Loop6、Loop7和Loop8参与副猪嗜血杆菌OmpP2抗血清的杀菌作用研究

Loop6,Loop7,and Loop8 participated in the bactericidal efficacy study of the H.parasuis OmpP2 antiserum

为研究副猪嗜血杆菌(HPS)外膜蛋白P2(OmpP2)不同表面环状结构(Loop)在HPS抗血清杀菌过程中发挥的作用。本研究以pBAD18-Km质粒和HPS SC096为模板,PCR扩增卡那霉素抗性基因(Km)片段和含ompP2 TTA终止密码子下臂基因片段。通过重叠延伸PCR扩增融合基因片段Km-ompP2克隆至自杀性质粒pK18mobsacB中,获得重组自杀性质粒pZY01。以HPS SC096为模板经PCR分别扩增ompP2 Loop1~Loop8上臂片段和ompP2 Loop1~Loop 8下游片段,并经重叠延伸PCR扩增各loop基因融合片段(分别缺失Loop1~Loop8基因序列的ompP2片段)并克隆至pZY01,构建各Loop基因缺失的自杀性质粒pZY02~pZY08,所有的自杀性质粒均经PCR鉴定。将正确构建的自杀性质粒pZY01~pZY08通过自然转化法转化至ompP2基因缺失的HPS SC096(SC096ΔompP2)中,构建了HPS ompP2ΔLoop缺失菌株,并均经PCR鉴定后在TSA中传10代,通过PCR方法检测其遗传稳定性。结果显示,正确构建了8个自杀性质粒pZY01~pZY08及缺失各Loop基因的HPS ompP2ΔLoop2、ompP2ΔLoop5、ompP2ΔLoop6、ompP2ΔLoop7和ompP2ΔLoop8,共5个HPS ompP2ΔLoop缺失菌株,且各缺失菌株的Loop基因均能够稳定缺失,表明其均具有较好的遗传稳定性。未获得HPS ompP2ΔLoop1、ompP2ΔLoop3和ompP2ΔLoop4缺失菌株。制备新鲜兔血清并分别测定不同HPS ompP2ΔLoop缺失菌株在50%和90%兔血清中的存活率,分析不同缺失菌株的抗兔血清中补体介导的杀菌作用。结果显示,与HPSΔompP2菌株相比,HPS ompP2ΔLoop2菌株在50%和90%兔血清中的存活率分别增加了4×10^(5)倍和1×10^(6)倍;HPS ompP2ΔLoop5菌株在50%和90%兔血清中的存活率分别增加了3×10^(5)倍和1×10^(6)倍;HPS ompP2ΔLoop6、ompP2ΔLoop7和ompP2ΔLoop8的存活率无显著变化。与野生型SC096株相比,在50%兔血清中,HPS ompP2ΔLoop2、ompP2ΔLoop5、ompP2ΔLoop6、ompP2ΔLoop7和ompP2ΔLoop8缺失菌株的存活率分别降低了53.7%、60.1%、99.8%、99.6%和99.9%;在90%兔血清中,上述HPS ompP2ΔLoop菌株的存活率分别降低了78.0%、84.4%、99.9%、99.9%和99.9%。以上结果表明Loop6、Loop7和Loop8参与OmpP2的抗血清杀菌作用,且这三者可能是OmpP2重要的活跃肽段。本研究为探究HPS ompP2的毒力机制提供参考。

To investigate the roles of different surface Loop structures(Loops)of outer membrane protein P2(OmpP2)of Haemophilus parasuis(HPS)in the resistance to serum bactericidal activities.In this study,pBAD18-Km plasmid and HPS SC096 were used as templates to amplify kanamycin resistance gene(Km)fragment and the downstream arm of ompP2,with its TTA stop codon included.The fusion gene fragment Km-ompP2 was amplified by overlap extension PCR and cloned into the suicide plasmid pK18 mobsacB to obtain the recombinant suicide plasmid pZY01(OmpP2 fragments lacking Loop1-Loop8 gene sequences,respectively).The upper arm fragment of ompP2 Loop1-Loop8 and the downstream fragment of ompP2 Loop1-Loop8 were amplified by PCR using HPS SC096 as a template,and the fusion fragment was cloned into pZY01 by overlap extension PCR to construct the suicide plasmid pZY02-pZY08 of the Loop gene,and all suicide plasmids were verified by PCR.The correctly constructed suicide plasmid pZY01-pZY08 was naturally transformed into SC096ΔompP2 to construct the HPS ompP2Δloop mutant strains,which were verified by PCR and passaged in TSA for 10 generations,and its genetic stability was detected by PCR.The results showed that eight suicide plasmids pZY01-pZY08 and five HPS ompP2ΔLoop2,ompP2ΔLoop5,ompP2ΔLoop6,ompP2ΔLoop7 and ompP2ΔLoop8 were correctly constructed,and a total of five HPS ompP2ΔLoop mutant strains had good genetic stability.HPS ompP2ΔLoop1,ompP2ΔLoop3 and ompP2ΔLoop4 mutant strains were not obtained.Compared with the HPSΔompP2 strain,the bacterial survival rates of the HPS ompP2ΔLoop2 and ompP2ΔLoop5 complementary strains were increased by 4×10^(5)-fold and1×10^(6)-fold,3×10^(5)-fold and 1×10^(6)-fold in 50%and 90%rabbit serum,respectively.However,the bacterial survival rates of ompP2ΔLoop6,ompP2ΔLoop7 and ompP2ΔLoop8 complementary strains did not change significantly in serum.Compared with wild-type SC096 strain,the bacterial survival rates of HPS ompP2ΔLoop2,ompP2ΔLoop5,ompP2ΔLoop6,ompP2ΔLoop7 and ompP2ΔLoop8 complementary strains were decreased by 53.7%,60.1%,99.8%,99.6%and 99.9%in 50%rabbit serum,respectively,and were reduced by 78.0%,84.4%,9.9%,99.9%and 99.9%in 90%serum,respectively.The above results indicated that Loop6,Loop7 and Loop8 of HPS OmpP2 are involved in the resistance to serum bactericidal activities,suggesting that Loop6,Loop7 and Loop8 of OmpP2 are important active peptides.Therefore,this study provides a reference for exploring the virulence mechanism of HPS OmpP2.