花䱻抗缪勒氏管激素基因克隆、抗体制备及其对外源雌激素的响应

Molecular cloning of anti-Müllerian hormone gene, polyclonal antibody preparation and response to exogenous estrogen in spotted steed (Hemibarbus maculatus Bleeker)

  • 摘要: 【目的】克隆花䱻抗缪勒氏管激素基因Amh,分析其序列特征及亚细胞定位,检测不同浓度雌二醇处理下的表达变化规律,为后续研究Amh基因的功能及其在卵巢发育中分子机制提供理论参考。【方法】基于花转录组文库测序结果,克隆Amh基因cDNA序列,并对其进行生物信息学分析。构建重组表达质粒pET32a-Amh,转化大肠杆菌BL21感受态细胞,用纯化的重组蛋白免疫小鼠制备多克隆抗体。通过免疫荧光技术检测荧光信号确定蛋白的亚细胞定位情况。同时,利用实时荧光定量PCR检测Amh基因在雌二醇处理后的表达情况。【结果】克隆的花䱻Amh基因cDNA序列全长为1849 bp,开放阅读框(ORF)为1671 bp,编码556个氨基酸残基。系统进化分析结果显示,花䱻Amh基因与斑马鱼、鲤鱼等鲤科鱼类Amh基因聚为一支,表明其亲缘关系较近。将纯化的AMH重组蛋白免疫小鼠以制备AMH多克隆抗体,通过酶联免疫方法(ELISA)检测其抗体效价为2.4×106以上,Western blotting检测显示抗体具有特异性。AMH蛋白在花卵巢颗粒细胞表达。利用不同浓度的雌二醇处理花䱻60 d后发现,低浓度雌二醇(100 μg/g)促进花䱻卵母细胞生长,卵巢内皮质液泡数量增多。经雌二醇处理后,卵巢中Amh基因的表达量降低,说明雌激素在卵巢发育中对Amh基因表达具有抑制作用。【结论】Amh基因含有TGF-β家族典型的结构域,在卵巢颗粒细胞表达,且响应雌激素的调控,可能在花䱻卵巢发育中发挥调控作用。

     

    Abstract: 【Objective】In the present study, the anti-Müllerian hormone gene(Amh) in Hemibarbus maculatus was cloned. The sequence characteristics and subcellular localization were analyzed. Meanwhile, the expression changes under different concentrations of estrogen treatment were detected. The results will provide further theorial basis for the study of Amh gene function and its molecular mechanism in ovarian development.【Method】 The cDNA sequence of Amh gene was cloned and bioinformatically analyzed based on the transcriptome sequencing of spotted steed. The recombinant expression plasmid pET32a-Amh was construc-ted, transformed into Escherichia coli BL21, and polyclonal antibodies were prepared by immunizing mice with the purified recombinant protein. The subcellular localization of the protein was determined by immunofluorescence technique. Meanwhile, quantitative real-time PCR was used to detect the expression of Amh gene after estradiol treatment.【Result】The full length cDNA sequence of spotted steed Amh gene was 1849 bp, with an open reading frame(ORF) of 1671 bp that encoding 556 amino acid residues. Phylogenetic analysis showed that the spotted steed Amh gene clustered with Cyprinidae fish such as zebrafish and common carp, indicating that they were closely related. The purified AMH recombinant protein was immunized in mice to prepare AMH polyclonal antibody, and its antibody potency was above 2.4×106 by enzyme-linked immunoassay(ELISA), and Western blotting showed that the antibody was specific.AMH protein was expressed in granulosa cells of spotted steed ovary. After treated with estradiol for 60 d, it was found that low concentration of estradiol(100 μg/g) promoted the growth of spotted steed oocytes and the number of cortical follicles. After treated with estradiol, the expression of Amh gene was reduced, indicating that estrogen inhibited Amh gene during ovary development.【Conclusion】Amh gene contains a structural domain typical of the TGF-β family, it is expressed in ovarian granulosa cells, and responds to estrogen regulation, and may play a regulatory role in spotted steed ovary development.

     

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