Abstract: In order to study the structure and function of Small-tailed Han sheep leukocyte antigen (OLA) class Ⅰ tetramer light chain β₂-microglobulin (β₂m).A pair of specific primers based on the published sequence of this gene was designed and the cDNA of full coding region for β₂m precursor was obtained by RT-PCR from the Small-tailed Han sheep whole blood.The Small-tailed Han sheep β₂m gene was cloned into the pMD18-T vector and the positive clones of pMD18T-OLAⅠ-β₂m were selected.After double digestion,the β₂m gene in positive clones was further ligated into the pET-28a(+) expression vector and transformed into E.coli BL21(DE3) to construct the recombinant strain of pET-28a(+)-OLAⅠ-β₂m.Then the targeted protein was detected by SDS-PAGE within induction of IPTG.The secondary structure of the OLAⅠ-β₂m protein was predicted and analyzed by SOMPA software.The PCR results showed that the length of OLAⅠ-β₂m was about 357 bp which was consistent with the theoretical value.The amplified fragment was successfully cloned into pMD18-T vector,and the positive clones were verified by EcoR Ⅰ and Hind Ⅲ digestion and DNA sequencing analysis.The cut β₂m gene from pMD18T-OLAⅠ-β₂m was inserted into pET-28a(+) and transformed into E.coli BL21(DE3) successfully.After IPTG induction,the expressed protein was detected by western blotting and SDS-PAGE,the results showed that the target protein (17.3 ku) was expressed efficiently with inclusion body.Finally,the secondary structure α-helix,extended strand and random coil of the target protein was 22.03%,22.03% and 55.93%,respectively.In this study,the recombinant tetramer precursor of OLAⅠ-β₂m light chain was constructed into pET-28a(+) expression line successfully,and the secondary structure of the OLAⅠ-β₂m protein was predicted and analyzed by SOMPA software,which would lay a solid foundation for constructing the OLAⅠtetramer of Small-tailed Han sheep.

Key words: β₂-microglobulin; Small-tailed Han sheep; major histocompatibility complex; inclusion body