Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (5): 1058-1070.doi: 10.3864/j.issn.0578-1752.2020.05.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

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:xubaozeng@hotmail.com

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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

Table 1

Primers sequences and real-time PCR amplification parameters"

基因
Gene		 GenBank登陆号
GenBank accession No.		 上下游引物 (5′-3′)
Primer		 产物长度
Product size (bp)		 退火温度
Annealing temperature (℃)
Aromatase NM_214427.1 CGCTCAGTCCTGGTCAAAGG
ACCAGGAGAGGGGATCTCAC		 141 60.0
P450scc NM_214429.1 ACCAGCATGGTGTCTGAAGTT
GACCTGGTATTGAAGATGTGTTTT		 109 60.0
StAR NM_213755.2 TTAAGCTGTGTGCTGGGAGC
CCATGACCCTGAGGTTGGAC		 128 60.0
3β-HSD NM_001004049.2 TCGTCCACTTGTTGCTGGAG
TGCTCTGGAGCTTAGAAAATTCC		 102 60.0
LHR NM_214449.1 ACATAACCACCGTACCAGCA
GGAAGGCGTCATTGTGCATC		 177 60.0
FSHR NM_214386.3 TCTGGATTTGGGGACCTGGA
GAAGGCATCAGGGTCGATGT		 150 60.0
GAPDH XM_021091114.1 GTATGATTCCACCCACGGCA
CACCCCATTTGATGTTGGCG		 122 60.0

Table 2

Antibody information table"

抗体名字
Name of antibody		 生产厂家
Manufacturer		 种属（单抗/多抗）
Species raised (Monoclonal or polyclonal)		 稀释比例
Dilution ratio
Smad2(L16D3) Cell Signaling Technology Mouse (M) 1:1000
p- Smad2(Ser465/467) Cell Signaling Technology Rabbit (M) 1:1000
Smad3(C67H9) Cell Signaling Technology Rabbit (M) 1:1000
p- Smad3(Ser423/425) Cell Signaling Technology Rabbit (M) 1:1000
P450 Aromatase BIO-RAD Mouse (M) 1:200
StAR(ab96637) abcam Rabbit (P) 1:1000
6X His tag(ab9108) abcam Rabbit(M) 1:1000
β-actin(49900) abcam Mouse (M) 1:10000
GAPDH(181602) abcam Rabbit (M) 1:5000
Goat Anti-Mouse (HRP) (ab6728) abcam Goat (P) 1:5000
Goat Anti-Rabbit (HRP) (ab6721) abcam Goat (P) 1:5000

Fig. 1

Amplification of ACTBA gene M. DNA Marker(DL2000); 1. PCR product of ACTBA"

Fig. 2

Identification of recombinant plasmid pcDNA4/ACTBA by single and double restriction endonuclease digestion M1. DNA Marker (DL10000); 1. Digested product of pcDNA4/myc-His with BamH I; 2. Digested product of pcDNA4/ACTBA with BamH I; 3. Digested product of pcDNA4/ACTBA with BamH I and Hind III; M2. DNA Marker (DL10000)"

Fig. 3

Analysis of nucleotide acid homology of CDS region of ACTBA genes"

Fig. 4

Analysis of phylogenetic tree based on the ACTBA gene"

Fig. 5

Transient expressions of ACTBA gene in CHO cells (×200) A1,A2,A3: The CHO cells transfected with pcDNA4/ACTBA (A1: Cell stained with DAPI, A2: Expression of ACTBA-His, A3: Merge);B1,B2,B3: The CHO cells transfected with pcDNA4/myc-His (B1: Cell stained with DAPI, B2: Negative control, B3: Merge)"

Fig. 6

Western blot detected the expression of recombinant plasmid in the cells of CHO 1: Sonication product of transfected recombinant plasmid pcDNA4/ACTBA of CHO; 2: Sonication product of transfected empty vector pcDNA4/myc- His of CHO"

Fig. 7

SDS-PAGE detected the expression of Activin A 1: Culture medium of transfected empty vector pcDNA4/myc-His of CHO; 2: Culture medium of transfected recombinant plasmid pcDNA4/ACTBA of CHO（SDS-PAGE loading buffer, non-reducing）"

Fig. 8

Western blot detected the expression of Activin A A: Culture medium of transfected recombinant plasmid pcDNA4/ACTBA of CHO (SDS-PAGE Loading buffer, non-reducing); B: Culture medium of transfected recombinant plasmid pcDNA4/ACTBA of CHO (SDS-PAGE loading buffer, reducing)"

Fig. 9

Precursor and mature forms of Activins"

Fig. 10

The effect of Activins A on the expression of SMAD signal pathway protein"

Fig. 11

Effects of Activins A on Aromatase and StAR expression in pig granulosa lutein cells A: P450 aromatase and StAR mRNA levels were measured by RT-qPCR; B: P450 aromatase and StAR protein levels were measured by Western blot"

Fig. 12

Effects of Activins A on P450scc, 3β-HSD, FSHR and LHR expression in pig granulosa lutein cells"

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