CN112326963A - 真核表达的A型流感病毒PB2cap蛋白的应用 - Google Patents
真核表达的A型流感病毒PB2cap蛋白的应用 Download PDFInfo
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Abstract
本发明属于基因工程领域,具体涉及A型流感病毒PB2cap蛋白的真核表达及其应用。本发明公开了真核表达的A型流感病毒PB2cap蛋白在检测A型流感病毒RNA帽结合活性中的应用。本发明构建了基于真核表达载体pCAGGS的真核表达质粒pCAGGS‑PB2cap(aa:318‑483),并经瞬时转染将PB2cap进行真核表达,通过该表达蛋白与m7GTP在体外的结合能力来评估流感病毒的RNA帽结合活性,可为A型流感病毒PB2蛋白功能的差异研究提供新的检测指标。
Description
技术领域
本发明属于基因工程领域,具体涉及A型流感病毒PB2cap蛋白的真核表达及其应用。
背景技术
流感病毒属于正黏病毒科,包括A、B、C、D四型;其中A型流感病毒(Influenza Avirus,IAV)的宿主谱最广,可感染人、禽、猪、马、犬等多种动物并能引起流感大流行,具有重要的公共卫生意义(郑腾,陈枝华.禽流感的历史和公共卫生意义[J].生物学通报,2002,37(4):13-15.)。IAV是单股、负链RNA病毒,其基因组分为8个节段,分别编码PB2、PB1、PB1-F2、PA、PA-X、HA、NP、NA、M1、M2、NS1、NS2等10余种病毒蛋白(Mostafa A,Abdelwhab EM,Mettenleiter TC,et al.Zoonotic Potential of Influenza A Viruses:AComprehensive Overview[J].Viruses,2018,10(9):497.)。
IAV的生命周期中,聚合酶蛋白(PB2、PB1、PA)与核蛋白(NP)共同催化宿主细胞核内病毒RNA的转录和复制(Reich S,Guilligay D,Pflug A,et al.Structural insightinto cap-snatching and RNA synthesis by influenza polymerase[J].Nature,2014,516(7531):361-366.)。转录过程中,PB2亚基首先与宿主mRNA 5’末端的帽(cap)结构结合(Fechter P,Mingay L,Sharps J,et al.Two aromatic residues in the PB2 subunitof influenza A RNA polymerase are crucial for cap binding[J].J BiolChem,2003,278(22):20381-20388.;Ulmanen I,Broni BA,Krug RM,et al.Role of two of theinfluenza virus core P proteins in recognizing cap 1structures(m7GpppNm)onRNAs and in initiating viral RNA transcription[J].Proc Natl AcadSci USA,1981,78(12):7355-7359.),随后被具有内切核酸酶活性的PA亚基在距5’帽结构下游8-14nt的位置切割(Dias A,Bouvier D,Crepin T,et al.The cap snatching endonuclease ofinfluenza virus polymerase resides in the PA subunit[J].Nature,2009,458(7240):914-918.;Fodor E,Crow M,Mingay LJ,et al.A single amino acid mutationin the PA subunit of the influenza virus RNA polymerase inhibitsendonucleolytic cleavage of capped RNAs[J].J Virol,2002,76(18):8989-9001.;Hara K,Schmidt FI,Crow M,et al.Amino acid residues in the N-terminal regionof the PA subunit of influenza