Expression Of Recombinant Human Beta-Defensin-2 In Mouse Fibroblast Cells And Its Antiseptic Activity

OBJECTIVE: To obtain and construct eukaryotic expression vector of hBD-2. To examine the expression of hBD-2 in L929 cells and observe the antimicrobial activity of recombinant hBD-2. It will provide a foundation for further study on the transfection and expression of hBD-2.METHODS:According to the sequence of the hBD-2 cDNA in Genebank ,the primers was designed. The total RNA was isolated from human cervical carcinoma tissue and hBD-2 cDNA fragments were obtained by RT-PCR amplification with specific primers.The gene was ligated with pGEM-T Easy vector ,transformed into the E.coli DH5α,and throughαcomplementary ,the positive recombinant was identified and sequenced.The positive recombinant and pCAGG vector were digested by EcoR I , ligated at 16℃, transformed into the E.coli DH5α,and then sequenced after the recombinant was identified. The expression of hBD-2 in L929 cells was examined by RT-PCR and Western-blot,the antimicrobial activity of recombinant hBD-2 was assessed by K-B disc agar diffusion test.Results:1. A fragment about 300bp appeared after RT-PCR.Sequencing analysis indicated that the hBD-2 cDNA obtained by RT-PCR was identical to the sequence reported in Genebank.2. DNA sequencing analysis demonstrated that the hBD-2 was inserted into the eukaryotic expression vector pCAGG, and the ligation was correct and could run gene transfection.3. The total RNA was extracted from L929 cells transfected by pCAGG-hBD-2,then the fragment about 300bp was obtained after RT-PCR .The protein band about 3.4 KD appeared on SDS-PAGE gel, Western-blot showed a significant hBD-2 expression in the cell lysates of the pCAGG-hBD-2-transfected L929 cells. It was proved that we successfully cloned the cDNA of hBD-2 and Recombinant expression vector of hBD-2 could be exactly expressed in L929 cells.4. Recombinant hBD-2 in supernate showed antiseptic activity by K-B disc agar diffusion test.Conclusion:The hBD-2 gene is constructed and expressed in E.coli DH5αsuccessfully. Recombinant expression vector of hBD-2 could be effectively expressed in L929 cells, and the recombinant hBD-2 in supernate showed obvious antiseptic effects toward some standard bacteria lines.