摘要
目的在大肠杆菌中表达人Id-2与谷胱甘肽-S转移酶(GST)的融合蛋白,并制备抗人Id-2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-2基因的编码序列,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-2蛋白,SDS-PAGE分析表达产物。通过亲和层析法纯化表达的GST-Id-2融合蛋白,Western-Blot检测重组抗原的免疫原性,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-2基因已正确克隆至pGEX-6P-1中,经IPTG诱导后,表达出相对分子质量40000的GST-Id-2融合蛋白。Western-Blot、ELISA和琼脂双向扩散实验鉴定所制备的多克隆抗体可以与GST-Id-2特异性反应。结论Id-2基因在在大肠杆菌中的成功表达及制备的多克隆抗体,为检测Id-2及其在各种组织中的表达提供了一种检测方法,也为分析Id-2分子结构及抗原表位奠定了基础。
Objective To express the fusion protein of glutathione S-transferase （GST） and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2. Methods The coding sequence of Id-2 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. The fusion protein GST-Id-2 expressed in E coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2. Results PCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40 000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion （AGP）. Conclusion The prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2009年第6期1094-1097,1110,共5页
Journal of Southern Medical University
基金
黑龙江省自然科学基金重点项目(ZJY-0603-02)
关键词
人Id-2基因
基因表达
多克隆抗体
inhibitor of DNA binding and differentiation, human
gene expression
polyclonal antibodies
