为探究微小扇头蜱P0基因序列特征,预测P0蛋白的理化性质和二、三级结构,筛选出P0蛋白的B、T优势抗原表位,本研究克隆了微小扇头蜱P0基因,运用Clustal Ⅹ软件分析P0基因序列特征,用在线软件EXPASY、PRABI和SWISS-MODEL预测P0蛋白的理化性质和二、三级结构,用在线软件ABCpred Prediction、Scratch、JEDB和NetCTL筛选P0蛋白的B、T优势抗原表位.试验结果显示:微小扇头蜱P0基因全长957 bp,碱基A含量为24.0％,T含量为20.3％,G含量为27.5％,C含量为28.2％,A+T含量为44.3％,G+C含量为55.7％,共编码318个氨基酸;P0蛋白分子量为34 ku,理论等电点(pI)为5.86,平均亲水系数为-0.153,不稳定指数为38.15;P0蛋白的二级结构含163个α螺旋(占比51.25％),130个无规卷曲(占比40.88％),25个延伸链(占比7.86％),其中以α螺旋为主要结构;P0蛋白的三级结构以α螺旋的含量最高,该蛋白的全局模型质量评估(global model quality estimation,GMQE)、定性模型能量分析(qualitative model en-ergy analysis,QMEAN)值分别为0.49和0.52±0.05,无信号肽和跨膜结构域,但存在40个磷酸化位点和1个糖基化位点;P0蛋白有13个B淋巴细胞优势抗原表位和6个T淋巴细胞优势抗原表位.综上所述,微小扇头蜱P0基因序列呈GC偏好,P0蛋白是以α螺旋为主要结构成分的亲水性酸蛋白,具有B、T淋巴细胞优势抗原表位,是今后研制防控微小扇头蜱疫苗的理想靶标.
Cloning of P0 gene in Rhipicephalus microplus and bioinformatics analysis of its encoded protein
In order to explore the sequence characteristics of Rhipicephalus microplus P0 gene,predict the physical and chemical properties,and secondary and tertiary structures of P0 protein,and screen the B and T dominant epitopes of P0 protein,the P0 gene of R.microplus was cloned and analyzed by Clustal Ⅹ software.The physical and chemical properties,secondary and tertiary structures of P0 protein were predicted by online software EXPASY,PRABI and SWISS-MODEL,and the B and T dominant epitopes of P0 protein were screened by online software AB-Cpred Prediction,Scratch,IEDB and NetCTL.The results showed that the P0 gene of R.microplus was 957 bp in length,with 24.0％A,20.3％T,27.5％G,28.2％C,44.3％A+T and 55.7％G+C,encoding 318 amino acids;The molecular weight of P0 protein was 34 ku,the theoretical isoelectric point(pI)was 5.86,the average hydrophilicity coefficient was-0.153,and the instability index was 38.15;The secondary structure of P0 protein contained 163 α-helices(51.25％),130 random coils(40.88％)and 25 extended strands(7.86％),and the α-helix was the main structure;The content of α-helix in tertiary structure of P0 protein was the highest.The value of global model quality estimation(GMQE)and qualitative model energy analysis(QMEAN)of P0 protein were 0.49 and 0.52±0.05,respectively.There was no signal peptide and transmembrane domain,but there were 40 phos-phorylation sites and 1 glycosylation site;There were 13 B lymphocyte dominant antigen epitopes and 6 T lymphocyte dominant antigen epitopes in P0 protein.In conclusion,the P0 gene sequence of R.microplus showed GC prefer-ence,and the P0 protein was a hydrophilic acidic protein with α-helix as the main structural component,and had the dominant antigen epitopes of B and T lymphocytes,which was an ideal target for the development of R.microplus vaccine in the future.
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