拟蜘蛛牵丝蛋白基因单体（S）原核表达及多克隆抗体制备

生物技术通报 ›› 2013, Vol. 0 ›› Issue (3): 134-138.

拟蜘蛛牵丝蛋白基因单体（S）原核表达及多克隆抗体制备

孟凡华,房君,王海涛,邢燕平,齐昱,周欢敏

内蒙古农业大学生命科学学院，呼和浩特 010018

收稿日期:2012-09-14 修回日期:2013-03-21 出版日期:2013-03-20 发布日期:2013-03-21
作者简介:孟凡华，博士研究生，讲师，研究方向：生物技术与发育生物学；E-mail ：fhmeng2003@yahoo.com.cn
基金资助:
国家转基因生物新品种培育重大专项（2009ZX08008-008）

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

Meng Fanhua, Fang Jun, Wang Haitao, Xing Yanping, Qi Yu, Zhou Huanmin

College of Life Science of Inner Mongolia Agricultural University,Hohhot 010018）

Received:2012-09-14 Revised:2013-03-21 Published:2013-03-20 Online:2013-03-21

摘要/Abstract

摘要： 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CD Ⅰ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。

关键词: 拟蜘蛛牵丝蛋白基因单体（S）, 原核表达, 多克隆抗体

Abstract: 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 CDⅠ. 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.

Key words: Intended spider dragline silk protein gene monomer（S）, Prokaryotic expression, Polyclonal antibody

孟凡华,房君,王海涛,邢燕平,齐昱,周欢敏. 拟蜘蛛牵丝蛋白基因单体（S）原核表达及多克隆抗体制备[J]. 生物技术通报, 2013, 0(3): 134-138.

Meng Fanhua, Fang Jun, Wang Haitao, Xing Yanping, Qi Yu, Zhou Huanmin. Prokaryotic Expression and Polyclonal Antibody Preparation of Intended Spider Dragline Silk-like Peptide Gene Monomer（S）[J]. Biotechnology Bulletin, 2013, 0(3): 134-138.

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