Expression of Recombinant J-HNP-1 Gene with C Terminal of Double Marks Myc and Poly-histidine in Transfected COS-7 Cells

OBJECTIVE To reconstruct the HNP-1 into the J-HNP-1 with a J chain,and explore to set up a mammalian cell expression system which can express and secret J-HNP-1,so that the products could be examined and purified conveniently. METHODS The J-HNP-1 cDNA fragments were produced by recombinant PCR.Then the J-HNP-1 was inserted into the mammalian expression vector pcDNA3.1(-)/Myc-His which had the double marks Myc and 6×His.The recombinant vector rpcDNA3.1(-)/Myc-His /J-HNP-1 was transfected into the COS-7 cell.The J-HNP-1 expression was analyzed at the mRNA and protein level.The germicidal activity of cell culture supernatant and cellular solution protein was assayed. RESULTS By the use of RT-PCR with special primers,a band of 786bp was amplified from COS-7 cells transfected by this recombinant plasmid.Western blot analysis with specific anti-histidines antibody revealed that the cell culture supernatant and cellular solution protein of COS-7 cells transfected by rpcDNA3.1(-)/Myc-His /J-HNP-1 had a strong band with relative molecular mass of about 24×10~3.Antibacterial activity assay showed that obvious bacterial inhibition occurred in both lysate and supernatant of COS-7 cells transfected by rpcDNA3.1(-)/Myc-His /J-HNP-1. CONCLUSIONS The J-HNP-1 recombinant is obtained and inserted into the mammalian expression vector then to be expressed in vitro.The expression product is found to have the anti-bacterial effect in vitro.