抗IL-4R单链抗体与蜂毒肽融合蛋白的构建、表达与纯化  

Construction,Expression and Purification of Fusion Protein of Anti-interleukin-4 Receptor Single-chain Antibody and Melittin

作　　者：杨光勇[1] 刘茜明 刘莉莉 王文佳[2] 何光志[2] Yang Guangyong;Liu Qianming;Liu Lili(Department of Function Laboratory,Basic Medical Sciences of Guiyang College of Traditional Chinese Medicine,Guiyang 550025,China;Department of Microbiology and Immunology, Basic Medical Sciences of Guiyang College of Traditional Chinese Medicine,Guiyang 550025,China)

机构地区：[1]贵阳中医学院基础医学院机能实验室,贵阳550025 [2]贵阳中医学院基础医学院微生物免疫教研室,贵阳550025

出　　处：《华中科技大学学报（医学版）》2018年第2期193-197,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：贵州省委组织部高层次人才科研条件特助经费资助项目(No.TZJF-2011-28);贵州省高等学校工程研究中心项目(No.20153377);贵州省教育厅创新群体重大研究项目(No.2016-036)

摘　　要：目的制备重组抗白细胞介素4受体(recombinant anti-interleukin-4receptor,anti-IL-4R)单链抗体(singlechain variable fragment,scFv)与蜂毒肽(melitin,MLT)的融合蛋白。方法设计并合成抗IL-4R单链抗体基因与蜂毒肽基因,再用1.5%琼脂糖凝胶电泳进行鉴定;通过重叠延伸PCR(gene splicing by over lap extension polymerase chain reaction,SOE-PCR)技术将抗IL-4R单链抗体基因与蜂毒肽基因连接成scFv-MLT融合基因,并以1%琼脂糖凝胶电泳进行鉴定;scFv-MLT融合基因连接pET-32a原核表达载体以构建重组质粒,并用EcoRⅠ和XhoⅠ内切酶进行酶切鉴定;将重组质粒转入BL21(DE3)原核表达菌感受态细胞,37℃环境下培养于含有羧苄青霉素(100μg/mL)的平板TB固体培养基表面,分离出单菌落;挑取单菌落接种于含有羧苄青霉素(200μg/mL)TB液体培养基中,于通气良好的37℃环境下培养,使其A600nm值达到0.4~0.6,用浓度为1mmol/L的诱导剂IPTG诱导表达scFv-MLT融合蛋白;3h后达到表达高峰,离心沉淀菌体并将其裂解,提取所有蛋白并进行scFv-MLT融合蛋白纯化和复性;通过SDS-PAGE分析scFv-MLT融合蛋白的分子量,运用Western blot检测表达蛋白的特异性。结果 scFv-MLT基因序列大小约1 000bp,IL-4R单链抗体与蜂毒肽重组蛋白的分子量在48~63kD之间,与预期分子量52kD相符,scFv-MLT融合蛋白具有较高的特异性。结论成功制备了scFv-MLT重组蛋白,为进一步研究抗IL-4R单链抗体与蜂毒肽的融合蛋白的靶向生物治疗奠定了工作基础。Objective To construct the fusion protein of recombinant anti-interleukin-4 receptor(IL-4R)antibody against single-chain variable fragment(scFv)and melittin(MLT).Methods Anti-IL-4R scFv gene and MLT gene were designed,synthesized,and then identified by 1.5%agarose gel electrophoresis.The two genes were linked by splicing by overlap extension polymerase chain reaction(SOE-PCR)to obtain the scFv-MLT fusion gene,which was later identified by electrophoresis on 1%agarose gel.Thereafter,the scFv-MLT fusion gene was connected with pET-32a plasmid to construct the recombinant plasmid vector,which was identified by the restriction enzyme digestion with Eco RⅠand XhoⅠ.Then the recombinant plasmid of scFv-MLT was transferred into BL21(DE3)bacterial competent cells,cultured at 37℃on the surface of the TB solid medium which contained carbenicillin(100μg/mL)for isolation of single colonies.A single colony was picked and incubated on the TB liquid medium containing carbenicillin(200μg/mL)at 37℃in good ventilation until the A 600 nm value reached 0.4-0.6,and then the IPTG inducer at a concentration of 1 mmol/L was used to induce the scFv-MLT fusion protein.Three hours later,the bacteria were harvested at the peak of the expression of scFv-MLT fusion protein,and they were centrifuged and cleaved for extraction of scFv-MLT fusion protein for purification and renaturation.The molecular weight of scFv-MLT fusion protein was analyzed by SDS-PAGE,and its specificity was detected by Western blotting.Results The gene sequence of scFv-MLT was approximately 1 000 bp.The molecular weight of the recombinant protein was between 48 kD and 63 kD,which was consistent with the expected value of 52 kD,and the fusion protein had high specificity.Conclusion scFv-MLT recombinant protein was successfully built,and our study provides a basis for further study on the targeted biological treatment of the fusion protein of anti-IL-4R scFv and MLT.

关 键 词：白细胞介素4受体 单链抗体 蜂毒肽 融合蛋白构建 原核表达 

分 类 号：R730.51[医药卫生—肿瘤]