摘要
目的 :降低鼠源性抗体的免疫原性及分子量 ,为其进一步研究、应用奠定基础。方法 :应用RT PCR技术 ,克隆SZ 2重链、轻链可变基因。应用基因重组技术构建SZ 2单链抗体表达载体pET2 2 2scFv ,导入大肠杆菌BL2 1(DE3)Plys ,诱导表达。流式细胞术、ELISA、Westernblot检测SZ 2scFv与血小板结合能力 ,瑞斯托霉素、凝血酶和ADP诱导血小板聚集试验对表达产物功能进行研究。结果 :克隆基因序列符合小鼠轻、重链可变区基因特征 ;pET2 2 2scFv表达质粒拼接正确 ;表达产物以包涵体形式为主 ,表达量占菌体总蛋白的 2 5 % ;纯化、复性后证明表达产物具有与血小板结合的活性 ,并可抑制瑞斯托霉素、凝血酶诱导的血小板聚集。结论 :成功表达了SZ 2单链抗体 ,该小分子抗体具有与血小板结合的活性 ,并可抑制瑞斯托霉素、凝血酶诱导的血小板聚集。
Objective:To express the recombinant single chain Fv(scFv) in E.coli and reduce immunogenicity and molecular weight of a monoclonal antibody specific for human platelet.Methods:The variable regions of the heavy and light chains of platelet specific antibody SZ 2 were amplified by reverse transcription and polymerase chain reaction.VH and VL gene segments were cloned into pUC Tm and joined together with a (gly 4ser) 3 linker.The resulting scFv was expressed in PET expression system.The expressed recombinant protein was characterized by its size on SDS PAGE,by Western blot,by flow cytometry and its functions.Results:The VH and VL genes were homologous with the published gene sequences of mouse antibody variable region.The recombinant scFv was expressed mostly in the form of inclusion bodies,and the yield was up to 25% of the total cell proteins.Functional studies showed that SZ 2 scFv could bind to platelet and could suppress platelet aggregation induced by ristocatin and thrombin.Conclusion:A recombinant SZ 2 scFv specific against platelet was developed and characterized.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2003年第4期243-247,252,共6页
Chinese Journal of Immunology
