CAJ | 학술논문

[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。
[목적]이용탑교PCR기술대단백질내함자HP진행개조。[방법]근거탑교PCR중과취핵감산련지간중첩적부분호상탑교、호위모판적원칙설계다대인물，통과다차PCR확증획득목적기인편단，진이실현대단백질내함자HP재기인수평상진행다개위점동시정점돌변화첨가Linker。[결과]탑교PCR산물극륭경DNA측서분석，증명단백질내함자HP내부적4개반광안산성공돌변성사안산，궤개PCR전체편단야무봉적융합성HP기인편단，이차몰유인진임하기타돌변；경DNA측서，46bp적DNALinker성공적삽입도설계호적HP기인서렬중，차몰유임하감기돌변화착배。[결론]해연구불부실현료대단백질내함자HP적쾌속개조，이차확대료탑교PCR적응용범위，동시야위단백질내함자적기출개조제공료저성본、간첩、고효적방법。
[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlapped and served as the template to the other DNA strand in amplification. Then, several rounds of over- lap PCR reaction were conducted to mutate the four cysteines in the HP intein into serines, and introduce a linker sequence containing a tag for product detection and purification. [Result] DNA sequencing analysis proved that the four cysteines in the HP intein were successfully mutated into serines; the PCR precursor fragments were also rejoined into an intact HP gene fragment, without introducing any other unex- pected mutation; the DNA linker of 46 bp was successfully inserted into the de- signed HP gene sequences, without any mutation and base pair mismatch. [Conclu- sion] The HP intein was rapidly reconstructed by overlap-extension PCR in this study, which not only expands the application of overlap PCR in protein engineering, but also provides a simple, efficient and highly accurate tool for the reconstruction and modification of intein.