2个表达单元间相互位置和转录方向对表达的影响

Effects on the Expression of Two Cassettes with Different Mutual Position and Transcriptional Directions

目的:研究质粒表达载体中2个表达单元间相互位置和转录方向关系对表达的影响,找出利于2个表达单元表达的优化的相互关系。方法:以全抗体为研究对象,构建了轻重链方向不同的2种相互关系的单质粒表达载体pIRESdhfrA和pIRESdhfrB,转染CHO-dhfr-细胞后,对瞬时表达水平进行了比较。以pIRESdhfrA和pIRESdhfrB为基础,构建了瞬时表达载体pIRESdhfrA-sv40ori和pIRESdhfrB-sv40ori,在COS-7细胞中进行了瞬时表达水平的比较。构建了基于CHO定点细胞系的稳定表达细胞株,对pIRESdhfrA和pIRESdhfrB进行稳定表达水平的比较。结果:在CHO-dhfr-的瞬时表达水平比较中,pIRESdhfrB转染的细胞表达水平是pIRESdhfrA转染细胞的2.18倍。与pIRESdhfrA-sv40ori在COS-7细胞的瞬时表达水平相比,pIRESdhfrB-sv40ori是其表达水平的2.3倍。pIRESdhfrB-FRT构建的CHO定点细胞平均表达水平是pIRESdhfrA-FRT构建的CHO定点细胞的11.6倍。结论:在构建的真核细胞质粒表达载体pIRESdhfr中,2个表达盒的启动子头-头转录方向关系比头-尾方向关系更利于重组蛋白的表达。

Objective： To investigate the effects of the two expression cassettes of one vector on the level of the expression in different cell lines, to determine the optimal mutual position and transcriptional directions of light and heavy chain cassettes to the expression. Methods： Two complete antibody expression vectors including pIRESdhfrA and pIRESdhfrB, have been constructed. They differ from each other by their mutual position and transcriptional directions of light and heavy chain cassettes. The expression levels of the two vectors in CHO cells were compared via transient expression. Two derived vectors pIRESdhfrA-sv40ori and pIRESdhfrB-sv40ori were constructed and transfected to COS-7 cells. Their transient expression levels in COS-7 were evaluated by sandwich ELISA. To be able to compare the expression levels of the pIRESdhfrA and pIRESdhfrB in engineered CHO cells, two gene-targeting vectors, pIRESdhfrA-FRT and pIRESdhfrB-FRT were constructed. These gene-targeting vectors were then transfected to the engineered CHO cell line, and their expression levels were compared. Results： Transient expression in CHO cell line shows that the expression level of pIRESdhfrB was as 2.18 fold higher than the expression level of pIRESdhfrA. In comparison to the expression level of pIRESdhfrA-sv40ori, the expression level of the pIRESdhfrB-sv40ori was 2.3 fold higher in COS-7. Furthermore, the ratio of expression levels of the two engineered CHO cell line which were targeted with pIRESdhfrA-FRT and pIRESdhfrB-FRT was 11.6 to 1. Conclusion： The mutual position and transcriptional directions of the two expression cassettes in the vector pIRESdhfr constructed, head-to-head is better than head-to-tail for the expression of recombinant protein.