华支睾吸虫富甘氨酸2a类抗原Cs4重组蛋白的免疫学特性与定位

目的 对华支睾吸虫富甘氨酸2a(glycine rich antigen 2a,GRA2a)类抗原Cs4重组蛋白(rCs4)进行免疫学特性分析,并对GKA2a类抗原进行组织定位.方法 使用蛋白质印迹(Western印迹)分析rCs4与华支睾吸虫病患者混合血清的免疫反应性,并分析GRA2a类抗原的分泌性.应用ELISA法检测rCs4免疫小鼠血清中抗rCs4特异性IgG水平,并动态检测华支睾吸虫感染兔0～44周的血清中抗rCs4特异性IgG水平.通过免疫荧光实验(immunofluorescence assay,IFA)对华支睾吸虫GRA2a类抗原进行组织定位.结果 纯化的rCsd可被华支睾吸虫病患者混合血清识别.rCs4单抗(mAb Cs4-31)与华支睾吸虫排泄分泌抗原和可溶性抗原在相对分子质量(Mr)26 000与55 000之间分别有13、15个反应条带,均呈阶梯状分布.ELISA检测rCs4免疫小鼠抗rCs4和ESA的IgG效价均达1:64 000,感染兔的抗rCs4特异性抗体平均水平自感染第4周开始上升,于第6周达到高峰,此后逐步递减,至第20周已呈较低水平.IFA检测发现GRA2a类抗原定位于华支睾吸虫成虫的表皮.结论 Cs4蛋白是华支睾吸虫在感染早期的分泌型蛋白,具较好的抗原性.GRA2a类抗原定位于华支睾吸虫成虫表皮.

Abstract：

Objective To study immunological characteristics of Cs4 recombinant protein(rCs4)of Clonorchis sinensis,one of the glycine rich antigen 2a(GRA2a)gene family member,and the histolocalization of GRA2a antigens. Methods The immunological reactivity of Clonorcihs sinensis Cs4 protein to pooled sera from clonorchiasis patients was investigated by using purified rCs4 in Western blotting.The secretory property of GRA2a antigens was also analyzed by Western blotting.The level of specific antibodies against rCs4 in the sera from mice immunized with rCs4,and the dynamic change of antibodies against rCs4 in the sera of C. sinensisinfected New Zealand rabbits(O-44 w post infection)were examined by ELISA.Immunofluorescence assay (IFA)was performed using the anti-rCs4 monoclonal antibody Cs4-31(mAb Cs4-31)to determine the localization of GRA2a antigens in C. sinensis adult worm.Results The purified rCs4 was recognized by pooled sera from clonorchiasis patients.13 and 15 bands appeared at the sites between M,26 000 and 55 000 after mAb Cs4-31 reacting with excretory-secretory antigen(ESA)and soluble adult worm antigen(AWA),respectively.The anti-rCs4 and anti-ESA serum antibody titers in mice immunized with rCs4 were 1:64 000.In C. sinensis infected rabbits,antibody level began to elevate at 4 w after infection,peaked at 6 w,and declined rapidly to a lower level at 20 w.IFA revealed that GRA2a antigens were localized in the tegument of C.sinensis adult worm.Conclusion The Cs4 protein was a secretory antigen appeared in the early stage of infection,and exhibited high antigenieity.GRA2a antigens were localized in tIle tegument of C. sinensis adult worm.

Immunological characterization and localization of Cs4 recombinant protein of Clonorchis sinensis,one of the glycine rich antigen 2a gene family member

Objective To study immunological characteristics of Cs4 recombinant protein(rCs4)of Clonorchis sinensis,one of the glycine rich antigen 2a(GRA2a)gene family member,and the histolocalization of GRA2a antigens. Methods The immunological reactivity of Clonorcihs sinensis Cs4 protein to pooled sera from clonorchiasis patients was investigated by using purified rCs4 in Western blotting.The secretory property of GRA2a antigens was also analyzed by Western blotting.The level of specific antibodies against rCs4 in the sera from mice immunized with rCs4,and the dynamic change of antibodies against rCs4 in the sera of C. sinensisinfected New Zealand rabbits(O-44 w post infection)were examined by ELISA.Immunofluorescence assay (IFA)was performed using the anti-rCs4 monoclonal antibody Cs4-31(mAb Cs4-31)to determine the localization of GRA2a antigens in C. sinensis adult worm.Results The purified rCs4 was recognized by pooled sera from clonorchiasis patients.13 and 15 bands appeared at the sites between M,26 000 and 55 000 after mAb Cs4-31 reacting with excretory-secretory antigen(ESA)and soluble adult worm antigen(AWA),respectively.The anti-rCs4 and anti-ESA serum antibody titers in mice immunized with rCs4 were 1:64 000.In C. sinensis infected rabbits,antibody level began to elevate at 4 w after infection,peaked at 6 w,and declined rapidly to a lower level at 20 w.IFA revealed that GRA2a antigens were localized in the tegument of C.sinensis adult worm.Conclusion The Cs4 protein was a secretory antigen appeared in the early stage of infection,and exhibited high antigenieity.GRA2a antigens were localized in tIle tegument of C. sinensis adult worm.