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| 牙鲆芳香化酶Cyp19a多克隆抗体制备及其应用 |
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舒畅1,2,3, 王丽娟1,2, 邹聪聪1,2,3, 邹玉霞1,2, 王国玉1,2, 吴志昊1,2, 李泽1,2,3, 尤锋1,2
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1.中国科学院海洋研究所 实验海洋生物学重点实验室海洋大科学中心 山东省实验海洋生物学重点实验室, 山东 青岛 266071;2.青岛海洋科学与技术试点国家实验室海洋生物学与生物技术功能实验室, 山东 青岛 266237;3.中国科学院大学, 北京 100049
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| 摘要: |
| 芳香化酶Cyp19a在鱼类性别决定和性别分化中起关键作用,通过调控体内雌、雄激素的转化来影响鱼类性别表型形成。为深入研究Cyp19a在牙鲆(Paralichthys olivaceus)性腺分化和发育过程中的表达规律及作用机制,本文从牙鲆cDNA中克隆获得1557bp的cyp19a基因编码序列,并成功构建了原核重组表达质粒。经体外重组与纯化获得较高纯度的重组Cyp19a蛋白;以此作为抗原免疫兔子,制备多克隆抗体。用间接酶联免疫吸附剂测定(ELISA)技术检测抗血清效价,评估其免疫原性。结果表明免疫结束后得到的抗体血清效价超过了1∶50000,且纯化后抗体具有较好活性。Western Blot(WB)检测结果表明Cyp19a兔多抗可以特异性识别牙鲆重组Cyp19a蛋白和内源性Cyp19a蛋白。蛋白水平的雌雄性腺差异表达分析显示,牙鲆Cyp19a蛋白在卵巢中高表达,在精巢中微量表达。利用Foxl2和Dmrt1重组蛋白处理牙鲆性腺分化期幼鱼可分别显著上调和下调Cyp19a的表达(P<0.05)。综上所述,本研究成功制备了牙鲆Cyp19a多克隆抗体并进行了应用,为深入研究牙鲆等鱼类性别分化机制提供有力工具。 |
| 关键词: 牙鲆(Paralichthys olivaceus) Cyp19a 重组蛋白 多克隆抗体 蛋白表达 |
| DOI：10.11759/hykx20220615001 |
| 分类号:S917.4 |
| 基金项目:国家自然科学基金项目(31872558);国家重点研发计划项目(2018YFD0900202);青岛海洋科学与技术试点国家实验室海洋生物学与生物技术功能实验室青年科学基金项目(YQ2018NO01);山东省重点研发计划项目(农业良种工程,2021LZGC029);山东省自然科学基金项目(ZR2022MC026) |
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| Polyclonal antibody preparation and application of aromatase Cyp19a in olive flounder Paralichthys olivaceus |
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SHU Chang1,2,3, WANG Li-juan1,2, ZOU Cong-cong1,2,3, ZOU Yu-xia1,2, WANG Guo-yu1,2, WU Zhi-hao1,2, LI Ze1,2,3, YOU Feng1,2
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1.CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China;3.University of Chinese Academy of Sciences, Beijing 100049, China
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| Abstract: |
| Aromatase Cyp19a plays a key role in fish sex determination and differentiation and could affect fish sex phenotype by regulating the conversion of estrogen and androgen. To further examine the expression and role of Cyp19a in gonadal differentiation and development of olive flounder Paralichthys olivaceus, the coding sequence of the cyp19a gene with the full length of 1, 557 bp was obtained from its cDNA, and the plasmid encoding recombinant Cyp19a protein was constructed. Following in vitro recombination and purification, high-purity recombinant Cyp19a protein was obtained. The protein was used as the antigen to immunize rabbits to prepare polyclonal antibodies. An indirect enzyme-linked immunosorbent assay was employed to detect the titers of the anti-serum and evaluate their immunogenicity. The titers of the poly-antibodies obtained were above 1: 50, 000, and the purified antibodies had good activity. Western blot detection revealed that the rabbit poly-antibodies of Cyp19a could specifically recognize the recombinant and endogenous Cyp19a proteins in flounders. The differential expression analysis of the female and male gonads at the protein level showed that Cyp19a protein was highly expressed in the ovary and slightly expressed in the testis. The treatment with the recombinant Foxl2 and Dmrt1 proteins could significantly upregulate and downregulate the expression of Cyp19a in undifferentiated gonads (P<0.05), respectively. In conclusion, this study successfully prepared and applied rabbit-derived polyclonal antibodies against Cyp19a of flounders, which provides a powerful tool for the in-depth investigation of the sex differentiation mechanism in flounders or other fish. |
| Key words: Paralichthys olivaceus Cyp19a recombinant protein polyclonal antibody protein expression |
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