Abstract: Objective　To analyze the sequence characteristics of Rhipicephalus microplus Enolase gene, and to predict the secondary and tertiary structure and antigenic epitopes of the Enolase protein. Methods　Sixty⁃two engorged female R. microplus were sampled from a yellow cattle breeding farm in Zhijiang County, Huaihua City, Hunan Province in June 25, 2022. Genomic DNA was isolated from R. microplus, and the Enolase gene was amplified using PCR assay, followed by cloning, sequencing and expression of the amplification product. The sequence characteristics of the Enolase gene were analyzed using the software Clustal X, and the gene sequence was translated into amino acid sequences. The secondary and tertiary structures of the Enolase protein were deduced using the software PRABI, and the physicochemical properties of the Enolase protein were analyzed using the software PRABI. In addition, the B⁃ and T⁃cell epitopes of the Enolase protein were predicted using the software ABCpred Prediction, Scratch, IEDB and NetCTL. Results　The R. microplus Enolase gene sequence was 1 323 bp in size, and the contents of A, T, G and C bases were 24.5%, 22.5%, 27.0% and 26.0%,with 47.0% of A + T content and 53.0% of G + C content. The R. microplus Enolase gene encoded 434 amino acids, and the Enolase protein had a molecular weight of 47.12 kDa. The secondary structure of the Enolase protein contained 186 α⁃helixes (42.86%), 32 β⁃turns (7.37%), 144 random coils (33.18%) and 72 extended strands (16.59%). The Enolase protein was most probably present in cytoplasm (76.7%), followed by in mitochondrion (39.1%) and nucleus (21.7%), and the Enolase protein had no signal peptide or transmembrane domain. In addition, the Enolase protein had 14 B⁃cell dominant epitopes and 8 T⁃cell dominant epitopes. Conclusions　The R. microplus Enolase gene sequence exhibits a GC preference, and its encoding Enolase protein is an acidic and hydrophilic protein, with α⁃helixes and random coils as its primary structure, and presenting B⁃ and T⁃cell dominant epitopes, which is a potential target for development of vaccines against R. microplus.

Key words: Rhipicephalus microplus, Enolase gene, Enolase protein, Protein structure, Antigenic epitope

摘要： 目的　分析微小扇头蜱Enolase基因序列特征，并预测其所编码Enolase蛋白二、三级结构及抗原表位。方法 　2022年6月25日在湖南省怀化市芷江县某黄牛养殖场采集62只雌性饱血微小扇头蜱，提取其DNA，PCR扩增其Enolase基因，PCR扩增产物克隆、测序并表达。采用软件Clustal X分析Enolase基因序列特征，并将基因序列翻译成氨基酸序列。采用PRABI软件推导出Enolase蛋白二、三级结构，并对其理化性质进行分析；采用ABCpred Prediction、Scratch、IEDB和NetCTL软件预测Enolase蛋白B、T细胞抗原表位。结果　微小扇头蜱Enolase基因序列全长1 323 bp，碱基A、T、G、C含量分别为24.5%、22.5%、27.0%、26.0%，A + T含量为47.0%、G + C含量为53.0%。该基因共编码434个氨基酸；Enolase蛋白分子量大小为47.12 kDa，其二级结构含186个（42.86%）α⁃螺旋、32个（7.37%）β⁃转角、144个（33.18%）无规卷曲、72个（16.59%）扩展链。Enolase蛋白存在于细胞质中的概率最大（76.7%），其次是线粒体（39.1%）和细胞核（21.7%），该蛋白无信号肽和跨膜结构域。Enolase蛋白预计有14个B细胞优势抗原表位和8个T细胞优势抗原表位。结论　微小扇头蜱Enolase基因序列呈GC偏好；其所编码的Enolase蛋白为酸性亲水性蛋白，以α⁃螺旋和无规卷曲为主要结构，且具有B、T细胞优势抗原表位，是微小扇头蜱疫苗研发的一种理想靶标。 　

关键词: 微小扇头蜱, Enolase基因, Enolase蛋白, 蛋白结构, 抗原表位