CN101481415A - 一种新型prrs病毒受体及该受体的阻断抑制剂 - Google Patents
一种新型prrs病毒受体及该受体的阻断抑制剂 Download PDFInfo
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Abstract
本发明涉及一个新型PRRS病毒的细胞受体,即非肌肉肌球蛋白II型重连A(nonmuscle myosin heavy chain II-A,NMHC II-A)蛋白以及非肌肉肌球蛋白II型(nonmuscle myosin II)的抑制剂-blebbistatin,作为抑制PRRS病毒感染细胞的药物。本发明提供了一种利用纯化的NMHC II-A蛋白和人工合成的多肽及其blebbistatin抑制PRRS病毒感染细胞的方法。本发明还提供了利用NMHC II-A蛋白和多肽产生的抗体。这种通过纯化得到的NMHC II-A蛋白或人工合成方法获得的多肽及其blebbistatin以及抗NMHC II-A蛋白和抗多肽抗体都对PRRS病毒感染细胞有抑制活性,可开发成防治PRRS病毒感染的药物。
Description
技术领域
本发明涉及发现一个新型PRRS病毒细胞受体以及利用受体蛋白和多肽及其衍生物进行对PRRS病毒感染的防治,属于生物学领域。
背景技术
猪繁殖与呼吸综合征(Porcine reproductive and res piratory syndrome,PRRS)又名蓝耳病,是由PRRS病毒(PRRSV)感染所致的一种猪的急性传染病。PRRSV侵入宿主细胞首先通过与细胞膜表面上的特异性受体结合。利用细胞内吞作用而感染易感细胞(Nauwynck,HJ,Duan X,Favoreel HW,etc.J.Gen.Virol.1999,80:297-305),如MA-104和PAM。到目前为止已报道的四个独立但功能相关的PRRSV细胞受体,它们是唾液酸粘附素(Sialoadhesin)(Vanderheijden N,Delputte PL,Favoreel HW,etc.J.Virol.2003,77:8207-8215),似肝磷脂蛋白(Heparinlike)(Delputte PL,Vanderheijden N,Nauwynck,HJ,etc.J.Virol.2002,76:4312-4321),波形蛋白(Vimentin)(Kim JK,Fahad AM,Shanmukhappa K,etc.J.Virol.2006,80:689-696),清道夫受体(CD163)蛋白(Calvert JG,Slade DE,Shields SL,etc.J.Virol.2007.81:7371-7379)。但是还存在有其他的PRRSV细胞受体,这些受体均可结合或参与结合PRRSV感染易感细胞,从而引起蓝耳病。因此,通过对PRRSV的研究,依然会不断的有新的病毒受体出现,从而扩大研究的范围。
发明内容
本发明涉及一个新型PRRSV的细胞受体,同时提供了一种利用纯化的NMHCII-A蛋白和人工合成的多肽及其blebbistatin作为受体阻断剂,抑制PRRSV感染细胞的方法,同时可以作为受体阻断剂的还有利用NMHC II-A蛋白和多肽免疫小鼠所得的抗体。上述的各种阻断剂都对PRRSV感染细胞有抑制活性,可开发成防治PRRSV感染的药物。
申请人利用模拟PRRSV的GP5膜抗原的单克隆抗独特型抗体首先从非洲绿猴肾细胞系(MA-104)和猪肺巨噬细胞(PAM)上分离和提纯出一个可溶性蛋白(Zhou EM,Xiao YH,Shi YF,etc.J.Virol.Methods.2008,149:300-308)。
利用这个模拟PRRSV GP5抗原的单克隆抗独特性抗体,申请人发现了一个新的PRRSV细胞受体,经过分析其氨基酸序列所得该受体为即非肌肉肌球蛋白II型重连A(nonmuscle myosin heavy chain II-A,NMHC II-A)蛋白,其蛋白的羧基端的氨基酸序列为Seq ID No:1所示。
同时发现了该受体阻断和抑制剂,它们是NMHC II-A蛋白、NMHC II-A多肽、抗NMHC II-A蛋白和多肽抗体,以及非肌肉肌球蛋白II型(nonmuscle myosinII,NMM-II)抑制剂,即(+)-blebbistatin:是NMM-II的选择性抑制剂[(±)-或(-)-blebbistatin]的无活性的对映异构体。其中:
(a)NMHC II-A蛋白,蛋白的羧基端的氨基酸序列为Seq ID No:1所示;
(b)NMHC II-A人工多肽,其氨基酸序列为Seq ID No:2所示;
