| 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(MHC classⅡmolecule transactiva-tor,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. |
| 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 |
| DOI：10.3724/SP.J.1008.2006.01014 |
| 投稿时间：2005-12-06修订日期：2006-05-22 |
| 基金项目:上海市科委联合利华研究与发展基金(200305). |
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| Construction of a middle fragment-deleted class Ⅱ molecule transactivator mutant by modified OE-PCR technique |
| GUAN Zheng,SHEN Qian |
| (第二军医大学长海医院实验诊断科,上海,200433) |
| Abstract: |
| 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 |
| Key words: overlap extension by polymerase chain reaction middle fragment deletion mutant MHC class Ⅱ molecule transaetivator |
