家蚕高迁移率蛋白的表达差异和亚细胞定位(英文)  

Differential expression and subcellular localization of BmHMGA in silkworm(Bombyx mori)

作　　者：张海花[1] 赵燕[1] 张涵铭[1] 李司[1] 全艳萍[1] 于威[1] 张耀洲[1] 童富淡[1] 张晓娟[2] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018 [2]陕西理工学院生命科学与工程学院,陕西汉中723001

出　　处：《浙江大学学报（农业与生命科学版）》2014年第3期237-248,共12页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：Supported by Major State Basic Research Development Program of China"973 Program"(No.2005CB121006);the National High Technology Research and Development Program"863 Project"(Nos.2007AA021703 and 2007AA120402);National Natural Science Foundation of China(No.31101831);the Education Department of Zhejiang Province(No.Y201226220);the Zhejiang Open Foundation of the Most Important Subjects and Natural Scientific Foundation of Zhejiang Province(No.Y3110354)

摘　　要：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(345bp),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∶12 800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BmN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。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.

关 键 词：家蚕 高迁移率蛋白 表达差异 亚细胞定位 免疫细胞化学 

分 类 号：Q78[生物学—分子生物学]