Abstract: Objective: To study the construction of pcDNA3.1-B7.1-IRES-B7.2 and its stable expression in mouse HCC cell line H22. Methods: The murine full-length cDNA of B7.1 and B7.2 genes were obtained and subcloned into pcDNA3.1-IRES. The recombinant named pcDNA3.1-B7.1IRES -B7.2 was constructed and identified with restriction enzyme digestion. Subsequently, the recombinant was transfected into H22 with Lipofectamine reagent, then Hygromycin-resistence colonies were acquired, and named H22-CD80/CD86+. RT-PCR was applied to determine whether there were stable mRNA expressions of objective genes in variants. The H22-CD80/CD86/CD137L+ cell line was set up by transfecting PCI-neo-CD137L into H22-CD80/CD86 +. FCM was applied to determine whether there were stable mRNA expressions of objective genes in variants. Results: pcDNA3.1-B7.1IRES-B7.2 was digested, then electrophoresis of the digested products showed fragments of 862bp (mB7.1) and 984bp (mB7.2), which indicated the construction was successful. The DNA sequence analysis is identical to the sequence of mB7.1/B7.2 in Genebank and did not reveal any mutation. RTPCR showed dual expressions of mB7.1/B7.2 gene with strong intensity in H22-CD80/CD86+. FCM showed co-expression of mB7.1/B7.2/4-1BBL gene with strong intensity in H22-CD80/CD86/CD137L+. Conclusions: pcDNA3.1-B7.1- IRES-B7.2 has been constructed successfully, and stable and effective expressions of B7.1, B7.2 and 4-1BBL genes also have been obtained in H22 variant.