Abstract: To screen for the interaction of target proteins with the virB4 gene in bovine embryo trophoblast cells that were infected with Brucella bacteria. A cell culture for bovine trophoblasts was established. The cDNA library of trophoblast cells infected with the vaccine strain RB51 was constructed and a Saccharomyces cerevisiae expression vector containing the gene virB4 was synthesized. Target proteins which interacted with virB4 were screened through a yeast two-hybrid system. The results showed that the cDNA library of bovine trophoblast cells infected with the vaccine RB51 strain was successfully constructed. A recombinant plasmid pGBKT7-virB4 was cloned successfully into the expression vector Y187. Thirteen different types of proteins that interacted with virB4 were screened through a yeast two-hybrid assay.