摘要
目的 用噬菌体展示肽库筛选登革 2型病毒 (DEN2 )E蛋白的抗原表位 ,并确定该抗原表位性质。方法 以DEN2型特异的E单克隆抗体作为筛选分子 ,生物淘洗噬菌体随机 12肽库 ,将筛选的噬菌体阳性克隆进行ELISA检测、DNA序列测定及展示肽的氨基酸序列推导 ,通过噬菌体展示肽序列与DEN2E蛋白的氨基酸一级结构的对比 ,初步确定E蛋白的抗原表位 ;用模拟该表位线性序列的合成十肽进行抗体结合试验、噬菌体竞争抑制试验及与DEN感染患者的血清学试验 ,确定其为免疫优势线性表位。结果 肽库淘洗获得的 11个ELISA阳性的噬菌体克隆有相似的结构基序WFKKGSS ,其展示肽与DEN2E蛋白 390～ 398AA序列有 3～ 5个氨基酸相同。对应于DEN2E蛋白390～ 399AA的合成十肽能与淘洗单抗特异反应 ,并可抑制噬菌体阳性克隆与该单抗结合。该合成肽与DEN2感染患者血清有较高的免疫反应性。结论 本实验通过噬菌体随机肽库的生物淘洗确定的DEN2E蛋白 (E390～ 398AA)线性序列为免疫优势表位 ,其对应的合成肽E10可望用于DEN2感染的快速诊断。
Objective To identify the B-cell epitopes of dengue virus type 2 (DEN2) envelope glycoprotein (E) from a random phage displayed peptide library (RPL) using a serotype-specific monoclonal antibody (McAb) of DEN2 NGC strain. Methods Biopanning of RPL-12 was done with a serotype-specific anti-DEN2 E protein McAb immobilized on plastic surface. After 3 rd round of biopanning, the selected phage clones were bound to the McAb for ELISA, and the immunopositive phage clones were selected for DNA sequencing. The epitope was identified by alignment of phage displayed peptides sequences with amino acid residues of the E protein of DEN2 NGC strain. An epitope-based peptide was synthesized to perform binding assays with DEN McAb and a competitive inhibition assay with the phage clone particles. This synthetic peptide was also tested with serum samples obtained from DEN-infected patients. Results 11 significantly reactive phage clones being bound to the McAb were selected and then used for DNA sequencing. These phage-borne peptides had a consensus motif WFKKGSS. Each of these phage-borne peptides had 3-5 amino acid residues similar to sequence 390 to 398 aa of the DEN2 E protein. A synthetic peptide (WFKKGSSIGQ, E10), corresponding to amino acid residues 390 to 399 of the E protein of DEN2, was found to bind to the McAb specifically and inhibit the binding of phage particles to the McAb in a competitive inhibition assay. Furthermore, this epitope-based peptide E10 could react with a high degree of specificity with serum samples obtained from DEN2-infected patients. Conclusion This study suggests that a linear immuno-dominant epitope exists in the N-terminal (390-398 aa) of DEN2 E protein and the epitope-based synthetic peptide E10 demonstrated its clinical diagnostic potential for developing DEN2 serologic test.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2003年第7期498-501,共4页
Chinese Journal of Microbiology and Immunology
关键词
登革2型病毒
包膜糖蛋白
抗原表位
噬菌体肽库
Dengue virus (DEN)
Envelope glycoprotein (Egp)
Phage display peptide library (RPL)
Epitopes mapping
