CN117070562B - 重组呼吸道合胞病毒蛋白疫苗及制备方法 - Google Patents
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Abstract
本发明涉及呼吸道合胞病毒技术领域,具体涉及重组呼吸道合胞病毒蛋白疫苗及制备方法。所述重组呼吸道合胞病毒蛋白疫苗以呼吸道合胞病毒的SH蛋白和G蛋白的融合蛋白为抗原成分。该融合蛋白通过重组表达载体于原核表达体系进行可溶性表达,便于收获抗原蛋白。该重组呼吸道合胞病毒蛋白疫苗能通过G和SH的抗原表位产生抗血清和免疫中和抗体,免疫原性高,产生的抗体滴度高。
Description
技术领域
本发明涉及呼吸道合胞病毒技术领域,具体涉及重组呼吸道合胞病毒蛋白疫苗及制备方法。
背景技术
呼吸道合胞病毒(respiratory syncytial virus,RSV),V属于副粘病毒科(Paramyxoviridae)肺病毒属(Pneumovirus),为具被膜的非节段单链RNA(负链)病毒。它能在呼吸道分泌物中存活4~7h,但在高于37℃、缓慢冻存和解冻过程中均易失活,但在快速冻存时稳定。RSV的基因组长度为15200nt,含有10个编码基因,共编码11个蛋白。其中多个蛋白的功能和序列都已明确。在这些蛋白中,只有被膜融合蛋白F(fusion protein)和黏附蛋白G(Gattachment glycoprotein)具有免疫原性,能诱导产生保护性免疫反应。G蛋白是一个高度糖基化的蛋白,目前,这两个蛋白的功能和序列都已比较清楚,是用于发展各种类型的RSV疫苗的优选抗原。其他的已经测序的蛋白还有磷蛋白PRNA聚合酶L等。其中,SH蛋白也是其编码包膜糖蛋白之一,但其功能尚不清楚。
根据RSV对不同的单克隆抗体的反应以及核苷酸序列差异,将其分成A、B两个亚型。两个亚型的RSV单独流行,但A型常占优势。A、B两亚型间的序列差异主要表现在G蛋白上,二者仅有53%的氨基酸同源性,而抗原相关性仅为5%。F蛋白在结构和抗原性方面变异不大,有89%的氨基酸同源性和53%的抗原相关性。值得注意的是,虽然G蛋白在A、B亚型间变异较大,但二者也存在相对保守的区域,而且已有试验证明,特定长度和区域的G蛋白片段具有诱导交叉保护的能力,因而成为发展各种类型的疫苗的首选抗原。然而,这些G蛋白或重组G蛋白疫苗由于其T细胞表位免疫缺陷,不能有效地产生体液免疫,保护作用单一。
发明内容
有鉴于此,本发明提供重组呼吸道合胞病毒蛋白疫苗及制备方法,以解决上述技术问题之一。
本发明的目的之一提供一种重组表达载体,所述重组表达载体含有编码呼吸道合胞病毒的SH蛋白和G蛋白的融合蛋白的核苷酸序列,所述核苷酸序列如SEQ ID NO.7所示。
本发明的目的之二提供所述的重组表达载体的制备方法,包括如下步骤:
(1)获得编码呼吸道合胞病毒的SH蛋白和G蛋白的融合蛋白的目的片段,所述目的片段如SEQ ID NO.7所示;
(2)将所述融合蛋白插入至表达载体中,以得到所述重组表达载体。
在所述制备方法中,所述表达载体为真核表达载体。进一步优选地,所述真核表达载体为pCDNA3.1或p3x-FLAG-CMV。
在所述制备方法中,步骤(1)包括:
(1-1)从呼吸道合胞病毒基因组RNA中反转录和扩增得到G基因目的片段和SH基因目的片段;所述G基因目的片段如SEQ ID NO.5所示,所述SH基因目的片段如SEQ ID NO.6所示;
(1-2)将所述G基因目的片段和所述SH基因目的片段混合进行互补延伸,以得到所述融合蛋白的目的片段。
