应用噬菌体肽库技术定位沙门氏菌鞭毛蛋白上属特异性共同抗原表位

Mapping of the Generically-specific Common Epitope on Salmonella Flagellins Using Phage-displayed Peptide Libraries

用生物素标记的沙门氏菌属特异性单克隆抗体de7作为分子探针 ,应用亲和筛选法从噬菌体肽库中筛选该单抗所针对的表位克隆 ,最终进行定位。经三轮筛选后 ,用斑点印迹法检测 ,得到良好的富集效果。通过免疫筛选法从第三轮筛选物中挑取了 2 4个阳性克隆 ,再以斑点印迹、竞争ELISA进一步鉴定阳性克隆 ,证实已获得了沙门氏菌鞭毛属特异性共同抗原的模拟表位。测得的序列为SRRSFTTTE ,将其与沙门氏菌鞭毛蛋白氨基酸序列相比较 ,同源性较小 ,但在不同血清型沙门氏菌鞭毛蛋白氨基酸序列的Ⅰ区高度保守序列中 ,发现 6 1 6 5位氨基酸的RSXXT序列与所得的线性表位有相同的排列 ,而大肠杆菌鞭毛蛋白氨基酸在此段的序列为RAXXT ,且这种序列排列的几率为 1/2 0 5,因此推断该单抗所针对的表位在该区段上。此外 ,以阳性克隆免疫Balb/c小鼠 ,并制备高免血清进行间接荧光检测和竞争ELISA鉴定 ,结果表明其具有优良的免疫原性和高度的特异性 ,这亦为模拟表位疫苗的研究提供了新思路。

We used a 9-mer phage peptide library to screen the mimicking epitopes by biotin-labelling monoclonal antibody (AcAb) de7 which is specific for the generically-specific common epitope of Salmonella flagellins.Three rounds of biopanning have been carried out.The result of dot blot showed that the enrichment of positive signals was gradually increased.In order to select and identify the specific single positive clones,24 clones were screened by affinity immunodetection,and then estimated by dot blot,and cELISA.The PVⅢ genes of bearing the inserts of one positive clone was sequenced successfully,the amino acid sequences were postulated as SRRSFTTTE.Compared the sequence with Salmonella flagellin showed little homology,so the epitopes are the mimicking epitopes of the native ones.Common sequence RSXXT is present in the hyper-conservative region I of Salmonella flagellin,while there is RAXXT in the E.coli flagellin.This suggests that the epitope for the McAb de7 is located at this region and including 61-65 AA.Furthermore,after immunization of BALB/c mice,the postive clone can elicits a strong specific humoral responses against the generically epitope of Salmenlla flagellin.The sera of mice can reacted with all salmonella tested,but can not bind to Echerichaia coli in cELISA and indirect immunofluorenscence test.These results indicated that the selected minotope of common epitopes on Salmonella flagellin has good immunogenicity and high specificity.It also supply some data for the development of the minotope vaccine.