Furebring C, Ohlin M, Pettersson S, Borrebaeck C A
Department of Immunotechnology, Lund University, Sweden.
Gene. 1997 Apr 1;188(2):191-8. doi: 10.1016/s0378-1119(96)00791-3.
A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH) genes has been designed. This expression vector contains the variable heavy chain (VH) promoter, the IgH intron enhancer (muE) and the IgH 3' enhancer (3'E). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a rearranged IgH gene was observed when using the pTIF-1 vector containing the 3'E compared to an expression vector lacking this enhancer. Interestingly, this positive effect on the expression level of the 3' enhancer appears to be position independent. The introduction of two recently identified Ig control elements, HS3 and HS4, to the vector cassette did not further elevate the expression level in the cell line tested. The pTIF-1 vector can be used for expression of any antibody specificity, using PCR amplification of the VDJ region of interest. Furthermore, the constant region can easily be exchanged, which further facilitates studies to dissect different effector functions of IgH constant genes.
一种用于生产免疫球蛋白重链（IgH）基因的新型哺乳动物真核表达载体已被设计出来。该表达载体包含可变重链（VH）启动子、IgH内含子增强子（μE）和IgH 3'增强子（3'E）。这种构建体被命名为pTIF-1，它被稳定转染到骨髓瘤细胞系J558L中。与缺乏该增强子的表达载体相比，当使用含有3'E的pTIF-1载体时，重排的IgH基因表达水平提高了五倍。有趣的是，3'增强子对表达水平的这种积极作用似乎与位置无关。将两个最近鉴定出的Ig控制元件HS3和HS4引入载体盒中，在测试的细胞系中并未进一步提高表达水平。pTIF-1载体可用于表达任何抗体特异性，通过对感兴趣的VDJ区域进行PCR扩增即可实现。此外，恒定区可以很容易地进行交换，这进一步便于研究剖析IgH恒定基因的不同效应功能。
