鸡催乳素受体胞外域的分子克隆及原核表达

Molecular Cloning and Prokaryotic Expression of Chicken Prolactin Receptor Extracellular Domain

根据家鸡催乳素受体基因胞外域序列（GenBank号：D13154）设计并合成1对覆盖编码区58～1249位核苷酸的引物，以家鸡垂体总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断，将该片断克隆入pMD18-T载体并转化感受态细菌Ecoli DH5α永久保存．通过设计第2对引物扩增PRLR胞外域cDNA片段，该片段经酶切后克隆到表达载体pRSETA的EcoRⅠ和HindⅢ两酶切位点之间，构建重组质粒pR-PRLR．该质粒在转化感受态细菌E．coli B121（DF．3）后，在IPTG诱导下促使重组菌表达了相对分子质量为53640左右的重组融合蛋白，表达量在0．1mmol／L IPTG诱导5h时达到最高，约占总菌体蛋白的17．6％左右．表达产物可经体积分数为50％的Ni-NTA凝胶纯化，说明重组蛋白中具有正确的His-tag标签及之后的鸡催乳素受体编码区胞外域序列．

A pair of primers were synthesized according to the sequence of chicken prolactin receptor （PRLR） gene extracellular domain （ECD） fragment （GenBank No. D13154）. Total RNA was extracted from the chicken pituitary tissue and used as template to amplify the PRLR ECD fragment by RT-PCR. The 1 200 bp PCR product was then cloned into the pMD18-T vector and the resultant plasmid was transformed into E. coli DH5α. A second pair of primers were synthesized for amplification and insertion of the ECD fragment into the EcoR Ⅰ and Hind Ⅲ sites of the pRSET A vector, generating the recombinant plasmid pR-PRLR which was subsequently transformed into E. coli BL21 （DE3）. The transformed bacteria were induced with IPTG and produced a recombinant protein of 53 640 relative molecular mass which accounted for 17.6% of the total bacterial protein. The recombinant product was purified by 50% NiNTA agarose, which indicated its composition of a correct His-tag amino acid sequence and the subsequent PRLR ECD.