日本蟾蜍聚集素cDNA 的克隆与序列分析

生物技术通报 ›› 2013, Vol. 0 ›› Issue (1): 156-161.

日本蟾蜍聚集素cDNA 的克隆与序列分析

袁进强徐跃杨仙玉诸葛慧张姝芳

浙江农林大学林业与生物技术学院，临安 311300

收稿日期:2012-06-13 修回日期:2013-01-31 出版日期:2013-01-30 发布日期:2013-01-30
作者简介:袁进强，男，硕士研究生，研究方向：野生动物资源保护与利用; E-mail: kk154home@163.com

Cloning and Sequence Analysis of Clusterin cDNA of Bufo japonicus formosus

Yuan Jinqiang, Xu Yue ,Yang Xianyu ,Zhuge Hui ,Zhang Shufang

School of Forestry and Biotechnology，Zhejiang Agriculture and Forestry University，Lin’an 311300

Received:2012-06-13 Revised:2013-01-31 Published:2013-01-30 Online:2013-01-30

摘要/Abstract

摘要： 为研究蟾酥、蟾衣、蟾皮中多肽类有效成分，通过菌落PCR 对日本蟾蜍（Bufo japonicus formosus）皮肤cDNA 质粒文库进行了筛选，获得了聚集素（clusterin，CLU）全长cDNA 序列（GenBank 登录号为JX035891）并对其进行了生物信息学分析。日本蟾蜍clu cDNA 全长为1 616 bp，包括1 332 bp 的开放阅读框（open reading frame，ORF）、5' 端30 bp 及3' 端254 bp 的非翻译区（untranslated region，UTR）。根据cDNA 序列推导的日本蟾蜍CLU 前体蛋白由443 个氨基酸残基组成，其中含20 个氨基酸残基组成的信号肽，6 个糖基化位点，10 个可形成二硫键的半胱氨酸残基和2 个卷曲螺旋区域。氨基酸序列同源性分析显示，日本蟾蜍CLU 与非洲爪蟾（Xenopus laevis）的同源性为65%，与其他7 种动物的同源性则介于41%-48% 之间。

关键词: 日本蟾蜍 , 聚集素 , 基因克隆 , 序列分析

Abstract: To study the polypeptide effective components included in Venenum Bufonis, the plasmid cDNA library of adult Japanesetoad Bufo japonicus formosus skin was screened. As the result, the clusterin cDNA was obtained(GenBank accession number ：JX035891).The transcript is 1 616 bp in length containing 30 bp 5', 254 bp 3' untranslated region(UTR)and an open reading frame(ORF)of 1 332bp encoding a polypeptide of 443 amino acids. There is a signal peptide consisted of 20 amino acid residues, six N-glycosylation sites, tencysteine residues for disulfide bond and two coiled-coil domains in the putative gene product. The homologous analysis indicates the similarityof B. japonicus formosus with Xenopus laevis was 65%, and 41%-48% with other species. The functional characteristics of CLU of B. japonicusformosus seem to be similar as that of human.

Key words: Bufo japonicus formosus , Clusterin , Gene cloning , Sequence analysis

袁进强徐跃杨仙玉诸葛慧张姝芳. 日本蟾蜍聚集素cDNA 的克隆与序列分析[J]. 生物技术通报, 2013, 0(1): 156-161.

Yuan Jinqiang, Xu Yue, Yang Xianyu, Zhuge Hui, Zhang Shufang. Cloning and Sequence Analysis of Clusterin cDNA of Bufo japonicus formosus[J]. Biotechnology Bulletin, 2013, 0(1): 156-161.

