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目的制备针对恶性疟原虫环子孢子蛋白(circumsporozoite protein,CSP)NANP四肽重复序列[Asn-Ala-AsnPro(NANP)repeated motif]的单克隆抗体。方法采用马来酰亚胺修饰的BSA与(NANP)5C偶联法及ADH-EDC介导的BSA与(NANP)5偶联法,将合成的NANP多肽与载体蛋白偶联,免疫BALB/c小鼠后,采用免疫后小鼠脾细胞与骨髓瘤细胞SP2/0杂交的细胞融合技术,获得分泌抗(NANP)5多肽单克隆抗体的阳性杂交瘤细胞株,间接ELISA法检测效价。通过免疫F1小鼠诱生腹水,采用50%硫酸铵沉淀法盐析纯化后,进行单克隆抗体亚类和特异性鉴定。结果采用2种方法制备了BSA-(NANP)5偶联蛋白,以该蛋白为抗原,获得10株抗(NANP)5多肽阳性杂交瘤细胞株,效价在1∶80 000~1∶640 000之间,均为Ig G1亚类,轻链类型为κ,可被(NANP)5及CSP抗原特异性识别。结论成功制备了抗(NANP)5多肽的单克隆抗体,该抗体可在恶性疟疾疫苗的研发中发挥重要作用。
Objective To prepare a monoclonal antibody directed against Asp-Ala-AsnPro (NANP) repeated motif of Plasmodium falciparum circumsporozoite protein (CSP) NANP tetrapeptide. Methods The conjugated NANP peptide and carrier protein were conjugated with maleic imide modified BSA and (NANP) 5C and ADH-EDC mediated BSA and (NANP) 5 to immunize BALB / c After the mice were immunized, the hybridomas secreting anti-NANP 5 polyclonal antibody were obtained by the cell fusion technique of immunized mice spleen cells with myeloma cell SP2 / 0, and the titer was tested by indirect ELISA. Ascites was induced by immunization of F1 mice and purified by salting-out with 50% ammonium sulfate precipitation. The subclass and specificity of monoclonal antibodies were identified. Results BSA- (NANP) 5 coupled protein was prepared by two methods. Ten anti-NANP peptide-producing hybridoma cell lines were obtained with the protein as antigen. The titer ranged from 1:80 000 to 1: 640 000 , All of Ig G1 subclass and light chain type κ, can be specifically recognized by (NANP) 5 and CSP antigen. Conclusion Monoclonal antibody against NANP 5 has been successfully prepared and the antibody can play an important role in the development of malaria vaccine.