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目的探索利用特异多克隆抗体结合噬菌体递呈（ｐｈａｇｅｄｉｓｐｌａｙ）随机肽库，进行病原体蛋白质的Ｂ细胞抗原表位研究。方法采取特异抗原→特异多克隆抗体→相应抗原表位的技术路线。所选研究对象为丙型肝炎病毒（ＨＣＶ）核心蛋白。实验分三步：（１）抗ＨＣＶ核心蛋白多克隆抗体的制备。用活化的Ｓｅｐｈａｒｏｓｅ４Ｂ耦联重组的ＨＣＶ核心蛋白，亲和层析纯化丙型肝炎病人血清中特异的抗核心蛋白多克隆抗体。（２）随机肽库的生物淘洗（ｂｉｏｐａｎｎｉｎｇ）。以纯化的多克隆抗体作为筛选分子，生物淘洗噬菌体递呈的随机七肽库。（３）阳性噬菌体克隆的鉴定。将筛选的噬菌体克隆进行ＥＬＩＳＡ检测、ＤＮＡ测序以及竞争抑制试验等，最后分析所获资料。结果七肽氨基酸序列分析表明，ＨＣＶ核心蛋白存在着至少３个Ｂ细胞抗原位点，其中１９～２５ａａ间（ＰＱＤＶＫＦＰ）的线性表位是该蛋白的优势表位，证实了本研究策略的可行性。结论本技术路线可以有效地进行ＨＣＶ核心蛋白的Ｂ细胞抗原表位研究。由此推论，此方法也可移植于其它病原微生物抗原或自身抗原的表位研究，继而为基于抗原表位水平的特异诊断试剂的研制、疫苗的设计提供依据
Objective To explore the use of specific polyclonal antibodies combined with phagedisplay random peptide library for antigenic study of B cell epitopes of pathogens. Methods Take the specific antigen → specific polyclonal antibody → corresponding epitopes of the technical route. The selected study was Hepatitis C virus (HCV) core protein. The experiment is divided into three steps: (1) Preparation of anti-HCV core protein polyclonal antibody. The purified anti-core protein polyclonal antibody in the serum of patients with hepatitis C was purified by affinity chromatography with activated Sepharose 4B conjugated recombinant HCV core protein. (2) Biopanning of Random Peptide Library. A purified polyclonal antibody was used as a screening molecule to bio-elute the random peptide library presented by phage. (3) Identification of positive phage clones. The selected phage clones were tested by ELISA, DNA sequencing and competition inhibition test. Finally, the data obtained were analyzed. Results The analysis of amino acid sequence of heptapeptide showed that there were at least 3 B-cell antigenic sites in HCV core protein. The linear epitope of PQDVKFP between 19 and 25aa was the dominant epitope of this protein, which confirmed the feasibility of this study . Conclusion This technical route can effectively study the B cell epitope of HCV core protein. It is concluded that this method can also be transplanted in other epitopes of the antigen or autoantigen of the pathogenic microorganisms, and then provide a basis for the development of a specific diagnostic reagent based on the antigen epitope level and the design of the vaccine