拟蜘蛛牵丝蛋白基因单体(S)原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of Intended Spider Dragline Silk-like Peptide Gene Monomer(S)

利用原核表达获得的拟蜘蛛牵丝蛋白制备多克隆抗体,将为我们在真核水平检测其表达情况奠定基础。以质粒pGEX-2T为骨架载体,在其多克隆位点处连接人工合成的拟蛛丝蛋白基因单体(S),构建含有GST标签的融合蛋白原核表达载体pGEX-S;利用IPTG诱导重组质粒pGEX-S在大肠杆菌感受态细胞BL21中表达,并利用GST标签亲和纯化重组蛋白,获得的S-GST蛋白与佐剂混合后,采用多点皮下免疫注射8只健康的CDⅠ小白鼠,免疫期结束后收集抗血清进行ELISA检测。序列分析后,确定原核表达载体pGEX-S编码正确;诱导表达后,Western blot检测显示在41 kD处有重组蛋白条带出现,与我们预期的结果相符;抗血清Elisa检测显示有2只小鼠A和F抗血清效价较高。采用原核表达生产的拟蜘蛛牵丝蛋白成功制备了多克隆抗体。

This research, through prokaryotic expression to get intended spider dragline silk protein to prepare specific antibody, would lay the foundation for detecting its expression in eukaryotic cell. Using plasmid pGEX-2T as original vector, we constructed the prokaryotic expression vector pGEX-S into the MCS （ multiple clone sites ） of an artificial synthesized spider dragline silk protein gene inserted. Then we induced the expression of the recombinant plasmid pGEX-S in Escherichia coli competent cells B21 with the utilization of IPTG. Recombinant protein was purified by the means of GST-tag-specific affinity to gain S-GST protein. The specific antibody was prepared by immunizing mouse CD Ⅰ . After immune collect serum, ELISA was employed. The expression vector pGEX-S coding sequence is right after analysis. The western blot Show the recombinant protein band appears at 41 kD. ELISA results indicated that two of mouse A and F have better immunoreactivity. The study proved the successful preparation of specific polyclonal antibody through prokaryotic expression.