(c)NMHC II-A人工多肽,其氨基酸序列为Seq ID No:3所示;
(d)NMHC II-A人工多肽,其氨基酸序列为Seq ID No:4所示;
上述病毒受体及受体阻断和抑制剂,所针对的PRRSV主要包括经典美洲株(代表毒株为VR-2332),中国分离株(代表毒株为Ch-1a),以及高致病性毒株(代表毒株为JXA-1)。
而上述的非肌肉肌球蛋白II型(nonmuscle myosin II,NMM-II)抑制剂:(+)-blebbistatin与NMM-II反应而阻断PRRSV经典美洲株、中国分离株,以及高致病性毒株感染细胞。与(±)-或(-)-blebbistatin不同的是,(+)-blebbistatin不抑制细胞依赖NMM-II的细胞***和增殖(Straight AF,Cheung A,Limouze J,etc.Science.2003,299:1743-1747)。而(±)-blebbistatin一直是脊椎动物细胞上NMM-II ATP酶活性的选择性抑制剂,而由于发明人将NMHC II-A蛋白作为了PRRSV受体,同时经发明人的长期试验,发现(+)-blebbistatin是可以作为阻断PRRSV感染细胞的受体阻断剂,这在之前的研究中也一直未见应用。
其病毒受体非肌肉肌球蛋白II型重连A(nonmuscle myosin heavy chainII-A,NMHC II-A)蛋白的具体分离及鉴定过程为:
利用模拟PRRSV GP5膜抗原的单克隆抗体独特型抗体从MA-104和PAM细胞中提纯出一个可溶性蛋白(Zhou EM,Xiao YH,Shi YF,et al.J.Virol.Methods,2008,149:300-308);50μg上述可溶性蛋白用7.5% SDS-PAGE分离,分离出的蛋白用胃蛋白酶水解,水解片段经微毛细管高压液相层析分离,将分离片段用纳米电喷射离子化,经离子捕获质谱仪分析。将获得的水解片段序列与GenBank氨基酸序列库对比获得受体蛋白为非肌肉肌球蛋白II型重连A(NMHC II-A)。
上述(b)或(c)或(d)人工多肽的合成,具体方法是:根据NMHC II-A重连的氨基酸序列,用DNA Star软件分析序列的抗原表位,设计出20个多肽序列,通过固相多肽合成方法合成多肽,合成的多肽通过MBS或戊二醛方法与载体蛋白匙孔虫戚血蓝蛋白(Keyhole Limpet Hemocyanin,KLH)偶联。同时利用上述合成的人工多肽通过PRRSV阻断试验,证明了NMHC II-A羧基端内的23个氨基酸为PRRSV结合区域,因此羧基端具有与序列表中Seq ID No:1一致的蛋白,可以作为病毒的受体并作为相应的阻断剂。
NMHC II-A蛋白和多肽-KLH偶联产物与铝佐剂混合免疫小鼠产生抗NMHCII-A蛋白和多肽抗体,可采用腹腔免疫小鼠的方法制得,也可采用其他的抗体制备方法制得该血清抗体。如:与其它佐剂混合,采用肌肉或皮下免疫方法制得。具体的免疫方法可采用现有生物学中的免疫方法。
综上所述,由于发明人将羧基端具有与序列表中Seq ID No:1一致的蛋白,特别是非肌肉肌球蛋白II型重连A(nonmuscle myosin heavy chain II-A,NMHCII-A)蛋白作为了PRRSV受体,因此与之相关的上述的各种物质,均可以都对PRRSV感染细胞有抑制活性,可开发成防治PRRSV感染的药物。从而为PRRS的研究和防治提供了一个全新的思路,扩大了研究的范围,对于实际生产有着极为重要的意义。
具体实施方式
实施例1 新型PRRSV细胞受体的发现和鉴定
利用模拟PRRSV GP5膜抗原的单克隆抗体独特型抗体从MA-104和PAM细胞中提纯出一个可溶性蛋白(Zhou EM,Xiao YH,Shi YF,et al.J.Virol.Methods,2008,149:300-308):50μg可溶性蛋白用7.5%SDS-PAGE分离,分离出的蛋白用胃蛋白酶水解,水解片段经微毛细管高压液相层析分离,将分离片段用纳米电喷射离子化,经离子捕获质谱仪分析。将获得的水解片段序列与GenBank氨基酸序列库对比获得受体蛋白为非肌肉肌球蛋白II型重连A(NMHCII-A)。
实施例2 NMHC II-A人工多肽合成
根据NMHC II-A重连羧基端序列制定的多肽序列通过固相多肽合成方法合成多肽,具体方法是:
将2-Chlorotrityl Chloride Resin树脂与DMF混合振荡30-60min进行树脂溶涨;通过沙芯抽滤掉DMF,加入3倍摩尔过量C端第一个氨基酸与树脂连接,再加入10倍摩尔过量的DIEA,最后加入DMF溶解,振荡30-60min;去掉DMF,加20%哌啶DMF溶液,反应5-15min;取十几粒树脂,用乙醇洗三次,加入茚三酮,KCN,苯酚溶液各一滴,105℃-110℃加热5min对反应物进行检测。依次用DMF、甲醇和DMF洗涤两次。通过下列方法之一对连接产物进行缩合。
