Cloning,Expression and Transcriptional Regulation of Interferon-α in Forest Musk Deer

doi:10.13560/j.cnki.biotech.bull.1985.2021-0177

Abstract

Abstract:

Interferon（IFN）has a high clinical application prospect in the prevention and treatment of viral diseases. In order to study and apply the IFN-α of forest musk deer（FMD）,nine IFN-α gene sequences of FMD were cloned for the first time via homologous cloning method. Bioinformatics analysis showed that all the sequences were 570 bp and encoding 189 amino acids. Amino acid 1 to 23 were signal peptides. All of them had 4 conserved cysteine residues and 5 conserved proline residues. Among the 9 subtypes,the nucleotide sequence homology was 97.0%-99.6%,and the amino acid sequence homology was 93.7%-99.5%. The secondary structure was mainly composed of 5 α-helices and random coil. The tertiary structure was highly similar to bovine and human IFN-α,and domain predicted that it contained interferon receptor IFNAR binding sites. The expression plasmid of FMD IFN-α was constructed for the first time,and the IFN-α was successfully expressed by Pichia pastoris. The optimal methanol concentration of 1% and the optimal induction time of 96 h were determined. qPCR was used to confirm that IFN-α of FMD regulated the transcription of interferon-stimulated gene in lung fibroblasts FMD-C1 in a dose-dependent manner,and the regulatory effect reached the maximum at 6 h. CCK-8 assay confirmed that FMD IFN-α inhibited the proliferation of FMD-C1 cells in a dose-dependent manner. These results provide a theoretical basis for the antiviral research and application of IFN-α from FMD.

Key words: forest musk deer, interferon, Pichia pastoris, transcriptional regulation

YANG Wei, WU Xi, CHENG Jian-guo, LUO Yan, WANG Yin, YANG Ze-xiao, YAO Xue-ping. Cloning,Expression and Transcriptional Regulation of Interferon-α in Forest Musk Deer[J]. Biotechnology Bulletin, 2022, 38(1): 194-204.

Figures/Tables 12

Table 1 Primer sequence

引物名Primer name	引物序列Primer sequence（5'-3'）	Tm/℃	产物长度Product length/bp
IFN-α	F：ATGGCCCCAGCCTGGTC R：TCAGTCCTTTCTCCTGAATCTCTCC	58	570
ExIFNα	F：CGGAATTCTGCCACCTGCCTCACAC R：ATAAGAATGCGGCCGCGTCCTTTCTCCTGAATCTCTCC	57	522
OAS	F：TGCTGACCTCGTCGTCTTCC R：TGGGGGACCTCAGCACAAAG	58	194
Viperin	F：TGGTGCCCGAGTCTAACCAG R：TCCATACATATTTCCCTCCTCGC	57	195
Mx1	F：TCAACCTCCACCGAACTG R：TCTTCTTCTGCCTCCTTCTC	56	172
ISG15	F：CAGCCAACCAGTGTCTG R：CCTAGCATCTTCACCGTCAG	56	79
ISG56	F：TGGACTGTGAGGAAGGATGG R：GGCGATAGACAACGATTGC	56	142
β-actin	F：GAATCCTGCGGCATTCACG R：TCTTCATCGTGCTGGGTGC	58	172

Fig. 1 Cloning and identification of FMD-IFNα A：RT-PCR of FMD-IFNα. M：DL2 000 maker（1：Negative control. 2：Target fragment）. B：Identification of recombinant plasmid pMD19-T-FMD-IFNα. （M：DL2 000 maker. 1：Negative control. 2：Target fragment）

Fig. 2 Homology of amino acid sequence of each subtype of FMD-IFNα The gray background amino acids are conserved cysteine residue positions. The black boxed amino acids are conserved proline positions. BosIFN-αA：NP_001017411.1, SusIFN-α4：NM_001166319.1, HomoIFN-α2：NM_000605.4

Fig. 3 Phylogenetic tree of IFNα amino acid sequence BosIFN-αA：NP_001017411.1, CapralIFN-α：NP_001272633.1, CervicapraIFN-α：ACR61636.1,OvisIFN-α：XP_004005368.4, SusIFN-α4：NP_001159791.1, HomoIFN-α2：NP_000596.2

Fig. 4 Comparison of the tertiary structure of interferon-α A-E：Mark 5 α-helical structures respectively

Fig. 5 Prediction of the conserved domains of FMD-IFNα

Fig. 6 Agarose gel electrophoresis of PCR products A：FMD-IFNα expression fragment（M：DL2000 maker. 1-4：Positive transformants. 5：Negative control）. B：Identification PCR product of pPICZα-IFNα（M：DL2000 maker. 1-5：Positive transformant. 6：Negative control）

Fig. 7 PCR identification results of positive Pichia pastoris transformants A：Identification result of ExIFNα-F/R（M：DL2 000 maker. 1-3：Positive transformants）. B：Identification result of AOX1-F/R（M：DL2 000 maker. 1：Negative control. 2-4：Positive transformants）

Fig. 8 Preliminary expression results of FMD-IFNα A：SDS-PAGE results（M：Color pre-stained protein molecular weight standard（15-120 kD）. 1：Recombinant yeast expression supernatant）. B：Western blot results（M：Color pre-stained protein molecular weight standard（15-120 kD）. 2：Recombinant yeast expression supernatant）

Fig. 9 Recombinant Pichia pastoris induced expression optimization results A：Time optimization results（M：Color pre-stained protein molecular weight standard（15-120 kD）. 1-6 are expression supernatant after induced 0 h,24 h,48 h,72 h,96 h,and 120 h,respectively）. B：Concentration optimization results（M：Molecular weight standard of color predyed protein（15-120 kD）. 1-5 are expression supernatants induced by methanol concentration 0.5%,1%,1.5%,2%,and 2.5%）

Fig. 10 Regulation results of FMD-IFNα on ISG transcription A：Dose-dependent results. B：Time-dependent results. *：P<0.05

Fig. 11 Inhibitory effect of FMD-IFNα on the proliferation of FMD-C1 cells

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