SARS相关冠状病毒Spike蛋白定点诱变

Site-Directed Mutation of the Spike Protein of SARS-CoV

目的分析确定SARS-CoV Spike蛋白序列中对SARS-CoV的感染能力相关性较强的位点,并进行定点诱变。方法比对不同来源株SARS-CoV的Spike蛋白的序列,计算其相应位点的突变墒值和突变几率,结合进一步的生物信息学分析,筛选出经预测可能影响Spike蛋白与其受体结合进而导致SARS病毒对宿主细胞的膜融合侵染能力的7个位点,对各个位点用重叠延伸PCR法或直接PCR法进行人工定点诱变,获7个突变片段,之后将各片段亚克隆至克隆载体PSP72中。结果经PCR和测序确认各片段点突变成功,并正确插入克隆载体PSP72中。结论成功实施Spike蛋白的定点诱变,获得7个突变片段的重组子,这些突变体可为研究这些突变位点在Spike蛋白与受体结合中的意义,进而可为阐述Spike蛋白的结构与功能的关系、揭示Spike蛋白变异与SARS病毒侵入(染)细胞能力的相关性提供坚实的实验基础。

Objective To determine the protein sequence homology of the Spike proteins of SARS-CoV,and perform functional analysis of the Spike protein using site-directed mutagenesis. Methods We align the sequences of Spike protein of various resources SARS-CoVs and calculate the mutation entropy and rate of every site. Combining more bio-information analyse, we select seven sites in the decades of mutation sites which may effect the binding between Spike protein and the receptor and then effect the ability of membrane fusion and mutate every site with overlap-extension PCR （OE-PCR） or mega-primer PCR method and sub-clone them into vector of PSP72. Results Site-directed mutations have been done successfully and they are inset into the vector, which is confirmed by PCR and sequence. Conclusion we acquire seven fragments of mutation which are the foundation of the study in the binding between the Spike and the receptor, and they can expound the relation between the construction and function and open out the relativity between the mutation of Spike protein and the viral ability of infection.