Preliminary Study On Recombinant Of J-HNP-1 And Anti-bacteria Effect In Vitro

The opportunist pathogens often come from the mucosal membrane surface of respiratory tract, intestinal lumen or reproductive tract, most of which are resistance to the antibiotics and difficult to prevent. The opportunist infection usually can be found in the patients suffering severe trauma, burn or AIDS. One of the common features of mucosal epithelial cell is that there are poly-IgA receptors (pIgR)on the cell membrane. The IgA antibody which has linked with J chain could be transported into the cells by the connecting of J chain and pIgR. In the study, we want to rebuild defensin into a germicidal molecule in which one end is J chain and another end is defesin so it is called as "pIgR ligand-like germicidal peptide". The peptide can connect with pIgR by J chain, so that by using pIgR as a "bridge" the defensin can be transported into the epithelial cell of mucus m embrane to kill the microorganisms intra-cell. The cDNA gene of HNP-1 belongs to the α -defensin is recombined with J chain cDNA , then the recombinant is inserted into the mammalian express system to express. Then we investigate the expressed product's anti-bacteria effect in vitro in order to provide the evidence for the further study on the anti-bacteria effect inside the epithelial cell of mucus membrane.Material and methodThe 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 express vector pcDNA3. l(-)/Myc-HisC. The recombinant vector was transfected to the COS-7 cell by lipid transfection method. The J-HNP-1 expression was investigated at the level of mRNA and protein by RT-PCR and Western-blot respectively. The anti-bacteria effects of cellular solution protein and cell culture supernatant were examined in vitro.Result1. The J-HNP-1 recombinant was obtained by connection of J chain and HNP-1cDNAbyPCR.2. The J-HNP-1 recombinant was inserted into the mammalian express vector pcDNA3.1(-)/Myc-HisC successfully, double label gene Myc and 6XHis were in the downstream. 6XHis was label marker for the J-HNP-1 expression product was easy to be examined.3. The recombinant plasmid rpcDNA3.1(-)/Myc-HisC/ J-HNP-1 was sequenced with the forward and backward probes. The J-HNP-1 sequence and inserting direction were examined, the recombinant with the correct inserting direction was obtained.4. From the RNA extract of COS-7 cell which had been transfected with rpcDNA3.1(-)/Myc-HisC/ J-HNP-1, 786bp fragment was amplified which met with the predicted result by RT-PCR. In the cellular solution protein and cell culture supernatant, 24KD peptide was found positive by western-blot with antibody to 6 X His.5. The cellular solution protein and cell culture supernatant of COS-7 cell which had been transfected with rpcDNA3.1(-)/Myc-HisC/ J-HNP-1, were found to have the effect of anti-bacteria in vitro.ConclusionThe -HNP-1 recombinant is obtained and inserted into the mammalian express vector then to be expressed in vitro. The expression product is found to have the effect of anti-bacteria in vitro.