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A perspective on fish gonad manipulation for biotechnical applications
  • ISSN号:1002-0470
  • 期刊名称:《高技术通讯》
  • 时间:0
  • 分类:Q78[生物学—分子生物学]
  • 作者机构:[1]中国科学院水生生物研究所淡水生态与生物技术国家重点实验室,武汉430072, [2]中国科学院研究生院,北京100049
  • 相关基金:973计划(2001CB109006)和国家自然科学基金(30430540,30130540)资助项目.
中文摘要:

采用逆转录-聚合酶链式反应(RT-PCR)方法,从鲤鱼脑垂体获得了两种GtHβ亚基的cDNA,克隆在pMD18-T载体。经测序确证后,将这两个基因克隆到原核表达载体pET-32(a)中,转化E.coli表达菌BL21(DE3),以IFFG诱导融合蛋白的高效表达。利用初步纯化后的抗原免疫新西兰大白兔,制备多克隆抗体。应用制备的兔抗血清与抽提的鲤鱼脑垂体的总蛋白分别进行Westem-blot及ELISA分析,结果显示获得的多抗能特异识别各自的天然蛋白。该结果为纯化天然GtH蛋白提供了有效的检测手段,为进一步制备GtH单市降抗体奠定了基础。

英文摘要:

The article gives a description of the cloning, expression and polyclonal antibody preparation of two different gonadotropin (GtH) beta subunits complementary DNAs (cDNAs) of the common carp ( Cyprinus carpio ). By RT-PCR, the cDNAs encoding GtH Ⅰand GtH Ⅱ beta subunits were obtained from common carp pituitary and cloned into pMD18- T vector. Confirmed by sequencing, two right cDNAs were cloned into pET-32(a) vector and then transformed into E. coli BL (21) DE3 to constnlct two expression strains. The recombinant GtH Ⅰand GtHⅡ beta subunits were efficiently expressed in the form of fusion protein after induction with IFrG. After being primarily purified, the recombinant proteins were injected into New Zealand White rabbits to produce polyclonal antibodies. Western blot analysis showed that these produced polyclonal antibodies could bind not only to the expressed antigens, but also to the denatured ones extracted from common carp pituitary specifically. Furthermore, ELISA analysis demonstrated that these polyclonal antibodies could recognize the natural GtH Ⅰand GtHⅡ beta subunits respectively. The results laid a foundation for the detection and purification of natural GtH as well as their monoclonal antibody preparation.

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期刊信息
  • 《高技术通讯》
  • 北大核心期刊(2011版)
  • 主管单位:中华人民共和国科学科技部
  • 主办单位:中国科学技术信息研究所
  • 主编:赵志耘
  • 地址:北京市三里河路54号
  • 邮编:100045
  • 邮箱:hitech@istic.ac.cn
  • 电话:010-68514060 68598272
  • 国际标准刊号:ISSN:1002-0470
  • 国内统一刊号:ISSN:11-2770/N
  • 邮发代号:82-516
  • 获奖情况:
  • 《中国科学引文数据》刊源,《中国科技论文统计与分析》刊源
  • 国内外数据库收录:
  • 美国化学文摘(网络版),荷兰文摘与引文数据库,日本日本科学技术振兴机构数据库,中国中国科技核心期刊,中国北大核心期刊(2004版),中国北大核心期刊(2008版),中国北大核心期刊(2011版),中国北大核心期刊(2014版),英国英国皇家化学学会文摘
  • 被引量:12178