在所述制备方法中,扩增所述G基因目的片段的引物对为:F1:tttttcgactaggatacctatttttGGGGCAAATGCAAACATG,小写序列为同源重组序列,如SEQ ID NO.1所示;R1:GCAAGCTTCTACTGGTTTGTTGATGTT,下划线为Hind III酶切位点。
在所述制备方法中,扩增所述SH基因目的片段的引物对为:F2:GCGAATTCACACGCCAGGCAAAATCAAC,下划线为EcoRI酶切位点,如SEQ ID NO.3所示,R1:tttttatccataggatcagctttttTGGCTTGCATGGTGAGATGT,小写序列为同源重组序列,如SEQ IDNO.4所示。
本发明的目的之三提供含有如权利要求1所述的重组表达载体的宿主细胞,所述宿主细胞为293T细胞、Hep-2细胞或DF-1细胞。
本发明的目的之四提供一种重组呼吸道合胞病毒蛋白疫苗的制备方法,包括以下步骤:
构建所述的表达载体;
将所述表达载体转入所述的宿主细胞;
培养和诱导宿主细胞,收获宿主细胞,纯化得到所述呼吸道合胞病毒的SH蛋白和G蛋白的融合蛋白;
以所述融合蛋白为抗原成分制备所述重组呼吸道合胞病毒蛋白疫苗。
本发明的目的之五提供所述的重组表达载体、所述的制备方法制得的重组表达载体或所述的宿主细胞在制备预防呼吸道合胞病毒感染的疫苗中的应用。
与现有技术相比,本发明至少具有以下有益效果之一:
本发明提供的重组呼吸道合胞病毒蛋白疫苗以呼吸道合胞病毒的SH蛋白和G蛋白的融合蛋白为抗原成分。该融合蛋白通过重组表达载体于真核表达体系进行可溶性表达。该重组呼吸道合胞病毒蛋白疫苗能通过G和SH的抗原表位产生抗血清和免疫中和抗体,免疫原性高,产生的抗体滴度高。
另外,本发明提供的重组蛋白疫苗还能有效激活特异性细胞毒性T淋巴细胞(CTL)反应;还能通过呼吸道合胞病毒糖蛋白的抗原表位直接阻断免疫抑制途径,提高调节性T细胞的活性;弥补G蛋白疫苗的T细胞表位免疫缺陷,以通过体液免疫有效地产生T淋巴细胞,增加保护模式,保护和预防力度更广,安全性更高。
最后,相比单一靶点治疗,本发明提供的重组蛋白疫苗能更全面、多途径预防呼吸道合胞病毒感染、消除呼吸道合胞病毒及其相关的疾病和肿瘤,利于疫苗在临床中的实际应用。
附图说明
图1为G基因目的序列(泳道1)、SH基因目的序列(泳道2)以及SH-G目的序列(泳道3)的电泳图。
图2为pUC18(泳道1)、pUC18-SH-G(泳道2)、pcDNA3.1(+)(泳道3)、pcDNA3.1(+)-SH-G(泳道4)、pcDNA3.1(+)-G(泳道5)、pcDNA3.1(+)-SH(泳道6)的电泳图。
图3为融合基因SH-G的相关SDS-PAGE图;泳道M为BSA;泳道1为融合基因诱导表达物经冷冻破碎和离心处理的上清液的条带;泳道2为融合基因诱导表达物经冷冻破碎和离心处理的上清液再经亲和层析所得蛋白条带;泳道3为融合基因诱导表达物经冷冻破碎和离心处理的沉淀物的条带;泳道4为融合基因诱导表达物经冷冻破碎和离心处理的沉淀物再经亲和层析所得蛋白条带。
图4为免疫印迹检测各重组表达蛋白的WB图;泳道1为融合基因诱导表达物经冷冻破碎和离心处理的上清液再经亲和层析所得蛋白条带;泳道2为融合基因诱导表达物经冷冻破碎和离心处理的沉淀物再经亲和层析所得蛋白条带;泳道3为G基因诱导表达物经冷冻破碎和离心处理的上清液再经亲和层析所得蛋白条带;泳道4为SH基因诱导表达物经冷冻破碎和离心处理的上清液再经亲和层析所得蛋白条带。
图5为实验组、对照组I、对照组II和阴性组的免疫血清抗体和中和抗体水平;图中“**”表示与阴性组(P<0.01)、对照组I(P<0.05)和对照组II(P<0.05)相比均具有统计学显著差异;“*”表示与阴性组相比具有统计学显著差异(P<0.05)。
