BPI N端优势抗原表位的模拟合成及其抗血清的制备

目的通过生物信息学模拟合成杀菌/渗透增强蛋白氨基端(BPIN端)优势抗原表位肽,免疫动物获得相应抗血清。方法利用生物信息学分析BPIN端(1-199)氨基酸序列的抗原性、亲水性、可塑性、表面可及性和二级结构等理化特性,据此设计合成TA/IK两条多肽,将其与钥孔戚血蓝素(KLH)偶联后,免疫家兔获得相应抗血清;采用间接ELISA法鉴定多肽的抗原性、测定血清抗体效价,Westernblot鉴定抗血清特异性。结果人工合成BPIN端TA/IK两个B细胞表位肽;ELISA检测证实TA/IK抗原肽能与商品化兔抗人BPI55多克隆抗体结合;所获TA/IK抗血清效价分别为1∶51200和1∶25600;Westernblot证实TA/IK抗血清能与BPI55标准品特异性结合。结论模拟合成的TA/IK抗原肽确为BPIN端优势抗原表位,相应抗血清可用于BPIN端功能性片段的检测鉴定。

Synthesis of dominant epitopes on N-terminal part of BPI and preparation of corresponding antisera

AIM: To synthesize B cell dominant epitopes on N-terminal part of bactericidal/permeability increasing protein (BPI) and prepare the corresponding antisera. METHODS: The antigenicity, hydrophilicity, flexibility, surface probability and secondary structure of N-terminal amino acids 1-199 on BPI were predicted by bioinformatics applications. Two antigen peptides TA/IK were designed and synthesized on the basis of the above analysis. Then the TA/IK were respectively conjugated to keyhole limpet hemocyanin (KLH) and injected into rabbits to prepare corresponding antisera. Indirect ELISA was performed to analyze the antigenicity of TA/IK and to test the titer of the antisera. And Western blot was used to identify the specificity of the antisera. RESULTS: (1) Two B cell epitope-based peptides TA/IK were successfully synthesized; (2) the peptides could bind to commercial polyclonal antibody, anti-BPI(55); (3) titers of the antisera against TA/IK were up to 1:51,200, 1:25,600, respectively; (4) Western blot analysis revealed that these antisera could specifically react with the standard sample of BPI(55). CONCLUSION: The two synthetic antigen peptides TA/IK are indeed dominant epitopes of BPI N-terminal part, and the corresponding antisera are competent for detecting and identifying the N-terminal fragments of BPI.