人pEGFP-C1-IL-8真核表达载体的构建与表达  

Construction and expression of human pEGFP-C1-IL-8 eukaryotic expression vectors

作　　者：黄素辉[1] 马晓霞 屈野[1] 郭小芹[1] 王越[1] HUANG Su-hui;MA Xiao-xia;QU Ye;GUO Xiao-qin;WANG Yue(Department of Pathogenic Biology,Logistics University of PAP,Tianjin 300309,China)

机构地区：[1]武警后勤学院卫生勤务学系病原生物教研室,天津300309 [2]天津医科大学基础医学院免疫学系,天津300070

出　　处：《武警后勤学院学报（医学版）》2017年第12期1039-1043,共5页Journal of Logistics University of PAP（Medical Sciences）

基　　金：国家自然科学基金项目(81502256)

摘　　要：【目的】构建人pEGFP-C1-IL-8真核表达载体,为研究白介素8(interleukin-8,IL-8)在卵巢癌及其他肿瘤中的作用奠定基础。【方法】PCR法扩增IL-8全基因序列后,将其克隆入含增强型绿色荧光蛋白(enhenced green fluorescent protein,EGFP)表达载体pEGFP-C1,通过菌落PCR鉴定、双酶切鉴定及测序鉴定后,将构建好的表达载体与其空载体分别转染至人卵巢癌细胞系A2780中,荧光显微镜下观察转染效率,用RT-PCR和ELISA法检测细胞转染后细胞IL-8的表达水平。【结果】经过菌落PCR鉴定、双酶切鉴定及测序鉴定,重组人IL-8真核表达载体构建正确。pEGFP-C1-IL-8和pEGFP-C1分别转染入A2780细胞后,转染成功的细胞能够表达GFP,转染效率约为75%~80%。转染空载体的A2780细胞未检测到IL-8 m RNA表达,而转染重建载体的A2780细胞表达水平较高,细胞上清液IL-8蛋白表达水平约为前者的2.91倍(P<0.05)。【结论】成功构建了真核表达载体pEGFP-C1-IL-8,重组质粒能使人卵巢癌细胞系A2780过表达IL-8。【Objective】To construct eukaryotic recombinant vector expressing green fluorescent protein（GFP） and interleukin-8（IL-8）for studying the role of IL-8 in the ovarian cancer and other tumors.【Methods】IL-8 full length gene were amplified by reverse transcription polymerase chain reaction（RT-PCR） and cloned into an eukaryotic expression vector pEGFP-C1 whose expression product can be observed in a fluorescence microscope. After identified by bacterial colony PCR, restriction endonuclease digestion and the nucleotide sequencing of the target gene,the recombinant plasmid pEGFP-C1-IL-8 was transfected into human ovarian cancer cell line A2 780 transiently via lipofectamine method. The m RNA and protein levels of IL-8 in the transfected cells were detected by RT-PCR and enzyme-linked immuno-sorbent assay ELISA.【Results】The recombinant eukaryotic vectors named by pEGFP-C1-IL-8, were constructed and identified. After being successfully transfected A2780 express GFP which can be observed in a fluorescence microscope, the IL-8 m RNA apparently expressing in pEGFP-C1-IL-8/A2 780 but not pEGFP-C1/A2780,and the protein levels of IL-8 were increased by 2.91 times（P〈0.05）.【Conclusion】The eukaryotic expression vectors pEGFP-C1-IL-8 has been constructed successfully,and the recombinant plasmid can promote the expression of IL-8 in the human ovarian cancer line A2780.

关 键 词：白介素8 绿色荧光蛋白 真核表达载体 卵巢癌细胞系 

分 类 号：R392[医药卫生—免疫学]