鼻疽诺卡菌Nfa34810蛋白的抗原表位筛选与鉴定

Screening and identification of epitopes of Nfa34810 protein of Nocardia farcinica

目的制备鼻疽诺卡菌IFM10152的Nfa34810蛋白的单克隆抗体并进行B细胞抗原表位的筛选与鉴定。方法首先将Nfa34810蛋白截成相互重叠20个氨基酸的P1和P2,使用重组Nfa34810蛋白兔多抗血清与鼻疽诺卡菌IFM10152的鼠多抗血清,通过Western Blot方法检测P1和P2,初步定位抗原表位,对初步确定表位所在片段的P2进行纯化,并使用纯化的P2和Nfa34810蛋白分别免疫BALB/c小鼠,制备单克隆抗体。采用Western Blot方法鉴定获得的单克隆抗体,应用肽扫描法进一步用单克隆抗体对逐步截短表达的短肽进行筛选,从而确定Nfa34810蛋白的B细胞抗原表位。结果成功制备了5株针对Nfa34810完整蛋白和4株针对P2的单克隆抗体,抗体能与P2和重组Nfa34810蛋白发生特异性抗原抗体反应。通过肽扫描法成功筛选到1个抗原表位和2个抗原表位区域。结论本研究成功制备了Nfa34810蛋白的单克隆抗体,并对抗原表位进行定位,为开展鼻疽诺卡菌病原学检测、流行病学研究等奠定基础。

Objective To prepare monoclonal antibodies against Nfa34810 protein of Nocardia farcinica IFM10152 and screen and identify the B cell epitopes.Methods First,Nfa34810 protein was truncated into P1 and P2 with 20 amino acids overlapping with each other.P1 and P2 were detected to initially map the epitope region by Western Blot using recombinant Nfa34810 protein rabbit polyclonal antibody serum and the mouse polyclonal antibody against N.farcinica IFM10152.Then the P2 where the epitope was located was purified,and BALB/c mice were immunized with the purified P2 and Nfa34810 protein respectively to prepare monoclonal antibodies.The antibodies were identified by Western Blot.Nfa34810 was gradually truncated and expressed and the series of proteins were screened and identified by Western Blot to determine the B cell epitopes of Nfa34810 protein using the prepared monoclonal antibodies by peptide scanning.Results The monoclonal antibodies of Nfa34810 were prepared and they could react with truncated protein P2 and full-length Nfa34810 protein.One epitope and two epitope regions were successfully screened by peptide scanning.Conclusion In this study,the monoclonal antibodies of Nfa34810 protein was successfully prepared and the B cell epitopes were mapped.This study laid a foundation for pathogen detection and epidemiological research of N.farcinica.