【摘要】 为鉴定猪链球菌（Streptococcus suis） A型青霉素结合蛋白PBP1b在S.suis中的细胞定位，本研究经PCR扩增S.suis pbp1b基因融合位点上下游序列UP和DN、强启动子P_eno基因片段及绿色荧光蛋白（GFP） gfp基因片段，再通过重叠延伸PCR扩增构建融合片段UP-P_eno-GFP-DN。利用同源重组的方法将UP-P_eno-GFP-DN转化含反向筛选标记的PSS-pbp1b中间菌株，经含7%蔗糖的TSAS平板筛选，挑单菌落进行PCR及测序鉴定。PCR及测序鉴定结果显示，获得的重组菌株中P_eno-gfp片段替换了PSS-pbp1b中的PSS片段，表明融合强启动子P_eno、GFP及PBP1b （GFP-PBP1b）的重组菌株P-GFP-pbp1b正确构建。以S.suis 05ZYH33株和本实验室前期构建的GFP-pbp1b为对照，培养P-GFP-pbp1b菌株并测定OD_600nm值，绘制各菌株的生长曲线；采用western blot检测P-GFP-pbp1b菌株中GFP-PBP1b融合蛋白的表达水平，并与GFP-pbp1b菌株进行比较；利用Alexa Fluor 633-WGA染色，经超高分辨显微镜观察GFP-pbp1b和P-GFP-pbp1b菌株形态及GFP-PBP1b融合蛋白在S.suis细胞中的定位。生长曲线结果显示，S.suis融合菌株GFP-pbp1b和P-GFP-pbp1b均正常生长；western blot结果显示，GFP-pbp1b和P-GFP-pbp1b菌株均能够表达GFP-PBP1b融合蛋白，但强启动子驱动的P-GFP-pbp1b菌株中GFP-PBP1b蛋白表达量较GFP-pbp1b菌株提高了6倍（P<0.01）；超高分辨显微镜观察结果显示，GFP-pbp1b和P-GFP-pbp1b菌株形态均正常，GFP-pbp1b菌株中GFP荧光较弱，难以定位GFP-PBP1b，而P-GFP-pbp1b菌株中GFP荧光较强，能够明显观察到融合蛋白GFP-PBP1b定位在S.suis的细胞横隔和两极。本研究通过构建高表达GFP-PBP1b融合蛋白的P-GFP-pbp1b菌株首次观察到PBP1b在S.suis中的细胞定位，为进一步研究PBP1b在S.suis细胞壁肽聚糖合成中的作用奠定了基础。更多还原

【Abstract】 In order to observe the cellular localization of class A penicillin-binding protein PBP1b in Streptococcus suis（S.suis）,PCR was used to amplify the upstream and downstream sequences （UP and DN） of the fusion site of S.suis pbp1b,the strong promoter P_enogene fragments,and the gfp gene fragments using indicated templates and primers.Then,the UP-P_eno-GFP-DN fusion fragment was constructed by overlapping extended PCR amplification.This fragment was transformed into the PSS-pbp1b intermediate strain containing reverse screening label by homologous recombination method,and screened on TSAS plate containing 7%sucrose.Single colonies were identified using PCR and sequencing.PCR and the sequencing results confirmed that the PSS fragment in PSS-pbp1b were replaced by the Peno-gfp gene fragment in the obtained recombinant strain,indicating that the recombinantstrain P-GFP-pbp1b fusing strong promoter P_enoand GFP was correctly constructed.The strain was designated as P-GFP-pbp1b.Next,using the wild type （WT） and previously constructed GFP-pbp1b strains as contrast,P-GFP-pbp1b strains was cultured and the growth curve was drawn with OD_600nmvalues.Western blot was used to detect the expression level of GFP-PBP1b fusion protein in P-GFP-pbp1b,and compared it with that in GFP-pbp1b.The morphology of GFP-pbp1b and P-GFP-pbp1b strains,as well as the cellular localization of the GFP-PBP1b fusion protein in both strains were observed using super resolution microscope after staing the cells with Alexa Fluor 633-WGA.The growth curves showed that GFP-pbp1b and P-GFP-pbp1b strains grow normally.Western blot results showed that both GFP-pbp1b and P-GFP-pbp1b strains could express GFP-PBP1b fusion protein,but the expression of GFP-PBP1b in P-GFP-pbp1b strain driven by strong promoter was 6 times higher than that of GFP-pbp1b strain（P<0.01）.Super resolution microscopic observations showed that the cell shape of both GFP-pbp1b and P-GFP-pbp1b strains appeared to be as normal as the WT strain.The fluorescence signal in the GFP-pbp1b strain was too weak to locate GFP-PBP1b.In contrast,the P-GFP-pbp1b strain exhibited strong fluorescence signal,allowing easily localization of GFP-PBP1b at the cell septum and cell poles.This study firstly locallizted PBP1b protein in S.suis by constructing gene-fusion strain with enhanced expression of the fusion protein GFP-PBP1b,facilitating the further study of PBP1b protein function in cell wall peptidoglycan synthesis of S.suis.更多还原