摘要
使用SignalP-4.0软件预测信号肽剪切位点,设计引物扩增建鲤(Cyprinus carpio var.jian)leptin基因(jlLEP-A1,jlLEP-B)不含信号肽的ORF区域。扩增产物分别连接到pET-32a(+)和pGex-4T-1原核表达载体,构建重组质粒,测序验证插入位点的正确性。将重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达重组蛋白pET-32a(+)/jlLEP-A1,pET-32a(+)/jlLEP-B和pGex-4T-1/jlLEP-A1,pGex-4T-1/jlLEP-B。结果表明,在37℃和1 mmol/L IPTG的条件下诱导5 h,重组蛋白表达量最大。SDS-PAGE电泳显示重组蛋白主要以可溶蛋白形式存在。这一结果为后续的建鲤leptin基因功能研究奠定了基础。
The signal peptide cleavage of Cyprinus carpio var. jian LEP-A1 （jlLEP-A1） and LEP-B （jlLEP-B） sites were identified by SignalP-4.0 software. And pairs of primers were designed to amplify the jlleptiA1 and jlleptinB ORF region without the signal peptide sequence. The amplification products were ligated into the prokaryotic expression vector of pET-32a （＋） and pGex-4T-1 respectively, and the recombination plasmida were constructed. The plasmid sequence was confirmed by sequencing and transferred into the host bacteria, Escherichia coli BL21 （DE3）. The recombinant proteins of pET-32a（＋）/jlLEP-A1, pET-32a（＋）/jlLEP-B, pGex-4T-1/jlLEP-A1 and pGex-4T-1/jlLEP-B were highly expressed by induction with IPTG. The optimum concentration of IPTG was 1 mmol/L for 5 h at 37℃. The recombinant proteins mainly existed in soluble form as revealed by SDS-PAGE electrophoresis analysis. The results provided useful information for research on jlleptin function.
出处
《动物学杂志》
CAS
CSCD
北大核心
2014年第1期51-56,共6页
Chinese Journal of Zoology
基金
863项目(No.2011AA100401)
农业部公益性行业科研专项(No.200903045)
中央级公益性科研院所基本科研业务费专项资金项目(No.2013JBFM03)
关键词
建鲤
LEPTIN
载体
原核表达
Cyprinus carpio var.jian Leptin Vector Prokaryotic expression
