中國農業科學 ?? 2020, Vol. 53 ?? Issue (5): 1058-1070.doi: 10.3864/j.issn.0578-1752.2020.05.016

? 畜牧·獸醫·資源昆蟲 ? 上一篇    下一篇

梅花鹿激活素A重組蛋白真核表達、純化 及其生物活性的測定

張宇飛,曹滿園,王麗英,趙偉剛,李曉霞,常彤,許保增()   

  1. 中國農業科學院特產研究所/中國農業科學院特種經濟動物分子生物學重點實驗室,長春 130112
  • 收稿日期:2018-12-22 接受日期:2019-10-08 出版日期:2020-03-01 發布日期:2020-03-14
  • 通訊作者: 許保增 E-mail:[email protected]
  • 作者簡介:張宇飛,Tel:17743401925;E-mail:[email protected]
  • 基金資助:
    國家重點研發計劃(2018YFC1706601-03);中國農業科學院創新工程;吉林省國際合作項目(20170414049GH);中央級公益性科研院所基本科研業務費專項(1610342017025)

Eukaryotic Expression, Purification and Biological Activity of Recombinant Cervus Nippon Activin A Protein

ZHANG YuFei,CAO ManYuan,WANG LiYing,ZHAO WeiGang,LI XiaoXia,CHANG Tong,XU BaoZeng()   

  1. Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences/State Key Laboratory for Molecular Biology of Special Economic Animal, Chinese Academy of Agricultural Sciences, Changchun 130112
  • Received:2018-12-22 Accepted:2019-10-08 Online:2020-03-01 Published:2020-03-14
  • Contact: BaoZeng XU E-mail:[email protected]

摘要:

【目的】體外真核表達梅花鹿(Cervus nippon)激活素 A重組蛋白并測定其生物活性,為進一步明確激活素 A在梅花鹿卵母細胞體外成熟過程中的生理學功能提供基礎。【方法】利用RT-PCR技術克隆梅花鹿激活素βA (Activin βA, ACTBA)亞基基因的cDNA 全長序列,同時利用生物信息學對梅花鹿激活素βA的基因特征進行分析。在NCBI 數據庫下載其他物種激活素βA同源序列,利用Clustalx和MEGA 4 軟件進行同源比對并構建進化樹。構建pcDNA4/ACTBA重組質粒并轉染至CHO細胞內,進行目的蛋白的體外表達。采用免疫熒光及Western blot 技術對目的蛋白表達情況進行檢測,并通過Ni-NTA 親和層析柱對蛋白產物進行純化。采用Western blot檢測了梅花鹿激活素 A重組蛋白處理后的豬顆粒細胞中的SMAD2和SMAD3的磷酸化水平。最后采用熒光定量PCR及Western blot檢測了梅花鹿激活素 A重組蛋白處理后的豬顆粒細胞中類固醇激素合成相關酶的表達量。【結果】克隆得到的梅花鹿ACTBA亞基基因含有1 278 bp 的堿基,編碼426個氨基酸。同源性比較發現梅花鹿ACTBA基因與牛的ACTBA基因同源性最高達98.4%同源。進化分析表明該基因與同為偶蹄目動物的反芻獸牛和山羊親緣關系最為接近。經酶切、PCR和測序方法鑒定,成功構建真核表達質粒pcDNA4/ACTBA。免疫熒光顯示該質粒在CHO細胞中主要定位在細胞質。Western blot 結果顯示激活素 A的前體蛋白分子量約為58 kD左右。鎳親和層析純化后的激活素 A重組蛋白顯著增加SMAD2和SMAD3的磷酸化,表明激活素 A可以激活SMAD信號通路。同時成熟的激活素 A重組蛋白可以誘導豬顆粒細胞芳香化酶(Aromatase)蛋白表達量上調,類固醇生成急性調節蛋白(steroidogenic acute regulatory protein, StAR)表達量下調,FSH受體基因表達量升高,LH受體基因表達量降低,但膽固醇側鏈裂解酶(Cholesterol side-chain cleavage enzyme, CYP11A1 or P450scc)及3β-羥基類固醇脫氫酶(3β-Hydroxysteroid dehydrogenase, 3β-HSD)基因表達量不變。結果表明梅花鹿激活素 A重組蛋白可以增強FSH對顆粒細胞的生物學作用,也可以減弱LH對顆粒細胞的生物學作用。【結論】成功構建了激活素 A蛋白的真核表達載體,并獲得了較高純度的、具有生物學活性的梅花鹿激活素 A重組蛋白,為下一步激活素 A蛋白的生物學功能和生理學機制研究奠定了基礎。

