关键词: 环氧合酶, 反义技术, 重组载体, 增殖

Abstract: BACKGROUND AND AIM: To construct the antisense expression vector of human COX-2 gene and explore the relationship between COX-2 gene and tumor proliferation. MATERIALS AND METHODS: COX-2 gene fragment cleaved from plasmid pBOSNeo COX-2 with EcoRⅠ, restriction enzyme cutting sites EcoRⅠand Bgl II was added to its two ends in two reverse directions artificially. The target fragment was inserted into the polyclone site of plasmid eukaryotic expression vector pIRES2-EGFP. The constructed recombinant was identified by agarose gel electrophoresis. The transfection of antisense COX-2 recombinant was made to gastric cancer cell line named SGC-7901 which with high expression of COX-2 gene mediated by liposome. The transfectants were screened by G418 and idendified by detection of exogenous NEO resistant gene with PCR technique. PCR and Western Blot were used to determine if the expression of COX-2 was inhibited by antisense gene in the transfectant. MTT essay and colony formation test were used to observe the effect to the proliferation of the transfectant. RESULTS: Agarose gel electrophoresis confirmed that the sense and antisense target fragment were successfully bound to pIRES2-EGFP. The test of exogenous gene showed we obtained stable transfectants. The expression of COX-2 was downregulated in transfectant with antisence gene. MTT essay and clone formation test showed the proliferation of transfectant was inhibited. CONCLUSION: The antisense eukaryotic expression vector of COX-2 was constructed successfully by means of reversely inserting the target fragment into pIRES2-EGFP. COX-2 gene could control proliferation of SGC-7901.

Key words: cyclooxygenase, antisense technology, recombinant vector, proliferation