2021 年第 9 期 第 16 卷

人鼠嵌合弓形虫免疫球蛋白M重组抗体制备及J链对其聚体形成的影响

Preparation of human and mouse chimeric toxoplasma-immunoglobulin M recombinant antibody and the effect of J chain on its polymer formation

作者：杜伟鹏1崔亚敏2田晓平2王素娟2付光宇3赵巧辉2

英文作者：Du Weipeng1 Cui Yamin2 Tian Xiaoping2 Wang Sujuan2 Fu Guangyu3 Zhao Qiaohui2

单位：1河南省南阳市中心医院检验科473009；2郑州伊美诺生物技术有限公司研发部450016；3郑州安图生物工程股份有限公司研发部450016

英文单位：1Department of Clinical Laboratory Nanyang Central Hospital Henan Province Nanyang 473009 China; 2Department of Research and Development Zhengzhou Immuno Bio-Tech Co. Ltd. Zhengzhou 450016 China; 3Department of Research and Development Zhengzhou Autobio Diagnostic Co. Ltd. Zhengzhou 450016 China

关键词：人鼠嵌合弓形虫免疫球蛋白M抗体；J链；DNA重组技术

英文关键词：Humanandmousechimerictoxoplasma-immunoglobulinMantibody;Jchain;DNArecombinationtechnology

• 摘要：

• 目的 体外制备人鼠嵌合弓形虫免疫球蛋白M（TOX-IgM）抗体，并验证加入J链对IgM抗体蛋白聚体、效价及稳定性的影响。方法 从美国国家生物信息中心数据库获取抗体的重链可变区、轻链可变区以及J链序列，通过DNA重组技术分别将TOX抗体轻重链可变区和人源恒定区进行拼接，构建人鼠嵌合IgM表达载体，转染HEK 293F细胞进行表达。观察J链相关效应。结果 J链有利于抗体聚体形成（聚体占比由11.3%提升至75.5%），抗体效价由1∶40提升至1∶160；在37 ℃热稳定性实验及反复冻融实验中，TOX-IgM（J+）稳定性与TOX-IgM（J-）相当。通过酶联免疫吸附试验鉴定蛋白活性，本实验成功制备人鼠嵌合TOX-IgM抗体，经聚丙烯酰胺凝胶电泳还原显示，重链大小约为75 000，轻链大小约为25 000，符合预期；效价、稳定性、基质效应均满足需求。结论 在连接链J链（15 000蛋白）存在下，IgM抗体更易形成五聚体，即5个IgM单体通过二硫键和J链彼此之间相互连接，且经评价效价和稳定性均有了明显提升。

• Objective To prepare human and mouse chimeric toxoplasma-immunoglobulin M(TOX-IgM) antibody, and to verify the effects of addition of J chain on protein clusters, potency and stablilty of IgM antibody. Methods The variable region of heavy chain, variable region of light chain and J chain sequence of the antibodies were obtained from National Center for Biotechnology Information of America. The variable region and the constant human region of the TOX antibodies were spliced by DNA recombination technology respectively, the human and mouse chimeric IgM expression vectors were constructed and HEK 293F cells were transfected for expression. The related effects of J chain were observed. Results J chain was beneficial to the formation of antibody polymer (the proportion of polymer increased from 11.3% to 75.5%), and the antibody potency increased from 1∶40 to 1∶160; the stability of TOX-IgM (J+) was equivalent to TOX-IgM (J-) in accelerated stability experiment at 37 ℃ and repeated freeze-thaw experiment. Human and mouse chimeric TOX-IgM antibody was successfully prepared, the protein activity was indentified by enzyme-linked immunosorbent assay, the polyacrylamide gel electrophoresis reduction showed that the heavy chain size was about 75 000 and the light chain size was about 25 000, which met the expectation, and its potency, stability and matrix effect were all satisfied. Conclusions In the presence of J chain(15 000 protein), IgM antibody is more likely to form pentamer that is 5 IgM monomers connected to each other through disulfide bond and J chain and the potency and stability are significantly improved by evaluation.