CXXC5多克隆抗体的制备及表达研究

A Preparation of Polyclonal Antibody and Expression of Human CXXC5 Gene

CXXC5基因是从人类胚胎心脏的cDNA文库中克隆出来的一个人类锌指基因,包含zf-CXXC5结构域,该基因编码322个氨基酸,在物种进化上高度保守。为了进一步研究该基因的功能,需要获得CXXC5蛋白并制备其抗体.通过PCR扩增方法扩增得到了CXXC5部分编码区序列,然后将其连接到PGEX-4T-1上,转化到大肠杆菌BL21(DE3)中,再通过自身诱导法诱导表达重组质粒的融合蛋白.通过割胶回收纯化融合蛋白。免疫新西兰大白兔制备多克隆抗体,Western blot检测抗体活性。结果表明,实验获得了高质量的多克隆抗体。

CXXC5 was cloned from a human heart cDNA library and identified as human zf-CXXC-related zinc finger gene encoding a 322 amino acid protein. CXXC5 is an evolutionarily conserved protein. Obtianing the CXXC5 protein and it＇ s antibody is essential for the further studies. A cDNA fragment of partial coding sequence of CXXC5 was amplified by PCR, then the PCR product was cloned into the expression vector pGEX-4T-1 and transformed into Escherichia coli BI21 （DE3）. The fusion protein of the recombinant expression plasmid was expressed by a way of auto-induction. The fusion protein was separated with SDS-PAGE and recovered by gel extraction. New Zealand big white rabbits were immunized with the separated protein and the antibody titer and specification was identified by Western blot. The result showed that the high quality muhi-clonal antibody was obtained.