鸡瘦素受体胞外域成熟肽重组蛋白质的表达以及多克隆抗体的制备

Expression of recombinant protein of chicken leptin receptor extracellular domain mature peptide and the preparation of its polyclonal antibody

根据鸡瘦素受体基因(LEPR)编码区序列(Gen Bank:NM_AB033383)设计并合成2对引物,以鸡肝脏c DNA为模板扩增鸡LEPR胞外域2段c DNA序列。然后将这2段序列克隆到表达载体pRSET-A的Bam H I和Hind III酶切位点之间,构建重组表达载体(pR-LEPR1和pR-LEPR2)并转化大肠杆菌E.coli BL21(DE3)。转化菌在IPTG诱导后分别表达了分子量为2.62×104的LEPR1和分子量为2.86×104的LEPR2重组蛋白质。经凝胶NiNTA纯化后的重组蛋白质与矿物油佐剂混合免疫生长鸡,经过3次免疫之后,获得高效价的抗LEPR1和抗LEPR2抗血清。

Two pairs of primers designed based on the chicken leptin receptor gene ( LEPR) mature peptide se-quence (GenBank:NM AB033383) were used to amplify two cDNA sequence fragments of chicken LEPR extracellu-lar domains ( ECD) mature peptides with chicken liver cDNA as the template. The two cDNA sequence fragments were cloned into the BamH I and Hind III sites of the plasmid pRSET-A to generate the expression vector pR-LEPR1 and pR-LEPR2, which were further transformed into bacterium Escherichia coli BL21(DE3). The transformed bacte-ria were induced to produce recombinant proteins LEPR1 and LEPR2 with the expected molecular mass of 2. 62×104 and 2. 86×104 by IPTG. The recombinant LEPR1 and LEPR2 were purified and mixed into mineral oil adjuvant to immunize growing chickens to produce anti-LEPR1 and anti-LEPR2 antibodies. Antisera with high anti-LEPR1 and anti-LEPR2 titers were obtained after three immunizations.