【摘要】 目的通过构建基于SM基因的分泌工程化改造的CHO-AV-SM细胞株,探讨在悬浮培养条件下,过表达转运蛋白SM基因对分泌蛋白表达的影响。方法首先从人HEK293FT细胞中克隆出sly1和munc18c基因,然后构建含有SM基因的真核表达载体pBSM;随后将pBSM质粒稳定转染入CHO-AV细胞并鉴定,获得了基于SM基因的分泌工程化改造的CHO-AV-SM细胞株;在此基础上对CHO-AV-SM细胞株进行悬浮培养适应,并测定了CHO-AV和CHO-AV-SM细胞株的生长和蛋白表达参数。结果过表达转运蛋白SM基因,CHO-AV-SM细胞株的抗体表达量较CHO-AV抗体表达量提高约75%,且测得第6天细胞数达到最大值,此时CHO-AV的表达量约为45.4μg/ml,CHO-AV-SM的表达量为79.5μg/ml。结论通过构建全抗稳定表达细胞,在细胞中过表达SM基因,并悬浮培养适应,建立了悬浮培养条件下基于转运蛋白过表达的分泌工程改造方法,为转运蛋白改造CHO细胞,提升全抗表达能力提供了有力支持。更多还原

【Abstract】 Objective The aim of this study is to investigate the improvement of secreted protein expression by constructing antibody-producing CHO cells based on molecular engineering of secretion, which includes SM gene over-expression and cell adaptation to suspension culture. Method In this study, we first cloned sly1 gene and munc18 c gene from human cells and constructed the eukaryotic co-expression vector. We then successfully transfected the sly1 and munc18 c genes into CHO-AV cells, and identified the SM protein expressing CHO-AV-SM cells. We next suspended the adapted CHO-AV-SM cells in culture and determined the growth and the expression parameters of CHO-AV and CHO-AV-SM cells. Results Due to SM transporter protein gene over-expression, antibody expression of CHO-AV-SM cell line was increased by 75%,as compared with CHO-AV cells. On the sixth day the cell number reached the maximum, and the antibody production of CHO-AV and CHO-AV-SM cells was about 45.4 μg/ml and 79.5 μg/ml, respectively. Conclusion This study improves the antibody production of CHO cells by molecular engineering of secretion, which includes SM gene over-expression and cell adaptation to suspension culture. This provides supports to molecular engineering of CHO-cell secretion and antibody production improvement.更多还原