伊氏锥虫伊犁株扩繁及其PFR基因克隆表达和生物信息学分析

doi:10.16431/j.cnki.1671-7236.2023.02.005

摘要/Abstract

摘要： 【目的】本研究旨在研究锥虫入侵宿主细胞的机制并为其检测方法的建立奠定基础。【方法】采用已分离保存的伊氏锥虫在昆明白小鼠体内进行培养繁殖,对其鞭毛束旁棒(PFR)基因进行克隆,并构建系统发育树。通过生物信息学方法分析和预测PFR蛋白的理化特性、亲疏水性、跨膜区结构、二级结构和三级结构;构建原核表达载体pET28a-PFR,用Western blotting检测PFR重组蛋白的反应原性。【结果】在昆明白小鼠体内成功扩繁伊氏锥虫伊犁株,第5天染虫率达到最高;PFR基因PCR扩增片段大小为834 bp,与冈比亚布氏锥虫PFR基因(XP_011775815.1)相似性为99.52%,基于PFR蛋白氨基酸序列的进化树显示,该虫株与冈比亚布氏锥虫的亲缘关系也最近。PFR蛋白分子式为C₁₄₁₆H₂₂₈₆N₄₁₆O₄₄₂S₁₁,理论等电点为5.74,是一种碱性、亲水性及不稳定蛋白,没有跨膜区和信号肽,有10个潜在抗原表位;主要定位于细胞质中;PFR蛋白二级结构主要由α-螺旋组成(占92.34％),三级结构与二级结构一致。成功构建伊氏锥虫PFR基因表达质粒pET28a-PFR,经诱导时间、温度和IPTG浓度等条件优化后发现,浓度为1 mmol/L IPTG于37 ℃诱导重组菌液6 h时,PFR蛋白表达量最高,并且以包涵体的形式表达;Western blotting结果显示,重组蛋白与伊氏锥虫阳性血清反应为阳性。【结论】本试验成功在昆明白小鼠体内扩繁出伊氏锥虫,克隆了马伊氏锥虫PFR基因,构建了PFR原核表达载体,诱导表达出PFR重组蛋白,该蛋白具有良好的反应原性,为后续该蛋白功能和马伊氏锥虫的致病相关机制研究提供了理论依据。

关键词: 伊氏锥虫; 鞭毛束旁棒(PFR); 原核表达; 遗传进化; 生物信息学分析

Abstract: 【Objective】 The aim of this study was to study the mechanism of trypanosome invasion into host cells and to lay the foundation for the establishment of detection methods.【Method】 The isolated and preserved Trypanosoma evansi was used for culture propagation in Kunming White mice,and its paraflagellar fasciculus rod (PFR) gene was cloned and a phylogenetic tree was constructed.The physical and chemical properties,hydrophobicity,transmembrane region structure,secondary structure and tertiary structure of PFR protein were analyzed and predicted by bioinformatics methods.The prokaryotic expression vector pET28a-PFR was constructed,and the reactogenicity of PFR recombinant protein was detected by Western blotting.【Result】 The Yili strain of Trypanosoma evansi was successfully expanded in Kunming White mice,and the highest infection rate was reached on the 5th day.The PCR amplified fragment size of PFR gene was 834 bp,and the similarity with PFR gene of Trypanosoma brucei gambiense (XP_011775815.1) was 99.52%,and the phylogenetic tree based on the amino acid sequence of the PFR protein also showed that the strain had the closest genetic relationship with Trypanosoma brucei gambiense.The molecular formula of PFR protein was C₁₄₁₆H₂₂₈₆N₄₁₆O₄₄₂S₁₁,and the theoretical isoelectric point was 5.74.PFR protein was an alkaline,hydrophilic and unstable protein,without transmembrane region and signal peptide,and had 10 potential antigen epitopes,which were mainly located in cytoplasm.The secondary structure of PFR protein was mainly composed of alpha helix (92.34%),the tertiary structure was consistent with the secondary structure.The expression plasmid pET28a-PFR of PFR gene of Trypanosoma evansi was successfully constructed.After optimization of induction time,temperature and IPTG concentration,it was found that when IPTG concentration was 1 mmol/L and recombinant bacterial solution was induced at 37 ℃ for 6 hours,PFR protein expression was the highest and expressed in the form of inclusion body.Western blotting results showed that the recombinant protein reacted positively with Trypanosoma evansi positive serum.【Conclusion】 In this experiment, Trypanosoma evansi was successfully propagated in Kunming White mice,the PFR gene of Trypanosoma evansi was cloned,the prokaryotic expression vector of PFR was constructed,and the recombinant protein of PFR was induced to express.The protein had good reactivity,which provided a theoretical basis for the subsequent research on the function of the protein and the pathogenesis of Trypanosoma evansi.

Key words: Trypanosoma evansi; paraflagellar rod (PFR); prokaryotic expression; genetic evolution; biological characteristics

中图分类号:

S855.9

甘露, 郑会珍, 诺明达来, 刘燕, 金敏, 何文文, 温丽翠, 李永畅, 巴音查汗·盖力克. 伊氏锥虫伊犁株扩繁及其PFR基因克隆表达和生物信息学分析[J]. 中国畜牧兽医, 2023, 50(2): 469-478.

GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.

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