Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (1): 194-204.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0177
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YANG Wei1(), WU Xi1, CHENG Jian-guo2, LUO Yan1(), WANG Yin1, YANG Ze-xiao1, YAO Xue-ping1
Received:
2021-02-23
Online:
2022-01-26
Published:
2022-02-22
Contact:
LUO Yan
E-mail:cnyw809462194@163.com;Lycjg@163.com
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.
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 |
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. 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%)
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