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目的 将IL 10基因定向克隆入pEGFP N2质粒 ,探讨采用构建的质粒转染支气管上皮细胞H2 92株 ,稳定表达IL 10和荧光蛋白可行性。方法 采用定向克隆技术 ,将IL 10基因克隆入pEGFP N2质粒 ,用构建成功的IL 10 pEGFP N2质粒在Fuge 6介导下转染培养的支气管上皮细胞株 (H2 92 ) ,利用免疫组化、RT PCR检测细胞中IL 10蛋白和mRNA的表达 ,并在荧光显微镜下观察荧光蛋白表达。结果 ①重组后的pEGFP N2质粒已成功载入IL 10基因。②IL 10 pEGFP N2质粒转染支气管上皮细胞H2 92株后 ,可在H2 92株内表达IL 10mRNA、IL 10蛋白和荧光蛋白。结论 重组的IL 10 pEGFP N2质粒可在上皮细胞内表达IL 10和荧光蛋白。由于两者共用一个启动子 ,荧光蛋白表达可报告IL 10表达 ,适用于动物实验气道内的IL 10转基因研究。
OBJECTIVE: To clone IL 10 gene into pEGFP N2 plasmid and investigate the feasibility of transfecting bronchial epithelial cell line H2 92 by using the constructed plasmid to stably express IL 10 and fluorescent protein. Methods IL 10 gene was cloned into pEGFP N2 plasmid by directional cloning technique. The transfected bronchial epithelial cell line (H2 92) was transfected with IL 10 pEGFP N2 plasmid mediated by Fuge 6 by immunohistochemistry, RT PCR was used to detect the expression of IL-10 protein and mRNA in the cells, and the fluorescent protein expression was observed under a fluorescence microscope. Results ① The recombined pEGFP N2 plasmid successfully loaded IL 10 gene. ② IL 10 pEGFP N2 plasmid transfected into bronchial epithelial cells H2 92, the expression of IL 10 mRNA, IL 10 protein and fluorescent protein in H2 92 strains. Conclusion The recombinant IL 10 pEGFP N2 plasmid can express IL 10 and fluorescent protein in epithelial cells. Because both share a single promoter, fluorescent protein expression reports IL-10 expression and is suitable for IL 10 transgene studies in animal airways.