Reconstruction of J Chain Gene with HNP-1 Gene and Construction of Its Expression Vector / 中华医院感染学杂志

OBJECTIVE To recombine J chain gene with HNP-1 into a new germicidal(molecule) J-HNP-1,which can connect with pIgR by J chain,so that by using pIgR as a "bridge" the J-HNP-1(germicidal) peptide can be transported into the epithelial cell of mucous membrane to kill the intracellular microorganisms,then the recombinant is inserted into the mammalian expression system.METHODS The J chain and HNP-1 cDNA were amplified from the plasmids respectively by PCR,then the two cDNA fragments were recombined into J-HNP-1 by recombinant PCR.The J-HNP-1 cDNA fragment was inserted into the(mammalian) expression vector pcDNA3.1(-)/Myc-HisC.RESULTS The J-HNP-1 recombinant was obtained by(connection) of J chain and HNP-1 cDNA by PCR.The recombinant J-HNP-1 cDNA was 786bp.CONCLUSIONS The recombinant J-HNP-1 cDNA and the construction of expression vector are the basis for the new bactericidal peptide production.

Expression of Recombinant J-HNP-1 Gene with C Terminal of Double Marks Myc and Poly-histidine in Transfected COS-7 Cells / 中华医院感染学杂志

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.

Construction and expression of eukaryotic expression vector harboring human neutrophil peptide 1 / 解放军医学杂志

Objective To explore the possibility of human neutrophil peptide 1 (HNP1) gene engineering, we construct the eukaryotic expression vector carrying HNP1 gene. Methods With RNA extracted from human neutrophil cell as template, cDNA encoding mature HNP1 was amplified by RT-PCR, and then it was inserted into vector pMD18-T. After restriction endonuclease digestion and DNA sequencing confirmation the gene was subcloned into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO to construct a recombinant expression plasmid pcDNA3.1/V5-His-TOPO/HNP1, then the recombinant plasmid was transfected into COS-7 cells by lipofectamine, and the expressed product was identified by biotin-avidin enzyme-linked immunosorbent assay ( BA-ELISA). Results The sequence of HNP1 completely matched those published in GenBank, thus eukaryotic expression vector pcDNA3.1/V5-His-TOPO/HNP1 was constructed correctly. The ELISA results showed that the eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO/HNP1 could temporarily express HNP1 in COS-7 cells. Conclusion The successful construction and expression of pcDNA3.1/V5-His-TOPO/HNP1 pave the way for the stable expression HNP1 in mammalian engineering cells.