陆俭,李萍,马亚茹,李刚,陈勇,蒋琳
LU Jian,LI Ping,MA Ya-ru,LI Gang,CHEN Yong,JIANG Lin

• 1:兰州生物制品研究所有限责任公司

摘要(Abstract)：

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 the monoclonal antibody(Mc Ab) against Asn-Ala-Asn-Pro(NANP) repeated motif of circumsporozoite protein(CSP)of Plasmodium falciparum. Methods Maleimide-modified BSA was coupled with(NANP)5C, while ADH-EDC mediated BSA with(NANP)5. The synthetic NANP was coupled with carrier protein and immunized to BALB / c mice. Hybridoma cell strain secreting Mc Ab against(NANP)5 was obtained by hybridization of spleen cells of immunized mice with bone marrow hybridoma SP2 / 0 cells, determined for titer by indirect ELISA, and immunized to F1 mice. The obtained ascites was purified by precipitation with 50% ammonium sulphate, and identified for subclass and specificity. Results BSA-(NANP)5 coupling protein was prepared by two methods and used as an antigen to prepare ten hybridoma cell strains secreting Mc Abs against(NANP)5 peptide. All the Mc Abs were Ig G1 with Ⅱκ chain and titers of1 ∶ 80 000 ~ 1 ∶ 640 000, which were recognized by(NANP)5 and CSP antigen. Conclusion The Mc Ab against(NANP)5 peptide was successfully prepared, which played an important role in development of malignant malaria vaccine.

关键词(KeyWords)： 疟疾;环子孢子蛋白;NANP;四肽重复序列;单克隆抗体
Malaria;Circumsporozoite protein;NANP;Tetrapeptide repeated motif;Monoclonal antibody(Mc Ab)

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 陆俭,李萍,马亚茹,李刚,陈勇,蒋琳
LU Jian,LI Ping,MA Ya-ru,LI Gang,CHEN Yong,JIANG Lin

DOI: 10.13200/j.cnki.cjb.001085

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