梅花鹿激活素A重组蛋白真核表达、纯化及其生物活性的测定

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

【目的】体外真核表达梅花鹿(Cervus nippon)激活素A重组蛋白并测定其生物活性,为进一步明确激活素A在梅花鹿卵母细胞体外成熟过程中的生理学功能提供基础。【方法】利用RT-PCR技术克隆梅花鹿激活素βA (ActivinβA, ACTBA)亚基基因的cDNA全长序列,同时利用生物信息学对梅花鹿激活素βA的基因特征进行分析。在NCBI数据库下载其他物种激活素βA同源序列,利用Clustalx和MEGA 4软件进行同源比对并构建进化树。构建pcDNA4/ACTBA重组质粒并转染至CHO细胞内,进行目的蛋白的体外表达。采用免疫荧光及Western blot技术对目的蛋白表达情况进行检测,并通过Ni-NTA亲和层析柱对蛋白产物进行纯化。采用Western blot检测了梅花鹿激活素A重组蛋白处理后的猪颗粒细胞中的SMAD2和SMAD3的磷酸化水平。最后采用荧光定量PCR及Western blot检测了梅花鹿激活素A重组蛋白处理后的猪颗粒细胞中类固醇激素合成相关酶的表达量。【结果】克隆得到的梅花鹿ACTBA亚基基因含有1 278 bp的碱基,编码426个氨基酸。同源性比较发现梅花鹿ACTBA基因与牛的ACTBA基因同源性最高达98.4%同源。进化分析表明该基因与同为偶蹄目动物的反刍兽牛和山羊亲缘关系最为接近。经酶切、PCR和测序方法鉴定,成功构建真核表达质粒pcDNA4/ACTBA。免疫荧光显示该质粒在CHO细胞中主要定位在细胞质。Western blot结果显示激活素A的前体蛋白分子量约为58 kD左右。镍亲和层析纯化后的激活素A重组蛋白显著增加SMAD2和SMAD3的磷酸化,表明激活素A可以激活SMAD信号通路。同时成熟的激活素A重组蛋白可以诱导猪颗粒细胞芳香化酶(Aromatase)蛋白表达量上调,类固醇生成急性调节蛋白(steroidogenic acute regulatory protein, StAR)表达量下调,FSH受体基因表达量升高,LH受体基因表达量降低,但胆固醇侧链裂解酶(Cholesterolside-chaincleavageenzyme,CYP11A1orP450scc)及3β-羟基类固醇脱氢酶(3β-Hydroxysteroid dehydrogenase, 3β-HSD)基因表达量不变。结果表明梅花鹿激活素A重组蛋白可以增强FSH对颗粒细胞的生物学作用,也可以减弱LH对颗粒细胞的生物学作用。【结论】成功构建了激活素A蛋白的真核表达载体,并获得了较高纯度的、具有生物学活性的梅花鹿激活素A重组蛋白,为下一步激活素A蛋白的生物学功能和生理学机制研究奠定了基础。

【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 pc DNA4/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(St AR). 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.