Cloning of equine β_2-microglobulin gene and its expression-in-Escherichia coli and purification

To construct an equine MHC Ⅰ tetramer,the total RNA was extracted from PBMC of(equine),and then a β_2microglobulin(β_2m) gene without the fragment encoding signal peptide was amplified by RT-PCR.The purified amplification product was cloned subsequently into a modified pET-28a(+) vector(after) being digested with BamHⅠ+XhoⅠ.The recombinant plasmid pETβ_2m was transformed into(E.coli) BL21(DE3) after sequence analysis,and a fusion protein was then expressed and purified. Results showed that the β_2m gene's specific fragment was 300bp in length,which contained an open reading frame and encoded the fusion protein of 99 aa,and the expressed product existed in inclusion bodies.The SDS-PAGE and Western-blotting analysis showed that the fusion protein was 15 ku in molecular weight and had immunological activity.