A virus RNA polymerase play a critical role inprotein stability,endonuclease activity,cap binding,and virion RNA promoterbinding[J].J Virol,2006,80(16):7789-7798.),而聚合酶蛋白的另一亚基PB1可进一步将这种短帽RNA延长,用于合成病毒自身mRNA(Biswas SK,Nayak DP,et al.Mutationalanalysis of the conserved motifs of influenza A virus polymerase basicprotein 1[J].J Virol,1994,68(3):1819-1826.;Poch O,Sauvaget I,Delarue M,etal.Identification of four conserved motifs among the RNA-dependent polymeraseencoding elements[J].EMBO J,1989,8(12):3867-3874.)。因此,PB2亚基的cap-bingding结构域(PB2cap)以其独特的“抓帽”机制起始病毒的转录和复制,具有至关重要的作用。
发明内容
为了解决现有技术存在的问题,本发明提供了构建pCAGGS-PB2cap真核表达质粒以及真核表达、鉴定、纯化PB2cap蛋白的方法,及真核表达的PB2cap蛋白的应用。
本发明公开了真核表达的A型流感病毒PB2cap蛋白在检测A型流感病毒RNA帽结合活性中的应用。
本发明中通过PCR扩增出PB2蛋白第318-483位氨基酸cap-binding结构域(aa:318-483)的基因片段(即PB2cap),并在3’端添加组氨酸标签(6xHis:CATCATCACCATCACCAC),将其与经EcoRⅠ、XhoⅠ双酶切后的线性真核表达载体pCAGGS连接。进一步将成功构建的真核表达质粒pCAGGS-PB2cap转染293T细胞,48h后收集细胞裂解物,利用镍(Ni+)亲和层析柱分离纯化得到PB2cap与组氨酸标签的真核表达融合蛋白。
本发明还公开了利用真核表达的PB2cap蛋白在体外与m7GTP的结合能力来评估流感病毒的RNA帽结合活性,为A型流感病毒PB2蛋白功能的差异研究提供新的检测指标。
具体的RNA帽结合实验方法,包括以下步骤:
1)吸取100μL m7GTP琼脂磁珠(Jena Bioscience:EDA-m7GTP-ATTO 488)转移至新1.5mL指形管;
2)500rpm离心30s沉淀琼脂磁珠,小心地弃去上清液;
3)加入500μL TBS(pH 8.0)重悬琼脂磁珠,重复步骤2)洗去琼脂磁珠保存液进行平衡,共洗涤重复平衡2次;
4)在平衡好的m7GTP琼脂磁珠中加入50μg真核表达PB2cap蛋白,4℃摇床缓慢晃动结合3h;
5)500rpm离心30s沉淀琼脂磁珠,小心地弃去上清液;
6)加入500μL洗涤液(50mM Tris-HCl pH8.0、200mM NaCl、2mM DTT)重悬琼脂磁珠,重复步骤5)洗去未结合蛋白,共重复洗涤5次;
7)为去除非特异性结合,加入600μL含1mM m7GTP(Sigma:7-Methylguanosine5′-triphosphate)的洗涤液,4℃摇床缓慢晃动结合1h;
8)500rpm离心30s沉淀琼脂磁珠,小心地弃去上清液;再次进行洗涤,弃去上清;
9)在琼脂磁珠中加入50μL 2x loading buffer,煮沸后进行SDS-PAGE考马斯亮蓝染色或Western-Blot分析。
本发明构建了基于真核表达载体pCAGGS的真核表达质粒pCAGGS-PB2cap(aa:318-483),并经瞬时转染将PB2cap进行真核表达,通过该表达蛋白与m7GTP在体外的结合能力来评估流感病毒的RNA帽结合活性,可为A型流感病毒PB2蛋白功能的差异研究提供新的检测指标。
附图说明
图1pCAGGS-MZ34-PB2cap、pCAGGS-SH14-PB2cap真核表达质粒的菌液PCR鉴定电泳图。
图2pCAGGS-MZ34-PB2cap真核表达质粒中***的外源基因MZ34-PB2cap区域测序结果。注:所***外源基因MZ34-PB2cap的两端pCAGGS载体序列用下划线表示。
图3pCAGGS-SH14-PB2cap真核表达质粒中***的外源基因SH14-PB2cap区域测序结果。注:所***外源基因SH14-PB2cap的两端pCAGGS载体序列用下划线表示。
