新型嵌合补体抑制分子HCI-3的设计构建及真核细胞表达的研究

目的：基于抑制补体活化的两个关键环节，即C3／C5转化酶及MAC的形成，设计、构建及制备一种新型、高效的补体抑制剂．方法：首先设计引物，通过PCR技术扩增重组可溶性CD46／CD55／CD59嵌合分子cDNA片段，重组于pcDNA3．1( )真核表达载体上，构建兼有三分子功能的CD46／CD55／CD59嵌合补体抑制分子，命名为HCI-3(Human Chimeric Complement Inhibitor,HCI-3)．分别利用COS-7细胞和CHO细胞进行表达，并用抗人CDl6多抗及抗人CD55，CD59单抗对表达产物进行Western Blotting鉴定．结果：DNA测序结果证实，前端带有CD59信号肽序列，后端融合有编码6个组氨酸碱基的HCl-3 cDNA片段的阅读框完整．免疫印迹结果显示，表达的重组蛋白分别能与抗人CD46多克隆抗体和抗人CD55，CD59单抗结合．结论：成功构建并在真核细胞内表达了重组可溶性HCl-3分子，为进一步研制和开发新一代多功能、多靶点补体抑制剂奠定了基础．

The Construction and Eukaryotic Expression of a Novel Chimeric Complement Inhibitor HCI-3

Objective To obtain novel efficient complement inhibitor based on inhibiting the formation of C3/ C5 and assembly of MAC. Methods Based on the cDNA of the membrane CD46/CD55/CD59, to be designed primer, soluble CD46/CD55/CD59 recombinant cDNA was obtained by PCR, soluble chimeric human CD46/CD55/CD59 was constructed and expressed in COS-7 celis and CHO celis. Recombinant proteins were identified with western blotting using CD46 polyclonal antibody and CD55, CD59 McAb. Results Results showed that the reading frame of chimeric CD46/CD55/CD59 molecules was correct. The recombinant products could be detected by CD46, CD55 and CD59 antibody in western blotting. Conclusion Successful construction and expression of the target molecules provide us a novel complement inhibitor, which can be potentially used as therapeutic agents in severe inflammation and autoimmune disease.