【摘要】 用PCR技术扩增环加氧酶2(COX-2)基因的C末端777 bp的片段,插入到pET-28b(+)中,构建原核表达载体.转入大肠杆菌BL21(DE3),经IPTG诱导产生包涵体形式his-COX-2融合蛋白.用镍离子螯合柱(Ni-NTA)纯化融合蛋白,获得纯度较高的原核表达蛋白.纯化后的蛋白免疫家兔制备多克隆抗体,用ELISA及West-ern blotting分别检测抗体.用此抗体Western blotting检测肝癌组织中的环加氧酶2的表达.ELISA及Westernblotting结果显示免疫家兔所得的环加氧酶2抗体具有很高的效价,并能够特异性的识别真核细胞中的环加氧酶2.用此抗体检测肝癌组织中的环加氧酶2的表达,证实肝癌组织中伴随有环加氧酶2的大量表达.同时所得的环加氧酶2抗体具有很高的效价和特异性,为进一步研究环加氧酶2的功能提供了条件.更多还原

【Abstract】 Expression vector carrying cyclooxygenase-2(COX-2) gene was constructed by inserting the C-terminal 777 bp fraction amplified by PCR into the vector pET-28b(+).The fusion protein was expressed in the form of inclusion body after induction by IPTG in BL21(DE3).Using Ni-NTA chelating column,the fusion protein was highly purified.The polyantibody was prepared by immunizing the rabbits with five injections of the purified protein.ELISA and Western Blotting were performed to test its specificity and sensitivity.Meanwhile,cyclooxygeanse-2 expression was detected in hepatocellular carcinoma and surrounding noncancerous liver tissues by Western Blotting using the antiserum.The polyantibody can recognize cyclooxygease-2 with high specificity and sensitivity and cyclooxygenase-2 was expressed at higher level in hepatocellular carcinoma compared to that in surrounding noncancerous liver tissues.Preparation of cyclooxygeanse-2 polyantibody make it possible to further study the biological functions.更多还原