不同信号肽对人鼠嵌合CMV-IgM分泌表达的影响

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

摘要:

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。

Abstract:

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.