参考文献

[1] Fritz IB, Burdzy K, Setchell B, et al. Ramrete testis fluid containsa protein（clusterin）which influences cell-cell interactions invitro[J]. Biolreprod, 1983, 28（5）：1173-1188.
[2] Shannan B, Seifert M, Leskov K, et al. Challenge and promise ：roles for clusterin in pathogenesis, progression and therapy ofcancer[J]. Cell Death and Differentiation, 2006, 13（1）：12-19.
[3] Konstantinos NA, Maria TV, John PC, et al. Circulating clusterin（apolipoprotein j）levels do not have any day/night variability andare positively associated with total and LDL cholesterol levels inyoung healthy individuals[J]. Clin Endocrinol Metab, 2011, 96（11）：E1871-5.
[4] Konstantin SL, Dmitry YK, Jing L, et al. Synthesis and functionalanalyses of nuclear clusterin, a cell death protein[J]. Biol Chem,2003, 13（278）：11590-11600.
[5] Wyatt AR, Yerbury JJ, Berghofer P, et al. Clusterin facilitates in vivoclearance of extracellular misfolded proteins cell[J]. Cell Mol LifeSci, 2011, 68（23）：3919-3931.
[6] Kurosu T, Chaichana P, Yamate M, et al. Secreted complement regulatoryprotein clusterin interacts with dengue virus nonstructural protein[J]. Biochemical and Biophysical Research Communications,2007, 362（4）：1501-1056.
[7] Chi KN, Hotte SJ, Yu EY, et al. Randomized phase ii study of docetaxeland prednisone with or without ogx-011 in patients with metastaticcastration-resistant prostate cancer[J]. Clin Oncol, 2010, 28（27）：4247-4254.图5 基于日本蟾蜍与其他8 种动物CLU 氨基酸序列构建的NJ 系统树
[8] Zoubeidi A, Chi K, Gleave M. Targeting the cytoprotective chaperone,clusterin, for treatment of advanced cancer[J]. Clin Cancer Res,2010, 16（4）：1088-1093.
[9] Mahon MG, Lindstedt KA, Hermann M, et al. Multiple involvement ofclusterin in chicken ovarian follicle development[J]. Biol Chem,1999, 274（7）：4036-4044.
[10] 张飞春, 孙文革, 高晓玲, 等. 蟾酥乙醇提取物抗肿瘤剂量效应关系实验研究[J]. 药物研究, 2011, 20（16）：23-24.
[11] 缪珠雷, 张康, 杨鸣泽, 等. 蟾蜕抗肿瘤及增强免疫效应研究[J]. 中国中药杂志, 2010, 25（2）：211-214.
[12] 曾洋, 张爱, 文筱. 干蟾皮的研究进展[J]. 中国医药科学,2011, 1（15）：19-31.
[13] Lars E, Chonn A, Ducrest D, et al. Murine clusterin ：molecularcloning and mRNA localization of a gene associated with epithelialdifferentiation processes during embryogenesis[J]. Cell Biol,1999, 122（5）：1119-1130.
[14] Yang CR, Yeh S, Leskov K, et al. Isolation of Ku70-binding protein（KUBs）[J]. Nucleic Acids Res, 1999, 27（10）：2165-2174.
[15] Choi-Miura NH, Takahashi Y, Nakano Y, et al. Identification of thedisulfide bonds in human plasma protein SP-40, 40（apolipoprotein-J）[J]. Biochem, 1992, 112（4）：557-561.
[16] Stewart EM, Aquilina JA, Easterbrook-Smith SB, et al. Effects ofglycosylation on the structure and function of the extracellular chaperoneclusterin[J]. Biochemistry, 2007, 46（5）：1412-1422.
[17] 赵晓航, 何洪智, 王堃, 等. 食管鳞癌中clusterin 的糖基化修饰和表达定位改变[J]. 第二军医大学学报, 2005, 26（12）：1333-1336.
[18] Comunale MA, Wang MJ, Rodemich-Betesh L, et al. Novel changesin glycosylation of serum Apo-J in patients with hepatocellularcarcinoma[J]. Cancer Epidemiology Biomarkers and Prevention,2011, 20（6）：1222-1229.
[19] Ammar H, Closset JL. Clusterin activates survival through thephosphatidylinositol 3-kinase/Akt pathway[J]. Biochem, 2008,283（19）：12851-12861.
[20] Trougakos IP, Lourda M, Antonelou MH, et al. Intracellularclusterin inhibits mitochondrial apoptosis by suppressing p53-activating stress signals and stabilizing the cytosolic Ku70-Baxprotein complex[J]. Clin Cancer Res, 2009, 15（1）：48-59.