關鍵詞: 梅花鹿, 激活素A, 顆粒細胞, 真核表達, 蛋白純化

Abstract:

【Objective】The objective of this study was to investigate the eukaryotic expression and biological activity of recombinant Cervus Nippon Activin A protein, which would provide an experimental basis for further clarification on the physiological function of Activin A in the maturation of the oocytes of Cervus Nippon. 【Method】 The full length cDNA of Activin βA (ACTBA) gene was acquired by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) technology. Bioinformatics tools were used to determine the characteristics of Activin βA sequence. The homologous sequences of Activin βA from other species were downloaded from NCBI, the deduced amino acid sequence of Activin βA was aligned by using the Clustal X (1.83) software, and the phylogenetic tree was constructed by using MEGA 4. Recombinant plasmids of pcDNA4/ACTBA were constructed, and then transfected into CHO cells to express target proteins in vitro. Target proteins were detected by Immunofluorescence technology and Western blot technology, and then purified by Ni-NTA affinity chromatography column. The effects of the treatment of purified Cervus Nippon Activin A on the phosphorylation of SMAD2 and SMAD3 proteins in porcine granulosa cells were investigated through Western blot, as well as the expression levels of steroid hormone-related enzymes in porcine granulosa cells treated with recombinant Cervus Nippon Activin A were detected by real-time PCR and Western blot. 【Result】 The Cervus Nippon Activin A was cloned, which contained 1 278 bp, encoding 426 amino acids. The homology comparison showed that the sequence of ACTBA gene in Cervus Nippon had the highest 98.4% identity with that in cattle. Phylogenetic analysis showed that it had the closest relationship with that in Bos taurus and Capra hircus. The data through endonuclease digestion, PCR and DNA sequencing showed the eukaryotic expression plasmid was constructed successfully. Immunofluorescent results showed that this plasmid expressed in CHO cells successfully, and Activin A protein mainly presented in the cytoplasm of CHO cells. Western blot data showed that the protein molecular weight of precursor Activin A was about 58 kD. Treatment with the purified recombinant mature Activin A through nickel affinity chromatography triggered the phosphorylation of SMAD2 and SMAD3 proteins in porcine granulosa cells, indicating that the functional Activin A could activate the SMAD signaling pathway. Treatment of primary porcine granulosa cells with mature Activin A increased the mRNA and protein levels of P450 aromatase and decreased the mRNA and protein levels of steroidogenic acute regulatory protein (StAR). Meanwhile, the treatment of mature Activin A also enhanced FSHR mRNA levels and decreased LHR mRNA levels in primary pig granulosa cells. Whereas it did not alter the mRNA levels of P450 side chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase in porcine granulosa cells. Thus, the recombinant protein of Cervus Nippon Activin A could enhance the biological effects of FSH in granulosa cells, and attenuate biofunctions of LH in granulosa cells. 【Conclusion】 The eukaryotic expression vector of Activin A protein was successfully constructed in our study. The recombinant Activin A protein had high purity and bioactivity, which provided a foundation for further study of the biological functions and physiological mechanisms of Activin A.

Key words: Cervus nippon, Activin A, granulosa cells, eukaryotic expression, protein purification

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