NOVEL VECTORS FOR THE EXPRESSION OF ANTIBODY MOLECULES USING VARIABLE REGIONS GENERATED BY POLYMERASE CHAIN-REACTION
NOVEL VECTORS FOR THE EXPRESSION OF ANTIBODY MOLECULES USING VARIABLE REGIONS GENERATED BY POLYMERASE CHAIN-REACTION
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DOI：
10.1016/0022-1759(92)90092-8
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发表时间：
1992-07-31
影响因子：
2.2
通讯作者：
MORRISON, SL
中科院分区：
文献类型：
--
作者：
COLOMA, MJ;HASTINGS, A;MORRISON, SL
已经产生了一个新的载体家族，其有利于通过聚合酶链反应（PCR）克隆的免疫球蛋白可变区的克隆和表达。该载体设计成表达与人恒定区连接的克隆的可变区，并利用前导序列中的引发，从而不会将氨基酸改变引入成熟抗体分子。重链和轻链载体都利用提供有EcoRV限制性位点的鼠V（H）启动子，从而扩增的可变区可以直接克隆到功能性启动子中。对于重链，在C（H）1的前两个氨基酸处产生NheI限制性位点，克隆的前导区和可变区直接融合至恒定区的C（H）1结构域。当轻链的前导区和可变区直接与C（κ）融合时，未观察到表达。因此，轻链表达载体设计有SalI限制性位点，用于克隆到可变区的3'剪接点;然后通过剪接将V（L）连接到C（κ）。两种载体都指导具有预期分子量的功能性、完全组装的免疫球蛋白分子的表达。使用冗余的寡聚体来引发PCR允许克隆和表达重组抗体，而无需关于其序列的任何先验信息，并且使得可以从任何单克隆抗体产生细胞系快速产生重组抗体。
A new family of vectors has been produced which facilitates the cloning and expression of immunoglobulin variable regions cloned by polymerase chain reaction (PCR). The vectors are designed to express the cloned variable regions joined to human constant regions and take advantage of priming in the leader sequence so that no amino acid changes will be introduced into the mature antibody molecule.Both the heavy chain and light chain vectors utilize a murine V(H) promoter provided with an EcoRV restriction site so that the amplified variable regions can be directly cloned into a functional promoter. For the heavy chain an NheI restriction site has been generated at the first two amino acids of C(H)1 and the cloned leader and variable region are fused directly to the C(H)1 domain of the constant region. When the leader and variable regions of the light chain were fused directly to C(kappa), no expression was observed. Therefore the light chain expression vector was designed with a SalI restriction site for cloning into a splice junction 3' of the variable region; V(L) then is joined to C(kappa) by splicing. Both vectors direct the expression of functional, fully assembled immunoglobulin molecules with the expected molecular weight. Use of redundant oligomers to prime the PCR permits the cloning and expression of recombinant antibodies without any prior information as to their sequence.and makes it possible to rapidly generate recombinant antibodies from any monoclonal antibody producing cell line.