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Published on: February 6, 2018
[Prokaryotic expression of HLA-B * 2705 heavy chain and identification for its activity]
Yan-Bo Li1, Yu-Ying Sun, Si-Qi Guo
1Department of Immunology, Affiliated Hospital of Academy of Military Medical Sciences, Laboratory of Immunoassy, National Center of Biomedical Analysis, Beijing 100039, China.
Abstract:
In order to investigate the expression of heavy chain of HLA-B * 2705 in prokaryotic system and identify its activity, the extra-membrane gene fragment of HLA-B * 2705 was amplified from full-length HLA-B*2705 cDNA by PCR and cloned into pGEM-T vector. After identification by sequencing, the prokaryotic expressing vector pET32a (+)-B * 2705 was constructed. The antigenic activity of expressed protein was identified by Western blot and antibody blocking reaction. The results indicated that the fused HLA-B * 2705 protein expression with high efficiency was obtained. The expressed product was more than 50% of the total bacteria protein. The antigenic activity of expressed protein was confirmed by Western blot and antibody blocking reaction. It is concluded that HLA-B * 2705 fusion protein are obtained as basis for the further studies.
Insights
Researchers successfully expressed the heavy chain of HLA-B*2705 in bacteria, confirming its antigenic activity. This provides a foundation for further studies on this important human leukocyte antigen.
Area of Science:
- Molecular Biology
- Immunology
- Protein Expression
Background:
- Human Leukocyte Antigen (HLA) B*2705 is associated with autoimmune diseases like ankylosing spondylitis.
- Efficient expression of HLA proteins in prokaryotic systems is crucial for functional studies and therapeutic development.
- Previous methods for expressing HLA proteins have limitations in yield and activity.
Purpose of the Study:
- To investigate the expression of the heavy chain of HLA-B*2705 in a prokaryotic system.
- To confirm the antigenic activity of the expressed HLA-B*2705 fusion protein.
- To establish a basis for further research on HLA-B*2705.
Main Methods:
- Amplification of the HLA-B*2705 extra-membrane gene fragment using PCR.
- Cloning into pGEM-T vector and subsequent construction of the prokaryotic expression vector pET32a(+)-B*2705.
- Identification of the expressed protein using Western blot and antibody blocking assays.
Main Results:
- High-efficiency expression of the fused HLA-B*2705 protein was achieved in the prokaryotic system.
- The expressed fusion protein constituted over 50% of the total bacterial protein.
- Antigenic activity of the expressed protein was confirmed through Western blot and antibody blocking reactions.
Conclusions:
- A robust method for expressing active HLA-B*2705 heavy chain fusion protein in a prokaryotic system has been established.
- The high yield and confirmed antigenic activity provide a valuable resource for further immunological and structural studies.
- This work lays the groundwork for investigating the role of HLA-B*2705 in disease pathogenesis and developing targeted therapies.
