早孕因子多克隆抗体的制备及应用

Preparation and application of polyclonal antibody of early pregnancy factor

通过含有家兔早孕因子的重组质粒EPF-pET-32a(+)转化表达宿主菌Escherichia coli Rosetta,IPTG诱导,融合表达硫氧还原蛋白-兔早孕因子Trx-EPF。亲和层析纯化Trx-EPF,并在柱上进行肠激酶酶切,得到EPF蛋白并进行复性。EPF免疫波尔山羊,制备羊抗兔家兔早孕因子抗血清,硫酸铵沉淀纯化多克隆抗体,用于家兔早孕检测。Western-blot结果表明,羊抗兔家兔早孕因子多克隆抗体对家兔早孕的检出率达到100%。

A stable inducible prokaryotic expression system was set up and the protein Trx-EPF was induced by IPTG after recombinant expression plasmid EPF-pET-32a（＋） was transformed into host bacteria Escherichia coli Rosetta.The expressed protein was then purified by Ni-NTA HisBind purification kit and renatured.EPF was prepared by the lysis of enterokinase on Ni-NTA affinity chromatography.Goat was immunized with EPF,and serum was pooled.The goat anti-rabbit polycolonal antibody was prepared through ammonium sulfate precipitation from the serum.Early pregnancy of rabbits were detected by goat anti-rabbit polycolonal antibody by Western-blot,with a positive rate of 100%.