CAJ | 학술논문

目的:利用PET101/D-TOPO蛋白表达系统,表达人IL-13和IL-33重组蛋白. 方法:以健康人外周血单个核细胞(PBMC)的mRNA为模板,采用RT-PCR扩增IL-13和IL-33开放阅读框,将扩增所得目的基因连接到质粒pET101/D-TOPO,构建出重组质粒并转化大肠杆菌BL21,诱导其表达IL-13和IL-33重组蛋白,纯化后进行鉴定.结果:RT-PCR扩增所得IL-13 cDNA为490 bp, IL-33 cDNA大小为800 bp,将其分别连接到质粒PET101/D-TOPO,构建出IL-13和IL-33重组质粒,分别转化大肠杆菌BL21,诱导其表达. SDS-PAGE电泳分析,发现目的条带大小分别为29 kDa和35 kDa左右,Western blotting结果证实重组表达正确. 结论:成功构建出IL-13和IL-33重组质粒,成功表达并纯化出IL-13和IL-33重组蛋白.
목적:이용PET101/D-TOPO단백표체계통,표체인IL-13화IL-33중조단백. 방법:이건강인외주혈단개핵세포(PBMC)적mRNA위모판,채용RT-PCR확증IL-13화IL-33개방열독광,장확증소득목적기인련접도질립pET101/D-TOPO,구건출중조질립병전화대장간균BL21,유도기표체IL-13화IL-33중조단백,순화후진행감정.결과:RT-PCR확증소득IL-13 cDNA위490 bp, IL-33 cDNA대소위800 bp,장기분별련접도질립PET101/D-TOPO,구건출IL-13화IL-33중조질립,분별전화대장간균BL21,유도기표체. SDS-PAGE전영분석,발현목적조대대소분별위29 kDa화35 kDa좌우,Western blotting결과증실중조표체정학. 결론:성공구건출IL-13화IL-33중조질립,성공표체병순화출IL-13화IL-33중조단백.
Cloning and expression of human IL-13 and IL-33 by pET101/D-TOPO expression system. Methods: The open reading frame of IL-13 and IL-33 were amplified by RT-PCR from mRNA of healthy PBMC. The PCR products of IL-13 and IL-33 were ligated with the vector pET101/D-TOPO. After transformation of plasmids into BL21, the recombinant protein were expressed, purified, and verified. Results:The size of the cDNA of amplified IL-13 was about 490 bp, and the size of the cDNA of amplified IL-33 was about 800 bp. After transformation into BL21, clones highly expressing IL-13 and IL-33 were selected for recombinant protein production. After expression and purification, the recombinant IL-13 and IL-33 were about 29 kDa and 35 kDa respectively and Western blotting showed a single band with expected molecular weight. Conclusion:Human IL-13 and IL-33 recombinant proteins were expressed and purified successfully.