| CXCR1is one of the members in chemokine receptor family, it is aglycoprotein which has only one peptide chain.Having the IL-8as its mainligand,CXCR1can combination with IL-8specialy.After it binding withligands,CXCR1mediate chemotaxis via signal transduction which active thechemotactic function of the corresponding ligand. CXCR1involves in thecell activation, proliferation, migration, angiopoiesis and releasingangiogenic precurosors. Reserch has found that CXCR1is connected withthe pathogenesis of a variety of diseases related to inflammation,tumour andimmuneoreaction. We construct the recombinant adenovirus of fusion geneencoding CXCR1,which can make the CXCR1expression in human cell.Objective： To construct the replication-deficient recombinantadenovirus-CXCR1by AdEasy system.Method:1.We obtained the gene encoding of CXCR1from GeneBankand amplified by PCR.2.The CXCR1gene was coloned to the shuttleplasmid pAdTrace. We excised the plasmid pAdTrace-CXCR1with KpnIand HindIII to cofirm the plasmid was correct constructed.3.We digested therecombinant adenovirus plasmids pAdTrace-CXCR1with PemI. Thelinearized shuttle plasmids and adenoviral backbone vector wereco-transformed to BJ5183cell. The adenoviral plasmids carried human CXCR1gene and they were confirmed by PCR.4.Linearized thepAd-CXCR1and transfected it to HEK-293cell with Lipofectamine2000.Collect the adenovirus plasmids and amplified by HEK-293.Result: The pAdTrace-CXCR1and pAdCXCR1were successfullyconstructed as described in the materials and methods. Restrictive enzymedigestion assay and the PCR confirmed the correct construction of theseplasmids. After transfected to HEK-293cell we got high titer of recombinantadenovirus plasmid.Conclusion: We have successfully constructed the recombinantadenovirus plasmid pAd-CXCR1.We can make further study of thebiological function of CXCR1protein and its mechanism in clinical disease. |
