抗人CD33单链抗体与CD80胞外区融合基因的构建、表达及其生物活性检测  

Construction,expression and functional detection of fusion gene of CD80 extracellular region and anti-human CD33 scFv

作　　者：陈小军[1] 梁璐 薛峰 隋涛 董春兰 

机构地区：[1]天津医科大学基础医学院免疫学系,天津300070 [2]中国医学科学院血液学研究所血液病医院,天津300020

出　　处：《细胞与分子免疫学杂志》2014年第2期179-183,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：天津市教委重点项目(2012ZD01);天津医科大学基金(2010ky07)

摘　　要：目的构建及表达CD80胞外区与抗人CD33单链抗体（scFv）融合基因，并初步检测融合蛋白的生物活性。方法扩增CD80胞外区，利用人血清白蛋白（HAS）第三结构域亲水性的403—427位氨基酸作为链间连接肽将其与抗人CD33scFv连接，并克隆至原核表达载体PET22b（＋）中，经IPTG诱导，在大肠杆菌Rosseta（DE3）内得到融合蛋白的表达。表达产物经过溶解包涵体，镍柱亲和层析纯化和体外复性过程，获得了高纯度的融合蛋白。并用间接免疫荧光法检测融合蛋白与人CD33抗原结合活性。结果克隆出CD80胞外区序列，大小为633bp，正确合成作为链间连接肽的人血清白蛋白（HAS）第三结构域的403～427位氨基酸。成功构建融合基因表达载体，SDS．PAGE和Westernblot分析结果表明，融合蛋白在Rosseta（DE3）中得到高效表达，融合蛋白相对分子质量（M1）约为55000，复性后经过间接免疫荧光检测表明具有与人CD33抗原结合活性。结论成功构建PET22b（＋）-CD80胞外区一CD33scFv（ExCD80-CD33scFv）融合基因表达质粒，融合蛋白在宿主菌Rosetta（DE3）中获得了表达，并初步检测其生物学活性。Objective To construct and express the CD80 extracellular region-anti-human CD33 single chain fragment of variable region （ExCDSO-CD33scFv） fusion gene, and detect the biological activity of the fusion protein. Methods Extracellular region of CDSO （ExCD80） was amplified and then linked with anti-CD33scFv using 403Aa-427Aa （hydrophilic fragments） in domain II of human serum albumin （HAS） as interlinker. The recombinant fusion gene was subcloned into the prokaryotic expression vector PET22b（＋） and expressed in E. coil Rosetta （DE3） after induction by IPTG. The purified fusion protein was obtained after a series of purification steps including cell lysis, inclusion body solubilization, Ni2＋ metal affinity chromatography and protein refolding. The biological activity of the fusion protein was detected with indirect technique. Results The ExCDSO （633bp） was amplified by PCR and 403aa-427aa from domain Ill of HAS as interlinker was synthesized correctly. SDS-PAGE and Western blotting demonstrated that ExCDSO-CD33scFv fusion gene expression vector was successfully constructed and expressed in E. coli Rosetta （DE3）. The relative molecular mass （Mr） of the fusion protein was 55 000, which has human CD33-binding specificity after renaturation as shown by indirect immunofluorescence technique. Conclusion Recombinant ExCDSO-CD33scFv fusion gene was successfully constructed and expressed in E. coli Rosetta （ DE3）, which could provide a foundation for the future target therapy to the myeloid leukemia.

关 键 词：CD33 SCFV CD80 原核表达 髓系白血病 

分 类 号：R392.11[医药卫生—免疫学] R392-33[医药卫生—基础医学]