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目的克隆苏黎世克罗诺杆菌(CT)DSM 18703株外膜蛋白A(Outer membrane protein A,OmpA)基因,并对其编码蛋白进行生物信息学分析。方法以CT DSM 18703株DNA为模板,PCR扩增OmpA基因,克隆入表达载体pMD19-T进行测序分析,利用生物信息学方法对编码OmpA蛋白的理化性质、保守结构域、亲水性/疏水性、信号肽、跨膜区、亚细胞定位、抗原决定簇、二级和三级结构及同源性进行预测分析。结果CT DSM 18703株OmpA基因核苷酸序列长度为1 044bp,其编码蛋白由347个氨基酸组成(基因登录号:AGQ57145.1),编码蛋白的分子质量单位为37.11ku,等电点值理论为5.20。该蛋白具有1个跨膜蛋白保守结构域,含有1个信号肽,不含跨膜区,是一种外膜蛋白;含11个与免疫相关的抗原决定簇,具有形成抗原表位的优势结构,可作为潜在的候选疫苗。二级结构中α-螺旋、β-折叠、β-转角和无规则卷曲的含量分别为29.97%、19.60%、8.93%和41.50%。多重序列比对和聚类分析显示,该蛋白序列与CT z2032株OmpA蛋白(CBA 29736.1)有高度同源性,且在系统发育树上与二者聚为一簇。结论成功克隆了CT DSM 18703株OmpA基因,生物信息学分析显示其编码蛋白OmpA含有潜在的抗原决定簇区域,可作为苏黎世克罗诺杆菌工程疫苗候选基因。
Objective To clone OmpA gene of Outer membrane protein A (OmpA) from Corynebacterium zikka (CT) strain DSM 18703 and analyze its encoded protein for bioinformatics analysis. Methods CTM18703 DNA was used as template to amplify OmpA gene by PCR and cloned into expression vector pMD19-T for sequencing analysis. Bioinformatics methods were used to analyze the physicochemical properties, conserved domains, hydrophilicity / hydrophobicity , Signal peptide, transmembrane region, subcellular localization, antigenic determinant, secondary and tertiary structure and homology. Results The nucleotide sequence of OmpA gene of CT DSM strain 18703 was 1 044 bp in length and contained a protein of 347 amino acids (gene accession number: AGQ57145.1). The molecular mass unit of the encoded protein was 37.11 ku. The isoelectric point value theory was 5.20. The protein has a transmembrane protein conserved domain, contains a signal peptide, does not contain the transmembrane region, is an outer membrane protein; contains 11 immune-related epitopes, with the formation of antigen epitopes of the dominant structure , Can be used as a potential candidate vaccine. The content of α-helix, β-sheet, β-turn and random coil in the secondary structure were 29.97%, 19.60%, 8.93% and 41.50%, respectively. Multiple sequence alignment and cluster analysis showed that this protein was highly homologous to the OmpA protein of CT z2032 strain (CBA 29736.1) and clustered with both in phylogenetic tree. Conclusion OmpA gene of CT DSM strain 18703 was successfully cloned. Bioinformatics analysis showed that OmpA encoded the protein OmpA region containing potential antigenic determinants, which could be used as a candidate gene of Cronobacterium Zurich engineering vaccine.