基于人DAF框架的酵母展示肽库的构建与鉴定

以hDAFcDNA为模板 ,用随机引物分别通过大引物PCR和交叠PCR方法引入突变 ,酶切后克隆到酵母表面展示载体pYD1 ,构建DAF pYD1重组质粒 ,转化酵母细胞 ,PCR鉴定随机性 ,流式细胞仪分析DAF的表达。结果表明 ,随机性达到理论要求 ,通过流式细胞仪检测到酵母细胞表面有DAF分子的表达。以上结果表明 ,成功构建了基于DAF框架的酵母表面展示肽库 ,为进一步筛选DAF的突变体库奠定了基础。

Construction and Identification of the Human Decay-Accelerating Factor (hDAF) Scafford-Based Yeast Displayed Peptide Library

In the present study, the cDNA of human decay-accelerating factor(hDAF) was used as template, and the PCR with mega-primer and the overlap extension PCR-based mutation was induced by random primers. The PCR products were cloned into yeast surface display vector pYD1 after digestion with enzymes, and the recombinant vector DAF-pYD1 was thus constructed. This recombinant vector was then transfected into yeast cells. Diversity of amino acids at the randomized positions was evaluated by PCR, and the expression of DAF on the surface of yeast cells was determined by flow cytometery. It was found that the library showed great diversity of amino acids at the randomized positions, and DAF was detected on the surface of yeast cells by flow cytometery. It is concluded that the yeast display peptide library on human decay-accelerating factor scafford was successfully constructed in the present study, and this might provide a favorable basis of further studies.