CN109206502B - 一种猫干扰素ω及其制备方法和在抗病毒中的应用 - Google Patents
一种猫干扰素ω及其制备方法和在抗病毒中的应用 Download PDFInfo
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- CN109206502B CN109206502B CN201811126498.0A CN201811126498A CN109206502B CN 109206502 B CN109206502 B CN 109206502B CN 201811126498 A CN201811126498 A CN 201811126498A CN 109206502 B CN109206502 B CN 109206502B
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Abstract
本发明公开了一种猫干扰素ω及其制备方法和在抗病毒中的应用。所述的猫干扰素ω的氨基酸序列如SEQ ID NO.2所示。优选的,编码所述的猫干扰素ω的核苷酸序列如SEQ ID NO.1所示。此外,本发明还公开了一种制备所述的猫干扰素ω的方法及其在制备抗病毒药物中的应用。本发明采用病毒刺激(猫肠道冠状病毒)结合干扰素诱生剂刺激(聚肌胞poly I:C)方法对宠物猫体进行刺激,进而诱导猫干扰素基因表达。然后通过PCR扩增得到了一条猫干扰素ω基因,细胞试验显示本发明获得的一种猫干扰素ω具有明显的抗病毒活性,并优于现有已知猫干扰素。本发明的提出为猫干扰素制品的进一步研发和应用奠定了基础。
Description
技术领域
本发明涉及一种新型的猫干扰素ω及其制备方法和在抗病毒中的应用。本发明属于兽药技术领域。
背景技术
干扰素是机体受病毒感染时产生的一种抗病毒细胞因子,由被感染细胞释放以抑制病毒的增殖,具有广谱抗病毒和增强免疫应答的作用。现已确定的干扰素有两大类:I型和II型。I型干扰素主要包括IFN-α、IFN-β和IFN-ω;II型干扰素主要有IFN-γ。目前,人用干扰素研究进展迅速,如基因工程重组人干扰素α已被广泛用于人类病毒感染的治疗,重组人干扰素ω也已用于SARS的治疗。
近年来,我国宠物猫饲养量在增长迅速,已成为人们休闲生活一部分,而诸多病毒病却严重危害着宠物猫的健康和生存。干扰素是治疗病毒感染性疾病最为有效的治疗药物,而目前对猫干扰素的研究和临床制剂的开发还相对滞后,同时已公布的猫干扰素序列也比较有限。
本发明公开了一种新型猫干扰素ω及其编码基因序列,并通过基因工程方法制备了该新型猫干扰素以及进行了抗病毒活性测定,本发明为猫干扰素生物制品的研发提供了物质基础。
发明内容
本发明的目的在于提供一种新型的猫干扰素ω及其制备方法。
为了达到上述目的,本发明采用了以下技术手段:
本发明采用病毒刺激(猫肠道冠状病毒)结合干扰素诱生剂刺激(聚肌胞poly I:C)方法对宠物猫体进行刺激,进而诱导猫干扰素基因表达。然后根据GenBank中已发布的猫干扰素ω基因序列,通过DNAMAN进行基因序列比对,使用Oligo6设计了一对简并引物用以扩增猫干扰素ω基因,最终本发明获得了一种新型的编码猫干扰素ω的基因序列,通过测序及BLAST比对发现,其与已发布的猫干扰素ω基因序列同源性较高,属于猫干扰素ω新成员,核苷酸序列如SEQ ID NO.1所示,编码的氨基酸序列如SEQ ID NO.2所示。本发明通过基因工程方法制备了该新型猫干扰素并对其进行了抗病毒活性测定,结果发现本发明的新型猫干扰素ω具有良好的抗病毒活性,有效抗病毒浓度为0.65mg/mL,优于已知猫干扰素,显示了良好的应用潜力。
在上述研究的基础上,本发明提出了一种新型的猫干扰素ω,所述的猫干扰素ω的氨基酸序列如SEQ ID NO.2所示。
编码所述猫干扰素ω的核苷酸序列、含有所述核苷酸序列的重组表达载体以及含有所述重组表达载体的宿主细胞也在本发明的保范围之内。其中,优选的,所述的核苷酸序列如SEQ ID NO.1所示。
进一步的,本发明还提出了所述的猫干扰素ω在制备抗病毒药物中的应用。
