COX-2的基因克隆和多克隆抗体制备

Cloning of Coding Gene and Preparation of Polyclonal Antibody of COX-2

环氧合酶 2 (COX 2 )是一种具有多种功能的神经调节物质 ,它的许多功能细节仍不十分清楚 ,还需要进一步深入研究 .用PCR技术从大鼠脑cDNA文库中 ,扩增到正常成年大鼠COX 2的cDNA序列 ,测序结果与已发表的序列一致 .通过PCR扩增和基因重组 ,分别构建COX 2编码基因全长和羧基端部分编码序列的表达载体 ,并导入大肠杆菌DH5α中 ,通过IPTG诱导表达重组融合蛋白 ,结果导入全长编码基因表达载体的DH5α无COX 2融合蛋白表达 ,而羧基端部分基因编码的COX 2融合蛋白在DH5α中以包涵体的形式进行表达 .经SDS 聚丙烯酰胺凝胶电泳分析 ,在相对分子质量 (Mr)为 44 0 0 0处有 1条特异的蛋白质条带 .对以包涵体形式表达的蛋白质进行变性、重折叠及纯化后 ,得到了高纯度融合蛋白 .以重组融合蛋白免疫新西兰种大白兔 ,制作了抗COX 2多克隆抗体 ,经酶联免疫吸附测定、蛋白质印迹和免疫细胞化学方法检测 ,此抗体具有较高效价和特异性 .COX 2基因和多克隆抗体的获得 ,为进一步研究COX

COX-2 is a multifunctional neuronal modulator, however, its many functions are still unclear and need to be investigated further. Normal adult rat COX-2 cDNA was amplified by PCR from rat brain cDNA library. Sequencing result showed that the cloned gene was identical with that having reported. Expression vectors of whole COX-2 coding gene and its partial coding sequence in carboxyl terminal were respectively constructed by PCR and gene recombination techniques and were transformed into E. coli DH5alpha, for IPTG-induced expression. The results showed that there was no COX-2 fusion proteins expressed in E. coli DH5a which were transformed with expression vectors of whole COX-2 coding gene, whereas in E. coli DH5alpha which were transformed with expression vectors of partial coding gene in carboxyl terminal, COX-2 fusion protein was expressed in the form of inclusion body. A protein band of M, being 44 000 appeared on SDS-PAGE gel. The protein expressed by carboxyl terminal coding sequence had a high purity after denaturation, refolding and purification. New Zealand rabbits were immunized with COX-2 fusion proteins to prepare a polyclonal antibody against COX-2. The COX-2 antiserum was obtained and characterized by ELISA, Western blot, immunohistochemistry and immunocytochemistry. Results showed that the antibody has high titer, affinity and specificity. The studies provide a favorable tool for further functional study of COX-2 in future.