自组装短肽K_2I_4K_2在PCR中提高Taq DNA聚合酶热稳定性  

Self-assembling peptide K_2I_4K_2 enhances the thermal-stability of Taq DNA polymerase in PCR system

作　　者：李萌萌[1] 徐晓帆[1] 陈振银[1] 张慧楠[1] 罗忠礼[1] 

机构地区：[1]重庆医科大学基础医学院分子与肿瘤研究中心,重庆400016

出　　处：《基础医学与临床》2016年第7期879-885,共7页Basic and Clinical Medicine

基　　金：国家自然科学基金(31540019);重庆市科委自然科学基金(cstc2015jcyj BX0072);重庆医科大学科研培育基金(自然科学类)(201417);重庆医科大学基础医学院苗圃基金(JC201514);四川省重点实验室抗生素及其相关研究实验室基金(ARRLKF14-01)

摘　　要：目的设计自组装短肽K_2I_4K_2作为新型表面活性剂来稳定Taq DNA聚合酶,探索其对Taq DNA聚合酶在高温下的半衰期以及对PCR扩增效率的影响,探讨其在PCR中应用的可行性。方法圆二色仪检测短肽K_2I_4K_2在不同理化条件下的二级结构;原子力显微镜检测其自组装形成的纳米结构特征;紫外可见光谱检测其对Taq酶在高温下半衰期的影响;普通PCR验证其在PCR中应用的可行性;RT-q PCR检测其对Taq酶扩增效率的影响。结果短肽K_2I_4K_2在盐离子溶液中能自组装形成纳米纤维结构,且在高温下拥有稳定的二级结构;短肽K_2I_4K_2使得Taq在95℃下的半衰期增加了约1.6倍;短肽K_2I_4K_2组装24 h后大大增加了Taq酶的扩增效率。结论短肽K_2I_4K_2较为显著地增加了Taq DNA聚合酶的热稳定性,能够很好地应用于PCR反应系统中。Objective To design a self-assembling peptide K214K2 as a new type of surfactant to stabilize Taq DNA polymerase, for exploring the half-life of Taq DNA polymerase at high temperature and the influence on PCR ampli- fication efficiency. Methods Circular dichroism spectrum was used to detect the secondary structure of short pep- tide K214K2 in different physical and chemical condition. Atomic force microscope was used to detect the nanostmc- ture characteristics formed by K214K2. UV-vis spectrum was used to examine the impact on the half-life of Taq en- zyme stabilized by K214K2 at high temperature. Ordinary PCR was used to verify the feasibility of the application of K214K2 in the PCR. RT-qPCR was used to detecte of K214K2s effect on the amplification efficiency of Taq polymer- ase. Results Peptide K214K2 can form nanofiber structure in salt solution, and shifted to a stable secondary struc- ture at high temperature. UV-vis spectrum result indicated that peptide KEI4K2 increase the half-life by 1.6-fold compared with the stability in buffer alone. Furthermore, RT-qPCR indicated that peptide K214K2 greatly increasedthe amplification efficiency of Taq polymerase. Conclusions Peptide K214K2 more significantly increases the ther- mal stability of the Taq DNA polymerase at a high temperature, and can be well used in the PCR system.

关 键 词：自组装短肽 纳米纤 Taq酶半衰期 PCR扩增效率 

分 类 号：Q503[生物学—生物化学]