图4pCAGGS-MZ34-PB2cap(A)与pCAGGS-SH14-PB2cap(B)真核表达质粒的质粒图谱。注:环形质粒图谱右下角灰色阴影部分为***外源基因MZ34-PB2cap或SH14-PB2cap区域。
图5MZ34-PB2cap与SH14-PB2cap真核蛋白表达的Western-Blot鉴定图。
图6真核表达的不同毒株PB2cap蛋白的帽结合能力。
图7MZ34与SH14毒株PB2基因诱导的聚合酶活性。
图8含MZ34-PB2基因的重组病毒rMZ34与含SH14-PB2基因的重组病毒rMZ34-SH14PB2其PB2蛋白表达水平。
具体实施方式
以下实施例中所使用的实验方法若无特殊说明,均为常规方法。所用材料、试剂若无特殊说明,均可从商业途径得到。所用的材料和试剂如下:
高保真酶(2×TransStart FastPfu Fly PCR SuperMix)、同源重组试剂盒(pEASY-Uni Seamless Cloning and Assembly Kit)购自北京全式金公司;转染试剂购自Qiagen公司;Western细胞裂解液、His标签蛋白纯化试剂盒、BCA蛋白浓度测定试剂盒购自碧云天公司;m7GTP-immobilized agarose(EDA-m7GTP-ATTO 488)购自Jena Bioscience;真核表达载体pCAGGS购自BioVector质粒载体菌种细胞蛋白抗体基因保藏中心-NTCC典型培养物保藏中心。
1.同源重组法构建不同毒株(MZ34与SH14)PB2cap的真核表达质粒
根据相关文献报道的Cap-binding结构域范围(Schrauwen EJ,de Graaf M,Herfst S,et al.Determinants of virulence of influenza A virus[J].Eur JClinMicrobiol Infect Dis,2014,33(4):479-490.),分别以病毒株MZ34(A/chicken/Anhui/MZ34/2016(H5N6),PB2基因GenBank登录号为KY005860.1)、SH14(A/chicken/Shanghai/14/2001(H9N2),PB2基因GenBank登录号为EU753319.1)的pHW2000重组质粒为模板(刘娇,郝小利,李秀丽,等.G1-like与F/98-like进化谱系的PB2、M基因在H5N6亚型禽流感病毒重配中的竞争优势研究[J].中国预防兽医学报,2019,41(11):987-993),利用高保真DNA聚合酶和表1的克隆引物,PCR扩增质粒的PB2Cap-binding结构域(aa:318-483)(即PB2cap对应的基因编码区),并在3’端添加His标签(6xHis:CATCATCACCATCACCAC),回收扩增产物。按同源重组试剂盒说明书,将回收的目的片段与经EcoRⅠ、XhoⅠ双酶切后的线性真核表达载体pCAGGS连接,转化后,分别提取疑似阳性质粒进行菌液PCR鉴定和测序验证,最后分别构建pCAGGS-MZ34-PB2cap与pCAGGS-SH14-PB2cap这2个重组真核表达质粒。
表1同源重组法构建MZ34与SH14毒株PB2cap真核表达质粒的引物
注:同源臂用下划线表示;添加的His标签用斜体表示。
菌液PCR鉴定结果(图1)显示,真核表达重组质粒pCAGGS-MZ34-PB2cap、pCAGGS-SH14-PB2cap利用构建引物均能扩增出500bp左右的外源基因片段,而进一步的测序验证(或图2(SEQ ID NO.5)、图3(SEQ ID NO.6)表明外源基因无突变,***位置准确,二者均构建成功,质粒图谱如图4所示。
2.真核表达纯化MZ34-PB2cap与SH14-PB2cap蛋白
将成功构建的真核表达质粒pCAGGS-MZ34-PB2cap、pCAGGS-SH14-PB2cap分别取24μg转染至细胞培养皿(直径:10cm)中的293T细胞。转染48h后,弃去上清,使用Western细胞裂解液制备细胞裂解物。按His标签蛋白纯化试剂盒说明书,将裂解物离心后的上清液与Ni+柱4℃混合作用1h,混合液加入纯化管并经洗涤液洗涤8~10次后,用洗脱液进行蛋白洗脱3~5次,洗脱后的蛋白经PBS(pH 8.0)透析后利用BCA试剂盒检测蛋白浓度,最后用His标签的一抗进行蛋白验证。
Western-blot结果显示,MZ34与SH14毒株的真核表达PB2cap蛋白均表达成功(图5)且后期纯化效果较好,无杂蛋白。
