Construction Of Prokaryotic Expression Vector Of Human B7h Gene And Its Expression

Objective To construct prokaryotic expression vector carrying human B7h geneand express it in E. coli BL21(DE3) in that the recombinant B7h protein is verycritical to the study on the function of ICOS/B7h pathway and to the preparation ofantibodies to B7h protein.Methods1. Dendritic cells were prepared by incubation of human peripheral bloodmononucleat cells (PBMC) isolated by Ficoll-Hypaque in the presence of IL-2,IL-4,GM-CSF. Total RNA was isolated with Trizol reagent from the dendriticcells, and the isolated total mRNA was used for preparation of B7h cDNART-PCR.2. The B7h gene was cloned by insertion of B7h cDNA into vector pTZ57R/T usingInsT/Aclone^TM PCR Product Cloning Kit and the recombinant pTZ57R/T-B7hwas amplified by expansion of E. coli JM109 transformed with the recombinantpTZ57R/T-B7h. The pTZ57R/T-B7h was identified by PCR, digestion with theenzyme EcoR I and enzyme Not I, and sequence analysis.3. A prokaryotic expression system, pGEX-4T-1-B7h, was constructed with thecloned B7h gene and pGEX-4T-1, and it was used to transform E. coliBL21(DE3). The fusion protein expressed in transformed E. coli BL21(DE3) wasidentified by SDS-PAGE and Western blot.Results1. The B7h cDNA produced by RT-PCR was found to be 909bp in size asdemonstrated by agarose electrophoresis.2. The B7h cDNA was used to clone B7h gene by insertion of it into vectors pTZ57R/T. PCR and endonucleases digestion showed that the size of cloned B7hgene was found to be the same as the B7h cDNA. Sequencing demonstrated thecloned B7h gene was of 99.6% identity to the B7h sequence from the database ofGenBank. These results indicated that the gene cloned in the experiment was B7hgene.3. The B7h DNA contained in pGEX-4T-1-B7h was also of 99.6% identity to theB7h sequence from the database of GenBank. When transformed withpGEX-4T-1-B7h, expressed by the transformed E. coli BL21(DE3) expressed afusion protein of 56 kD as indicated by SDS-PAGE. Western blot showed that theexpressed fusion protein was recognized by the monoclonal antibody specific toB7h.Conclusion1. The gene cloned in the present study is human B7h gene.2. The recombinant plasmid pGEX-4T-1-B7h constructed in the present study is afunctional prokaryotic expression system.3. The fusion protein expressed in E. coli BL21(DE3) transformed withpGEX-4T-1-B7h is B7h protein.