CAJ | 학술논문

目的：构建白介素－37（interleukin-37，IL-37）与增强型绿色荧光蛋白（enhanced green fluorescent protein， EGFP）融合基因真核表达载体，并检测其在293T细胞中的表达。方法：通过PCR分别扩增出IL-37和EGFP基因，经酶切和测序鉴定正确后，克隆入真核表达载体 pcDNA3．1，用 Linker（G4S）3连接2个目的基因片段，构建 IL-37-GFP 融合基因真核表达质粒 pcDNA3．1／IL-37-EGFP，将其转染到293T 细胞中，观察荧光产生情况，用实时荧光定量RT-PCR和Western blot检测IL-37的表达情况。结果：经酶切和测序鉴定，由linker连接的IL-37-GFP 融合基因正确克隆入表达载体pcDNA3．1，重组表达质粒 pcDNA3．1／IL-37-EGFP转染293T 细胞后，用荧光显微镜检测显示荧光蛋白在细胞中表达，RT-PCR 与 Western blot 均检测到高水平IL-37 mRNA 和IL-37蛋白的表达。结论：成功构建了IL-37-融合报告基因 EGFP 的真核表达载体，并在293T细胞中得到了有效表达，为进一步研究IL-37的功能和表达调控奠定了实验基础。
목적：구건백개소－37（interleukin-37，IL-37）여증강형록색형광단백（enhanced green fluorescent protein， EGFP）융합기인진핵표체재체，병검측기재293T세포중적표체。방법：통과PCR분별확증출IL-37화EGFP기인，경매절화측서감정정학후，극륭입진핵표체재체 pcDNA3．1，용 Linker（G4S）3련접2개목적기인편단，구건 IL-37-GFP 융합기인진핵표체질립 pcDNA3．1／IL-37-EGFP，장기전염도293T 세포중，관찰형광산생정황，용실시형광정량RT-PCR화Western blot검측IL-37적표체정황。결과：경매절화측서감정，유linker련접적IL-37-GFP 융합기인정학극륭입표체재체pcDNA3．1，중조표체질립 pcDNA3．1／IL-37-EGFP전염293T 세포후，용형광현미경검측현시형광단백재세포중표체，RT-PCR 여 Western blot 균검측도고수평IL-37 mRNA 화IL-37단백적표체。결론：성공구건료IL-37-융합보고기인 EGFP 적진핵표체재체，병재293T세포중득도료유효표체，위진일보연구IL-37적공능화표체조공전정료실험기출。
Objective To construct the eukaryotic expression vector for IL-37 fused with the enhanced green fluorescent protein (EGFP) and express the fusion protein IL-37-EGFP in 293T cells. Methods The IL-37 and EGFP gene were amplified by PCR, respectively. Then the gene fragments of IL-37 and EGFP were in-serted into the pcDNA3.1 vector to construct the recombinant eukaryotic expression vector pcDNA3.1/IL-37-EGFP. The pcDNA3.1/IL-37-EGFP recombinant expression vector was identified by enzymatic digestion , DNA sequencing, PCR identification. Then the recombinant plasmid pcDNA3.1/IL-37-EGFP was used to transfect 293T cells , the expression of EGFP in 293T cells was detected by fluorescence microscope , the expression of IL-37 in 293T cells was detected by RT-PCR and Western blot. Results The results of enzymatic digestion and DNA sequencing showed that the fusion gene of IL-37 and EGFP linked with (G4S)3 was cloned correctly into pcDNA3.1 vector. The green fluorescence was observed in 293T cells transfected with pcDNA3.1/IL-37-EGFP by fluorescence microscope. RT-PCR and Western blot showed that the expression with high level of IL-37 mRNA and IL-37 protein in 293T cells transfected with pcDNA3.1/IL-37-EGFP , respectively. Conclusion The recom-binant eukaryotic expression vector for IL-37 fused with EGFP, pcDNA3.1/IL-37-EGFP, was constructed suc-cessfully and expressed effectively in 293T cells.