牛支原体PepO原核表达及功能验证

Study on Mycoplasma bovis PepO Prokaryotic Expression and Function Determination

牛支原体是严重危害养牛业的重要病原体,阐明其毒力因子和致病机制有助于防控技术的研发。内肽酶O(PepO)参与肺炎链球菌在猪体的致病过程,本研究旨在探讨PepO在牛支原体中是否具有毒力相关功能。利用巢式PCR将牛支原体PepO中的色氨酸密码子TGA突变为大肠杆菌的色氨酸密码子TGG,进一步克隆至大肠杆菌原核表达载体,成功表达重组蛋白PepO。利用重组蛋白免疫BALB/c小鼠制备针对PepO的高免血清,经Western-blot分析显示出多抗血清结合,可激活PLG的结构域,从而发挥降解特异性底物S-2251的纤溶酶功能。比较分析牛支原体PepO突变菌株T5.37与野毒株HB0801的生长及对牛肺上皮细胞EBL的黏附特性,其结果显示PepO缺失显著降低牛支原体对EBL的黏附。本研究证实了牛支原体PepO具有活性,参与宿主细胞黏附过程,是一个潜在的毒力相关因子,为进一步研究阐明牛支原体致病机制奠定了基础。

Mycoplasma bovis(M.bovis)is an important pathogen by causing great loss in cattle industy.To elucidate the virulence factors and pathogenesiswill help to pathogenesis of Streptococcus pneumonia in pigs.This study was aimed to determine whether this protein is active in M.bovis.To obtain the protein expressed in E.coli,the M.bovis tryptophan codon TGA was transformed into E.coli tryptophan TGG with nested PCR and cloned into E.coli prokaryotic expression vector.The recombinant rPepO was expressd and purified.Then the three BALB/c mice were immunized with purified rPepO and the antiserum against rPepO was developed and Western-blot assay demonstrated this protein could react with the antiserum very well.In addition,the enzyme activity of r was demonstrated by the evidence that rPepO could react with Plasminogen(PLG)and subsequently reactivate PLG to degrade specifically substrate S-2251.The mutant T5.37 was obtained from mutant library.The growth assay indicated that there was no difference in growth curves between the wild type strain M.bovis HB0801.However,the binding ability assay of M.bovis strains to embryonic bovine lung(EBL)cell line showed that the mutant binding was significantly decreased(P<0.05).In conclusion,this study confirms that PepO is active in M.bovis,and contributed to M.bovis adhesion to EBL cells,thereby a potential virulence related factor of M.bovis.This finding provides a basis for further study of M.bovis pathogenesis.