抗日本血吸虫生殖功能分子SIEA26～28kDa单链抗体与EGFP的融合表达及靶向性研究

Fusion expression and targeting ability of the single-chain Fv antibody against the anti-fecundity immunity molecule,soluble immature egg antigen SIEA26-28kDa from Schistosoma japonica

目的体外观察抗日本血吸虫生殖功能分子SIEA26-28kDa单链抗体与日本血吸虫各阶段的靶向部位，探讨单链抗体在抗血吸虫病疫苗研究中的应用价值。方法扩增增强型绿色荧光蛋白（EGFP）基因．克隆至PET32a／SIEA26～28kDa-scFv质粒，构建PET32a／EGFP-scFv质粒。转化至感受态E．coli BL21（DE3）中。并诱导表达单链抗体融合蛋白。分析单链抗体融合蛋白与SIEA26～28kDa的结合特异性。单链抗体融合蛋白与日本血吸虫成虫、虫卵切片孵育，荧光显微镜下观测GFP信号。确定特异性单链抗体的靶向性。结果重组质粒PET32a／EGFP-scFv构建成功．Trx-EGFP-scFv融合蛋白高效表达．与SIEA26-28kDa特异性结合。GFP信号主要集中在未成熟卵卵胚．雌虫卵巢、卵黄腺及与生殖系统邻近的肠腔组织。结论SIEA26-28kDa单链抗体与血吸虫未成熟卵胚胎及雌虫生殖系统具有较强的靶向性，具有潜在抗卵胚发育、抗雌虫生殖作用，为SIEA26-28kDa单链抗体在抗日本血吸虫病疫苗免疫靶向方面的研究奠定了基础。

To observe the binding sites of the single-chain Fv antibody（scFv） against the anti-fecundity immunity molecule, soluble immature egg antigen SIEA26-28kDa with different stages of Schistosoma japonica in vitro, in order to clarify its application value on the study of anti-schistsomiasis vaccines, enhanced green fluorescence protein（EGFP） coding gene was amplified and cloned to plasmid pET32a/SIEA26-28kDa-scFv and then transformed to the competent E. coli BL21（DE3） to induce the expression of seFv fusion protein. The specific antigen-binding activity of the scFv fusion protein against SIEA26-28kDa was analyzed and its targeting ability was determined by the observation of the GFP signals when the scFv fusion protein was incubated with the tissue sections of adult worms and eggs and examined under fluorescent microscopy. Under these ways, the recombinant plasmid pET32a/EGFP-scFv was successfully constructed, and the Trx-EGFP-scFv fusion protein was corrected expressed. This fusion protein could react specifically with SIEA26-28kDa molecule. The GFP signals were mainly enriched in the embryo of immature eggs, ovaries, vitellarium and the lining membrane tissue of the gut lumen of the female worms. From these observation, it is evident that the specific SIEA26-28kDa scFv has good targeting ability with embryo of immature eggs and the genital system of adult worms, thus paving a way for the targeting interference of S. japonica by using the specific SIEA26-28kDa scFv directly.