肠炎沙门氏菌SEF14菌毛的表达及其对该菌的特异性检测研究

Expression of SEF14 fimbriae protein and research on specific diagnostic technology of Salmonella enteritidis

为获得肠炎沙门氏菌SEF14菌毛蛋白并检测其生物学功能,本研究以肠炎沙门氏菌标准株SE50336为模板,PCR扩增其含分泌信号肽的sef14操纵子基因片段,并克隆至表达载体pBR322中,构建重组质粒pBR322-sef14,将该重组质粒转化至经修饰且不含任何菌毛的大肠杆菌SE5000M株中,获得重组菌pBR322-sef14/SE5000M。将重组菌pBR322-sef14/SE5000M与本实验室制备的SefA(SEF14菌毛的主要亚基)单克隆抗体(MAb)凝集;利用透射电镜和免疫电镜观察上述重组菌并通过SDS-PAGE和western blot鉴定,利用该重组菌pBR322-sef14/SE5000M与肠炎沙门氏菌阳性鸡血清及其他菌的阳性鸡血清结合。结果显示,本实验室制备的SefA MAb能够特异性识别该重组菌,并产生明显的肉眼可见的凝集反应。重组菌表达的SEF14菌毛产物大小约为14.3 ku,与菌毛的主要亚基SefA的大小一致,电镜观察到重组菌表面有明显的菌毛结构。在肠炎沙门氏菌特异性靶向检测中,该重组菌表达的菌毛蛋白仅与肠炎沙门氏菌阳性鸡血清反应,能够在2 min内用肉眼观察到清晰的凝集颗粒,而不与鸡白痢、鸡伤寒、鼠伤寒沙门氏菌阳性鸡血清反应。本研究首次克隆了肠炎沙门氏菌sef14操纵子基因并在体外成功表达并进行了功能鉴定,上述研究结果为进一步研究SEF14菌毛及建立特异性诊断技术提供基础。

In order to obtain the SEF14 fimbriae protein of Salmonella enteritidis and verify its biological function,the sef14 operon gene fragment containing the secretion signal peptide was amplified by PCR using the standard strain of Salmonella enteritidis SE50336 as a template and cloned into expression vector pBR322 to construct recombinant plasmid pBR322-sef14.The recombinant plasmid was transformed into Escherichia coli SE5000M strain without any fimbriae,and the recombinant strain pBR322-sef14/SE5000M was obtained.The recombinant strain pBR322-sef14/SE5000M was combined with the monoclonal antibody against SefA(the main subunit of SEF14 fimbriae) prepared in our laboratory,and the recombinant bacteria were observed by transmission electron microscope and immunoelectron microscope and identified by SDS-PAGE and western blot.The recombinant strain p BR322-sef14/SE5000M was used to bind to the positive chicken serum of Salmonella enteritis.The results showed that the monoclonal antibody against Sef A(the main subunit of SEF14 fimbriae) could specifically recognize the recombinant bacteria and produce obvious agglutination reaction visible to the naked eye.The size of SEF14 fimbriae expressed by recombinant bacteria was about 14.3 ku,which is consistent with the size of the main subunit Sef A of the fimbriae.Obvious fimbriae structure was observed on the surface of recombinant bacteria by electron microscope.In the specific targeted detection of Salmonella enteritidis,the fimbriae protein expressed by the recombinant strain only reacted with Salmonella enteritidis positive chicken serum,and clear agglutinated particles could be observed with the naked eye within two minutes,but p BR322-sef14/SE5000M did not react with Salmonella gallinarum-positive,Salmonella pullorum-positive and Salmonella typhimurium-positive chicken serum.In this study,the sef14 operon gene of Salmonella enteritidis was cloned for the first time,successfully expressed and verified in vitro.The above results provide a basis for further study of SEF14 fimbriae,establishment of specific diagnostic technology platform.