嗜吞噬细胞无形体msp4蛋白的生物信息学分析及表达鉴定

Expression and Bioinformatics Analysis of the msp4 Protein of Anaplasma Phagocytophilum

分析预测嗜吞噬细胞无形体msp4蛋白的抗原性,对嗜吞噬细胞无形体的msp4蛋白采用生物信息学对其进行分析二级结构,亲水性,疏水性,B细胞线性表位;根据分析的优势抗原表位区,进行基因合成并亚克隆至p ET32a表达载体,在大肠埃希菌中获得重组蛋白。生物信息学分析结果显示msp4蛋白由283个氨基酸组成,分子量为29.8 ku,理论等电点为6.05,不稳定系数为32.79,总平均疏水性为0.063;二级结构预测msp4蛋白主要以无规卷曲、延伸链、α-螺旋为主;B细胞表位预测msp4蛋白有14个线性表位;根据分析的结果选取msp4蛋白亲水性高的膜外区的28-158位序列克隆至p ET32a进行原核表达,在34 ku出有目的蛋白的表达。成功对嗜吞噬细胞无形体的msp4蛋白原核表达及纯化,为无形体病血清学检测方法的建立奠定了物质基础。

The purpose of this study was to analysis the antigenicity of msp4 protein of anaplamsa phagocytophilum. The informatics analysis was used to predict and analyze the important parameters of the msp4 protein, such as the physical and chemical properties, protein secondary structure, and its hydrophilicity, hydrophicity, bepipred linear epitope. According to the advantage epitope, we selected the high hydrophilic region of protein membrane outside domains, and cloned to pET32a prokaryotic expression vector. The bioinformatics analysis revealed that the msp4 protein was composed of 283 amino acids, and that its isoelectric point was 6.52. The msp4 protein was an stable hydropathilic protein（the instablility coefficient was 32.79, the grand average of hydropathicity was 0.063）. The secondary structure of the msp4 protein was mainly composed of random coils, extended strands, and alpha helixes. B cell epitope predicted that the msp4 protein has 14 linear epitopes. From the result of bioinformatics analysis, we selected the the 28-158 aa of the high hydrophilic region of protein membrane outside domains, and cloned to pET32a prokaryotic expression vector, the msp4 protein was successfully expressed and purified. The expressed and purified the recombinant msp4 protein lays a material foundation for the establishment of serological methods for anaplasma phaagocytophilum detection.