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目的构建和鉴定日本血吸虫(Schistosoma japonicum,Sj)紫外线(UV)-照射致弱尾蚴单链抗体(single chain Fv,ScFv)表达文库,并以此作为高通量的库源筛选出针对有潜在保护力的Sj尾蚴66-68 kDa抗原(Schistosoma japonicum cercariae antigen 66-68kDa,SCA66-68kDa)的单链抗体。方法选择最佳紫外灯照射条件,构建UV-致弱尾蚴小鼠模型,通过其血清被动转移实验证实其是否具有抗血吸虫攻击感染的保护效果;随后提取该小鼠脾脏mRNA,利用噬菌体展示技术构建UV-照射致弱尾蚴单链抗体库,并从库容量、多样性等方面进行鉴定;运用直接菌落挖集法以SjSCA66-68kDa筛选该抗体库,获得阳性克隆进行蛋白表达,再与SCA进行反应,验证其与SjSCA66-68kDa反应的特异性。结果构建UV-致弱尾蚴动物模型中的血清转移至小鼠体内得到42.5%的减虫率,显著高于日本血吸虫感染血清转移组的减虫率(28.0%);日本血吸虫UV-致弱尾蚴单链抗体库的库容量测定为1.9×108,重组率为100%;运用抗原直接菌落挖集法筛选共获得6个SCA66-68kDa阳性克隆,经SDS-PAGE、Western-blot分析结果显示可溶性ScFv获得了正确表达,且与相应抗原SjSCA66-68kDa特异性结合,证实成功获得了特异性抗SjSCA66-68kDa单链抗体。结论成功构建了高效日本血吸虫UV-致弱尾蚴单链抗体库,从该库筛选共获得6个阳性克隆,均能诱导表达SjSCA66-68kDa特异性可溶性单链抗体。
Objective To construct and identify the expression library of Schistosoma japonicum (Sj) ultraviolet (UV) - irradiated single chain Fv (ScFv) and use it as a high - Sj-cercariae 66-68 kDa antigen (Schistosoma japonicum cercariae antigen 66-68 kDa, SCA 66-68 kDa). Methods The UV-induced mouse model of weak cercariae was established by choosing the best UV lamp irradiation condition. The passive transfer assay was used to confirm whether it has the protective effect against the infection of schistosomiasis. The spleen mRNA of the mouse was extracted and then phage display technology was used to construct UV-irradiated cercariae single-chain antibody library, and from the library capacity, diversity and other aspects of identification; using direct colony scavenging SjSCA66-68kDa screening of the antibody library to obtain positive clones for protein expression, and then reacted with SCA , To verify its specificity with SjSCA66-68kDa reaction. Results The serum of mice immunized with UV-induced weak cercariae was 42.5%, which was significantly higher than that of the Schistosoma japonicum serogroups (28.0%). The UV-induced cercariae of Schistosoma japonicum The scFv library capacity was 1.9 × 108 and the recombination rate was 100%. Six SCA66-68kDa positive clones were screened by antigen direct colony-picking method. The results of SDS-PAGE and Western-blot showed that soluble ScFv Obtained the correct expression, and with the corresponding antigen SjSCA66-68kDa specific binding, confirmed the successful acquisition of a specific anti-SjSCA66-68kDa single chain antibody. Conclusions A single-chain antibody against UV-induced cercariae of Schistosoma japonicum has been successfully constructed. Six positive clones were screened from this library and all of them can express specific SjSCA66-68kDa soluble single chain antibody.