Construction of Prokaryotic Expression Vector for Soluble HLA-A*0203-BSP and Its High Yield Expression in Escherichia coli

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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.

Key words： ectodomain fusion protein prokaryotic expression inclusion body human leukocyte antigen

Received: 15 March 2006 Published: 25 September 2006

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. Construction of Prokaryotic Expression Vector for Soluble HLA-A*0203-BSP and Its High Yield Expression in Escherichia coli. China Biotechnology, 2006, 26(09): 5-10.

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https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2006/V26/I09/5

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