癌症转移相关基因Syntenin1多克隆抗体的制备与鉴定

Preparation and Identification of the Syntenin1 Polyclonal Antibody,a Cancer Metastasis Related Gene

目的获得原核表达GST-Syntenin1融合蛋白,制备高效价的抗Syntenin1多克隆抗体。方法RT-PCR扩增Syntenin1 cDNA开放阅读框(CDS)序列,亚克隆到编码GST的pGEX-4T-2原核表达载体上,将编码Syntenin1的pGEX-4T-2-Syntenin1重组质粒化学转化BL21(DE3)感受态细胞,IPTG诱导GST-Syntenin1融合蛋白表达,对融合蛋白亲和纯化。SDS-PAGE鉴定后,将纯化的融合蛋白辅以弗氏佐剂,免疫新西兰大白兔制备多克隆抗体,并用Western blot检测抗体效价及特异性。结果成功制备GST-Syntenin1融合蛋白及多克隆抗体。Western blot结果表明Syntenin1兔多克隆抗体效价可达到1∶20 000,且与Syntenin1蛋白特异结合。结论成功制备Syntenin1多克隆抗体,为进一步研究Syntenin1基因的功能奠定了基础。

Objective To express the GST fusion protein,GST-Syntenin1 in E.coli,and to prepare the polyclonal antibody of Syntenin1. Methods CDS fragment of Syntenin1 was obtained by RT-PCR from normal mouse brain and subcloned into pGEX-4T-2 to generate pGEX-4T-2-Syntenin1 recombinant.The confirmed recombinant was transformed into the BL21 competent cells and induced with IPTG.The recombinant fusion protein was purified with immobilized Glutathione Sepharose and confirmed by SDS-PAGE.The purified fusion protein was mixed with the Freund＇s adjuvant,and then injected into New Zealand white rabbits by hypodermic injection.The polyclonal antibody titer and specification were identified by Western blot.Results Syntenin1 polyclonal antibody bind Sytenin1 protein specifically and the antiserum tiger reached to 1∶20 000.Conclusion The Syntenin1 polyclonal antibody with high titer and high specificity was prepared successfully.This will be very helpful for the further study on Syntenin1 function and molecule mechanism of cancer metastasis.