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目的在耻垢分枝杆菌中克隆表达噬菌体D29LysinA基因,用生物信息学分析LysinA基因所编码蛋白的生物学特性及进化特征。方法增殖、收集噬菌体D29,提取其基因组DNA,PCR扩增LysinA基因后对产物进行测序和比对分析;采用ProtParam软件预测LysinA编码蛋白的结构、特性及进化特征。结果噬菌体D29LysinA基因扩增产物与预期值相符,大小约为1 482bp;测序表明LysinA基因序列与GenBank中已发表的完全一致。LysinA蛋白有493个氨基酸,分子式为C2419H3739N715O725S12,分子质量单位为54.8ku,理论等电点为5.79,在哺乳动物体外的半衰期约为30h;平均疏水系数为-0.443,总体上为亲水性蛋白;二级结构包含α-螺旋(占36.92%)、β-转角(占11.15%)、β-折叠(占17.04%)和无规则卷曲(占34.89%);预测的B细胞、CTL细胞及Th细胞抗原表位分别为17、15和21个,并模拟了LysinA蛋白进化树显示噬菌体D29与Chy5亲缘关系较近。结论成功表达了噬菌体D29LysinA蛋白,经生物信息学分析LysinA蛋白具良好免疫原性,为疫苗开发和结核病治疗奠定理论基础。
Objective To clone and express bacteriophage D29LysinA gene in Mycobacterium smegmatis and analyze the biological characteristics and evolution characteristics of LysinA gene by bioinformatics analysis. Methods The phage D29 was collected and its genomic DNA was extracted. The LysinA gene was amplified by PCR and sequenced. The structure, characteristics and evolution of LysinA protein were predicted by ProtParam software. Results The amplified product of phage D29LysinA gene was consistent with the expected value, which was about 1 482 bp in length. Sequencing showed that the sequence of LysinA gene was exactly the same as published in GenBank. LysinA protein has 493 amino acids, the molecular formula is C2419H3739N715O725S12, the molecular mass unit is 54.8ku, the theoretical isoelectric point is 5.79, and the half-life in mammals is about 30h. The average hydrophobic coefficient is -0.443, which is generally hydrophilic protein. The secondary structure contains α-helix (36.92%), β-turn (11.15%), β-sheet (17.04%) and irregular curl (34.89%). The predicted B cells, CTL cells and Th cells Epitopes were 17, 15, and 21, respectively. The phylogenetic tree of LysinA was simulated to show that the phage D29 had a close genetic relationship with Chy5. Conclusion The bacteriophage D29LysinA protein was successfully expressed. The bioinformatics analysis of LysinA protein showed good immunogenicity and laid the theoretical foundation for vaccine development and tuberculosis treatment.