研究报告 |
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Construction of Prokaryotic Expression Vector for Soluble HLA-A*0203-BSP and Its High Yield Expression in Escherichia coli |
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Abstract Objective: To clone the cDNA of HLA-A*0203 heavy chain and to construct the prokaryotic expression vector for the ectodomain of HLA-A*0203 fused with a BirA substrate peptide (BSP) at its carboxyl terminus ( HLA-A*0203-BSP ) and express the recombinant protein in Escherichia coli (E.coli). Methods: The cDNA for HLA-A*0203 heavy chain was cloned by RT-PCR from the PBMC of three HLA-A2+ donors and confirmed by DNA sequencing. DNA fragment encoding HLA-A*0203-BSP was amplified by PCR with the sequence-verified cDNA as a template. It was then cloned into pET-3d vector. After confirmed by DNA sequencing once again, the prokaryotic expression vector was transformed into BL21(DE3) strain. The recombinant protein was expressed in E. coli after IPTG induction. SDS-PAGE and Western blot analyses were employed to detect the expressed fusion protein. Results: DNA sequence analysis showed that the cDNA encoding the heavy chain of HLA -A*0203 was cloned from donor 2, which had been confirmed to be HLA-A2+ by flow cytometry. The expression vector for the recombinant HLA-A*0203-BSP fusion protein was then constructed and verified by DNA sequencing. SDS-PAGE analysis revealed that the fusion protein was highly expressed in E. coli and accounted for 30% of total bacterial proteins. Furthermore, Western blotting showed that all the recombinant protein existed in the inclusion bodies. The fusion protein had a molecular weight of about 34 kD, which is in accordance with the theoretical value. Conclusion: The cDNA of HLA-A*0203 heavy chain was cloned and the prokaryotic expression vector for the fusion protein of HLA-A*0203-BSP was constructed. The recombinant protein was highly expressed as the form of inclusion bodies in E. coli, which would facilitate the preparation of soluble HLA-A*0203 tetramer.
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Received: 15 March 2006
Published: 25 September 2006
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