[Cloning and expression of HLA-A*0201-BSP]

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• High-yield production of the HLA-A*0201 heavy chain is crucial for preparing HLA-A2 tetramers.
• Developing efficient methods for recombinant protein expression is essential in immunology research.

Purpose of the Study:

• To construct an expression vector for a recombinant HLA-A*0201-BSP fusion gene.
• To establish a method for the high-yield production and purification of HLA-A*0201 heavy chain for HLA-A2 tetramer preparation.

Main Methods:

• Cloning the extracellular region of HLA-A*0201 via RT-PCR.
• Adding a biotin-protein ligase substrate peptide (BSP) to the HLA-A*0201 heavy chain.
• Cloning the fusion gene into the pBV220 vector and transforming E. coli DH5alpha.
• Inducing expression at 42°C, purifying inclusion bodies using Triton X-100, urea, and Sephacryl S-300 HR.

Main Results:

• Successful construction and sequence confirmation of the pBV220-HLA-A*0201-BSP recombinant plasmid.
• Efficient expression of the HLA-A*0201-BSP fusion protein in E. coli DH5alpha, forming inclusion bodies constituting over 28% of total cell proteins.
• Purification of the fusion protein to over 90% purity.

Conclusions:

• The HLA-A*0201-BSP fusion gene was successfully cloned and expressed in E. coli DH5alpha.
• This work provides a convenient and efficient approach for the large-scale purification of recombinant HLA-A*0201-BSP.
• This method lays the foundation for the preparation of HLA-A2 tetramers.