CAJ | 학술논문

高迁移率蛋白 A(high mobility group A,HMGA)与染色质重塑及癌症转移有密切关系.在胚胎期广泛表达,然而在成年期表达量很低,甚至不表达.以本实验室保存的家蚕蛹 cDNA 为模板,克隆家蚕高迁移率蛋白（Bombyx mori high mobility group A,BmHMGA)开放阅读框(345 bp),构建重组质粒 pET-28a(+)-BmHMGA ,并在 BL21Star(DE3)中正确表达融合蛋白 His-BmHMGA.用 Ni-NTA 柱亲和层析纯化融合蛋白,免疫新西兰兔,获得多克隆抗体；抗体效价1∶12800以上,且具有较高特异性.实时荧光定量 PCR 检测结果表明：基因 BmHMGA从卵、5龄幼虫、蛹到蛾,转录水平依次下降；在5龄幼虫期,BmHMGA 基因的转录水平在头、表皮、卵巢、睾丸、脂肪体、气管、马氏管、丝腺和肠中从高到低分布.通过免疫印迹(Western blotting)法和酶联免疫法半定量分析结果显示.BmHMGA 蛋白在家蚕卵中含量最高,蛾中最低；5龄幼虫头部的 BmHMGA 蛋白含量明显高于其他组织,中肠、马氏管中则没有明显的信号,与 RT-PCR 的结果基本一致.家蚕 Bm N 细胞的免疫细胞化学实验结果显示, BmHMGA 蛋白在细胞质中均匀分布.
고천이솔단백 A(high mobility group A,HMGA)여염색질중소급암증전이유밀절관계.재배태기엄범표체,연이재성년기표체량흔저,심지불표체.이본실험실보존적가잠용 cDNA 위모판,극륭가잠고천이솔단백（Bombyx mori high mobility group A,BmHMGA)개방열독광(345 bp),구건중조질립 pET-28a(+)-BmHMGA ,병재 BL21Star(DE3)중정학표체융합단백 His-BmHMGA.용 Ni-NTA 주친화층석순화융합단백,면역신서란토,획득다극륭항체；항체효개1∶12800이상,차구유교고특이성.실시형광정량 PCR 검측결과표명：기인 BmHMGA종란、5령유충、용도아,전록수평의차하강；재5령유충기,BmHMGA 기인적전록수평재두、표피、란소、고환、지방체、기관、마씨관、사선화장중종고도저분포.통과면역인적(Western blotting)법화매련면역법반정량분석결과현시.BmHMGA 단백재가잠란중함량최고,아중최저；5령유충두부적 BmHMGA 단백함량명현고우기타조직,중장、마씨관중칙몰유명현적신호,여 RT-PCR 적결과기본일치.가잠 Bm N 세포적면역세포화학실험결과현시, BmHMGA 단백재세포질중균균분포.
Summary The high mobility group A (HMGA) proteins have a close relation with chromatin remodeling and cancer transformation.They are widely expressed during embryogenesis,whereas their expression is absent or very low in adult tissues.The full open reading frame(ORF) of Bombyx mori high mobility group A (BmHMGA) was cloned with the cDNA of B.mori pupae,and the recombinant plasmid,pET-28a(+)-BmHMGA was constructed.The fusion protein His-BmHMGA was correctly expressed in BL21Star (DE3).New Zealand rabbits were immunized with fusion protein purified by Ni-NTA affinity chromatography and the polyclonal antibody was obtained.The antibody had a high specificity and a titer of over 1 ∶ 12 800.The results of real-time PCR (RT-PCR) showed that the transcription levels of the BmHMGA gene declined with developmental stage in the order egg,fifth instar larva,pupa and moth.In the fifth instar larva stage,the transcription levels of the BmHMGA gene varied between tissues,declining in the order head,epidermis, ovaries,testis,fat body,stigma,Malpighian tubules,silk glands and midgut.The results of Western blotting and semi-quantitative analysis by enzyme-linked immuno sorbent assay(ELISA)showed that the BmHMGA content was the highest in the egg and the lowest in the moth,and that in the fifth instar larva,the protein levels were obviously higher in the head than that in other tissues.No signals were detected in the midgut or Malpighian tubules.These results were identical to those of RT-PCR.The result of immunocytochemistry in BmN cells showed that the proteins were evenly distributed in the cytoplasm.