Cui Yamin, Tian Xiaoping, Zhao Qiaohui, Li Guilin
Zhengzhou Immuno Bio-Tech Co., Ltd, Zhengzhou 450016, Henan, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Jun 25;36(6):1223-1231. doi: 10.13345/j.cjb.190424.
In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.
为了在体外制备人鼠嵌合巨细胞病毒免疫球蛋白M（CMV-IgM）并研究不同信号肽对CMV-IgM分泌的影响，使用RLM-RACE从杂交瘤细胞系中扩增基因以构建嵌合CMV-IgM的表达载体。然后，通过PCR方法将SigF自身的信号肽替换为五种不同的分泌信号肽（SigA-SigE），并选择CHO细胞作为体外表达的宿主细胞。进行SDS-PAGE、SEC-HPLC和ELISA实验以评估纯化的CMV-IgM的蛋白表达水平和免疫反应性。成功制备了一种910 kDa的重组蛋白，信号肽（SigA-SigE）使表达的CMV-IgM增加，分别比CMV 6#细胞信号肽SigF高6.72、5.19、1.44、1.85和1.98倍。总之，这项工作为开发人鼠嵌合CMV-IgM提供了理论基础，并为提高CMV-IgM的表达水平提供了一条新途径。
