杜氏利什曼原虫假定蛋白PA5的原核表达与生物信息学分析

The prokaryotic expression and bioinformatics analysis of the Leishmania donovia conserved hypothetical protein PA5

目的扩增杜氏利什曼原虫假定蛋白PA5基因,原核表达该假定蛋白并预测其结构与功能。方法以杜氏利什曼原虫前鞭毛体基因组DNA为模板,巢式PCR法扩增该基因,将其克隆入pQE30质粒构建pQE30-PA5重组质粒,经IPTG诱导表达目的蛋白。采用生物信息学方法对该蛋白进行同源性分析、信号肽及跨膜区预测、蛋白质结构与B细胞抗原肽位点分析。结果成功扩增出PA5基因,序列全长为765 bp,构建的pQE30-PA5重组质粒,经诱导后目的蛋白以包涵体形式表达。所获得的蛋白含254个氨基酸,相对分子质量为27 065,等电点为5.71的亲水性不稳定蛋白,不含信号肽及跨膜区。二级结构以α-螺旋及不规则卷曲为主,共含5个B细胞抗原肽片段。结论成功表达了杜氏利什曼原虫PA5蛋白,该亲水蛋白具有体液免疫刺激潜能,具有利什曼病潜在免疫诊断价值。

Objective To express the Leishmania donovia hypothetical protein PA5 in prokaryotic expression system and predict its structure and function. Methods The promastigote of Leishmania genomic DNA was used as the template for the amplification of PA5 by nested PCR method, and then it was cloned into pQE30 plasmid to construct pQE30-PA5 recombinant plasmid, which was induced by IPTG to express the target protein. Bioinformatics was used for homology analysis,signal peptide prediction, transmembrane region prediction, protein structure and B cell antigen peptide analysis.Results The PA5 gene was successfully amplified, and the sequence length of PA5 was 765 bp. The pQE30-PA5 recombinant plasmid was constructed, and the target protein was expressed in the form of inclusion body. The obtained protein contains 254 amino acids, the relative molecular weight was 27 065, and the isoelectric point was 5.71. It is a hydrophilic unstable protein without signal peptide and transmembrane region. Secondary structure is given priority to with alpha-helix and random coil,also there are five strong B cell antigen peptide fragments in PA5. Conclusion Leishmania donovia hypothetical protein PA5 was successfully expressed, and the hydrophilic protein has the potential of strong humoral immunity, which indicate a potential immunological diagnosis value of leishmaniasis.