[Effects of different signal peptides on the secretion of human-mouse chimeric CMV-IgM].

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. 为了实现在体外制备巨细胞病毒-免疫球蛋白M (CMV-IgM) 和探讨不同信号肽对CMV-IgM 表达的影响，通过RLM-RACE 技术钓取杂交瘤细胞基因序列，构建人鼠嵌合CMV-IgM 表达载体，并通过PCR 方法将5 种不同的分泌型信号肽 (SigA–SigE) 代替CMV-IgM 自身信号肽 (SigF)，利用中国仓鼠卵巢细胞 (CHO) 作为宿主细胞进行体外表达。对纯化后的CMV-IgM 进行SDS-PAGE、SEC-HPLC 和酶联免疫吸附试验 (ELISA) 确定其蛋白表达水平与免疫反应性，最终成功制备出910 kDa 的重组蛋白，且研究表明，5 种信号肽 (SigA–SigE) 的人鼠嵌合CMV-IgM 表达量较CMV 6#细胞株自身信号肽SigF 相比，分别提高了6.72、5.19、1.44、1.85、1.98 倍。这将对人鼠嵌合CMV-IgM 的开发提供理论基础，且为提高CMV-IgM 表达量的研究工作提供了新思路。.