Abstract: To reconstruct a hybrid molecule composed of chicken major histocompatibility complex heavy chain（α chain） extracelular domains gene and β2microglobulin (β2m) mature protein gene in vitro, chicken MHC Ⅰ α chain extracellular domains gene and β2m mature protein gene were amplified by RT-PCR respectively. There are 15 neucleartides overlap between reverse primer of the first pair of primers for α chain extracellular domains gene and upper primer of the second pair of primers for β2m mature protein gene. Then, using MHC α Ⅰ chain extracellular gene PCR product and β2m mature peptide gene PCR product mixture as a template, we reconstructed soluble expression plasmid pMAL-p2X which contained major histocompatibility complex Ⅰ by linking MHCⅠ α chain extracellular domain gene and β2m mature protein gene through a 45 nucleotides linker. Electrophoresis analysis showed that the specific single sequence of the two target products could be amplified by RT-PCR, and the product of interest was accord with expectation. Sequencing result proved that expression reading frame of recombinant plasmid was composed of the expected MHC Ⅰ α chain extracellular domains sequence of interest and β2m mature protein gene target sequence, which linked by a limp linker, and there was no base malposition. In conclusion, splicing overlap extension by PCR is a convenient method to obtain recombinant chicken MHC Ⅰ in vitro.