鸽子瘦素基因成熟肽重组蛋白的表达及多克隆抗体的制备

Cloning of pigeon leptin mature peptide cDNA sequence and preparations of the recombinant protein and polyclonal antibody

根据鸽子瘦素基因(leptin)编码区序列(Gen Bank:HG797022)设计并合成鸽子leptin基因编码胞外域成熟肽的c DNA序列,然后将这段序列克隆到表达载体p GEX-4T-1的Eco R I与Not I酶切位点之间,构建重组表达载体(p GEX-4T-1-leptin)并将该载体转化到大肠杆菌Arctic Express中。转化菌经IPTG诱导后表达了分子量为44 270的鸽子leptin重组蛋白。经镍柱亲和层析纯化后的重组蛋白质与矿物油佐剂混合,免疫日本大耳白兔,制备抗leptin多克隆抗体。经过4次免疫之后,获得高效价的抗leptin多克隆抗体。

According to the pigeon leptin gene mature peptide sequence （ GenBank ： HG797022） ,the leptin genewere synthesized and cloned. The synthesis leptin gene was cloned into the sites of the plasmid pGEX-4T-1 betw-een Eco R I and Not I to generate the expression vector pGEX-4T-1-leptin,which was further transformed into bacteria Arctic Express.The transforned bacteria were induced to produce recombinant leptin proteins with the expected molecular mass of 44 270 by IPTG. The recombinant leptin were purified by 50% Ni-NTA agrose chromatography and homogenized into mineral oil adjuvant. The mixers were used to immunize Japanese big ear rabbit to produce anti-leptin antibodies. Antisera with high anti-leptin titers were obtained after four immunizations.