寄生虫与医学昆虫学报
寄生蟲與醫學昆蟲學報
기생충여의학곤충학보
ACTA PARASITOLOGICA ET MEDICA ENTOMOLOGICA SINICA
2013年
3期
139-144
,共6页
张颖%孔令聪%张静%赵权%尹继刚
張穎%孔令聰%張靜%趙權%尹繼剛
장영%공령총%장정%조권%윤계강
隐孢子虫%Apple结构域%单克隆抗体
隱孢子蟲%Apple結構域%單剋隆抗體
은포자충%Apple결구역%단극륭항체
Cryptosporidium parvum%Apple domain%McAbs
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。
採用PCR技術從已構建的微小隱孢子蟲cDNA文庫中擴增含Apple 結構域基因,構建pGEX-4T-1-Apple重組質粒,併進行原覈錶達及純化,得到分子質量大小約為34 kDa的GST-Apple重組蛋白,以其為免疫原,免疫Balb/c小鼠,利用雜交瘤技術製備單剋隆抗體,測定其免疫毬蛋白亞類及效價,併用蛋白質印跡法(Western-blot)鑒定其特異性。結果共篩選齣能穩定分泌抗重組Apple單剋隆抗體的2株雜交瘤細胞株,抗體亞類均為IgG1, Western-blot分析顯示2株單抗均能與重組蛋白GST-Apple髮生特異性結閤。為研究Apple結構域在隱孢子蟲侵入過程中的功能提供有效工具。
채용PCR기술종이구건적미소은포자충cDNA문고중확증함Apple 결구역기인,구건pGEX-4T-1-Apple중조질립,병진행원핵표체급순화,득도분자질량대소약위34 kDa적GST-Apple중조단백,이기위면역원,면역Balb/c소서,이용잡교류기술제비단극륭항체,측정기면역구단백아류급효개,병용단백질인적법(Western-blot)감정기특이성。결과공사선출능은정분비항중조Apple단극륭항체적2주잡교류세포주,항체아류균위IgG1, Western-blot분석현시2주단항균능여중조단백GST-Apple발생특이성결합。위연구Apple결구역재은포자충침입과정중적공능제공유효공구。
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.