Construction of a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene and its transfection into vaginal epithelial cells / 中国组织工程研究

ABSTRACT

BACKGROUND:LL37, the only antimicrobial peptide of the cathelicidin family identified from the human, not only promotes the proliferation of endothelial cel s, but also plays an important role in angiogenesis and re-epithelialization. OBJECTIVE:To construct a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene, and to detect the expression and secretion of LL37 after transfected into the canine vaginal epithelial cells. METHODS:The cDNA encoding LL37 was amplified by PCR. Recombinant adenovirus expression plasmid encoding LL37 and green fluorescent protein (EGFP) was constructed, and identified using restriction endonuclease technology and DNA sequencing method. Adenovirus particles were generated by cotransfecting the 293-packaging cel line. The adenovirus were col ected, amplified and concentrated, and viral titers were determined by end-point dilution assay by applying serial dilutions of the purified viruses to 293 cel s. Primary cultured canine vaginal epithelial cel s were transfected by the recombinant adenovirus. The transfection efficacy was observed by fluorescence microscope, and the cultured supernatant was col ected to determine the expression of LL37 by ELISA method at 1, 2, 3, 5 and 7 days after transfection. RESULTS AND CONCLUSION:The adenovirus vector GV314-LL37 with the titer of 3×109 pfu/mL was successful y constructed and identified by DNA sequencing methods. Canine vaginal epithelial cel s were successful y isolated and cultured and grew stably. After transfection, vaginal epithelial cel s could express the EGFP and LL37 efficiently in a time-dependent manner detected by fluorescence microscope and ELISA method. The transfection efficacy of EGFP reached to 89%at 72 hours. The level of LL37 in the cel culture supernatant in the transfection group was significantly higher than that in the control group, the highest expression of LL37 was found at 3 days that lasting for 7 days. In conclusion, the recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene is successful y constructed, which can express and secrete LL37 after transfected into canine vaginal epithelial cel s, providing a foundation for constructing the tissue-engineered vagina possessing anti-infection and neovascularization.