论文部分内容阅读
[目的]利用搭桥PCR技术对蛋白质内含子HP进行改造。[方法]根据搭桥PCR中寡聚核苷酸链之间重叠的部分互相搭桥、互为模板的原则设计多对引物,通过多次PCR扩增获得目的基因片段,进而实现对蛋白质内含子HP在基因水平上进行多个位点同时定点突变和添加Linker。[结果]搭桥PCR产物克隆经DNA测序分析,证明蛋白质内含子HP内部的4个半胱氨酸成功突变成丝氨酸,几个PCR前体片段也无缝的融合成HP基因片段,而且没有引进任何其他突变;经DNA测序,46bp的DNALinker成功的插入到设计好的HP基因序列中,且没有任何碱基突变和错配。[结论]该研究不仅实现了对蛋白质内含子HP的快速改造,而且扩大了搭桥PCR的应用范围,同时也为蛋白质内含子的基础改造提供了低成本、简捷、高效的方法。
[Objective] The purpose of this study was to modify the protein intron HP by using the bypass PCR technique. [Method] According to the principle that the overlapping parts of the oligonucleotide strands of the bypass PCR were bridged with each other and designed each other as templates, multiple pairs of primers were designed. The target gene fragments were obtained by multiple PCR amplification, and then the protein intron HP At the genetic level, multiple site-specific site-directed mutagenesis and Linker addition were performed. [Result] The result of DNA sequencing showed that the four cysteines in protein intron HP were successfully mutated to serine, and several PCR precursor fragments were fused into HP gene fragment without any defects Introduced any other mutations; DNA sequencing, 46bp DNALinker successfully inserted into the designed HP gene sequence, and without any base mutation and mismatch. [Conclusion] The study not only realized rapid transformation of protein intron HP, but also expanded the scope of application of bypass PCR and provided a low-cost, simple and efficient method for the basic transformation of protein introns.