抗IL-4R单链抗体原核表达载体的构建与表达  

Construction and expression of anti IL-4R single antibody of prokaryotic expression vector

作　　者：杨光勇[1] 刘茜明 刘莉莉 王文佳[1] 何光志[1] YANG Guang-yong LIU Qian-ming LIU Li-li WANG Wen-jia HE Guang-zhi(Basic Medical School of Guiyang College of Traditional Chinese Medicine, Guiyang 550002, Chin)

机构地区：[1]贵阳中医学院基础医学院,550002

出　　处：《天津医药》2017年第9期897-901,共5页Tianjin Medical Journal

基　　金：贵州省教育厅创新群体重大研究项目(黔教合KY字2016036);贵中医科院内[2016]31号;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);贵州省高等学校工程研究中心项目(黔教合KY字20153377);2012年贵州省科学技术基金(黔科合[2012]2075号)联合资助

摘　　要：目的通过BL21(DE3)原核表达系统制备抗白细胞介素-4受体(IL-4R)鼠抗人单链抗体(scFv)。方法在前期研究结果的基础上优化抗IL-4R scFv序列,优化后分析scFv序列,构建重组质粒pET-32a-scFv,将该重组质粒酶切鉴定,将其转入BL21(DE3)原核表达菌进行诱导表达,并进行SDS-PAGE分析检测,对表达蛋白进行纯化和复性,通过SDS-PAGE分析抗IL-4R单链抗体的分子质量,运用Western blot检测融合蛋白的特异性。结果插入pET-32a载体的scFv序列长度761 bp,抗IL-4R单链抗体的分子质量在45 ku左右,重组蛋白具有较高的特异性。结论本实验成功构建pET32a-scFv原核表达系统,重组蛋白具有较高的免疫反应性,为进一步研究抗IL-4R单链抗体作为药物靶点提供了工作基础。Objective To construct anti-IL-4R murine anti-human single-chain variable fragment （scFvs） antibodies through BL21 （DE3） prokaryotic expression system. Methods The anti-IL-4R scFv sequence was optimizated on the basis of previous findings. The optimized scFv sequence was analyzed. The recombinant plasmid pET-32a-scFv was constructed. The recombinant plasmid was detected through enzyme identification, and was turned into BL21 （DE3） prokaryotic expression bacteria to express the pET-32a-scFv recombinant protein in E.coli BL21 （DE3）. The purification and renaturation were researched, and SDS-PAGE analysis was studied. The molecular weight of ScFv against IL-4R was analyzed by SDS-PAGE. The expression of the fusion protein was detected by Western-blot assay. Results The length of fusion gene scFv-MLT sequence was 761 bp. The molecular weight of the recombinant expression of proteins of anti-IL-4R single antibody was approximately 45 ku. The recombinant proteins showed high specificity with anti-6 × His-tag antibody. Conclusion This experiment successfully constructs pET-32a-scFv prokaryotic expression system of recombinant protein with high immune reactivity, which provides the basis for further study of anti-IL-4R single chain antibody as drug target.

关 键 词：受体 白细胞介素4 重组蛋白质类 电泳 聚丙烯酰氨凝胶 单链抗体 原核表达 INTERLEUKIN-4 

分 类 号：R34[医药卫生—基础医学]