促凋亡蛋白BID多克隆抗体的制备与鉴定

目的制备抗BID的多克隆抗体,用于检测凋亡信号转导过程中BID蛋白的表达。方法采用PCR技术合成编码BID特异性肽段的基因,构建GST融合基因表达载体,在E.coli BL21中诱导表达GST-BID多肽的融合蛋白,经Glutathione Sepharose 4B纯化后免疫家兔,免疫血清经纯化后,得到抗BID的多克隆抗体,经Western blot鉴定其特异性。结果通过PCR扩增获得了BID肽段的编码基因;pGEX-6P-1-BID多肽重组质粒经酶切鉴定及测序证明构建正确;在E.coli BL21中表达了GST-BID多肽融合蛋白;纯化后蛋白纯度达95%;制备的抗BID多克隆抗体能够特异地识别BID蛋白。结论所制备的抗BID多克隆抗体可特异性检测BID蛋白。

Preparation and Identification of Polyclonal Antibody against Pro-apoptotic Protein BID

Objective To prepare the polyclonal antibody against pro-apoptotic protein BID for determination of BID expression during apoptosis signal transduction.Methods The gene encoding BID-specific peptide was synthesized by PCR and cloned downstream to GST encoding region of prokaryotic expression vector pGEX-6P- 1.The constructed recombinant plasmid pGEX-6P-1-BID was transformed to E. coli BI221 for expression under induction of IPTG. Purity the expressed product by Glutathione Sephamse 4 B chromatography. Immunize rabbits with the purified GST-BID fusion protein and collect the sera to prepare polyclonal antibody.Identify the specificity of prepared polylonal antibody by Western blot. Results The gene encoding BID peptide was amplified by PCR. Restriction analysis and sequenang proved that recombinant plasmid pGEX-6P-1-BID was correctly constructed. GST-BID fusion protein was expressed in E. coli and reached a purity of 95% after purification. The prepared polyclonal antibody recognized BID protein specifically. Conclusion The polyclonal antibody against pro-apoptotic protein BID was successfully prepared.