3.体外帽结合实验
吸取100μL m7GTP后用TBS(pH 8.0)洗涤2次,在平衡好的m7GTP中分别加入50μg真核表达的pCAGGS-MZ34-PB2cap与pCAGGS-SH14-PB2cap蛋白,4℃摇床缓慢晃动结合3h后,500μL洗涤液(50mM Tris-HCl pH8.0、200mM NaCl、2mM DTT)重复洗5次,为去除非特异性结合,加入600μL含1mM m7GTP的洗涤液4℃摇床缓慢晃动结合1h后,再次进行洗涤,弃去上清,在琼脂磁珠中加入50μL 2x loading buffer,煮沸后进行SDS-PAGE考马斯亮蓝染色或Western-Blot分析。
Western-Blot及SDS-PAGE考马斯亮蓝染色均表明以pCAGGS作为载体进行真核表达的MZ34-PB2cap蛋白在体外同m7GTP的结合能力显著高于SH14-PB2cap蛋白(图6)。
值得注意的是,此结果也与其他测定MZ34、SH14毒株PB2蛋白功能差异的指标结果相一致:如图7所示,在其他RNP质粒(PB1、PA、NP)均为MZ34毒株来源的同等条件下,MZ34-PB2在293T细胞上24h诱导的聚合酶活性显著强于SH14-PB2;如图8所示,在重组病毒的其他7个基因片段(PB1、PA、HA、NP、NA、M、NS)均为MZ34毒株来源的同等情况下,含MZ34-PB2基因的重组病毒rMZ34在CEF细胞上感染前期3h、6h诱导的PB2蛋白表达水平强于含SH14-PB2基因的重组病毒rMZ34-SH14PB2。因此,我们认为本发明采用真核表达的PB2cap蛋白与mRNA的5’端帽子结构(m7GTP)进行结合能力的测定来在体外评估流感病毒的RNA帽结合活性的方法,可为PB2蛋白功能的差异研究提供新的检测指标。
最后应说明的是,以上所述仅为本发明的优选实施例而已,并不用于限制本发明。尽管参照前述优选实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述记载的技术进行修改,或对其中部分技术特征进行同等替换。凡在本发明的精神和原则以内所做的修改、等同替换等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 扬州大学
<120> A型流感病毒PB2cap蛋白的真核表达及其应用
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ccatgcatca actcctgagg catttccaaa aggatgcaaa ggtcctgttc caaaactggg 420
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Claims (4)
1.真核表达的A型流感病毒PB2cap蛋白在检测A型流感病毒RNA帽结合活性中的应用。
2.如权利要求1所述的应用,其特征在于,所述真核表达的A型流感病毒PB2cap蛋白通过下述方法得到:在cap-binding结构域的3’端添加组氨酸标签构建真核表达质粒,并通过将重组质粒转染293T细胞48h后,收取细胞裂解物,利用镍亲和层析柱分离纯化得到PB2cap与组氨酸标签的融合蛋白。
3.如权利要求2所述的应用,其特征在于,构建真核表达质粒过程中,真核表达载体为pCAGGS,酶切位点为EcoRⅠ、XhoⅠ。
4.如权利要求1所述的应用,其特征在于,用真核表达的PB2cap蛋白在体外与m7GTP进行帽结合实验,步骤如下:
1)吸取100μL m7GTP琼脂磁珠转移至新1.5mL指形管;
2)500rpm离心30s沉淀琼脂磁珠,小心地弃去上清液;
3)加入500μLTBS,pH8.0重悬琼脂磁珠,重复步骤2)洗去琼脂磁珠保存液进行平衡,共重复平衡2次;
4)在平衡好的m7GTP琼脂磁珠中加入50μg真核表达PB2cap蛋白,4℃摇床缓慢晃动结合3h;
5)500rpm离心30s沉淀琼脂磁珠,小心地弃去上清液;
6)加入500μL洗涤液重悬琼脂磁珠,重复步骤5)洗去未结合蛋白,共重复洗涤5次;
7)为去除非特异性结合,加入600μL含1mM m7GTP的洗涤液,4℃摇床缓慢晃动结合1h;
8)500rpm离心30s沉淀琼脂磁珠,小心地弃去上清液;
9)在琼脂磁珠中加入50μL2x loadingbuffer,煮沸后进行SDS-PAGE考马斯亮蓝染色或Western-Blot分析。
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