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目的初步探索人巨细胞病毒耐药相关蛋白PUL97局部序列(329～572aa)的三维模拟结构并进行突变的耐药分析。方法采用巢式PCR方法扩增UL97基因776 bp片段并测序,以此发现非同义突变;然后利用SWISS-MODEL结构模拟综合服务器和其他相关软件比对模式对野生型PUL97蛋白片段进行同源模建并优化,通过各种指标或服务器来评估模拟结构的优劣并作筛选。将筛选所得的最佳模拟结构用于非同义新突变型的初步耐药分析。结果最终筛选出的模拟结构是以1YDTE为模板,以L1.FASTA为比对文件所得的Model A。整个分子的能量最低,ProQ的预测分数最高。分子对接分析也发现了潜在的ATP和GCV结合口袋。利用已知的阴阳性耐药点突变验证了该结构的合理性。同时根据突变前后结构信息的改变,提示C518Y单点突变以及联合突变(E517G和C518Y)呈现高度可疑的耐药特性。结论 PUL97野生型蛋白计算机结构模拟和突变分析法是初步评估新突变型耐药特性的又一方便快捷的方法 ,可用于实验验证前的筛选;但模拟结构本身的准确性是成功运用该方法的前提。
Objective To explore the three-dimensional (3D) mimicry structure of PUL97 local sequence of human cytomegalovirus resistance-related protein (329-572aa) and to analyze its resistance to mutation. Methods The 776 bp fragment of UL97 gene was amplified by nested PCR and sequenced to find non-synonymous mutations. Then, the homology model of wild-type PUL97 protein fragment was amplified by SWISS-MODEL software and other related softwares. Built and optimized to assess and evaluate the pros and cons of simulation structures through various metrics or servers. The best simulated structure obtained from screening was used for the preliminary drug resistance analysis of non-synonymous new mutants. The results of the final screening of the simulation structure is 1YDTE as a template to L1.FASTA as a model file obtained from Model A. The whole molecule has the lowest energy and ProQ has the highest predicted score. Molecular docking analysis also found potential ATP and GCV binding pockets. The known rationality of this structure is verified by the known point mutation of yin-yang resistance. At the same time, according to the changes of the structural information before and after the mutation, it is suggested that the single-point mutation of C518Y and the combined mutation (E517G and C518Y) present highly suspicious resistance characteristics. Conclusion The computer simulation of structure and mutation analysis of PUL97 wild-type protein is a convenient and rapid method for preliminary evaluation of new mutant drug resistance characteristics, which can be used for screening before experimental verification. However, the accuracy of simulation structure itself is successfully applied premise.