其中,优选的,所述的药物用于治疗或预防猫的病毒感染性疾病。
其中,优选的,所述的病毒包括猫肠道冠状病毒和猫细小病毒。
更进一步的,本发明还提出了一种制备所述的猫干扰素ω的方法,包括以下步骤:
(1)获得编码所述猫干扰素ω的核苷酸序列,并且在该序列的两端连接Kpn I和BamH I酶切位点;
(2)重组表达载体pCold-TF-ω的构建
分别将表达载体pCold-TF及步骤(1)获得的序列经限制性内切酶Kpn I和BamH I双酶切处理,采用DNA胶回收试剂盒回收目的基因片段,进而将步骤(1)获得的序列经T4连接酶连入表达载体pCold-TF,构建重组表达载体,命名为pCold-TF-ω;
(3)重组菌pCold-TF-ω/BL21的构建
将重组表达载体pCold-TF-ω转化BL21感受态细胞,涂布于含有氨苄抗性的LB琼脂平板进行培养,挑选阳性重组菌,命名为pCold-TF-ω/BL21;
(4)诱导表达
过夜涂板活化重组菌pCold-TF-ω/BL21,挑取单菌落接种于含浓度为100μg/ml氨苄的LB液体培养基中,37℃震荡培养过夜;取新培养菌液按体积比1:10比例转接至含氨苄抗性的LB液体培养基中,37℃培养至OD600 0.4-0.6时,加入终浓度为1mmol/L的IPTG,37℃诱导6h;取诱导后的重组菌液,离心,弃上清,加入PBS重悬菌体并进行超声处理,然后加入5×SDS样品裂解液,沸水浴10min,离心,取上清,将重组融合蛋白经His-tag标签亲和纯化,再经HRV 3C蛋白酶处理及His-tag标签亲和纯化,最终获得所述的猫干扰素ω。
其中,优选的,所述的编码所述猫干扰素ω的核苷酸序列如SEQ ID NO.1所示。
相较于现有技术,本发明的有益效果是:
1、本发明公开了一种新型的猫干扰素ω及其编码基因序列,并基因工程方法制备了该猫干扰素ω,满足了市场上对于猫干扰素的需求。
2、细胞试验显示本发明公开的一种新型猫干扰素ω具有明显的抗病毒活性,并优于现有已知猫干扰素。本发明为猫干扰素制品的进一步研发和应用奠定了基础。
附图说明
图1为PCR扩增结果;
M:DNA Marker DL2000;1:PCR产物;2:阴性对照;
图2为新型猫干扰素ω的三级结构;
图3为重组表达载体pCold-TF-ω的鉴定结果;
M:DNA Marker 8000;1:重组质粒pCold-TF-ω经Kpn I-BamH I双酶切结果;2:阴性对照;
图4为猫干扰素ω的诱导表达图;
A.重组蛋白可溶性表达:M.蛋白标准分子量;1.重组菌pCold-TF-ω/BL21经诱导结果;2.空载体菌pCold-TF/BL21经诱导结果;3.未经诱导重组菌pCold-TF-ω/BL21.B.猫干扰素ω蛋白的纯化结果:M.蛋白标准分子量;1.本发明猫干扰素ω蛋白;
图5为本发明猫干扰素ω的有效抗病毒活性;
图6为猫干扰素α的有效抗病毒活性;
图7为猫干扰素ω2的有效抗病毒活性;
图8为猫干扰素ω9的有效抗病毒活性。
具体实施方式
下面结合具体实施例对本发明做进一步说明,但下述实施例不会在任何方面限制本发明的保护范围。
实施例1新型猫干扰素ω的获得
1、引物设计与序列分析
根据GenBank中已发布的猫干扰素ω基因序列,通过DNAMAN进行基因序列比对,使用Oligo6设计了一对简并引物用以扩增猫干扰素ω基因全长,在引物5′端和3′端引入BamHI和Kpn I限制性酶切位点(用于构建表达猫干扰素的重组表达载体)。引物序列见表1所示,引物由吉林省库美生物科技有限公司合成。
表1引物序列
2.猫基因组RNA的提取
本发明采用病毒刺激(猫肠道冠状病毒)结合干扰素诱生剂刺激(聚肌胞poly I:C)方法对宠物猫体进行刺激,进而诱导猫干扰素基因表达。具体操作如下:
