中国黑睡牛卵黄原蛋白的克隆、表达分析及检测

IF 0.7 4区 农林科学 Q4 FISHERIES
Yangyang Zhang, K. Meng, Tingting Zhou, Lei Chen, Tiansheng Chen, Min Long, Kefu Zhou
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引用次数: 0

摘要

以雌激素为代表的海洋环境中的内分泌干扰物会导致与健康相反的现象。为了获得一种更有效的反映和检测环境雌激素污染的方法,我们建立了一种获得17β-雌二醇(E2)溶液诱导中华白杨卵黄原蛋白基因全长cDNA编码的方法。我们从NCBI数据库中下载了16条鱼类基因序列,并设计了相应的PCR引物。基于实时荧光定量PCR (qRT-PCR)方法,我们分析了低、中、高剂量组不同E2暴露时间下基因表达的差异。全长cDNA由4738个核苷酸和一个编码1540个氨基酸残基的阅读框组成。构建体外重组质粒,转移至大肠杆菌BL21中表达卵黄蛋白原。在16℃下通过IPTG获得高效融合表达,表达的目标蛋白(680个氨基酸,75 kDa)处于可溶性状态,占可溶性蛋白总量的25%以上。我们利用已建立的免疫组织化学方法制备单克隆抗体,检测性成熟雌鱼卵黄蛋白原的表达位点。我们的研究表明,Vg在性成熟雌鱼中的表达位点主要分布在鱼尾、肝胰脏、肠、肌肉、卵巢和肾原肾。综上所述,中华白鲟卵黄原蛋白可作为环境雌激素的生物标志物,在海洋环境中实现快速检测,并可建立后续的胶体金条开发实验,为环境污染检测提供一种高效便捷的检测方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Cloning, Expression Analysis, and Detection of the Vitellogenin in the Chinese Black Sleeper Bostrychus sinensis
Endocrine disruptors in marine environments represented by estrogens lead to reverse health phenomena. To obtain a more effective way to reflect and detect environmental estrogens pollution, a method was developed to obtain the full-length cDNA coding vitellogenin gene in B. sinensis, induced by 17β-estradiol (E2) solution. We have downloaded 16 fish gene sequences from the NCBI database and designed PCR primers accordingly. Based on the quantitative real-time PCR method (qRT-PCR), we analyze the differences in gene expression under the conditions of different E2 exposure times in the low, middle, and high-dose groups. The full-length cDNA consists of 4738 nucleotides with a reading frame encoding 1540 amino acid residues. In vitro recombinant plasmids were constructed and transferred to E. coli BL21 for vitellogenin expression. Efficient fusion expression was obtained by IPTG at 16°C, and the expressed target protein (680 amino acids, 75 kDa) existed in a soluble state, accounting for more than 25% of the total soluble protein. We prepared monoclonal antibodies using established immunohistochemistry to detect vitellogenin expression sites in sexually mature female fish. Our study shows that the expression sites of Vg in sexually mature female fish are mainly distributed in the fishtail, hepatopancreas, intestine, muscle, ovary, and pronephric kidney. In conclusion, the vitellogenin from B. sinensis could be used as a biomarker of environmental estrogens to achieve rapid detection in the marine environment and the subsequent experiments of development in colloidal gold strips after this research would be established to provide a highly efficient and convenient detection method for environment pollution.
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来源期刊
Journal of Applied Ichthyology
Journal of Applied Ichthyology 生物-海洋与淡水生物学
CiteScore
2.30
自引率
11.10%
发文量
73
审稿时长
3-8 weeks
期刊介绍: The Journal of Applied Ichthyology publishes articles of international repute on ichthyology, aquaculture, and marine fisheries; ichthyopathology and ichthyoimmunology; environmental toxicology using fishes as test organisms; basic research on fishery management; and aspects of integrated coastal zone management in relation to fisheries and aquaculture. Emphasis is placed on the application of scientific research findings, while special consideration is given to ichthyological problems occurring in developing countries. Article formats include original articles, review articles, short communications and technical reports.
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