CAJ | 학술논문

目的构建展示角质细胞生长因子(KGF)噬菌体活性肽,检测其促表皮细胞增殖的作用.方法选择4个KGF序列,设计引物；用反转录-PCR法获得3个KGF序列(P1、P2和P4),直接合成1个KGF序列(P3)；将KGF序列亚克隆至噬菌粒pComb3中；用噬菌体展示技术,将KGF基因片段展示于噬菌体表面；用四甲基偶氮唑盐(MTT)法检测KGF噬菌体活性肽促表皮细胞增殖的作用,测定其在570 nm的吸光度(A)值,用免疫荧光法检测其细胞亲和力.结果获得4种KGF基因,构建在噬菌粒pComb3中；通过噬菌体展示技术将其表达于噬菌体的表面.MTT检测的吸光度(A)值结果显示阴性对照组(0.293±0.017)与KGF对照组(0.520±0.043)及4种KGF噬菌体活性肽组(P1 ～ 4) (0.469±0.057、0.441±0.048、0.438±0.035、0.446±0.037)间差异均有统计学意义(均P＜0.01),免疫荧光检测结果显示KGF和4种KGF噬菌体活性肽与表皮细胞具有较好的亲和力.结论构建展示的KGF噬菌体活性肽能够显著促进表皮细胞增殖.
목적 구건전시각질세포생장인자(KGF)서균체활성태,검측기촉표피세포증식적작용.방법 선택4개KGF서렬,설계인물；용반전록-PCR법획득3개KGF서렬(P1、P2화P4),직접합성1개KGF서렬(P3)；장KGF서렬아극륭지서균립pComb3중；용서균체전시기술,장KGF기인편단전시우서균체표면；용사갑기우담서염(MTT)법검측KGF서균체활성태촉표피세포증식적작용,측정기재570 nm적흡광도(A)치,용면역형광법검측기세포친화력.결과 획득4충KGF기인,구건재서균립pComb3중；통과서균체전시기술장기표체우서균체적표면.MTT검측적흡광도(A)치결과현시음성대조조(0.293±0.017)여KGF대조조(0.520±0.043)급4충KGF서균체활성태조(P1 ～ 4) (0.469±0.057、0.441±0.048、0.438±0.035、0.446±0.037)간차이균유통계학의의(균P＜0.01),면역형광검측결과현시KGF화4충KGF서균체활성태여표피세포구유교호적친화력.결론 구건전시적KGF서균체활성태능구현저촉진표피세포증식.
Objective To construct and display the keratinocyte growth factor (KGF) phage active peptides so as to detect the promoting effects of epidermal cell.Methods KGF sequences were chosen and their primers were designed.The selected genes of P1,P2 and P4 were obtained by reverse transcription (RT)-PCR.P3 was obtained by direct synthesis.And the KGF genes were subcloned into pComb3 vector.The technique of phage display was employed to display the genes on phage surface.Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the promoting effects of KGF phage active peptides on the proliferation of epidermal cell.Optical density (A) was determined at 570 nm.Immunofluorescent assay was employed to evaluate the cell affinity of KGF phage active peptides.Results The four KGF genes were obtained and subcloned into pComb3 vector.The proteins of the KGF genes were expressed on the surface of the pComb3 vector.The MTT data of optical density (A) showed that significant differences existed between the negative control and KGF control (0.293 ± 0.017 vs 0.520 ± 0.043) and KGF phage active peptide groups (0.293 ±0.017 vs0.469 ±0.057,0.441 ±0.048,0.438 ±0.035,0.446 ±0.037) (all P＜0.01).The results of immunofluorescent assay indicated that KGF and KGF phage active peptides had excellent cell affinity.Conclusion KGF phage active peptides are successfully constructed and displayed and they may promote the proliferation of epidermal cell.