학술논문
抗微小隐孢子虫 Apple 结构域单克隆抗体的制备 / PREPARATION MONOCLONAL ANTIBODIES AGAINST APPLE DOMAIN PROTEIN OF CRYPTOSPORIDIUM PARVUM
Document Type
Academic Journal
Author
Source
寄生虫与医学昆虫学报 / Acta Parasitology et Medica Entomologica Sinica. (3):139-144
Subject
Language
Chinese
ISSN
1005-0507
Abstract
采用PCR技术从已构建的微小隐孢子虫cDNA文库中扩增含Apple 结构域基因，构建pGEX-4T-1-Apple重组质粒，并进行原核表达及纯化，得到分子质量大小约为34 kDa的GST-Apple重组蛋白，以其为免疫原，免疫Balb/c小鼠，利用杂交瘤技术制备单克隆抗体，测定其免疫球蛋白亚类及效价，并用蛋白质印迹法（Western-blot）鉴定其特异性。结果共筛选出能稳定分泌抗重组Apple单克隆抗体的2株杂交瘤细胞株，抗体亚类均为IgG1， Western-blot分析显示2株单抗均能与重组蛋白GST-Apple发生特异性结合。为研究Apple结构域在隐孢子虫侵入过程中的功能提供有效工具。
Apple domain gene was amplified from cDNA library of Cryptosporidium parvum by PCR method. The recombinant plasmid pGEX-4T-1-Apple were constructed and then expressed in E. coli. The molecular weight of the recombination protein is 34 kDa.BALB/c mice were immunized with purified recombinant protein to prepare McAbs .The subtype and specificity of McAb were identified by kit and Western-blot respectively.Two hybridoma cell lines secreting monoclonal antibodies aganist Apple domain were obtained.The subtype of both McAbs are IgG1.Western-blot showed that the two McAbs reacted strongly and highly specific against GST-Apple recombination proteins.It would provide effective material to study the function of Apple domain during parasite attachment and host cell invasion.
Apple domain gene was amplified from cDNA library of Cryptosporidium parvum by PCR method. The recombinant plasmid pGEX-4T-1-Apple were constructed and then expressed in E. coli. The molecular weight of the recombination protein is 34 kDa.BALB/c mice were immunized with purified recombinant protein to prepare McAbs .The subtype and specificity of McAb were identified by kit and Western-blot respectively.Two hybridoma cell lines secreting monoclonal antibodies aganist Apple domain were obtained.The subtype of both McAbs are IgG1.Western-blot showed that the two McAbs reacted strongly and highly specific against GST-Apple recombination proteins.It would provide effective material to study the function of Apple domain during parasite attachment and host cell invasion.