全人源scFv-Fc真核表达载体的构建与鉴定  

Construction and identification of recombinant eukaryotic vector for expression of human scFv-Fc

作　　者：叶迎春[1] 年四季[1] 刘佳佳[1] 王栩[1] 高燕[1] 袁青[1] 

机构地区：[1]泸州医学院基础医学院,泸州646000

出　　处：《中国免疫学杂志》2014年第2期226-229,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金(31000415);四川省青年基金(2012JQ0018);泸州市科技局项目(13ZB0262)

摘　　要：目的:构建全人源抗IL-33 scFv-Fc重组载体,转染中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO),表达并鉴定融合抗体蛋白。方法:RT-PCR扩增人IgG1 Fc段并插入真核表达载体pcDNA3.1中,构建pcDNA3.1/Fc重组载体;将合成的信号肽(SP)序列插入重组质粒pcDNA3.1/Fc,构建重组pcDNA3.1/SP-Fc通用载体;最后将本实验室前期筛选的抗IL-33 scFv插入重组质粒pcDNA3.1/SP-Fc中,构建重组pcDNA3.1/SP-scFv-Fc载体,测序正确后转染CHO细胞。RT-PCR、Western blot检测目的基因的转录及表达。结果:重组质粒测序结果表明成功地构建了pcDNA3.1/SP-scFv-Fc重组真核表达载体,插入的SP-scFv-Fc目的基因大小为1 560 bp左右。转染CHO细胞后,RT-PCR结果显示目的基因在CHO细胞中成功转录,Western blot显示表达的蛋白可以和羊抗人IgG1 Fc抗血清发生特异性免疫反应。结论:成功构建了pcDNA3.1/SP-scFv-Fc重组真核表达载体,以便于后续在CHO细胞中表达。Objective：To construct the anti-human IL-33 scFv-Fc recombinant vector and transfection of Chinese hamster ovary cells （CHO）,then express and identify the fusion protein.Methods：The IgG1 Fc was amplified and inserted into eukaryotic expression plasmid pcDNA3.1 to construct recombinant vector pcDNA3.1/Fc.The signal peptide （SP） was synthesized and inserted into plasmid pcDNA3.1/Fc to construct recombinant vector pcDNA3.l/SP-Fc.Finally,the human anti-IL33 scFv which was selected in our previous work was inserted into plasmid pcDNA3.1/SP-Fc to construct recombinant vector pcDNA3.1/SP-scFv-Fc.After sequencing,the CHO cell was transfected by the correct recombinant plasmid pcDNA3.1/SP-scFv-Fc and the level of transcription and translation of target SP-scFv-Fc was identified by RT-PCR and Western blot.Results：The sequencing results showed that the recombinant vector pcDNA3.1/SP-scFv-Fc was successfully constructed and the size of inserted SP-scFv-Fc was about 1 560 bp.The RT-PCR results showed that the target SP-scFv-Fc was successfully transfected into CHO cells and the Western blot results indicated that the expressed protein had the specific binding reactions with goat anti human IgG1 Fc antibody.Conclusion：the SP-scFv-Fc eukaryotic expression vector was successfully constructed and could be used for the protein expression in CHO cells.

关 键 词：scFv-Fc CHO细胞 真核表达 IL-33 

分 类 号：R392.11[医药卫生—免疫学]