Cloning of P0 gene in Rhipicephalus microplus and bioinformatics analysis of its encoded protein
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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.
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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