摘要
目的在大肠杆菌中表达人Id-3与谷胱甘肽-S转移酶(GST)的融合蛋白,并制备抗人Id-3的多克隆抗体。方法从乳腺癌组织中提取总RNA,用RT-PCR扩增出Id-3的编码序列,克隆至表达载体pGEX-6P-1中,重组质粒经PCR、酶切、测序鉴定后,在大肠杆菌中经IPTG诱导表达获得GST-Id-3融合蛋白,SDS-PAGE分析表达产物,Western-Blot检测重组抗原的免疫原性。通过亲和层析法纯化表达的GST-Id-3融合蛋白,并以此为抗原制备多克隆抗体。结果经PCR、酶切、测序鉴定证明,Id-3基因已正确克隆至pGEX-6P-1中,经IPTG诱导后,表达出相对分子量为39000GST-Id-3融合蛋白。ELISA和琼扩试验鉴定所制备的多克隆抗体可以与GST-Id-3融合蛋白发生特异性反应。结论Id-3基因在大肠杆菌中的成功表达及制备的多克隆抗体,为检测Id-3及其在各种组织中的表达水平提供了一种检测途径,也为分析Id-3分子结构、抗原表位和生物学功能奠定了基础。
ObjectiveTo express the fusion protein of glutathione S-transferase(GST) and human Id-3 in E. coli. and prepare the polyclonal antibody against Id-3. MethodsThe coding sequence of Id-3 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR,restriction endonuclease digestion analysis and sequencing. And the fusion protein GST-Id-3 was expressed in E. coli. by IPTG induction. Then the expressed fusion protein was purified by glutathione-agarose a...
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2008年第23期3446-3449,共4页
China Journal of Modern Medicine
基金
黑龙江省自然科学基金重点项目(No:ZJY-0603-02)
关键词
人分化抑制因子3(Id-3)
表达
纯化
多克隆抗体
inhibitors of differentiation 3 of human（Id-3）
expression
purification
polyclonal antibody
