抗木瓜凝乳蛋白酶单链抗体的筛选、可溶性表达及鉴定  

Screening,soluble expression and identification of scFvs against chymopapain

作　　者：王舰平[1] 赵树进[2] 李脉[1] 许锋[1] 朱庆玲[1] 

机构地区：[1]广东食品药品职业学院实验中心,广东广州510520 [2]广州军区广州总医院药学部,广东广州510010

出　　处：《广东药科大学学报》2017年第4期531-535,共5页Journal of Guangdong Pharmaceutical University

基　　金：军队总后勤部科研基金资助项目(01Q022);海南原肽生物科技有限公司-广东食品药品职业学院技术服务项目(20161215)

摘　　要：目的筛选具有较好结合能力的抗木瓜凝乳蛋白酶单链抗体(scFv),并进行可溶性表达和鉴定。方法前期实验室构建了抗木瓜凝乳蛋白酶全套鼠源scFv噬菌体抗体文库,对抗体文库进行4轮富集后,采用ELISA筛选抗木瓜凝乳蛋白酶阳性scFv;将ELISA最强阳性克隆p FAB5C-scFv的重组噬粒切除gene 3 singal后进行表达,SDS-PAGE分析、双抗体夹心ELISA分析、鉴定表达产物,并将阳性克隆可变区序列与Gene Bank数据库进行同源性比较。结果经4轮噬菌体展示富集,筛选到3株亲和力相对较高的阳性克隆;去除gene 3 singal后的重组scFv在宿主菌E.coli XL1-blue中主要以可溶性的形式进行了表达,且对木瓜凝乳蛋白酶有较好的亲和力。基因序列分析表明阳性克隆所包含的重链可变区基因(VH)与小鼠免疫球蛋白γ重链基因同源性达96%,轻链可变区基因(VL)和小鼠免疫球蛋白轻链κ基因同源性达98%。结论成功筛选出结合能力较好的抗木瓜凝乳蛋白酶scFv并进行了可溶性表达,所克隆的scFv基因符合小鼠抗体序列的特征。Objective To screen, express and identify specific scFv against chymopapain. Methods The scFv library of mouse source was constructed in our previous study, and here the scFv library was enriched for four rounds and the positive scFv was screened by ELISA. The positive clone was ligated with the expression vector FAB5C and the construct was excised gene 3 signal for soluble expression in E.coli XL1-Blue. The expressed scFvs were identified by SDS-PAGE and DAS-ELISA, and gene homology of variable region of the positive clone was compared with the gene bank database. Results After four rounds of enrichment, the three specific scFvs with better affinity were selected, and the scFv with gene 3 signal excised was mainly expressed in soluble form. Gene sequence analysis showed that the gene homology between VH of the scFv and VH ofγchain of mouse immunoglobulin reached 96%, while VL of the scFv and VL of κchain of mouse immunoglobulin reached 98%. Conclusion The specific scFvs against chymopapain with better affinity have been successfully screened and expressed mainly in soluble form, and the scFv sequence is in line with that of mouse antibody.

关 键 词：木瓜凝乳蛋白酶 单链抗体 噬菌体抗体库 可溶性表达 

分 类 号：R392-33[医药卫生—免疫学]