方法a:加入三倍过量的溶于DMF的保护氨基酸(FOMC-Asp-OH)和HBTU,再加入十倍过量的NMM,反应30-60min;
方法b:加入三倍过量的溶于DMF的保护氨基酸(FOMC-Asp-OH)和HOBt,再加入三倍过量的DIC,反应30-60min。
之后依次用DMF、甲醇和DMF洗涤两次。重复以上步骤,依次连接序列中的氨基酸。最后一个氨基酸连接后,依次用DMF、甲醇、DMF和DCM洗涤两次,抽干10-20min。用切割液(TFA 94.5%,水2.5%,EDT 2.5%,TIS 1%)切割120min将多肽从树脂上切割出来,用氮气吹干,用***洗六次,常温挥干。粗制的多肽用纯水或者加少量乙腈溶解,按照下列条件用HPLC纯化多肽。层析柱为VenusiMRC-ODS C18,30 x 250mm;A泵溶液为0.1%的Trifluoroacetic溶于100%的纯水;B泵溶液为0.1%的trifluoroacetic溶于100%的acetonitrile。最后将纯化后的多肽冻干保存-20度保存。
或通过其它方法也可合成多肽,如利用tert-butoxy-carbonyl或9-fluorenylmethoxycarbonyl的固相合成方法(Merrifield RB.J.AM.Chem.Soc.,1963,85:2149-2154;Chang CD and Merenhofer J.Int.J.Pept.ProteinRes.1978,11:246-249)。
实施列3 多肽与载体蛋白匙孔虫戚血蓝蛋白(Keyhole Limpet Hemocyanin,KLH)偶联
每个多肽通过下列方法之一与KLH偶联:
(1)MBS(M-Maleimidobenzoyl-N-hydroxysuccinimide)偶联剂偶联:5mg KLH用1mL PBS(0.1M,pH6.0)溶解,加入50uL DMSO(含1mg MBS)反应1小时,反应产物用HPLC分离,加入5mg多肽,反应3小时,真空冷冻干燥制得。
(2)戊二醛偶联:5mg KLH溶于去离子水,加入一定量的戊二醛以及5mg多肽,反应5-6小时,用碳酸氢钠和碳酸钠缓冲液透析24小时,真空冷冻干燥制得。
实施例4 抗NMHC II-A蛋白和人工多肽抗体的制备
蛋白和多肽-铝佐剂制备:NMHC II-A蛋白或多肽-KLH偶联产物溶于0.005MPBS,pH 6.2(终容积10mL),缓慢加入溶于0.005M PBS(pH 6.2)的硫酸钾铝(比例为50μg蛋白/0.4mg铝),调升pH至6.8-7.3,搅拌2小时,离心800g 10分钟,蛋白-铝佐剂沉淀用生理盐水洗涤一次,用0.1M的PBS溶解制得。蛋白和多肽也可与其它佐剂使用,如:福氏佐剂。
免疫程序:每只小鼠经腹腔免疫50μg NMHC II-A蛋白或人工多肽,共免疫三次,采集小鼠血清检测和鉴定抗体。抗NMHC II-A蛋白或人工多肽的抗体也可使用其它动物(如:家兔,山羊等)以及采用其它途径免疫(如:肌肉或皮下)。产生抗体的小鼠也可利用其脾脏通过细胞融合制备单克隆抗体。
实施例5 NMHC II-A蛋白、人工合成多肽及抗体的鉴定
间接ELISA法:首先,NMHC II-A蛋白及各个多肽用PBS(0.01M,pH7.2)包被ELISA板,每孔200ng,4C过夜。ELISA板用PBS-T(PBS含0.5%体积的Tween-20)洗涤3次后,用封闭液(2.5%浓度的脱脂奶粉溶于PBS-T,200μl/孔)封闭1小时,洗涤3次,每孔加入稀释的小鼠血清反应1小时,洗涤3次,每孔加入辣根过氧化物酶(HRP)标记的羊抗鼠IgG反应1小时,洗涤3次,每孔加入100μl HRP底物(TMB)反应15分钟,每孔加入50μl 1M硫酸终止反应,用酶标仪在450nm下读取每孔的OD值。检测结果见表1和表2。小鼠免疫之前的血清作为阴性对照血清,免疫后小鼠血清的OD值大于阴性对照的3倍则为阳性。由表1结果证实NMHC II-A蛋白具有免疫原性,血清抗体具有与NMHC II-A蛋白结合特性,其抗体效价可以达到1:100000。同时,这些血清抗体可以与人工合成多肽结合,其抗体效价可以达到1:10000。由表2结果证实人工合成的3个多肽所免疫的小鼠不但可以结合免疫用的多肽,还可以结合NMHC II-A蛋白,其抗体效价可以达到1:10000。
表1.抗NMHC II-A血清抗体的鉴定结果
