中国免疫学杂志2017，Vol.33Issue(4)：498-501,506,5.DOI:10.3969/j.issn.1000-484X.2017.04.004
I-Ad/IgG2b Fc二聚体融合蛋白的构建表达及鉴定
Construction,expression of I-Ad/IgG2b Fc dimer fusion protein and its identification
摘要
Abstract
Objective:To construct I-Ad/IgG2b Fc baculovirus expression vector and express I-Ad/IgG2b Fc dimer fusion protein in Sf9 insect cells.Methods:I-Ad α,I-Ad β and IgG2b Fc gene sequences were amplified from BALB/c mouse lymphocytes by RT-PCR.I-Ad α and I-Ad β were connected with the leucine zipper sequence Fos and Jun respectively by overlapping PCR to form I-Ad α-Fos and I-Ad β-Jun.I-Ad α-Fos and IgG2b Fc fragments were ligated by restriction sites Xba I to form I-Ad α-Fos-IgG2b Fc recombination sequence.I-Ad α-Fos-IgG2b Fc and I-Ad β-Jun fragments were inserted to PPH and PP10,which were the downstream of the promoters in the plasmid pFastBacTMDual,to form pFastBacTMDual+[I-Ad/IgG2b Fc] recombinant plasmids.The constructed vector was identified by PCR,restriction endonuclease and sequencing.The recombinant plasmids pFastBacTMDual+[I-Ad/IgG2b Fc] was transferred into the DH10Bac competent cell to form recombinant baculovirus Bacmid+[I-Ad/IgG2b Fc].The recombinant baculovirus was transfected into Sf9 insect cells by liposome transfection reagent.After infected with Sf9 insect cells,the supernatant was collected and concentrated by PEG20000 to obtain I-Ad/IgG2b Fc dimer fusion protein.The fusion protein was detected by double-antibody sandwich ELISA and Western blot.Results:PCR,restriction enzyme digestion and sequencing confirmed that the recombinant vector pFastBacTMDual+[I-Ad/IgG2b Fc] had the correct sequence.The double antibody sandwich ELISA and Western blot showed that recombinant bacmid could successfully infect Sf9 insect cells,and the expressed fusion protein had the correct conformation.Conclusion:The pFastBacTMDual+[I-Ad/IgG2b Fc] baculovirus expression vector was successfully constructed and expressed in Sf9 insect cells,laying a foundation for the study of I-Ad-restricted T cells.关键词
I-Ad/二聚体/昆虫细胞/杆状病毒表达载体Key words
I-Ad/Dimer/Insect cell/Baculovirus expression vector分类
医药卫生引用本文复制引用
罗智环,钱航,徐成伟,闵新文,陈俊..I-Ad/IgG2b Fc二聚体融合蛋白的构建表达及鉴定[J].中国免疫学杂志,2017,33(4):498-501,506,5.基金项目
本文受国家自然科学基金青年项目(81400288)和湖北省教育厅科学研究计划(B2015495)资助. （81400288）
