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中文摘要：
目的以增强型绿色荧光蛋白（EGFP）为示踪物,建立一种简便、经济、有效的随机多肽文库,为进一步筛选出可上调内源性人SOD1表达多肽的药物奠定基础。方法采用点突变技术,在pET-14b-His-Tat-EGFP载体多克隆位点XhoI之后插入12个随机多肽的36个随机碱基序列。用阳离子脂质体LipofectamineTM2000包裹已构建的重组质粒pDsRed1-1-SOD1p后,转染NIH/3T3细胞,荧光显微镜观察SOD1启动子驱动的红色荧光蛋白的表达。用G418筛选稳定表达SOD1启动子驱动的红色荧光蛋白的细胞系,荧光显微镜检测红色荧光蛋白的表达及定位。结果在示踪载体的基础上构建的随机多肽文库理论上至少包含了107个独立克隆,且几乎所有克隆插入的随机片段都是36个碱基。稳定转染3个月后,重组质粒转染的细胞质及细胞膜上均有携带SOD1启动子的红色荧光蛋白的表达。结论成功建立随机多肽表达文库,获得了稳定表达SOD1启动子驱动的红色荧光蛋白的NIH3T3细胞系。
英文摘要：
Objective To construct a simple,economical and effective random peptide library by constructing a random peptide expression library coding 12 amino acids in pET-14b-His-TAT-EGFP plasmid with the enhanced green fluorescent protein （EGFP） as a tracer.To lay a foundation for further study on searching peptides which up-regulate the expression of endogenous human copper-zinc superoxide dismutase （Cu/Zn-SOD,SOD1）,the NIH 3T3 cell line that can stably express human SOD1 promoter-controlled red fluorescent protein was constructed.Methods Thirty-six random base sequences were inserted after the multiple clone site Xho I of pET-14b-His-TAT-EGFP vector by using site-directed mutagenesis.The constructed plasmid pDsRedl-1-SOD1p entrapped in cationic liposome LipofectamineTM 2000 was transfected into NIH/3T3 cells,and the expression of SOD1 promoter-controlled red fluorescent protein was then observed under fluorescence microscope.The cell lines that can stably express human SOD1 promoter-controlled red fluorescent protein were screened by using G418,and the expression and location of red fluorescent protein were then observed under fluorescence microscope.Results The random peptide library,constructed on the basis of the tracer carrier,contained at least 107 independent clones,nearly all of the clones had been inserted were with 36 base pairs.The expression of red fluorescent protein carrying SOD1 promoter was found 3 months after stable transfection both in cytoplasm and membrane of cells transfected with the constructed plasmid.Conclusion A random peptide expression library has been successfully constructed.The NIH/3T3 cell line has been acquired that can stably express human SOD1 promoter-controlled red fluorescent protein.The library and NIH/3T3 cell line can be used for the study on screening polypeptide drugs which up-regulate the expression of endogenous SOD1.Thereby,the purpose of controlled removal of excessive oxygen free radicals can be achieved.
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