关键词: 抗菌肽, 聚合酶链式反应, 真核重组质粒, 细胞原代培养, 阴道上皮细胞

Abstract: Objective：The eukaryotic expression vectors of human defensin 5 (HD5) and LL37 were built and transfected primary human vaginal epithelial cells. To observe the expression of HD5 and LL37 after transfection. Method: ① Total RNA was extracted from human vaginal epithelial cells using 1 ml of Trizol Reagent (Invitrogen Life Technologies, USA). The cDNA encoding the HD5 and LL37 were amplified through RT-PCR. Then inserted into pcDNA3.1(+)-EGFP , a expression vector. ② The vaginal epithelial cells from female vagina were culture primarily by Serum-Free Keratinocyte Medium and the tissue pieces culture method. These two kinds of recombinant eukaryotic plasmids were separately transfected or co-transferred into vaginal epithelial cells. At 6, 12, 24 and 48h the transfection efficacy was observed by fluorescence microscope，and the supernatant of every group was collected for determine the expression of HD5 and LL37 by ELSIA method. Result: This study constructed pcDNA3.1 (+) / HD5-EGFP and pcDNA3.1 (+) / LL37-EGFP eukaryotic expression vector, to achieve the expression of HD5 and LL37 in the vaginal epithelial cells. The highest expression of HD5 and LL37 protein in the cell culture supernatant was at transfected 24h by using ELISA method. Conclusion: We successfully constructed with HD5 and LL37 cDNA eukaryotic expression plasmid, and successfully transfected into human vaginal epithelial cells. On this basis, we can further study the antibacterial function of recombinant HD5 and LL37and vaginal epithelial cell innate immune system.

Key words: Antimicrobial peptide, Polymerase chain reaction, Recombinant eukaryotic plasmid, Primary cell culture, Vaginal epithelial cell