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题名: POLYCLONAL ANTIBODY PREPARATION AND IMMUNOFLUORESENCE LOCALIZATION OF GIBEL CARP OOCYTE-SPECIFIC LECTIN
作者: Wang Hong-Ying (hy_wang0806@yahoo.com.cn) ; Zhou Li ; Gui Jian-Fang (jfgui@ihb.ac.cn)
通讯作者: Wang Hong-Ying ; Chinese Acad Sci, Inst Hydrobiol, Wuhan Ctr Dev Biol, State Key Lab Freshwater Ecol and Biotechnol, Wuhan 430072, Peoples R China
刊名: Acta Hydrobiologica Sinica
发表日期: 2008
卷: 32, 期:3, 页:295-300
收录类别: 其他
英文摘要: Lectins were defined as proteins that recognize specific carbohydrate structures and agglutinate cells by binding to cell surface glycoproteins and glycoconjugates. Many kinds of different lectins have been purified from fish eggs. Lectins may have a variety of biological function in fish eggs, such as block of polyspermy, regulation of carbohydrates metabolism, participation in the formation of fertilization envelope after binding with glycoproteins, antibacterial effect and opsonization of pathogens. Therefore, lectins are important components in oocytes and play important roles. Cortical granules were as a kind of secretory vesicles that unique to oocyte. They are synthesized and accumulated during oogenesis, translocated to the cell surface before fertilization and exocytosed after fertilization. Cortical granule contents have diverse components, such as proteases and lectins, and play important roles in egg fertilization and early embryogenesis.Gibel carp oocyte-specific lectin (GOL) had been previously Screened from subtractive hybridization cDNA libraries in fully-grown oocytes between gibel carp (Carassius auratus gebelio) and color crucian carp Carassius auratits). In this study, A pair of primers (5'-GGGAATTCCATATGAACTGTAACGTGGTGAAC-3' including Nde I sites and start codon ATG,5'-TAGTTACAGGATCCTCAGCGAGTGACACCAGT-3' including Bam H I sites and stop codon TGA) were used to amplify the cDNA fragment encoding for 21-214 amino acids (193 aa) and the amplified product was subcloned between the Nde I and Bam H I sites of pET-15 b vector. The recombinant vector was digested with Nco I and Bam H I. And then the digested fragment was blunted, added A by Taq polymerase, ligated into PinPoint T expression vector (Promega). An about 40 kDa fusion protein was expressed in BL21 (DE3). The fusion protein was purified, treated and used to prepare polyclonal antibody by immunizing white rabbit. Furthermore, cortical granules were labeled by Lens culinaris agglutinin biotin and Cy3-strepavidin fluorescence for co-location analysis with COL. Double immunofluoresence localization indicated that the COL localized in oocytes and fluorescence of GOL was overlapped with that of cortical granules. The data suggested that the lectin should be a component of cortical granules in fish oocytes. As the major components in verteblate cortical granules, one of the major functions of the components in cortical granule is to construct or modify the existing oocyte extracellular matrix to serve barriers to block additional sperm into oocyte. Hence, in the future, it was worth to study whether CB102 involved in the formation of egg envelopment to block polyspermy.
ISSN号: 1000-3207
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/342005/18763
Appears in Collections:中科院水生所知识产出(2009年前)_期刊论文

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Wang Hong-Ying (hy_wang0806@yahoo.com.cn) ; Zhou Li; Gui Jian-Fang (jfgui@ihb.ac.cn).POLYCLONAL ANTIBODY PREPARATION AND IMMUNOFLUORESENCE LOCALIZATION OF GIBEL CARP OOCYTE-SPECIFIC LECTIN,Acta Hydrobiologica Sinica,2008,32(3):295-300
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文件名: POLYCLONAL ANTIBODY PREPARATION AND IMMUNOFLUORESENCE LOCALIZATION OF GIBEL CARP OOCYTE-SPECIFIC LECTIN.pdf
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