将猫肾细胞F81接种于96孔细胞板,置于37℃、5%CO2培养箱培养至细胞长成单层,然后接入猫肠道冠状病毒,37℃吸附1h后加入维持液,置于37℃、5%CO2温箱继续培养,逐日观察细胞病变情况,待细胞病变(CPE)达85%以上,将细胞进行冻融处理,以3000r/min离心10min收获病毒液,测定病毒TCID50。取100TCID50的病毒液与0.5mg poly I:C混匀,对宠物猫体进行注射,连续3天,每天注射1次。第5天时,将猫实施安乐死,在无菌条件下获取猫脾脏,剪碎并经液氮冷冻研磨至粉末状,移入离心管中;加入2mL Trizol裂解细胞,吸取800μL经裂解后的细胞液置于1.5mL离心管中,加入1/5体积的预冷氯仿,振荡混匀15s,12000rpm、4℃离心15min;取上清,加入等体积的预冷异丙醇,4℃作用10min,12000rpm、4℃离心10min;弃上清,用预冷75%乙醇12000rpm、4℃离心10min;弃上清,室温干燥,加入30μL去离子水溶解沉淀,获得总RNA。然后,使用Superscript reverse transcriptase reagentkit将提取的总RNA反转录成cDNA,保存于-80℃冰箱中备用。
3、PCR扩增
以得到的cDNA为模板,进行猫干扰素基因的扩增。
PCR反应体系为:10×PCR Buffer 2.5μL、dNTPs 3μL、Taq DNA聚合酶0.5μL、引物对(Primer-F和Primer-R,10uM)2μL、cDNA模板5μL、ddH2O 12μL。
PCR反应条件:95℃5min;95℃30s,55℃45s,72℃30s,共35循环;72℃终延伸10min。
PCR产物经1%琼脂糖凝胶电泳进行检测,结果见图1所示。将PCR产物经DNA胶回收试剂盒回收,测序并对其序列进行BLAST比对,结果见表2所示,经PCR扩增获得的基因序列与已发布的猫干扰素ω基因序列同源性较高,属于猫干扰素ω新成员,其核苷酸序列如SEQID NO.1所示,编码的氨基酸序列如SEQ ID NO.2所示。
表2本发明基因序列与已知猫干扰素基因同源性BLAST比对结果
4.干扰素三级结构预测
使用SWISS-MODEL软件对本发明干扰素的蛋白质三级结构进行预测,预测结果如图2所示。
实施例2新型猫干扰素ω的大量表达
1、重组表达载体pCold-TF-ω的构建
分别将表达载体pCold-TF及实施例1获得的新型猫干扰素ω的PCR产物经限制性内切酶Kpn I和BamH I双酶切处理,采用DNA胶回收试剂盒回收目的基因片段,进而将新型猫干扰素ω编码基因PCR产物经T4连接酶连入表达载体pCold-TF,构建重组表达载体pCold-TF-ω。
2、重组菌pCold-TF-ω/BL21的构建
将重组表达载体转化BL21感受态细胞,涂布于含有氨苄抗性的LB琼脂平板进行培养,挑选阳性重组菌,采用酶切法(结果见图3所示)进行鉴定,重组菌命名为pCold-TF-ω/BL21。
3、诱导表达
过夜涂板活化重组菌pCold-TF-ω/BL21,挑取单菌落接种于5mL含浓度为100μg/ml氨苄的LB液体培养基中,37℃震荡培养过夜。取新培养菌液按1:10比例转接至20mL含氨苄抗性的LB液体培养基中,37℃培养至OD600约0.5时,加入终浓度为1mmol/L的IPTG,37℃诱导6h。取2mL诱导后的重组菌液,12000rpm离心5min,弃上清,加入400μL PBS重悬菌体并进行超声处理,然后加入100μL5×SDS样品裂解液,沸水浴10min,12000rpm离心10min,取上清进行12%SDS-PAGE分析,结果可见重组蛋白获得原核可溶性表达(见图4A)。将重组融合蛋白(标签蛋白+目的蛋白)经His-tag标签亲和纯化,再经HRV 3C蛋白酶处理及His-tag标签亲和纯化,最终获得本发明猫干扰素ω蛋白(见图4B)。
实施例3新型猫干扰素ω的抗病毒活性测定
以猫肠道冠状病毒和猫细小病毒为模式病毒,采用微量细胞病变抑制法测定本发明得到的新型猫干扰素ω的抗病毒活性。具体操作如下:
将猫肾细胞F81接种于96孔细胞板,置于37℃、5%CO2培养箱培养至细胞单层,分别加入2倍倍比稀释的本发明新型猫干扰素ω作用16h,然后每孔加入100TCID50的病毒液,同时设置阴性对照组以及已知猫α、ω2和ω9干扰素阳性对照组,24h后观察细胞病变情况。
表3干扰素抗病毒活性对比结果
注:+表示无细胞病变(即有抗病毒活性),-表示有细胞病变(即无抗病毒活性)。
