高表达GFP-PBP1b融合蛋白的猪链球菌菌株的构建及PBP1b蛋白细胞定位的研究  

Construction of Streptococcus suis fusion strain with enhanced expression of GFP-PBP1b fusion protein and cellular localization of PBP1b protein

作　　者：武柳君 卫东 高广娟 陈平 刘思国[2] 张跃灵 WU Liu-jun;WEI Dong;GAO Guang-juan;CHEN Ping;LIU Si-guo;ZHANG Yue-ling(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;State Key Laboratory for Animal Disease Control and Prevention,Division of Bacterial Diseases,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [2]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室/动物细菌病创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第11期1101-1107,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2021YFD1800401);国家自然科学基金(32172853);中央级公益性科研院所基本科研业务费专项(1610302022001)。

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Peno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-Peno-GFP-DN。利用同源重组的方法将UP-Peno-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Peno-gfp片段替换了PSS-pbp1b中的PSS片段,表明融合强启动子Peno、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OD600nm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。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 Peno gene fragments,and the gfp gene fragments using indicated templates and primers.Then,the UP-Peno-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 Peno and 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 OD600nm values.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 wa

关 键 词：猪链球菌 荧光蛋白融合菌株 A型青霉素结合蛋白 PBP1b 细胞壁肽聚糖合成 

分 类 号：S852.61[农业科学—基础兽医学]