摘要
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 a high efficient UV-attenuated Schistosoma japonicum cercariae single chain Fv（ScFv）phage display library and screen for the specific ScFv by the potential protective Schistosoma japonicum cercariae antigen 66-68kDa（SCA66-68kDa）,one natural molecular antigen,which based on the high protection of both the UV-attenuated cercariae vaccines and natural molecular vaccine.Methods After selected the optimum conditions of ultraviolet irradiation,the BALB / c mice were immunized with UV-attenuated Schistosoma japonicum cercariae,then the serum of these mice was transferred passively to other mice.In order to evaluate the protection potential of UV-attenuated cercariae serum,once we get the protective serum,the spleens of these mice was used to prepare RNA.Then we used the methods of phage display antibody library to construct the single-chain Fv（scFv） antibody library against UV-attenuated Schistosoma japonicum cercariae and characterized it from the way of capacity and diversity.scFv antibody library was screened against UV-attenuated Schistosoma japonicum cercariae with the purified antigen of
出处
《热带医学杂志》
CAS
2010年第8期907-914,917,共9页
Journal of Tropical Medicine
基金
国家基础研究(973)计划(No.2007CB513108)
国家高技术研究发展计划(863重大专项2004AA2Z3530)
湖南省"十一.五"重大专项(No.2006SK1001)
湖南省"十一.五"重点学科建设专项(No.07-985-2)
国家"十.五"重大科技专项课题(No.2002AA2Z3343)
关键词
日本血吸虫
致弱尾蚴
被动转移
单链抗体
噬菌体展示抗体库
菌落挖掘法
Schistosoma japonicum
UV-attenuated cercaria
passive transfer
ScFv
phage display antibody library
colony lift assay