抗病毒活性检测结果见表3以及图5-8,从该结果可以看出本发明的新型猫干扰素ω具有良好的抗病毒活性,有效抗病毒浓度为0.65mg/mL(见图5所示),本发明一种新型猫干扰素ω的抗病毒活性优于已知猫α干扰素(0.93mg/mL)(见图6所示)、已知猫ω2干扰素(0.87mg/mL)(见图7所示)及已知猫ω9干扰素(0.91mg/mL)(见图8所示)抗猫肠道冠状病毒和猫细小病毒活性,显示了良好的应用潜力。
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<110> 东北农业大学
<120> 一种猫干扰素ω及其制备方法和在抗病毒中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 609
<212> DNA
<213> cattus
<400> 1
atggccctcc tgctccctct gctgactcct ttggctttgt tgacttgtag accaggtcgt 60
tctttgggtt gcgccttgcc aggttccgac gctcaggttt ctagagacaa cctggttttg 120
ttgggtcaga tgagaagatt gtccccattc ttgtgcttga gagctagaaa ggatttcaga 180
ttccctagag agatgttgga gcgtggtcag ctgcgtgagg ctcaagccgc tgctcccgtt 240
ttgagagagc tgttgcaaca aaccttcaac ctgttgcata ccgagagatc ctctgctgcc 300
tggtctccag ctccattgca cggtctgaga tccggtttgc atagacagtt ggaagccttg 360
gatgcttgtt tgttgcaagc cactcgtgag ggtgagagag ctactggtga gggtgagaga 420
gccccaggta tgcacggtcc agttttggct attaagagat acttccagga cattagagtt 480
tacttggagg acgagggtta ctccgactgt gcttgggaga ttgttagatt ggagatcatg 540
agagctttgt tctcttctgc tactttgcaa gactccttcg ctattaagga tggagacctg 600
ggctcatct 609
<210> 2
<211> 203
<212> PRT
<213> cattus
<400> 2
Met Ala Leu Leu Leu Pro Leu Leu Thr Pro Leu Ala Leu Leu Thr Cys
1 5 10 15
Arg Pro Gly Arg Ser Leu Gly Cys Ala Leu Pro Gly Ser Asp Ala Gln
20 25 30
Val Ser Arg Asp Asn Leu Val Leu Leu Gly Gln Met Arg Arg Leu Ser
35 40 45
Pro Phe Leu Cys Leu Arg Ala Arg Lys Asp Phe Arg Phe Pro Arg Glu
50 55 60
Met Leu Glu Arg Gly Gln Leu Arg Glu Ala Gln Ala Ala Ala Pro Val
65 70 75 80
Leu Arg Glu Leu Leu Gln Gln Thr Phe Asn Leu Leu His Thr Glu Arg
85 90 95
Ser Ser Ala Ala Trp Ser Pro Ala Pro Leu His Gly Leu Arg Ser Gly
100 105 110
Leu His Arg Gln Leu Glu Ala Leu Asp Ala Cys Leu Leu Gln Ala Thr
115 120 125
Arg Glu Gly Glu Arg Ala Thr Gly Glu Gly Glu Arg Ala Pro Gly Met
