抗恶性疟原虫复合重组抗原单克隆抗体的制备、鉴定及其体外抑制试验

PRODUCTION AND IDENTIFICATION OF MONOCLONAL ANTIBODIES AGAINST HYBRID RECOMBINANT ANTIGENS OF PLASMODIUM FALCIPARUM ZAND INHIBITORY TEST

目的制备抗恶性疟原虫单克隆抗体，为疟疾诊断及疟疾疫苗等方面的研究提供良好的材料。方法以恶性疟原虫复合重组融合蛋白为免疫原，经细胞融合及ＥＬＩＳＡ方法筛选获得阳性杂交瘤细胞。对其中３株２Ｈ１２、３Ｄ５、４Ｅ５进行了染色体核型分析，对其分泌的单克隆抗体作ＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎｂｌｏｔ及Ｄｏｔ－ＥＬＩＳＡ鉴定，并初步观察了３株单抗对恶性疟原虫（海南分离株）的体外抑制作用。结果两次细胞融合共获得８株阳性克隆，其中３株即２Ｈ１２、３Ｄ５、４Ｅ５的ＯＤ值超过阳性对照，传至２０代以上经ＥＬＩＳＡ检测仍呈强阳性反应，将其腹腔接种给ＢＡＬＢ／ｃ小鼠获得血性腹水，抗体滴度为１∶１２８００。Ｗｅｓｔｅｒｎｂｌｏｔ及Ｄｏｔ－ＥＬＩＳＡ分析显示３株单抗均与相应蛋白发生特异性反应。抑制试验结果表明：３株单抗对恶性疟原虫的体外生长和发育均有一定的抑制作用，抑制率分别为４９％±３％、７６％±６％、４１％±２％。结论已获得稳定分泌抗恶性疟原虫复合重组抗原单克隆抗体的杂交瘤细胞株。

AimTo obtain monoclonal antibodies against Plasmodium falciparumZ,which would provide useful help on further study in fast diagnosis and vaccine development of malaria. Methods In this subject,hybrid recombinant antigen of plasmodium falciparum was expressed as a fusion protein and acted as immunogen.After cell fusion,positive hybridoma cell lines were screened by ELISA.McAbs of 2H12,3D5 and 4E5 were indentified by SDSPAGE,Western blot and Dot ELISA.Inhibitory effect of the three McAbs on the growth and development of P.falciparum in vitro was observed. Results Eight hybridoma cell lines were obtained after twice cell fusions,three lines with OD value higher than that of positive control exhibited strong positive reaction after 20 generations of incubation.Then,the three hybridoma cells were injected into BALB/c mice intraperitoneally,the bloody ascites were collected,the titer of which was up to 112800.Western blot and Dot-ELISA analysis demonstrated that 2H12,3D5 and 4E5 all strongly and specifically reacted to the expressed 65kDa protein.The three McAbs were able to partially inhibit the growth and development of P.falciparum and the inhibitory rates were 49%3%,76%6%,and 41%2% respectively. Conclusion The hybridoma cell lines which secreted stable monoclonal antibodies against hybrid recombinant antigen of Plasmodium falciparum have been obtained.