Eukaryotic expression and characterization of mouse TSLP and HIV-1 gp120 BAL V1/V2 fusion protein / 中国免疫学杂志

ABSTRACT

Objective:To express fusion protein of mouse thymic stromal lymphopoietin (TSLP) and HIV-1 gp120BAL V1/V2 subdomain in 293F cell.Methods:Full length of the V1V2 sequence from BAL isolate was fused with the C-terminus of mouse thymic stromal lymphopoietin (TSLP) and sub-cloned into pCEP-Pu vector to construct the eukaryotic expression plasmid-pCTV1V2BAL.The recombinant plasmid was confirmed by enzyme digestion and sequencing , then transfected into 293 F cells using PEI as a transfection reagent .The fusion protein was purified by metal chelate affinity chromatography and characterized by SDS -PAGE and Western blot . The epitopes of V1/V2 in fusion protein were identified by ELISA .Results:The SDS-PAGE and Western blot results showed that there were highly heterogeneous glycoprotein bands at the site between 35 kD and 55 kD, which reacted with anti-mTSLP rabbit polyclonal antibody and anti-His tag mouse monoclonal antibody .The ELISA analysis showed that antibodies to V 1/V2BAL existed in the sera of HIV-1 positive patients.Conclusion:The mTSLP-V1/V2 fusion protein was successfully expressed in 293F cells, which may be useful for HIV-1 vaccine research .