提高表达分选酶A的猪链球菌突变株的构建及初步评价  

Construction and preliminary evaluation of a Streptococcus suis mutant with increased expression of sortase A

作　　者：朱金鲁 陈平 黄萌萌 刘冉 卫东 刘思国[1] 张跃灵[1] ZHU Jin-lu;CHEN Ping;HUANG Meng-meng;LIU Ran;WEI Dong;LIU Si-guo;ZHANG Yue-ling(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/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》2021年第1期7-13,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2017YFD0500203);国家自然科学基金(31772757、31672575);黑龙江省自然科学基金(C2017075、C2017078)。

摘　　要：为了提高猪链球菌(Streptococcus suis,SS)05ZYH33菌株中分选酶A(SrtA)的表达量,本研究利用同源重组技术,将强启动子Peno、SrtA基因与红霉素抗性基因融合并转化05ZYH33野生菌株(05ZYH33-WT),构建SS SrtA提高表达的突变株05ZYH33-srtAe,并对05ZYH33-srtAe突变株和05ZYH33-WT进行生长特性分析和SrtA蛋白的western blot检测;进一步构建SS SrtA基因缺失株05ZYH33-srtA^(Δ)作为对照,通过检测05ZYH33-WT、05ZYH33-srtAe和05ZYH33-srtAΔ菌株表面蛋白SSU05_0630的N-乙酰氨基葡萄糖苷酶(NAG)的活性,初步评价SrtA的提高表达在SS表面蛋白锚定中的作用。Western blot结果显示,正确构建了SS SrtA提高表达的突变株05ZYH33-srtAe,且该菌株的生长速度和05ZYH33-WT基本相同,但SrtA的表达量却提高了2.4倍。菌株表面蛋白SSU05_0630的NAG活性检测结果显示,05ZYH33-srtA^(Δ)菌株的NAG活性和本实验室前期构建的SSU05_0630基因缺失株ssu05_0630^(Δ)相同,检测不到SSU05_0630蛋白的NAG活性,表明SSU05_0630蛋白是SrtA的底物,通过SrtA锚定到SS的表面。05ZYH33-srtAe和05ZYH33-WT菌株表面蛋白SSU05_0630的NAG活性无明显差异,即SrtA表达量的提高并没有提高SSU05_0630蛋白在SS表面的锚定量。本研究首次构建了提高SS SrtA表达量的突变株,并对SrtA表达量的提高是否对SS表面蛋白锚定产生影响进行了初步探究,为进一步研究SrtA表达量的提高对SS其他表面蛋白锚定量的影响,以及优化SS表面蛋白展示系统奠定了基础。In order to increase the expression of Sortase A(SrtA)in Streptococcus suis(S.suis,SS),in this study,using homologous recombination technology,the strong promoter Peno,SrtA encoding gene and erythromycin resistance gene were fused and transformed into 05ZYH33 wild-type strain(05ZYH33-WT)to construct SS SrtA expression-increasing mutant strain 05ZYH33-srtAe.The growth characteristics of 05ZYH33-srtAe mutant strain and 05ZYH33-WT were analyzed and SrtA protein was detected by western blot.A strain 05ZYH33-srtAΔwith a deletion of the SrtA encoding gene of SS was constructed as control,and by detecting the N-acetylglucosaminase(NAG)activity of the surface proteins SSU05_0630 of strains 05ZYH33-WT,05ZYH33-srtAe and 05ZYH33-srtA^(Δ),the role of high expression of SrtA in anchoring SS surface proteins was preliminarily evaluated.Identification by western blot showed that the SS SrtA expression-increasing mutant strain 05ZYH33-srtAe was successfully constructed,and its growth rate was basically the same as that of 05ZYH33-WT,but the expression of SrtA was increased by 2.4-fold.The NAG activity test results of the SSU05_0630 protein on the surface of the strains showed that the NAG activity of the 05ZYH33-srtA^(Δ)strain was the same as the SSU05_0630 gene deletion strain ssu05_0630^(Δ)constructed earlier in our laboratory,the NAG activity of SSU05_0630 protein was not detected on the surface of the strain,indicating that SSU05_0630 protein is a substrate of SrtA and fix the object to the surface of the SS by SrtA.There was no significant difference in the NAG activity of SSU05_0630 protein on the surface of 05ZYH33-srtAe and 05ZYH33-WT strains,suggesting that the increase of SrtA expression did not increase the amount of SSU05_0630 protein on the surface of SS.In this study,a mutant strain that increased the expression of SS SrtA was constructed for the first time,and the effect of increased SrtA expression on the anchoring of SS surface proteins was initially discussed.In order to further study the influenced of the

关 键 词：猪链球菌 分选酶A 提高表达 强启动子 胞壁锚定 细菌表面展示 

分 类 号：S852.61[农业科学—基础兽医学]