人γ干扰素转座子质粒构建及其在HEK293T细胞中的表达

Construction of transposon expression vector of human interferon γ and its expression in HEK293T cells

[目的]克隆人γ干扰素(hIFNγ)基因,借助自剪切肽P2A,构建hIFNγ-P2A-RFP(红色荧光蛋白)融合基因转座子表达载体,并在HEK293T细胞中检测其表达水平。[方法]首先,以脂多糖(LPS)激活人外周血淋巴细胞,提取总RNA,经RT-PCR扩增hIFNγ基因;再通过重叠延伸PCR,获得hIFNγ-P2A-RFP融合基因,并将其插入PB002G转座子载体。经双酶切和DNA测序鉴定,将重组质粒转染HEK 293T细胞,荧光显微镜观察RFP表达,Western Blotting检测hIFNγ蛋白表达。[结果]克隆了hIFNγ基因,成功构建了PB-hIFNγ-P2A-RFP重组质粒;荧光显微镜观察发现RFP在HEK 293T细胞表达,转染效率达39.3%;Western Blotting检测显示,特异性蛋白条带约为21 kDa,与预期的hIFNγ蛋白大小一致。[结论]成功构建了人γ干扰素PB转座子真核表达质粒,该质粒在HEK 293T细胞中高效表达。

[Objective] To clone human interferon-γ,and construct a transposon expression vector of hIFNγ-P2A-RFP fusion gene with a self-cutting peptide P2A,and further to validate expression of the fusion gene in HEK293T cells.[Method] Firstly, human peripheral blood lymphocytes were cultured and activated with addition of lipopolysaccharide in culture medium, and then total RNA was extracted to amplify hIFNγ by reverse transcription PCR(RT-PCR).Secondly, a fusion gene, or hIFNγ-P2A-RFP was generated by overlap extension PCR,then the fusion gene was subcloned to a piggyBac(PB) transposon vector(PB002G).Identified by double digestion and DNA sequencing, the recombinant plasmid and a PB transposase vector were cotransfected to HEK 293T cells.Finally, RFP expression was observed under a fluorescence microscopy, and hIFNγ protein expression was detected with Western Blotting assay.[Result] The hIFNγ gene was amplified, and the recombinant plasmid PB-hIFNγ-P2A-RFP was successfully constructed;RFP positive cells were seen under an inverted fluorescence microscopy after 48 hours of transfection, and transfection efficiency was 39.3%;the expression of hIFNγ in HEK 293T cells was further verified by Western Blotting assay, resulting from the fact that a specific band was detected, whose size is about 21 kDa, which is the same size as we expected.[Conclusion] PB transposon expression plasmid of hIFN was successfully constructed, and it was efficiently expressed in HEK 293T cells.