生物技术通报 ›› 2022, Vol. 38 ›› Issue (1): 194-204.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0177
杨威1(), 伍茜1, 程建国2, 罗燕1(), 王印1, 杨泽晓1, 姚学萍1
收稿日期:
2021-02-23
出版日期:
2022-01-26
发布日期:
2022-02-22
作者简介:
杨威,男,硕士,研究方向:预防兽医学;E-mail: 基金资助:
YANG Wei1(), WU Xi1, CHENG Jian-guo2, LUO Yan1(), WANG Yin1, YANG Ze-xiao1, YAO Xue-ping1
Received:
2021-02-23
Published:
2022-01-26
Online:
2022-02-22
摘要:
干扰素在病毒性疾病的防治上,具有很高的临床应用前景。为了林麝干扰素的研究与应用,使用同源克隆方法首次克隆得到9条林麝干扰素α基因序列,序列全长均为570 bp,编码189个氨基酸,前23个氨基酸为信号肽,具有4个保守的半胱氨酸残基和5个保守的脯氨酸残基。9种亚型之间,核酸序列同源性为97.0%-99.6%,氨基酸序列同源性为93.7%-99.5%。蛋白结构主要由5个α-螺旋和无规则卷曲构成,三级结构与牛和人干扰素α高度相似。结构域预测包含干扰素受体IFNAR结合位点。首次构建了林麝干扰素α表达质粒并成功使用毕赤酵母表达出林麝干扰素α,确定了1%的最佳甲醇诱导浓度与96 h的最佳诱导时间。使用qPCR首次确定了林麝干扰素α对林麝肺成纤维细胞FMD-C1干扰素刺激基因具有转录调控作用,并具有剂量依赖性,调控作用在6 h达到最强。CCK-8法确定了林麝干扰素α可以抑制FMD-C1细胞的增殖,抗增殖作用也具有剂量依赖性。以上结果为林麝干扰素α的抗病毒研究与临床应用提供了理论基础。
杨威, 伍茜, 程建国, 罗燕, 王印, 杨泽晓, 姚学萍. 林麝干扰素α基因克隆、表达及转录调控分析[J]. 生物技术通报, 2022, 38(1): 194-204.
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 |
表1 引物序列
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 |
图1 FMD-IFNα的克隆及鉴定 A:FMD-IFNα的RT-PCR扩增(M:DL2 000 maker;1:阴性对照;2:目的片段);B:重组质粒pMD19-T-FMD-IFNα鉴定(M:DL2 000 Maker:1:阴性对照;2:目的片段)
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)
图2 各亚型FMD-IFNα氨基酸序列同源性 灰色背景氨基酸为保守半胱氨酸残基位点;黑色方框氨基酸为保守脯氨酸位点;BosIFN-αA:NP_001017411.1;SusIFN-α4:NM_001166319.1;HomoIFN-α2:NM_000605.4
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
图3 IFNα氨基酸序列系统发生树
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
图6 PCR产物的琼脂糖凝胶电泳图 A:FMD-IFNα表达片段PCR产物(M:DL2000 maker;1-4:阳性转化子;5:阴性对照);B:pPICZα-IFNα的鉴定PCR产物(M:DL2 000 maker;1-5:阳性转化子;6:阴性对照)
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)
图7 阳性毕赤酵母转化子PCR鉴定结果 A:ExIFNα-F/R鉴定结果(M:DL2000 maker;1-3:阳性转化子);B:AOX1-F/R鉴定结果(M:DL2000 maker;1:阴性对照;2-4:阳性转化子)
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)
图8 FMD-IFNα初步诱导结果 A:SDS-PAGE结果图(M:彩色预染蛋白分子量标准(15-120 kD);1:重组酵母表达上清);B:Western blot结果图(M:彩色预染蛋白分子量标准(15-120 kD);2:重组酵母表达上清)
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)
图9 重组毕赤酵母诱导表达优化结果 A:时间优化结果(M:彩色预染蛋白分子量标准(15-120 kD);1-6分别是诱导0 h,24 h,48 h,72 h,96 h,120 h的表达上清液); B:浓度优化结果(M:彩色预染蛋白分子量标准(15-120 kD);1-5泳道分别是甲醇浓度0.5%,1%,1.5%,2%,2.5%诱导表达上清液)
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%)
图10 FMD-IFNα调控ISG转录结果 A:剂量依赖性结果;B:时间依赖性结果;*:P<0.05
Fig. 10 Regulation results of FMD-IFNα on ISG transcription A:Dose-dependent results. B:Time-dependent results. *:P<0.05
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