Expression And Purification Of Human β-defensin-2(hBD2) In Lung Adenocarcinoma Line A549 And Its Antimicrobial Activity Test

Background: With more and more antibiotic-resistant pathogenic bacteria,it is important to find methods to treat infection diseases.Defensins are broad-spectrum antimicrobial peptides with three or four intramolecular cysteine disulfide bonds and a large p-sheet structure. They are found in mammals, insects as well as plants and are thought to act as the first barrier against microorganism invasion.Based on the location and connectivity of their cysteine residues, defensins are divided into two categories: a- and p-defensins. p-defensins were reported to link the innate and adaptive immunity by ruining membrane of germs attracting immature dendritic cells and memory T cells and reinforcing monocyt in immune response. Discovered in 1997 in human skin, human p-defensin -2 (hBD2) is the second member of the human-defensin family. It is a cationic peptide with 41 amino acid residues. The exploration of its pharmaceutical potential and medical importance may be accelerated by producing it via recombinant techniques; however,only few efforts have been made to produce this antimicrobial peptide in Escherichia coli expression system.Although Escherichia coli yeast insect/baculoviruses and mammalian cell expression systems are routine expression systems of the genetic engineering, it is not suitable to produce the protein like hBD2 which was toxic to host cell.There are experiments which show that tumor cells can express foreign proteins.In this study we will try to construct fusion gene of CEA signal peptide fragment and hBD2 peptide ,then integrate it into human lung cancer cell line A549 and let the cell synthesize and secrete hBD2 itself.Objectives: The purposes of present study are:(1)to construct fusion gene CEA signal peptide-EK-hBD2.(2)to transfect the fusion gene into A549 cell. (3)to investigate the goal protein expression and isolate,purify goal the protein hBD2.(4)to test the antimicrobialactivity of the protein in vitro.Methods:The fusion gene CEA signal peptide-EK-hBD2 was constructed by routine method of PCR and recomninant DNA.The fusion gene was integrated into MCS of the retroviral expression vector pLNCX2.Because the hBD2 itself is a secretory protein, It is necessary to delete gene fragment of signal peptide of hBD2 in fusion.According to SignalP soft online internet,the signal peptide fragment is formed by 23 amino acids.So the upstream 69 base pairs of the sequences coding hBD2 in fusion gene in the expression vector must be deleted.The routine method of PCR and recomninant DNA were used again to delete hBD2 signal peptid.and at last retroviral expression vector pLNCX2-CEA signal peptide-EK-hBD2 mature peptide was constructed. Calcium phosphate method was used to transfect retroviral expression vector into PT67 cells, the cells were selected by the G418.After test of titer of virus with Nffl 3T3 cell, transfecting virus were used to infect the host cell A549.The fusion gene was tested by RT-PCR,and the successfully transfected cells were cultured for further use.The expression of hBD2 was tested by ELISA and western blot analysis,and the hBD2 was isolated and purified.Bacterial plate growing inhibition assay was used to test the antibacterial activity of hBD2 and antimicrobial spectrum test was also done.Results:A stable hBD2-expressing cell line was got from lung adenocarcinoma cell A549 that transfected with the fusion gene of CEA signal peptide and hBD2 peptide.The goal protein expression was verified by ELISA and Western blot analysis.After the procedures of isolution and purification,the last concentration of hBD2 is 580 g/ml,with 75% purity of total protein.The antibacterial activity of hBD2 was tested with bacterial plate-growing inhibition assay with the MIV from 36.3 mg/L to 72.5 mg/L for bacteria such as Staphylococcus aureue,E.coli,Bacillus pyocyaneue and Candida albicans.Conclusions: Human cancer cells could be used to express hBD2.The hBD2 expressed by human adenocarcinoma cell line A549 is biological functional based on the results of bacterial plate-growing assay for hBD2 and...