表2-1.抗人工多肽(Seq ID No:2)血清抗体的鉴定结果
表2-2.抗人工多肽(Seq ID No:3)血清抗体的鉴定结果
表2-3.抗人工多肽(Seq ID No:4)血清抗体的鉴定结果
间接免疫荧光(IFA)法:将3次免疫后的小鼠抗NMHC II-A蛋白及抗多肽抗体血清稀释后与MA-104和PAM单层细胞结合0.5-2小时,用荧光标记的羊抗鼠IgG检测抗体与细胞的结合,检测结果见表3。由表3结果证实血清抗体具有与细胞结合特性,其抗体效价可以达到1:320。
表3.血清抗体结合MA-104和PAM细胞的间接免疫荧光检测结果
实施例6 NMM-II抑制剂(blebbistatin)对细胞的作用
(±)-blebbistatin是脊椎动物细胞上NMM-II ATP酶活性的选择性抑制剂,它抑制细胞卵裂沟的收缩,快速和可逆阻断细胞起泡,阻断细胞迁移以及胞质***(Straight AF,Cheung A,Limouze J,etc.Science.2003,299:1743-1747)。(-)-blebbistatin是(±)-blebbistatin的活性对映异构体,(+)-blebbistatin是(±)-blebbistatin的无活性对映异构体。Marc-145细胞和PAM(105个细胞/ml)培养于DMEM培养基(含青霉素100U/ml,硫酸链霉素50μg/ml,庆大霉素50μg/ml,10%小牛血清)中,37℃,5%CO2培养箱中培养成单层。将(±)-blebbistatin,(S)-(-)-blebbistatin和(R)-(+)-blebbistatin(购自美国Tocris Cookson公司)溶解于DMSO中,再用PBS倍比稀释,加入单层细胞培养板,连续培养并观察细胞病变(CPE)。CPE在普通显微镜下观察所见为细胞表面粗燥,折光性强,最后细胞脱落。由表4结果表明(±)-blebbistatin和(S)-(-)-blebbistatin抑制细胞增殖并造成CPE。然而,(R)-(+)-blebbistatin不抑制细胞增殖,无CPE。因此(+)-blebbistatin可作为PRRSV受体阻断剂。
表4.Blebbistatin对MA-104和PAM细胞的作用结果
实施例7 PRRSV受体阻断试验
Marc-145细胞和PAM(105个细胞/ml)培养于DMEM培养基(含青霉素100U/ml,硫酸链霉素50μg/ml,庆大霉素50μg/ml,10%小牛血清)中,37℃,5%CO2培养箱中培养成单层。将采用实施例1、2、4的方法得到的NMHC II-A蛋白、人工合成的多肽、血清抗体以及(+)-blebbistatin溶于DMEM培养基中,过滤除菌后与固定浓度(103TCID50/ml)的不同毒株的PRRSV混合孵育1小时后,加入上述细胞中,每个稀释度做8个重复,37℃吸附1小时后,去掉混合物,加入DMEM培养基,连续培养,每天观察CPE。PRRSV感染细胞所致的CPE表现为细胞表面粗燥,折光性强,最后细胞脱落。表5所示结果为NMHC II-A蛋白、人工合成的多肽、血清抗体以及(+)-blebbistatin在每孔浓度依次为μg、μg、μL和μM时抑制CPE的程度。判断细胞CPE的指标如下:“-”代表无CPE;“+”代表25%的细胞出现CPE;“++”代表50%的细胞出现CPE;“+++”代表75%以上的细胞出现CPE。由表5结果表明NMHC II-A蛋白和人工合成多肽(Seq ID No:2,3,4)在2μg-100μg/孔、抗NMHCII-A蛋白和抗人工合成多肽的血清在5μL-100μL/孔以及(+)-blebbistatin在2μM-100μM/孔都可以完全抑制PRRSV对细胞造成的CPE;说明NMHC II-A蛋白及其衍生物能够阻断PRRSV感染细胞,可用于开发防治PRRSV的药物。
表5.PRRSV受体阻断试验结果
<110>山东农业大学
<120>一种新型PRRS病毒受体及该受体的阻断抑制剂
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Claims (5)
1.一种新型PRRS病毒受体,其特征在于:蛋白的羧基端的氨基酸序列为Seq ID No:1所示。
2.根据权利要求1所述的病毒受体,其特征在于:所述的受体为非肌肉肌球蛋白II型重连A(nonmuscle myosin heavy chain II-A,NMHC II-A)蛋白。
3.一种阻断PRRS病毒感染细胞的受体阻断剂,选自具有如下特征之一的阻断剂:
(a)NMHC II-A蛋白,蛋白的羧基端的氨基酸序列为Seq ID No:1所示;
