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目的:将抗癌胚抗原(CEA)单克隆抗体(mAb)C245(IgG4)转变成IgG1,增强其在抗肿瘤免疫中的作用。方法:采用PCR从C245(IgG4)基因中克隆VH和Vk基因,并插入到含有人IgG1恒定区(CH)基因的杆状病毒表达载体pAckCH3中。用构建的重组基因感染Sf9昆虫细胞,将获得的高滴度重组病毒用于最终表达重组蛋白。应用免疫亲和层析法,纯化培养上清中表达的重组蛋白C245(IgG1),并鉴定其与CEA结合的能力和CH的生物学功能CDC及ADCC。结果:经CH置换形成的重组蛋白C245(IgG1),既保留了对于表达CEA的肿瘤细胞的结合,又比原抗体C245(IgG4)的补体依赖的细胞毒作用(CDC)和抗体依赖的淋巴细胞的细胞毒作用(ADCC)强得多。结论:成功地应用基因工程的方法制备了置换CH的抗CEA的人抗体C245(IgG1),使其在肿瘤的免疫治疗中具有更广泛的应用价值。
OBJECTIVE: To transform the anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) C245 (IgG4) into IgG1 to enhance its anti-tumor immunity. Methods: The VH and Vk genes were cloned from the C245 (IgG4) gene by PCR and inserted into the baculovirus expression vector pAckCH3 containing the human IgG1 constant region (CH) gene. Sf9 insect cells were infected with the constructed recombinant gene and the resulting high titer recombinant virus was used for the final expression of the recombinant protein. Immunoaffinity chromatography was used to purify the recombinant protein C245 (IgG1) expressed in the culture supernatant and its ability to bind to CEA and the biological functions of CHC, such as CDC and ADCC, were identified. Results: The recombinant protein C245 (IgG1) formed by CH substitution retains both the binding of CEA-expressing tumor cells and the complement-dependent cytotoxicity (CDC) of the original antibody C245 (IgG4) and the antibody-dependent lymphocytes The cytotoxicity (ADCC) is much stronger. CONCLUSION: The recombinant human anti-CEA antibody C245 (IgG1) that has been substituted for CH has been successfully used in genetic engineering and has been widely used in the immunotherapy of tumors.