【作者】 江艳；

【导师】 童富淡； 张耀洲；

【作者基本信息】 浙江理工大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 细胞周期依赖性蛋白激酶(Cdk7)是后生动物Cdk激酶(CAK)重要的催化、调节亚基,而Cdk的激活需要自身结合一个调节亚基,也就是细胞周期蛋白H(cyclin H)。在哺乳动物体内,cyclin H与Cdk7、MAT1形成的三元复合物具有CAK激酶的活性,它能磷酸化某些Cdk T环内保守的苏氨酸残基,继而行使Cdk的作用。目前,国内外对cyclin H的研究大多集中在脊椎动物上,而对无脊椎动物的研究较少。通过RT-PCR的方法从家蚕蛹中扩增到家蚕cyclin H基因完整的ORF序列,GenBank No. AV406047,将其命名为家蚕细胞周期蛋白H(Bombyx mori Cyclin H,BmCycH)。该基因序列长为753 bp,编码250个氨基酸残基,预测分子量为28.34 kD。将扩增到的BmCycH基因克隆到重组表达载体pET-28a(+)相应的酶切位点上,构建重组蛋白表达质粒pET-28a(+)-BmCycH,经PCR、酶切鉴定以及序列分析证明重组正确。将该重组子转化大肠杆菌BL21(DE3),用终浓度为1 mmol/L的IPTG诱导工程菌表达重组蛋白,裂解菌体作SDS-PAGE分析,在32 kD左右的位置有一条特异性蛋白条带,与预期值相符。重组蛋白以包涵体形式存在,将超声裂解菌体后12000 rpm离心所得到的沉淀溶于N-十二烷基肌氨酸钠中,并以亲和层析的方法纯化目的蛋白His-tag BmCycH。以纯化蛋白为抗原免疫雄性新西兰兔,制备了该重组蛋白的多克隆抗体,ELISA检测该抗血清的效价可达1:12800以上。利用荧光定量PCR技术,分析家蚕各个发育时期,以及五龄幼虫不同组织的靶mRNA的量。实验结果表明,BmCycH蛋白在家蚕各个时期都有表达,蛾、蛹、幼虫和卵表达量依次降低;BmCycH蛋白在家蚕五龄幼虫不同组织也均有表达,在头部表达量最高,丝腺表达量最低。提取家蚕各个时期以及五龄幼虫不同组织的蛋白,用于Western blotting实验。实验结果表明各个时期蛋白表达量与荧光定量PCR结果大致相同;而不同组织中蛋白表达量略有不同。这些研究表明BmCycH蛋白在家蚕发育分化过程中可能起到一定作用。利用免疫荧光化学法进行亚细胞定位,结果表明,该蛋白主要存在细胞核中。更多还原

【Abstract】 The catalytic subunit of the metazoan Cdk-activating kinase(CAK) is cyclin-dependent kinase 7(Cdk7). Activation of Cdk7 requires its association with a regulatory subunit, cyclinH. In mammalian cells, Cdk7, cyclin H and MAT1, function as the CAK which phosphorylates the conserved threonine residue within the T loop of several Cdks. However, most researches of cyclin H focused on vertebrate, seldom were done on invertebrate.The full open reading frame (ORF) of Bombyx mori cyclin H was obtained from silkworm pupa by RT-PCR and named BmCycH (Bombyx mori cyclin H), of which the accession number was AV406047. After cloned into pET-28a(+) and expressed in E.coli BL21(DE3), the BmCycH was fused with His-tag and purified on chelating columns. The molecular weight of this fusion protein was 32.0 kD, characterized by SDS-PAGE. This result proved that His-tag BmCycH was expressed in the right form. After being purified with Ni2+ affinity chromatography the recombinant protein was as antigen against which the antibody was raised by immune rabbit. The titer of the polyclonal antibody reaches 1:12800 measured by ELISA.We also compared the quantity of BmCycH mRNA in different stages of the fifth instar larva and different tissues of silkworm. The expression level in the moth was highest, but in the egg was the lowest; and in the different tissues, the expression level in the head was the highest and the silk gland was the lowest. Western blot analysis revealed that the protein was expressed in all silkworm tissues and 4 developmental phases examined, with highest expression in the moth, pupa and larvae, lowest in egg. It may suggest that BmCycH plays a role in the differentiation and development of silkworm. Using fluorescent antibody for subcellular localization, we preliminarily localized the protein in the nucleus in BmN.更多还原