西藏小型猪Leptin及其受体胞外区片段的克隆及原核表达

目的克隆及原核表达西藏小型猪瘦素（Leptin）成熟肽及瘦素受体胞外区片段。方法根据西藏小型猪瘦素序列（GenBank号：GQ240885.1）和猪瘦素受体基因胞外域序列（GenBank号：AF167719.1）分别设计并合成两对引物扩增瘦素、瘦素受体基因胞外域编码区1654-2319位片段,以西藏小型猪组织总RNA为模板,经反转录-聚合酶链反应（RT-PCR）方法获得了特异性片段。再以该两个特异性片段为模板,另外设计两对带有BanHⅠ和HidⅢ酶切位点的套式引物分别扩增瘦素64-504位（成熟肽编码区）和瘦素受体基因胞外域编码区1655-2314位的cDNA片段,将该两片段克隆入pMD18-T载体并转化感受态细菌E.coli DH5α测序并永久保存。此两片段经酶切后克隆到表达载体pRSET A的BamHⅠ和HindⅢ两酶切位点之间,构建重组质粒pR-OB和pR-OBR-a并在大肠杆菌E.coli BL21（DE3）中表达,SDS-PAGE电泳鉴定表达产物。结果在IPTG诱导下促使重组菌pR-OB表达了相对分子质量约18×103左右的融合蛋白;重组菌pR-OBR-a表达了相对分子质量约27×103左右的融合蛋白。结论说明重组质粒pR-OB、pR-OBR-a在大肠杆菌BL21（DE3）中分别可表达西藏小型猪瘦素成熟肽、瘦素受体片段蛋白,为进一步研究瘦素、瘦素受体功能和应用提供了基础。

Molecular Cloning and Prokaryotic Expression of Leptin and a Partial Leptin Receptor Extracellular Domain in Tibet Minipig

Objective To clone the prokaryotic expression of leptin mature peptide and a partial leptin receptor extracellular domain in Tibet minipig.Methods Two pairs of primers,amplifying the 64-504 bp leptin mature peptide coding region,and 1654-2319 bp leptin receptor（OBR） extracellular domain（ECD）,were designed and synthesized according to the sequences of swine leptin（GenBank No.GQ240885.1） and OBR（GenBank No.AF167719.1） genes.Total RNA was extracted from the Tibet minipig liver tissue,and was used as the template in RT-PCR to amplify the cDNA fragments coding leptin mature peptide and OBR ECD.The two fragments were then cloned into the pMD18-T vector and the resultant plasmid was transformed into competent E.coli strain DH5α.The fragments were digested and cloned into the BamHⅠand HindⅢ sites of the expression vector pRSETA to form the recombinant plasmids pR-OB and pR-OBR-a,which were subsequently transformed into E.coli strain BL21（DE3） for expression of the recombinant proteins.Results The transformed bacteria pR-OB and pR-OBR-a were induced with IPTG and produced recombinant proteins of the relative molecular mass of 17.5×103 and 27×103,respectively.Conclusions pR-OB and pR-OBR-a can express recombinant porcine leptin mature peptide and a partial OBR ECD of Tibet minipig in E.coli.strain BL21（DE3）.This work provided the basis for further research on pig leptin and OBR.