(b)人工多肽,其氨基酸序列为Seq ID No:2所示;
(c)人工多肽,其氨基酸序列为Seq ID No:3所示;
(d)人工多肽,其氨基酸序列为Seq ID No:4所示;
(e)利用(a)或(b)或(c)或(d)制得的血清抗体;
(f)非肌肉肌球蛋白II型(nonmuscle myosin II,NMM-II)抑制剂,即(+)-blebbistatin。
4.根据权利要求3所述的阻断PRRS病毒感染细胞的受体阻断剂,其特征在于:结合PRRS病毒或结合细胞受体,阻断PRRS病毒感染MA-104和PAM。
5.权利要求3所述的阻断PRRS病毒感染细胞的受体阻断剂,用于开发成防治PRRS疾病的药物。
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CN111450248A (zh) * | 2020-04-29 | 2020-07-28 | 西北农林科技大学 | 一种用于预防和治疗prrsv感染的抗体药物 |
CN111518194A (zh) * | 2020-05-07 | 2020-08-11 | 山东农业大学 | 具有抑制猪繁殖与呼吸综合症病毒感染活性的ecl2多肽及其应用 |
WO2020200272A1 (zh) * | 2019-04-04 | 2020-10-08 | 中国医学科学院阜外医院 | 人心肌细胞分离试剂、培养基、分离方法和培养方法 |
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CN110755612B (zh) * | 2019-10-22 | 2021-11-19 | 深圳市第三人民医院 | 蓝光激活(S)-blebbistatin分子杀灭耐药性鲍曼不动杆菌的应用及方法 |
CN110812481B (zh) * | 2019-11-01 | 2021-11-23 | 深圳市第三人民医院 | 以蓝光激活的blebbistatin类似物杀灭耐药细菌的应用及方法 |
CN111471104B (zh) * | 2020-04-29 | 2022-03-25 | 西北农林科技大学 | 一种prrsv广谱中和单克隆抗体及其应用 |
CN115006390B (zh) * | 2022-06-27 | 2024-04-19 | 国药集团动物保健股份有限公司 | 一种病毒减毒制剂、其制备方法以及应用 |
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CA2501222A1 (en) * | 2002-10-04 | 2004-10-21 | Mitsubishi Pharma Corporation | Antibody recognizing antigen |
WO2005035003A2 (en) * | 2003-09-22 | 2005-04-21 | Dihedron Corporation | Compositions and methods for increasing drug efficiency |
CN101125146B (zh) * | 2007-07-02 | 2011-05-04 | 中国药科大学 | 一种防治炎症相关心脑血管疾病的药物靶标及其抑制剂 |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2020200272A1 (zh) * | 2019-04-04 | 2020-10-08 | 中国医学科学院阜外医院 | 人心肌细胞分离试剂、培养基、分离方法和培养方法 |
CN111450248A (zh) * | 2020-04-29 | 2020-07-28 | 西北农林科技大学 | 一种用于预防和治疗prrsv感染的抗体药物 |
CN111450248B (zh) * | 2020-04-29 | 2023-02-28 | 西北农林科技大学 | 一种用于预防和治疗prrsv感染的抗体药物 |
CN111518194A (zh) * | 2020-05-07 | 2020-08-11 | 山东农业大学 | 具有抑制猪繁殖与呼吸综合症病毒感染活性的ecl2多肽及其应用 |
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US20120107932A1 (en) | 2012-05-03 |
CN101481415B (zh) | 2010-05-12 |
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