CAJ | 학술논문

目的构建鼠白细胞介素-33(IL33)真核表达质粒,建立并检测稳定表达IL-33的细胞株GL261.方法从Genebank找出鼠IL-33基因全长序列(801 bp),人工合成、装载于表达载体pEZ-M03,扩增后酶切鉴定.将鉴定正确的质粒转染的小鼠胶质瘤细胞GL261,并用浓度为200 mg/L的G418筛选出阳性克隆,采用实时定量聚合酶链反应(Real-time PCR)检测目的基因表达量；Western blot法检测目的蛋白的表达量,并观察转染后的GL261细胞体外生长状况.结果成功构建鼠IL-33真核质粒pEZ-M03-mIL33,经过浓度为20 mg/L的G418工作液筛选出了稳定高表达鼠IL-33的细胞株GL261,且其生长状态未受到转染基因的影响(P＞0.05).Real-time PCR检测目的基因在瞬转、稳转细胞株中皆为高表达(P＜0.01).Western blot检测到稳转细胞株中目的蛋白明显高表达(P＜0.01).结论本研究构建的鼠IL-33真核质粒能于GL261中获得稳定高表达.
목적 구건서백세포개소-33(IL33)진핵표체질립,건립병검측은정표체IL-33적세포주GL261.방법 종Genebank조출서IL-33기인전장서렬(801 bp),인공합성、장재우표체재체pEZ-M03,확증후매절감정.장감정정학적질립전염적소서효질류세포GL261,병용농도위200 mg/L적G418사선출양성극륭,채용실시정량취합매련반응(Real-time PCR)검측목적기인표체량；Western blot법검측목적단백적표체량,병관찰전염후적GL261세포체외생장상황.결과 성공구건서IL-33진핵질립pEZ-M03-mIL33,경과농도위20 mg/L적G418공작액사선출료은정고표체서IL-33적세포주GL261,차기생장상태미수도전염기인적영향(P＞0.05).Real-time PCR검측목적기인재순전、은전세포주중개위고표체(P＜0.01).Western blot검측도은전세포주중목적단백명현고표체(P＜0.01).결론 본연구구건적서IL-33진핵질립능우GL261중획득은정고표체.
Objective To construct the eukaryotic plasmid of mouse interleukin-33 (IL-33),and establish GL261 cell line stably expressing IL-33.Methods The full sequence (801 bp) of mouse IL-33 was found from Genebank,then synthesized artificially and loaded in the expression vector pEZ-M03.The recombinant plasmid with the target gene was identified by digestion.The GL261 cells were transfected with pEZ-M03-mIL33 and positive clones were screened in the presence of G418 (200 mg/L).The expression of the gene was detected by using real-time polymerase chain reaction (Real-time PCR) and Western blotting.Also growth kinetics of transfected GL261 cells was observed in vitro.Results The eukaryotic plasmid of mouse IL-33 was successfully constructed and GL261 cell line stably expressing mouse IL-33 was screened out by G418 (200 mg/L).Simultaneously the growth of the transfected GL261 cells was not influenced by the transfected gene in vitro (P ＞ 0.05).Real-time PCR revealed that the expression of the target gene was high after both stable and transient transfection (P ＜ 0.01).A significantly higher expression of mouse IL-33 was detected in stable expression clones (P ＜ 0.01).Conclusion The mouse IL-33 eukaryotic plasmid can be stably and highly expressed in the GL261 cells.