林麝干扰素α基因克隆、表达及转录调控分析

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

摘要/Abstract

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。

关键词: 林麝, 干扰素, 毕赤酵母, 转录调控

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

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.

图/表 12

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4 干扰素α三级结构对比 A-E：分别标注5个α-螺旋结构

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

图5 FMD-IFNα成熟蛋白质的保守结构域预测

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

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

图11 FMD-IFNα对FMD-C1细胞增殖的抑制作用

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

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