I-Ad / IgG2b Fc 二聚体融合蛋白的构建表达及鉴定

目的:构建I-Ad / IgG2b Fc 杆状病毒表达载体,使其在Sf9 昆虫细胞内表达。方法:利用RT-PCR 从BALB/ c小鼠淋巴细胞中扩增出I鄄Ad α、I-Ad 茁和IgG2b Fc 目的基因序列,运用重叠PCR 将I-Adα和I-Ad β分别与Fos 和Jun 的亮氨酸拉链序列相连,形成I-Adα和I-Ad βJun;利用酶切位点Xba玉将I-Ad -Fos 与IgG2b Fc 段相连,形成I-Ad –Fos-IgG2bFc 重组序列;分别将I-Ad 琢-Fos-IgG2b Fc 和I-Ad 茁-Jun 插入杆状病毒表达载体pFastBacTM Dual 的PPH 和PP10 两个启动子的下游,构建出重组载体pFastBacTM Dual+[I-Ad / IgG2b Fc];采用PCR 及限制性内切酶对所构建的载体进行鉴定并测序;将pFastBacTMDual+[I-Ad / IgG2b Fc]转入DH10Bac 感受态,使其在转座子的作用下形成重组杆粒病毒Bacmid+[I-Ad / IgG2b Fc],利用脂质体转染试剂将重组表达载体转染至Sf9 昆虫细胞,P4 后大量感染Sf9 昆虫细胞,收集上清通过PEG20000 浓缩可得到I-Ad / IgG2b Fc 二聚体融合蛋白,通过双抗体夹心ELISA 及Western blot 对表达的蛋白进行检测。结果:PCR 及酶切鉴定以及测序结果证实所构建的pFastBacTMDual+[I-Ad / IgG2b Fc]重组载体具有正确的序列;双抗体夹心ELISA 和Western blot 结果表明重组杆粒能成功感染Sf9 昆虫细胞,并且表达的融合蛋白具有正确构象。结论:成功构建pFastBacTM Dual+[I-Ad / IgG2b Fc]杆状病毒表达载体,并在Sf9 昆虫细胞中表达,为研究I-Ad 限制性的T 细胞奠定了基础。

Construction,expression of I-Ad / IgG2b Fc dimer fusion protein and its identification

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 ,I-Ad and IgG2b Fc gene sequences were amplified from BALB/ c mouse lymphocytes by RT-PCR.I-Ad and I-Ad were connected with the leucine zipper sequence Fos and Jun respectively by overlapping PCR to form I-Ad -Fos and I-Ad -Jun.Ad -Fos and IgG2b Fc fragments were ligated by restriction sites Xba I to form I-Ad –Fos-IgG2b Fc recombination sequence.I-Ad –Fos-gG2b Fc and I-Ad -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 pFastBacTM Dual+[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 re-combinant 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.