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 PDF

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CN112326963A
CN112326963A CN202011236849.0A CN202011236849A CN112326963A CN 112326963 A CN112326963 A CN 112326963A CN 202011236849 A CN202011236849 A CN 202011236849A CN 112326963 A CN112326963 A CN 112326963A
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顾敏
刘娇
刘秀梵
王晓泉
胡娇
胡顺林
刘晓文
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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

真核表达的A型流感病毒PB2cap蛋白的应用
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
Figure BDA0002766986530000041
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。
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<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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<213> 人工序列（artificial sequence）
<400> 2
ggaaaaagat ctgctagctc gattagtggt gatggtgatg atgcatttta ctaactctca 60
ctcctc 66
<210> 3
<211> 44
<212> DNA
<213> 人工序列（artificial sequence）
<400> 3
catcattttg gcaaagaatt atgaggatca gttcatcttt tagc 44
<210> 4
<211> 66
<212> DNA
<213> 人工序列（artificial sequence）
<400> 4
ggaaaaagat ctgctagctc gattagtggt gatggtgatg atgcatttta ctaactctca 60
ctcctc 66
<210> 5
<211> 557
<212> DNA
<213> 人工序列（artificial sequence）
<400> 5
catcattttg gcaaagaatt atgaggatca gttcatcctt cagcttcgga ggtttcactt 60
ttaaaaggac aagtgggtca tctgtcaaaa gggaagaaga agtgctcaca ggcaacctcc 120
aaacattgaa aataagagta catgaaggat atgaggaatt cacaatggtt gggagaagag 180
caacagccat tctaaggaaa gcaaccagaa gactgattca actgatagtg agtgggaaag 240
acgagcaatc aatcgccgag gcaatcatag tggcaatggt gttctcacaa gaggattgta 300
tgataaaggc agtgagaggt gatttgaact ttgtcaacag agcgaaccag cggctaaatc 360
ccatgcatca actcctgagg catttccaaa aggatgcaaa ggtcctgttc caaaactggg 420
gaattgagcc cattgacaat gtaatgggga tgatcggaat attgcctgac atgaccccca 480
gcacagagat gtccttgaga ggagtgagag ttagtaaaat gcatcatcac catcaccact 540
aatcgagcta gcagatc 557
<210> 6
<211> 557
<212> DNA
<213> 人工序列（artificial sequence）
<400> 6
catcattttg gcaaagaatt atgaggatca gttcatcttt tagctttgga ggattcactt 60
tcaaaagaac aagtgggtca tctgtcaaga aggaagagga agtgcttacg ggcaacctcc 120
aaacactgaa aataagagta catgaggggt atgaggaatt cacaatggtt gggcgaagag 180
caacagctat cctgaggaaa gcaaccagaa ggctgattca gctgatagta agtggaagag 240
atgaacaatc aatcgctgaa gcgatcattg tagcaatggt gttctcacag gaagattgca 300
tgataaaagc agtcagaggt gatctgaatt tcgtaaatag agcaaatcaa aggttaaacc 360
ccatgcatca actccttagg cacttccaaa aagatgcaaa ggtgctattt cagaactggg 420
gaattgaacc tattgacgat gtcatgggga tgatcggaat attacctgac atgactccaa 480
gcacagagat gtcactgaga ggagtaagag ttagtaaaat gcatcatcac catcaccact 540
aatcgagcta gcagatc 557

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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