Preparation of a novel human platelet/T cell activation antigen 1 (PTA1) IgG Fc fusion protein, a potential tools for identification of PTA1 ligand
Preparation of a novel human platelet/T cell activation antigen 1 (PTA1)IgG Fc fusion protein, a potential tools for identification of PTA1 ligand$$$$ SUN Kai, JIN Boquan, FENG Qi, et al. Department of Immunology, The Fourth Military Medical University, Xian 710032 【 Abstract 】 Objective To express and purify the PTA1/IgG Fc fusion protein as a tool for further research on PTA1 ligand and its function. Methods The mRNA of PTA1 was extracted from TPA activated Jurkat cells and used as a template for reversetranscription. After PCR amplification, a 793bp fragment including extracellular region and the splice donor sequence “ACTTACCTGT” was obtained and cloned into fusion expression vector pIG. The recombinant vector was named pPTA1/Ig. The pPTA1/Ig vector was transfected in COS7 cell by DEAEDextran method and expression of a secreting fusion protein was identified by sandwich ELISA with antiPTA1 McAb and HRPconjugated antihIg McAb. Results About 1.3mg PTA1/Ig fusion protein could be obtained from 1L COS7 culture supernatants by antiPTA1 affinity chromatography purification. SDSPAGE showed that the molecular weight of PTA1/Ig fusion protein was 83kD and the protein could be recognized by antiIgG polyclonal antibody in Western blot. Conclusions Both PTA1 extracellular domain and IgG Fc domain could be recognized respectively by antiPTA1 and antihIg Fc McAb, suggesting that PTA1/Ig could mimic the nature PTA1 molecule and be a potential tool in the research of PTA1 ligand and its functions. 【 Subject words 】 Platelet/T cell activation antigen Fusion protein Gene expression