摘要
目的构建抗人细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)嵌合抗体的真核表达质粒,在CHO-dhfr-细胞中进行表达,并检测其生物学活性。方法采用RT-PCR法从特异性分泌抗人ICAM-1单克隆抗体的杂交瘤细胞株3F2中扩增抗体VH和VL基因,从人淋巴细胞RNA中扩增人κ、IgG1的轻、重链恒定区序列,再通过重叠延伸PCR连接鼠源性可变区基因片段与人源性恒定区基因片段,获得人-鼠嵌合抗体基因;将轻、重链基因连接至pIRES双表达载体,构建嵌合抗体真核表达质粒;将质粒在脂质体LipofectAMINE介导下转染CHO-dhfr-细胞进行表达,表达的嵌合抗体经Protein A-Sepharose 4B亲和层析柱纯化后,紫外分光光度法测定抗体浓度,ELISA法检测嵌合抗体的特异性抗原结合活性及人源性,并进行还原性SDS-PAGE分析、Western blot分析及抑制细胞间黏附活性分析。结果嵌合抗体真核表达质粒pIRES-anti-ICAM-1经双酶切鉴定构建正确;嵌合抗体在真核细胞CHO-dhfr-中高效表达,培养上清中表达量可达0.5 mg/L;纯化的嵌合抗体经10%还原性SDS-PAGE分析,可见相对分子质量分别约为25 000的IgG轻链和50 000的IgG重链,相对分子质量约25 000的蛋白可被羊抗人IgGκ链所识别,而相对分子质量约50 000的蛋白可被羊抗人IgGγ链所识别;纯化的嵌合抗体可与羊抗人κ链或羊抗人IgG多抗呈强阳性反应,可与人ICAM-1抗原特异结合;该嵌和抗体具有良好的抑制内皮细胞与单核细胞黏附的活性。结论成功制备了抗人ICAM-1嵌合抗体,并在真核细胞中实现高表达,该抗体减少了鼠源性成分,降低了其免疫原性,为ICAM-1相关的炎性疾病的抗体治疗奠定了基础。
Objective To construct the eukaryotic expression vector for anti-human intercellular adhesion molecule-1（ICAM-1）chimeric antibody,express in CHO-dhfr- cells and determine the biological activity of expressed product.Methods VHand VLgenes were amplified by RT-PCR from hybridoma cell 3F2 strain secreting anti-human ICAM-1monoclonal antibody,while CH（IgG1）and CL（κ）genes from human lymphocyte RNA. The amplified genes were spliced by overlap extension PCR and then cloned into pIRES bicistronic expression vector. The constructed recombinant plasmid was transfected into CHO-dhfr-cells in mediation of LipofectAMINE. The expressed chimeric antibody was purified by Protein A-Sepharose 4B affinity chromatography,then determined for concentration by ultraviolet spectrophotometry,for specific antigen-binding activity and humanization level by ELISA,identified by reduced SDS-PAGE and Western blot,and analyzed for intercellular adhesion activity. Results Restriction analysis proved that recombinant plasmid pIRESanti-ICAM-1 was constructed correctly. The expression level of chimeric antibody in culture supernatant of CHO-dhfr-cells reached 0. 5 mg / L. Light and heavy chains of IgG,with relative molecular masses of about 25 000 and 50 000,were observed on reduced 10% SDS-PAGE profile of purified chimeric antibody,and recognized by the κ and γ chains of goat anti-human IgG,respectively. The purified chimeric antibody showed strong positive reactions with goat anti-human κchain or goat anti-human IgG polyclonal antibody,specific binding to human ICAM-1 antigen,and high activity in inhibiting the adhesion of endothelial cells to monocytes. Conclusion Chimeric antibody against human ICAM-1 was successfully prepared and highly expressed in eukaryotic cells,which was expected to be less immunogenic due to the decrease of components from mouse origin. It laid a foundation of antibody therapy of ICAM-1 associated inflammatory diseases.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第7期918-922,926,共6页
Chinese Journal of Biologicals
基金
黑龙江省教育厅高职高专院校科研项目(12515233)
关键词
抗人细胞间粘附分子-1
嵌合抗体
真核细胞
基因表达
生物学活性
Intercellular adhesion molecule-1（ICAM-1） Chimeric antibody Eukaryotic cells Gene expression Biological activity
