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胸膜肺炎放线杆菌三聚体自转运黏附素茎部功能区表达及其与猪肺组织结合蛋白的筛选分析
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国家自然科学基金项目(No. 31520103917)


Functional region expression and its binding protein analysis of trimeric autotransporter adhesin in Actinobacillus pleuropneumoniae
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    摘要:

    【目的】胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)可引发猪的传染性胸膜肺炎,给世界养猪业造成了巨大损失。黏附是APP在致病过程中的第一步,新型黏附分子——三聚体自转运黏附素(trimeric autotransporter adhesin,TAA)是该菌感染肺组织的重要毒力因子,茎部区2 464?2 574氨基酸(BD3)序列区域在细菌的黏附中发挥重要作用,但其如何结合肺脏组织尚属未知。本文表达TAA的茎部功能区,筛选其在肺组织中的结合蛋白。【方法】原核表达及纯化TAA功能区BD3蛋白,与猪肺组织共孵育,通过免疫共沉淀技术捕获BD3的结合蛋白并质谱鉴定。构建猪cDNA文库获取该蛋白核酸序列,进行生物信息学分析。【结果】经质谱分析获得与BD3结合的猪肺组织蛋白的肽段,数据库搜寻比对发现角蛋白1为TAA BD3区与猪肺组织结合的蛋白;构建cDNA文库筛选并测序后获知其基因序列。生物信息学分析显示该序列与猪源及人源角蛋白核酸序列相似性低,猪源细胞角蛋白1作为一个单独的进化分支,与其他角蛋白差异较大;该蛋白在8?100 aa处有一个跨膜区;主要二级结构元件为α螺旋和β折叠;该蛋白在82?362 aa处存在一个G蛋白α亚单位,在515?552 aa处存在一个TSP1区域(凝血酶敏感蛋白1型重复区域)。【结论】与TAA茎部BD3结合的猪肺组织角蛋白1的发现,为TAA黏附肺组织细胞的研究奠定基础,有助于揭示APP专嗜肺组织的致病机制。

    Abstract:

    [Objective] Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia, leading to great losses for the pig industry worldwide. Adhesion is the first step in the pathogenic process of APP. The newly discovered trimeric autotransporter adhesion (TAA) is an important pathogenic virulence factor of APP, and the 2 464 to 2 574 amino acids of TAA stalk region, named BD3, plays an important role in the bacterial adhesion. However, it is still unclear how APP combines with lung tissues. We express the stalk functional region of TAA, screen and analyze its binding protein in swine lung cells. [Methods] BD3 protein was expressed in prokaryotic expression system, purified, then incubated with swine lung tissue protein. The binding protein of BD3 was picked out by co-immunoprecipitation and analyzed by mass spectrometry. The cDNA library was constructed to get the sequence of the binding protein and then the bioinformatics method was done. [Results] Mass spectrometry analysis revealed that the peptide sequence of the binding protein, and the blast result in the database showed that Keratin 1 was the binding protein. cDNA library was constructed to know the sequence of swine Keratin 1. Bioinformatics analysis showed that low sequence homology of Keratin 1 existed between swine and human and it was a separate evolutionary branch, different from other keratins of swine or human, Keratin 1 had a transmembrane domain at 8 to 100 aa and the secondary structure mainly consisted of α-helix and β-fold. It contained α subunit of G protein at 82 to 362 aa and a TSP1 domain (thrombin-sensitive protein type I repeat domain) at 515 to 552 aa. [Conclusion] The finding of Keratin 1 that is in swine lung cells and binds with BD3 of TAA, lays a foundation for researching the mechanism of TAA adhesion to lung tissue, and has great significance to understand the pathogenic mechanism of APP specially colonized in lung tissues.

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鲍春彤,计群,刘建方,朱日宁,雷连成. 胸膜肺炎放线杆菌三聚体自转运黏附素茎部功能区表达及其与猪肺组织结合蛋白的筛选分析[J]. 微生物学通报, 2017, 44(12): 2923-2932

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  • 在线发布日期: 2017-12-05
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