NF-κB受体激活因子配体的表达及其多克隆抗体的制备

Expression of porcine receptor activator for nuclear factor-κB ligand in Escherichia coli and preparation of its polyclonal antibody

为获取猪源NF-k B受体激活因子配体(RANKL)及其抗体,根据GenBank中猪源RANKL的序列设计引物,将合成的RANKL基因克隆至pMAL-C4x表达载体,转化到大肠杆菌DH5α。经PCR和双酶切鉴定后测序分析,将阳性克隆转化至大肠杆菌BL21后通过IPTG诱导表达。优化表达条件,确定是包涵体表达,蛋白大小为78.1 ku。使用纯化的RANKL为抗原免疫兔制备多克隆抗体。构建重组质粒pCMV-RANKL,表达目的蛋白并使其与多克隆血清反应。经ELISA、Western-blot和IFA鉴定,证实抗RANKL多克隆抗体有良好的抗原特异性,为后续黏膜免疫的研究奠定了基础。

In order to obtain porcine nuclear factor-κB ligand(RANKL) and its antibodies,primers were designed according to the sequence of porcine RANKL in Gen Bank.The RANKL gene was cloned into p MAL-C4x expression vector and transformed into Escherichia coli DH5α.After identification by PCR,double enzyme digestion and sequencing,the positive clones were transformed into E.coli BL21 and induced by IPTG.After optimization of the induction conditions,the protein was expressed in inclusion bodies and the molecular weight was about 78.1 ku.Subsequently,the purified RANKL protein was used to immunize rabbit subcutaneously for preparation of anti-RANKL polyclonal antibody(Pc Ab).Coding RANKL was subcloned into pCMV-Myc plasmid,the expressed protein reacted with polyclonal antibody.Identified by ELISA,Western-blot and IFA,anti-RANKL PcAb has good antigen specificity,it is helpful for the follow-up study of mucosal immunity.