| 中华鳖StAR基因的克隆、表达及多克隆抗体制备 |
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| 引用本文： | 刘倩,马晓,岑双双,田雪,王璐明,吴利敏,刘慧芬,李学军.中华鳖StAR基因的克隆、表达及多克隆抗体制备[J].水产学报,2020,44(11):1813-1823. |
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| 作者姓名： | 刘倩 马晓 岑双双 田雪 王璐明 吴利敏 刘慧芬 李学军 |
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| 作者单位： | 河南师范大学水产学院,河南 新乡 453007 |
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| 基金项目： | 国家自然科学基金；河南省重点科技攻关项目 |
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| 摘 要： | 摘要 为探索StAR基因在中华鳖（Pelodiscus sinensis）性腺发育过程中的作用，利用RACE技术克隆获得中华鳖StAR基因全长cDNA，采用Real-time PCR分析其在不同组织及胚胎不同发育时期的表达情况，并制备多克隆抗体，采用western blot检测StAR在不同组织的表达，通过免疫组化对其在细胞中的表达进行定位，同时检测芳香化酶抑制剂来曲唑处理雄性中华鳖后StAR在精巢中的表达情况。结果显示：该基因全长2377bp，开放阅读框903bp，编码300个氨基酸。氨基酸序列比对及系统进化分析表明，中华鳖StAR与龟类亲缘关系最近。荧光定量PCR结果表明，StAR基因在精巢中表达量最高，显著高于其他组织；StAR基因于中华鳖胚胎发育16期已有表达，20期表达量最高，提示StAR可能参与中华鳖早期性腺分化;来曲唑处理的中华鳖精巢中，StAR表达量显著降低。本研究利用原核表达系统构建中华鳖StAR基因原核重组表达载体，经蛋白纯化后免疫小鼠，获得中华鳖StAR多克隆抗体。Western blot检测结果显示StAR在不同组织的表达量与荧光定量结果基本一致。免疫组化结果显示，StAR蛋白在卵巢间质细胞、精巢的间质细胞，精原细胞及胞质中表达。研究表明，StAR基因可能参与中华鳖性腺发育过程。
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| 关 键 词： | 中华鳖 StAR基因 克隆 表达 性腺 多克隆抗体 |
| 收稿时间： | 2019/10/29 0:00:00 |
| 修稿时间： | 2020/4/20 0:00:00 |
Molecular cloning, expression analysis and polyclonal antibody preparation of StAR gene in Chinese soft-shelled turtle (Pelodiscus sinensis) |
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| LIU Qian,MA Xiao,CEN Shuangshuang,TIAN Xue,WANG Luming,WU Limin,LIU Huifen,LI Xuejun.Molecular cloning, expression analysis and polyclonal antibody preparation of StAR gene in Chinese soft-shelled turtle (Pelodiscus sinensis)[J].Journal of Fisheries of China,2020,44(11):1813-1823. |
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| Authors: | LIU Qian MA Xiao CEN Shuangshuang TIAN Xue WANG Luming WU Limin LIU Huifen LI Xuejun |
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| Institution: | College of Fisheries Henan Normal University,College of Fisheries Henan Normal University,College of Fisheries Henan Normal University,College of Fisheries Henan Normal University,College of Fisheries Henan Normal University,College of Fisheries Henan Normal University,College of Fisheries Henan Normal University,College of Fisheries Henan Normal University |
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| Abstract: | Abstract To gain a better understanding of the role in gonadal development of StAR in Pelodiscus sinensis, we cloned P. sinensis StAR cDNA fragment of 2377bp, containing an open reading frame (ORF) for 300 amino acid residues. The predicted P. sinensis StAR was maximally identical with turtle StAR protein. The RT-PCR result showed that the P. sinensis StAR mRNA was absent in all somatic tissue, but abundant in adult testis. The StAR mRNA had been expressed at embryonic stage 16th and the expression increased with the development of embryo before the 20th stage.The expression of StAR in testis treated by letrozole was significantly decreased. The recombinant plasmid was constructed and the StAR polyclonal antibody was obtained by immunizing mice. Western blot analysis showed that the expression of StAR in different tissues was consistent with RT-PCR result. Immunohistochemical results showed that StAR protein was expressed in ovarian stromal cells. Therefore, the P. sinensis StAR gene might play an important role in gonadal differentiation and testis development. |
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| Keywords: | Pelodiscus sinensis StAR gene cloning expression gonad polyclonal antibody |
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