CAJ | 학술논문

目的探讨柯萨奇病毒B组3型(CVB3) woodruff病毒2B蛋白对细胞自噬的诱导作用,及其自噬相关基序的鉴定.方法分别构建增强型绿色荧光蛋白(EGFP)与CVB3 woodruff病毒2B蛋白及其9种截短蛋白的融合蛋白(EGFP-2B、EGFP-2B1-249、EGFP-2B1-201、EGFP-2B1-153、EGFP-2B1-105、EGFP-2B1-57、EGFP-2B106.201、EGFP-2B106-249、EGFP-2 B205-297、EGFP-2B106-201)真核表达载体,红色荧光蛋白(mCherry)与微管相关蛋白轻链3(LC3)的融合蛋白真核表达载体pmCherry-LC3;应用激光共聚焦与蛋白质免疫印迹(Western blot)检测病毒2B蛋白对宫颈癌细胞(HeLa) LC3表达的影响;荧光显微镜观察9种截短蛋白在HeLa细胞中的表达;Western blot检测pEGFP-2B和pEGFP-2B106-249转染细胞后LC3的表达;观察自噬抑制剂3-甲基腺苷(3-MA)处理后,pEGFP-2B和pEGFP-2B106-249诱导细胞自噬的情况.结果 CVB3攻击HeLa细胞后,mCherry-LC3呈现细胞核周点状聚集表达,Western blot检测出现清晰LC3-Ⅱ条带;pEGFP-2B与pmCherry-LC3共转染后也可见细胞核周围绿色荧光与红色荧光均成点状聚集并相互重叠,且LC3-Ⅱ条带明显;9种截短融合蛋白质粒分别转染HeLa细胞后,其中可见细胞核周围绿色荧光点状聚集的最短截短蛋白为EGFP-2B106-249;pEGFP-2B和pEGFP-2B106-249转染细胞后可检测出现清晰LC3-Ⅱ条带;3-MA处理后,pEGFP-2B和pEGFP-2B106-249分别与pmCherry-LC3共转染的细胞均未见绿色和红色荧光的核周点状聚集.结论 CVB3 woodruff病毒2B蛋白可以诱导宿主细胞发生自噬,其中截短蛋白2B106-249是病毒蛋白2B诱导HeLa细胞发生自噬的功能基序.
목적 탐토가살기병독B조3형(CVB3) woodruff병독2B단백대세포자서적유도작용,급기자서상관기서적감정.방법 분별구건증강형록색형광단백(EGFP)여CVB3 woodruff병독2B단백급기9충절단단백적융합단백(EGFP-2B、EGFP-2B1-249、EGFP-2B1-201、EGFP-2B1-153、EGFP-2B1-105、EGFP-2B1-57、EGFP-2B106.201、EGFP-2B106-249、EGFP-2 B205-297、EGFP-2B106-201)진핵표체재체,홍색형광단백(mCherry)여미관상관단백경련3(LC3)적융합단백진핵표체재체pmCherry-LC3;응용격광공취초여단백질면역인적(Western blot)검측병독2B단백대궁경암세포(HeLa) LC3표체적영향;형광현미경관찰9충절단단백재HeLa세포중적표체;Western blot검측pEGFP-2B화pEGFP-2B106-249전염세포후LC3적표체;관찰자서억제제3-갑기선감(3-MA)처리후,pEGFP-2B화pEGFP-2B106-249유도세포자서적정황.결과 CVB3공격HeLa세포후,mCherry-LC3정현세포핵주점상취집표체,Western blot검측출현청석LC3-Ⅱ조대;pEGFP-2B여pmCherry-LC3공전염후야가견세포핵주위록색형광여홍색형광균성점상취집병상호중첩,차LC3-Ⅱ조대명현;9충절단융합단백질립분별전염HeLa세포후,기중가견세포핵주위록색형광점상취집적최단절단단백위EGFP-2B106-249;pEGFP-2B화pEGFP-2B106-249전염세포후가검측출현청석LC3-Ⅱ조대;3-MA처리후,pEGFP-2B화pEGFP-2B106-249분별여pmCherry-LC3공전염적세포균미견록색화홍색형광적핵주점상취집.결론 CVB3 woodruff병독2B단백가이유도숙주세포발생자서,기중절단단백2B106-249시병독단백2B유도HeLa세포발생자서적공능기서.
Objective To identify the 2B protein of coxsackievirus B3 woodruff strain inducing autophagy,and further,identify the motif of 2B protein related to autophagy.Method The eukaryotic recombinant plasmids (pEGFP-2B) expressing the fusion protein of 2B protein and enhanced green fluorescent protein(EGFP) was constructed.The 9 kinds of recombinant plasmids expressing the fusion proteins of brachytmema proteins and EGFP were constructed.The plasmid (pmCherry-LC3) which can express the fusion protein of red fluorescent protein (mCherry) and microtubule-associated protein light chain 3 (LC3) was structured.The expression of mCherry-LC3 in the HeLa cells,tansfected with pmCherry-LC3 and infected with CVB3,was detected by Laser Confocal Scanning Microscope (LCSM) and Western blotting.The status of autophagy in the HeLa cells co-transfected with plasmids pEGFP-2B and pmCherry-LC3 was observed by LCSM,the expression of LC3 was detected by Western blotting.The HeLa cells were transfected with 9 kinds of plasmids expressing the brachytmema fusion proteins,respectively,the expression of fusion proteins was detected by fluorescence microscope.The HeLa cells dealing with 3-methyladenine (3-MA) were co-transfected with plasmids pEGFP-2B or pEGFP-2B106-249 and pmCherry-LC3,and the fluorescins were observed under fluorescence microscope.Results The mCherry-LC3 fusion protein expressed in punctuate pattern around the nucleus in HeLa cells infected by CVB3.The band of LC3-Ⅱ was distinct according to the viral inoculation by Western blotting.The green and red fluorescin expressed in punctuate pattern around the nucleus in HeLa co-transfected with pEGFP-2B and pmCherry-LC3,and the two kinds of the fluorescins were interloped.The distinct band of LC3-Ⅱ was detected by Western blotting.The HeLa cells were transfected with 9 kinds of plasmids expressing brachytmema proteins,respectively,the shortest brachytmema protein which can induce green fluorescence in punctuate pattern around the nucleus was EGFP-2B106-249,while no punctuate patterns of the green and red fluorescin were observed in HeLa cells dealing with 3-MA.The LC3-Ⅱ levels increased according to pEGFP-2B106-249 transfection by Western blotting.Conclusions 2B protein of CVB3 woodruff strain can induce autophagy in HeLa cells ; The motif of 2B protein related to autophagy located on 2B106-249.