摘要
目的：以构建缺失第109～226位氨基酸密码子的MHCII类分子反式激活因子（MHCclass Ⅱ molecule transactivator，CIITA）突变体为例探索一种简便、稳定的构建中间缺失突变体的方法。方法：首先按照传统重叠延伸PCR方法的原理扩增缺失片段两端的基因片段，再将两片段混合，在不加入外围引物的情况下进行8次PCR循环以有效完成重叠延伸，然后再加入引物进行目的片段指数扩增，将扩增产物直接克隆至真核表达载体pIRES。结果：成功地构建出缺失第109～226位氨基酸密码子的CⅡTA突变体。结论：优化后的重叠延伸PCR法（OE—PCR）克服了传统方法的诸多弊端，非常适合于构建中间缺失突变体，值得推广。
Objective：To develop a simple and efficient method for constructing a middle fragment-deleted mutant of MHC class Ⅱ molecule transactivator （C ⅡTA ）mutant with the 109^th to 226^th amino acid codons deleted. Methods： Two gene fragments at each end of the deleted C Ⅱ TA 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 C Ⅱ TA 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.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2006年第9期1014-1017,共4页
Academic Journal of Second Military Medical University
基金
上海市科委联合利华研究与发展基金(200305)~~
关键词
重叠延伸PCR法
中间缺失突变体
MHCⅡ类分子反式激活因子
overlap extension by polymerase chain reaction
middle fragment deletion mutant
MHC class Ⅱ molecule transaetivator
