斯氏副柔线虫GP基因的克隆、抗原表位预测及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2018.06.028

摘要/Abstract

摘要：

试验旨在克隆斯氏副柔线虫糖蛋白（glycoprotein，GP）抗原基因，并对其进行生物信息学分析。提取斯氏副柔线虫组织RNA，反转录合成cDNA，设计特异性引物以扩增GP基因CDS区，同时将其插入到克隆载体pMD19-T后测序，并对该基因编码蛋白的抗原表位、理化性质、信号肽、跨膜结构域等进行生物信息学预测。结果表明，GP基因开放阅读框（ORF）全长1 149 bp，编码382个氨基酸，其分子式为C₁₇₆₀H₂₈₇₈N₄₅₈O₅₉₀S₁₇₆₀，理论分子质量约为40.15 ku，等电点为4.37，无信号肽，总平均亲水性为0.032，不稳定系数为42.43，是疏水、不稳定蛋白；其磷酸化位点分布位于丝氨酸（Ser）、苏氨酸（Thr）和酪氨酸（Tyr）残基上；二级结构分析显示，斯氏副柔线虫GP蛋白以α-螺旋和无规则卷曲为主，与三级结构预测结果一致；抗原表位预测表明，GP蛋白可能有6个B细胞抗原表位和8个T细胞抗原表位，有望用作免疫诊断抗原和疫苗候选抗原。本研究成功克隆了斯氏副柔线虫GP基因，同时进行了系统的生物信息学分析和抗原表位预测，为斯氏副柔线虫病iELISA诊断方法的建立和DNA疫苗的研究提供理论依据。

关键词: 斯氏副柔线虫; GP基因; 抗原表位; 生物信息学

Abstract:

This study was aimed to clone and carry out bioinformatics analysis on the sequence of glycoprotein (GP) gene of Parabronema skrjabini.RNA was extracted from Parabronema skrjabini and cDNA was obtained by reverse transcription.A pair of specific primers was designed to amplify the coding region of GP gene.The target gene was linked into cloning vector pMD19-T to be sequenced.The antigen epitopes,physical and chemical properties,signal peptide and transmembrane domain of GP gene encoding protein were predicted according to the bioinformatics analysis.The bioinformatic analysis results showed that GP gene contained a 1 149 bp open reading fram (ORF),which encoding a hydrophobic protein 382 amino acids with a predicted theoretical molecular weight of 40.15 ku and the isoelectric point (pI) of 4.37.Amino acid sequence analysis showed that the GP protein was hydrophobin and unstable whose formula was C₁₇₆₀H₂₈₇₈N₄₅₈O₅₉₀S₁₇₆₀,the grand average of hydropathicity (GRAVY) was 0.032,and the coefficient of instability was 42.43.The protein was absence of signal peptide cleavage sites and the phosporylation site located in serine,threonine and tyrsine residue.The secondary structure of GP was mainly composed of alpha-helix and random curl,consistent with the results of tertiary structure prediction.Antigen epitopes prediction showed that GP protein might have six B-cell epitopes and eight T-cell epitopes.The GP antigen had promise as a candidate for immune diagnostics and a vaccine against Parabronema skrjabini.The GP gene of Parabronema skrjabini was cloned successfully and the structure and antigen epitopes of its coding protein were predicted by bioinformation software.This research would provide the theoretical basis for establishment of iELISA diagnostic method and development of DNA vaccine.

Key words: Parabronema skrjabini; GP gene; antigen epitopes; bioinformatics

中图分类号:

S852.73⁺1

赵学亮, 王梦雅, 孙柯, 冯陈晨, 王文龙. 斯氏副柔线虫GP基因的克隆、抗原表位预测及生物信息学分析[J]. 《中国畜牧兽医》, 2018, 45(6): 1645-1652.

ZHAO Xueliang, WANG Mengya, SUN Ke, FENG Chenchen, WANG Wenlong. Cloning, Prediction of Epitopes and Bioinformatic Analysis of GP Gene in Parabronema skrjabini[J]. , 2018, 45(6): 1645-1652.

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