马泰勒虫和驽巴贝斯虫半胱氨酸蛋白酶截短基因的克隆表达及其生物信息学分析

doi:10.11843/j.issn.0366-6964.2020.10.023

摘要/Abstract

摘要： 旨在克隆、表达马泰勒虫（Theileria equi）和驽巴贝斯虫（Babesia caballi）半胱氨酸蛋白酶（cystein protease，CP）基因，并对其进行生物信息学预测和分析。提取马泰勒虫和驽巴贝斯虫的DNA，以此为模板扩增目的基因。利用克隆技术和原核表达系统克隆表达CP，用尿素透析法进行纯化。通过Dot blot验证其与相应阳性血清特异性反应。应用MAGA软件和Prot Param、TMHMM、SignalP-5.0、Antibody Epitope Prediction、SYFPEITHⅡ、ProPred及EzMol^2.1等在线分析软件分析T.equi-CP和B.caballi-CP的基因及其蛋白序列。成功构建了重组质粒GST-T.equi-CP和GST-B.caballi-CP，经SDS-PAGE试验结果显示该基因表达的蛋白以包涵体形式高效表达，并能与相应阳性血清特异性反应。对T.equi-CP和B.caballi-CP基因编码蛋白进行系统发育分析发现，与其他原虫物种相比，系统发育分析结果与其原生动物学分类结果一致。经生物信息学分析预测显示，两个CP蛋白为亲水性蛋白，无跨膜区，不属于跨膜蛋白，无Th细胞表位，在细胞质、线粒体和细胞核中定位较多。其中T.equi-CP蛋白的相对分子质量为29 948.60，等电点为5.53，稳定系数为22.50。B.caballi-CP蛋白的相对分子质量为14 603.62，等电点为8.54，稳定系数为47.69，有信号肽区域。T.equi-CP和B.caballi-CP蛋白具有良好的反应原性，作为亲水性膜外蛋白，无Th细胞表位，在细胞质、线粒体和细胞核中定位较多等，可能说明CP在马梨形虫逃避宿主免疫反应，参与增殖、分化及程序性细胞死亡等发挥作用。本研究为T.equi-CP和B.caballi-CP蛋白的功能研究与马梨形虫的致病机制研究提供了理论依据。

关键词: 马泰勒虫, 驽巴贝斯虫, 半胱氨酸蛋白酶, 原核表达, 进化树分析, 生物信息学分析

Abstract: This study aimed to clone and express cysteine proteinase (CP) gene of Theileria equi and Babesia caballi. The CP proteins were analyzed using bioinformatics tools and online databases. The total DNA of T. equi and B. caballi as the template sequence for PCR. The CP genes were cloned, expressed using the cloning technique and prokaryotic expression system, respectively. Then the recombinant CP (rCP) proteins were purified by Urea dialysis. Finally the specific reaction of polyclonal horse anti-CP serum with rCPs was analyzed through Dot blot method. The CPs were predicted and analyzed by the software MAGA, Prot Param, TMHMM, SignalP-5.0, Antibody Epitope Prediction, SYFPEITHⅡ, ProPred and EzMol^2.1 respectively. The CP genes were successfully amplified from DNA of T. equi and B. caballi and expressed in the inclusion body fractions, Dot blot assay indicated that the recombinant protein could react with polyclonal horse anti - CP serum. Compared with T. equi-CP and B. caballi-CP, different protozoon CP genes were highly conserved in evolution, and phylogenetic analysis results were consistent with their protozoon taxonomy. The bioinformatics analysis revealed that two CPs are the hydrophilic protein instead of a transmembrane one, no transmembrane domain, no Th cell epitope was found, and more localized in the cytoplasm, mitochondria and nuclei. The relative molecular weight (MW) of T. equi-CP was 29 948.60, the theoretical isoelectric point (pI) was 5.53, stability coefficient was 22.50; B. caballi-CP’MW was 14 603.62, the theoretical pI was 8.54, stability coefficient was 47.69, but signal peptide was contained. The CPs have good reactionogenicity, as the hydrophilic extracellular proteins with no Th cell epitope, and more localized in the cytoplasm, mitochondria and nuclei, which helped T. equi and B. caballi avoid host immune response outside the cell membrane and the CPs involved in proliferation, differentiation and programmed cell death. In conclusion, a theoretical basis was provided for the study on CP’s functions and the pathogenesis of T. equi and B. caballi.

Key words: Theileria equi, Babesia caballi, cysteine proteinase, prokaryotic expression, phylogenetic analysis, bioinformatics

中图分类号:

S852.723

宋瑞其, 呼尔查, 翟雪洁, 樊新丽, 李敏, 张梦圆, 宋晶晶, 阿尔曼·海热, 吾力江·卡马力, 巴音查汗·盖力克. 马泰勒虫和驽巴贝斯虫半胱氨酸蛋白酶截短基因的克隆表达及其生物信息学分析[J]. 畜牧兽医学报, 2020, 51(10): 2547-2556.

SONG Ruiqi, Huercha, ZHAI Xuejie, FAN Xinli, LI Min, ZHANG Mengyuan, SONG Jingjing, HAIRE Arman, KAMALI Wulijiang, GAILIKE Bayinchahan. Prokaryotic Expression of the Cysteine Proteinase Gene of Xinjiang Strain of Theileria equi/Babesia caballi and Its Bioinformatics Analysis[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2547-2556.

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