草鱼IgM、IgD和IgZ的抗体制备与组织表达分析
PREPARATION OF POLYCLONAL ANTIBODY AGAINST IGM, IGZ AND IGD IN GRASS CARP (CTENOPHARYNGODON IDELLUS) AND EXPRESSION ANALYSIS IN TISSUES
查看参考文献17篇
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文摘
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根据已报道的草鱼免疫球蛋白IgM、IgZ和IgD的序列设计表达引物进行PCR扩增,将扩增片段克隆至表达载体pET-32a,并在大肠杆菌Rosetta-gami(DE3)中进行诱导表达。利用亲和层析法纯化表达的重组蛋白,然后免疫日本大耳白兔,获得兔抗IgM、IgZ和IgD的抗血清。经免疫印迹检测,表明IgM、IgZ和IgD的表达产物能够被兔多克隆抗体特异性识别。应用兔抗草鱼IgM和IgZ的多克隆抗体,对草鱼多种器官、组织提取的总蛋白进行免疫印迹检测,在肠、头肾、中肾、皮肤、脾脏、脑、鳃和血液中都检测到IgM和IgZ的表达。 |
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其他语种文摘
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The grass carp(Ctenopharyngodon idellus) is one of the most important freshwater fish in aquaculture industry of China.However,grass carp breeding is plagued by a number of diseases,mostly caused by viruses and bacteria,so the understanding of its immune system is essential for its aquaculture.In this paper,the expression,purification,and polyclonal antibody preparation of the IgM,IgZ and IgD gene were described.The expression primers were designed based on the reported open reading frame(ORF) of IgM,IgZ and IgD gene from grass carp,and incorporated with KpnI and HindIII restriction sites.PCR cycling conditions were as follows: 95°C for 5min;95°C for 45s,56°C for 1min,72°C for 1min for 30 cycles;72°C for 6min.The IgM,IgZ and IgD gene were cloned into pET-32a expression plasmid at the KpnI and HindIII restriction sites,and the recombinant plasmids were verified by sequencing.The recombinant plasmids were transformated into Escherichia coli Rosetta-gami(DE3) strain.Sodium dodecyl sulfate-polyacrylamide gel elactrophoresis(SDS-PAGE) analysis showed that recombinant protein were satisfactorily expressed by optimizing the concentration and induction time of Isopropyl β-D-1-thiogalactopyranoside(IPTG).All recombinant proteins were expressed as inclusion bodies in agreement with the expected molecular weight,and were purified successfully by using affinity chromatography in the denatured condition.The purified proteins were then injected into Japanese white rabbits in order to obtain polyclonal antisera.Western blot analysis showed that the purified IgM,IgZ and IgD recombinant proteins were recognized by the rabbit antisera,respectively.By using the rabbit antisera of IgM and IgZ,Western blotting showed that IgM and IgZ expressed in intestine,head kidney,trunk kidney,skin,spleen,brain,gill and blood. |
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来源
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水生生物学报
,2012,36(1):35-40 【核心库】
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关键词
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草鱼
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IgM
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IgZ
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IgD
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表达
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抗体
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地址
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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
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语种
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中文 |
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文献类型
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研究性论文 |
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ISSN
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1000-3207 |
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学科
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分子生物学 |
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基金
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国家973计划
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文献收藏号
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CSCD:4496767
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