重组白细胞介素4受体(IL-4R)噬菌体单链抗体库构建及筛选  

Construction and panning of scFv phage display library against recombinant interleukin 4 receptor

作　　者：杨光勇[1] 郭海涛[1] 刘茜明 何光志[1] 田维毅[1] 蔡琨[1] 王平[1] 王文佳[1] 

机构地区：[1]贵阳中医学院,贵州贵阳550002

出　　处：《细胞与分子免疫学杂志》2016年第6期829-833,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：2011年贵州省社会发展科技攻关项目[黔科合SY字(2011)3020];贵州省委组织部高层次人才科研条件特助经费(TZJF-2011年28号);2014年度贵阳中医学院"2011协同创新中心"联合资助

摘　　要：目的应用噬菌体展示技术构建重组白细胞介素4受体（IL-4R）噬菌体单链抗体（sc Fv）库,并从抗体库中进行筛选。方法利用重组人IL-4R（rh IL-4R）蛋白免疫BALB/c小鼠,提取总RNA,反转录PCR扩增V_H和V_L基因,经重叠延伸PCR扩增出带有限制性酶切位点的sc Fv,用限制性内切酶SfiⅠ和NotⅠ分别双酶切sc Fv和p CANTAB5E载体,利用T4连接酶进行连接,转入感受态细菌E.coli TG1中制备转化子;在培养基中培养得到噬菌体单链抗体库;通过3轮富集和筛选,选择抗原亲和力最强的克隆进行测序分析。结果经过3轮筛选,构建了库容为2×10~8的rh IL-4R单链抗体库,测序结果显示抗rh IL-4R蛋白sc Fv基因全长741 bp,编码247个氨基酸。通过VBASE2数据库分析,抗rh IL-4R蛋白的V_H和V_L基因序列都存在3个互补决定区和4个骨架区。结论成功构建了抗rh IL-4R蛋白噬菌体sc Fv库。Objective To construct the recombinant human interleukin 4 receptor（ rh IL-4R） single-chain Fv（ sc Fv） antibody library by phage display technique to obtain the anti-IL-4R sc Fv clones selected from the library. Methods Total RNA was extracted from splenocytes of the BALB/c mice immunized with rh IL-4R. Complementary DNA fragments of variable heavy（ V_H） and variable light（ V_L） chains of the antibodies were prepared by reverse transcription PCR and assembled into sc Fv by splice overlap extension PCR（ SOE-PCR）. Both sc Fv and the p CANTAB5 E vector were respectively double-digested with restriction endonuclease Sfi Ⅰ and Not Ⅰ,connected with T4 ligase,and then transformed into the competent cells E. coli TG1; it was cultured in medium to obtain the phage sc Fv antibody library; after three rounds of enrichment and panning,the specific antigen sc Fv with high affinity was selected for the sequencing. Results After three rounds of panning,we obtained a diversity of approximately 2 × 10~8anti-rh IL-4R sc Fv antibody library. Sequencing analysis of one positive clone showed that the anti-rh IL-4R sc Fv was 741 bp and coded 247 amino acids. The analysis of VBASE2 database indicated that V_Hand V_L gene sequences of anti-rh IL-4R protein all had three complementarity determining regions and four backbone areas.Conclusion The anti-rh IL-4R sc Fv was obtained from the sc Fv antibody library.

关 键 词：重组人白细胞介素4(rhIL-4R) 噬菌体展示技术 单链抗体 

分 类 号：R392.11[医药卫生—免疫学] Q789[医药卫生—基础医学]