摘要
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（Pl、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（MTF）法检测KGF噬菌体活性肽促表皮细胞增殖的作用，测定其在570nm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。MTr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。
Objective To construct and display the keratinocyte growth factor （KGF） phage active peptides so as to detect the promoting effects of epidermal eel1. 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 subeloned 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 MT＇F 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 vs 0.469±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.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2013年第14期1058-1062,共5页
National Medical Journal of China
基金
国家自然科学基金（30670571、81071560、81201467）
北京市自然科学基金（7122138）
山东省自然科学基金（ZR2011HM027）
关键词
细菌噬菌体
肽库
角质细胞生长因子
表皮细胞
Bacteriophages
Peptide library
Keratinocyte growth factor
Epidermal cell
