一种新型结缔组织生长因子人源单链抗体的可溶性表达以及分离纯化  

Soluble expression and purification of a novel single-chain Fv antibody against connective tissue growth factor(CTGF/CCN2)

作　　者：范小波[1] 苟丽霞[2] 刘昭[2] 沈子龙[1] 刘乃丰[2] 吴国球[2] 奚涛[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009 [2]东南大学附属中大医院临床检验中心,江苏南京210009

出　　处：《东南大学学报（医学版）》2010年第4期361-366,共6页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(No.30070300;No.30970809)

摘　　要：目的:研究单链抗体(scFv)在不同载体和菌株中的表达以及表达产物的免疫学活性评价。方法:将人源化单链抗体的基因通过酶切、连接等生物技术的方法克隆到pET28a、pET32a和pET-EI:X3种不同的载体中,并选用不同的表达菌株进行表达,观察单链抗体的表达情况并对抗体的可溶性表达进行优化,采用亲和层析和分子筛层析对目的蛋白进行纯化,并在体外研究目的蛋白的生物学活性。结果:抗结缔组织生长因子单链抗体在pET28a/BL21(DE3)中不能表达,在pET32a/BL21(DE3)和pET-EI:X/BLR(DE3)中均成功表达,但是在pET-EI:X/BLR(DE3)中scFv抗体无法从内含肽(intein)上裂解。scFv在pET32a/BL21(DE3)中表达量占总蛋白的30%左右,经过两步层析纯化后纯度达到85%以上。体外免疫试验显示,目的蛋白具有良好的免疫学活性。结论:人源化抗结缔组织生长因子单链抗体scFv在大肠杆菌中的表达需要分子伴侣,通过低温表达策略在pET32a/BL21(DE3)中成功得到表达。Objective：To investigate the expression of a noval anti-CTGF single-chian Fv（scFv） antibody within different plasmid and/or host cell and the in vitro immunoreactivity of this scFv antibody.Methods：In this study,a scFv antibody against CTGF/CCN2 with full human sequence was cloned into three different vectors,pET28a,pET32a and pET-EI：X,and expressed in BL21（DE3）,BL21（DE3）,BLR（DE3）,respectively.In addition,we optimized the temperature for soluble expression of the scFv.The expressed target protein was then purified by using molecular filter and affinity chromatography,and the bioactivity was also analyzed.Results：The scFv anti-body could not be expressed in pET28a/BL21（DE3） while it was expressed well in pET32a/BL21（DE3） and pET-EI：X/BLR（DE3）.However,the scFv antibody could not be cleaved from the ELP-intein-scFv fusion.After optimization,the fusion protein was overexpressed mainly as soluble form in pET32a/BL21（DE3） and approximately achieved 30% of total proteins.After purified with Ni-Sepharose affinity chromatography,the fusion protein was cleaved with thrombin protease.Finally,the scFv was separated from the fusion partner by gel filtration chromatography,and the purity could reach over 85%.Immunoreactivity study demonstrated that this scFv we obtained had a special affinity towards CTGF/CCN2.Conclusion：The results show that this scFv should be expressed with a fused partner to achieve satisfied amount in E.coli.It is well expressed and purified in pET32a/BL21（DE3）.

关 键 词：人源单链抗体 结缔组织生长因子 弹性蛋白 分离纯化 

分 类 号：Q512.6[生物学—生物化学] Q511