抗人整合素ανβ_3单链抗体的构建和表达  

Construction and expression of anti-human integrin ανβ_3 scFv

作　　者：王臣[1] 侯利华[1] 张彦明[2] 李建民[1] 廖振林[1] 杜桂鑫[1] 陈薇[1] 孙启鸿[1] 童贻刚[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071 [2]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《细胞与分子免疫学杂志》2004年第2期159-162,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划 ( 86 3)资助 (No .2 0 0 1AA2 1 536 1 )

摘　　要：目的 :利用基因工程抗体技术构建抗人整合素ανβ3 单链抗体(scFv)。方法 :从分泌抗人整合素ανβ3 单抗 (mAb)的杂交瘤细胞E10总RNA中 ,用RT PCR扩增VH 和VL 基因 ,对其核苷酸序列分析后 ,通过PCR在VH 和VL 基因间插入柔性连接子(Gly4Ser) 3,组装成scFv基因 ,并克隆至原核表达载体pTIG TRX中。以重组子转化大肠杆菌BL2 1(DE3 )诱导目的基因表达。结果 :转化菌可表达相对分子质量 (Mr)为 3 10 0 0的scFv。Westernblot证实 ,具有His6标签蛋白的表达。经低剂量的IPTG诱导和较低温度培养 ,scFv获得了可溶性形式表达。表达产物经Ni NTA琼脂糖层析纯化后纯度达 91%以上。ELISA鉴定证实 ,scFv具有良好的抗原结合活性。结论 :成功地构建并表达抗人整合素ανβ3 的scFv ,为进一步临床研究奠定了基础。AIM: To construct single chain antibody (scFv) gene of mAb E10 against h uman integrin ανβ 3. METHODS: The V H and V L genes were a mplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with th e use of linker (Gly 4Ser) 3 to assemble scFv gene. The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E.coli BL21 (D E3). RESULTS: SDS-PAGE analysis showed the expressed recombina nt protein with relative molecular mass(M r) being 31 000. Western blot con firmed that the protein was labeled with His6. scFv protein was expressed as sol uble protein under the condition of a small amount of IPTG induction and culture at lower temperature. The purity of the protein purified through Ni-NTA agaros e metal affinity resin column was over 91%. The purified protein could bind to t he human interin ανβ 3 by ELISA confirmation. CONCLUSION: sc Fv against human integrin ανβ 3 has been successfully constructed and exp ressed,which lays the foundation for further clinical research.

关 键 词：单链抗体 整合素αυβ3 构建 表达 

分 类 号：R392.11[医药卫生—免疫学]