人源中和性抗汉滩病毒单克隆抗体Fab段基因的获得和表达

GENERATION OF HUMAN NEUTRALIZING MONOCLONAL ANTIBODY Fab FRAGMENTS TO HANTAAN VIRUS BY USING PHAGE DISPLAY TECHNOLOGY

运用噬菌体表面表达（Ｐｈａｇｅｄｉｓｐｌａｙ）技术，获得人源中和性抗汉滩病毒汉滩型Ｇ１基因工程单克隆抗体（单抗）Ｆａｂ段基因及其表达，并同时获得抗汉滩病毒核蛋白（ＮＰ）的Ｆａｂ抗体。从肾综合征出血热疫区恢复期病人抗凝血中分离到的外周淋巴细胞中，提取了总细胞ＲＮＡ。通过ＲＴ－ＰＣＲ方法，用一组人ＩｇＧＦａｂ基因特异性引物，从合成的ｃＤＮＡ中经ＰＣＲ扩增了一组轻链和重链Ｆａｂ段基因，将轻链和重链先后插入噬菌体载体ｐＣｏｍｂ３，成功地建立了抗汉滩病毒抗体基因库，并用纯化的汉滩病毒颗粒及抗汉滩病毒糖蛋白鼠单抗捕捉糖蛋白抗体原的方法，对此抗体库进行了富集筛选，在短期内成功地获得了抗汉滩病毒核蛋白和糖蛋白Ｇ１的人源单抗Ｆａｂ段基因，并在大肠杆菌中获得有效表达。核苷酸序列分析证实，所获得的基因为人源ＩｇＧＦａｂ基因。用特异性放射免疫沉淀（ＩＰ）、ＩＦＡＴ和ＥＬＩＳＡ以及空斑减少中和试验鉴定表明，表达的人Ｆａｂ抗体能识别汉滩病毒结构蛋白，其中抗Ｇ１人Ｆａｂ抗体具有体外中和活性。人源抗汉滩病毒基因工程抗体的获得，为今后可能的临床应用提供了良好前景。

human neutralizing monoclonal antibody Fab fragments to the glycoprotein G1 and a human Fab antibody to the nucleocapsid protein (NP) of hantaan virus have been developed by using phage display technique. The human IgG Fab genes of heavy and light chains were amplified from a HFRS patient in convalescent phase. The combinatorial phage antibody library was prepared by inserting both heavy and light chain Fab genes into phagemid vector pComb3 and followed by the help of phage infection. The library was selected by panning phages which expressed the specific human antibody Fabs on their surface. Total 5 rounds of panning, with alternately using the purified hantaan virus virions and the non-neutralizing Mabs captured glycoproteins, were performed and the specific human Fab fragments to hantaan virus were selected and expressed in bacteria. The protein specificity of the expressed human derived monoclonal antibody Fab fragments were characterized by immune precipitation of radiolabeled hantaan viral proteins, three of the expressed human Fabs showed the G1 specificity and another one was specific to NP of hantaan virus. The specific bindings of the Fab antibodies to hantaan virus were demonstrated by their specific reactions with hantaan virus in ELISA and IFAT. The expressed and purified human Fabs to hantaan virus G1 protein clearly showed the neutralizing activity to HTN virus in plaque reduction neutralization test (PRNT). The results provide poten-tial promise for future use in the prophylaxis or therapy of serious HFRS caused by hantaan virus.