓度1mmol/L,诱导5小时的表达量最高,融合蛋白占细胞总蛋白的40%。采用几丁质结合的预装柱对表达产物进行纯化,产率约为5mg/L培养液。
The gene（2330bp）and cDNA（563 bp） of Ghrelin were cloned from the stomach of black-feather chicken. The gene contained 4 exons and 3 introns. Both the gene and cDNA of Ghrelin encoded a preproghrelin of 116 amino acids. Two different mRNA molecules,ghrelin and obestatin,are predicted to be produced by alternative splicing of the gene. Although the similarity of Ghrelin gene between black-feather chicken and other broiler chickens was 99.1%-99.3% with 13 to 18 nucleotides substitution,the preproghrelin was just the same. It demonstrated the conservation of Ghrelin in chicken. The gene encoding preproghrelin was cloned from the cDNA and expressed in Escherichia coli 2 566 by pTYB11 prokaryotic expression plasmids. The optimized expression condition of the pTYB11/G recombinant plasmid,was determined to be induced by 1 mmol/L IPTG （OD600=0.5）for 5 hours. The fusion protein was purified by chitin prepacked column,and the yield was about 5 mg/L of medium.
出处
《中国家禽》
北大核心
2008年第7期25-28,共4页
China Poultry
基金
国家自然基金(30560104)
贵州省优秀科技教育人才省长资金项目