130 135 140
His Gly Pro Val Leu Ala Ile Lys Arg Tyr Phe Gln Asp Ile Arg Val
145 150 155 160
Tyr Leu Glu Asp Glu Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg
165 170 175
Leu Glu Ile Met Arg Ala Leu Phe Ser Ser Ala Thr Leu Gln Asp Ser
180 185 190
Phe Ala Ile Lys Asp Gly Asp Leu Gly Ser Ser
195 200
Claims (9)
1.一种猫干扰素ω,其特征在于,所述的猫干扰素ω的氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述的猫干扰素ω的核苷酸序列。
3.如权利要求2所述的核苷酸序列,其特征在于,所述的核苷酸序列如SEQ ID NO.1所示。
4.一种重组表达载体,其特征在于,含有权利要求2或3所述的核苷酸序列。
5.一种宿主细胞,其特征在于,含有权利要求4所述的重组表达载体。
6.权利要求1所述的猫干扰素ω在制备抗病毒药物中的应用,其特征在于,所述的药物用于治疗或预防猫的病毒感染性疾病。
7.如权利要求6所述的应用,其特征在于,所述的病毒包括猫肠道冠状病毒和猫细小病毒。
8.一种制备权利要求1所述的猫干扰素ω的方法,其特征在于,包括以下步骤:
(1)获得编码权利要求1所述的猫干扰素ω的核苷酸序列,并且在该序列的两端连接Kpn I和BamH I酶切位点;
(2)重组表达载体pCold-TF-ω的构建
分别将表达载体pCold-TF及步骤(1)获得的序列经限制性内切酶Kpn I和BamH I双酶切处理,采用DNA胶回收试剂盒回收目的基因片段,进而将步骤(1)获得的序列经T4连接酶连入表达载体pCold-TF,构建重组表达载体,命名为pCold-TF-ω;
(3)重组菌pCold-TF-ω/BL21的构建
将重组表达载体pCold-TF-ω转化BL21感受态细胞,涂布于含有氨苄抗性的LB琼脂平板进行培养,挑选阳性重组菌,命名为pCold-TF-ω/BL21;
(4)诱导表达
过夜涂板活化重组菌pCold-TF-ω/BL21,挑取单菌落接种于含浓度为100μg/ml氨苄的LB液体培养基中,37℃震荡培养过夜;取新培养菌液按体积比1:10比例转接至含氨苄抗性的LB液体培养基中,37℃培养至OD6000.4-0.6时,加入终浓度为1mmol/L的IPTG,37℃诱导6h;取诱导后的重组菌液,离心,弃上清,加入PBS重悬菌体并进行超声处理,然后加入5×SDS样品裂解液,沸水浴10min,离心,取上清,将重组融合蛋白经His-tag标签亲和纯化,再经HRV 3C蛋白酶处理及His-tag标签亲和纯化,最终获得所述的猫干扰素ω。
9.如权利要求8所述的方法,其特征在于,编码权利要求1所述的猫干扰素ω的核苷酸序列如SEQ ID NO.1所示。
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CN112500472B (zh) * | 2020-12-04 | 2022-09-23 | 广州源博医药科技有限公司 | 一种猫ω干扰素突变体及其制备方法和应用 |
CN112717123A (zh) * | 2020-12-29 | 2021-04-30 | 泰州博莱得利生物科技有限公司 | 一种猫鼻支滴眼液及其应用 |
CN112695053B (zh) * | 2021-02-05 | 2022-03-29 | 华中农业大学 | 一种重组猫干扰素的制备方法 |
CN113121672B (zh) * | 2021-04-20 | 2023-01-03 | 甘肃农业大学 | 猫干扰素γ的可溶性原核表达和纯化方法及应用 |
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