Construction of a middle fragment-deleted class Ⅱ molecule transactivator mutant by modified OE-PCR technique
- VernacularTitle:用优化的OE-PCR法构建CⅡTA中间缺失突变体
- Author:
Zheng GUAN
;
Qian SHEN
- Publication Type:Journal Article
- Keywords:
overlap extension by polymerase chain reaction;
middle fragment deletion mutant;
MHC classⅡ molecule transactivator
- From:
Academic Journal of Second Military Medical University
2001;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To develop a simple and efficient method for constructing a middle fragment-deleted mutant of MHC class Ⅱ molecule transactivator(CⅡTA)mutant with the 109~(th)to 226~(th) amino acid codons deleted.Methods: Two gene fragments at each end of the deleted CⅡTA gene were obtained by OE-PCR method and were mixed together for 8 PCR cycles without primers to achieve effective overlapping,then 2 primers was added for amplification of the desired fragments.The amplification products were subsequently cloned into eukaryotic vector pIRES for identification.Results: A mutant of CⅡTA with the 109~(th)to 226~(th) amino acids deleted was successfully constructed.Conclusion: This modified OE-PCR technique overcomes some shortcomings of traditional method and is very suitable for constructing mutants with middle fragment deletion,making it worth to be popularized.
