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

Abstract

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

CLC Number:

S852.723

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.

References 20

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Prokaryotic Expression of the Cysteine Proteinase Gene of Xinjiang Strain of Theileria equi/Babesia caballi and Its Bioinformatics Analysis

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