图6为实验组、对照组I、对照组II和阴性组免疫血清对RSV感染的Hep-2细胞的抑制率;图中“**”表示与阴性组(P<0.01)、对照组I(P<0.01)和对照组II(P<0.01)相比均具有统计学显著差异;“*”表示与阴性组相比具有统计学显著差异(P<0.05)。
图7为实验组、对照组I、对照组II和阴性组的免疫小鼠肺部匀浆液中的INF-γ含量和IL-4含量比值结果;图中“**”表示与阴性组(P<0.01)、对照组I(P<0.01)和对照组II(P<0.01)均具有统计学显著差异。
图8为安全性研究中的实验组(A)、对照组I(B)、对照组II(C)和阴性组(D)的免疫小鼠肺部切片HE染色图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。本发明中未详细单独说明的试剂均为常规试剂,均可从商业途径获得;未详细特别说明的方法均为常规实验方法,可从现有技术中获知。
下述实施例中所用的方法如无特别说明均为常规方法,具体步骤可参见:《Molecular Cloning:A Laboratory Manual》(Sambrook,J.,Russell,David W.,Molecular Cloning:A Laboratory Manual,3rd edition,2001,NY,Cold SpringHarbor)。
实施例1:融合蛋白目的序列的获得
1、材料
本研究所用病毒为RSVA亚型(Long strain,ATCC VR-26),在Hep-2 cells(ATCCCCL-23)上增殖。克隆用的质粒和菌株为pUC18(产品编号:D2303-100μg,碧云天)。限制性内切酶、dNTP、砌DNA酶等购自TaKaRa公司;DEPc购自Sigma公司,病毒RNA分离试剂盒(Mini Kit,Cat.No.74104)购自Qiagen公司;DNA连接酶、cDNA合成试剂盒(Universal/>cDNA Synthesis System,Cat.No.4360)
2、Hep-2细胞的培养
取Hep-2冻存细胞管,置于4℃环境解冻2min,然后置于37℃温水中至完全解冻,转移至培养瓶,置于37℃培养4h后换液一次。待细胞长至单层后,进行传代培养后,备用。
3、RSV病毒的培养和收集
取生长至均匀覆盖50%~60%的Hep-2细胞;将病毒原液从-70℃冰箱中取出,于30℃左右温水中迅速阶段后,按10-1、10-2、10-3、10-4等将病毒样品维持液用细胞培养基作倍比稀释;先将lmL细胞培养基加入T75细胞瓶中,轻轻转动细胞瓶,使得细胞培养基均匀覆盖绸胞,然后将稀释均匀的病毒液lmL加入至细胞培养瓶中,健病毒液均匀细胞;置于37℃吸附lh,期间每隔15min摇动一次,使病毒充分感染细胞;补加8~10mL培养基,摇匀,37℃培养,每天至少观察1次细胞病变情况;在细胞病变数量于50%~70%时收获病毒,将收获的病毒液分装于l.5mLEp管中,保存于-70℃冰箱或者液氮气相备用。
4、RSV病毒的基因组RNA分离
本操作按照QiagenRNA提取试剂盒进行,具体步骤为:
(1)用0.25%胰酶上述收获的病毒液,500rpm离心5min,收集沉淀。
(2)加入350μL的RLT缓冲液裂解细胞(缓冲液使用前补加巯基乙醇),充分混匀裂解细胞以完全释放病毒;
(3)加入350μL70%乙醇,充分混匀,过柱,10000rpm离心15s,收集沉淀;
(4)加入700μL Rwl缓冲液洗挂,10000rpm离心15s,弃液体;然后再用500μL RPE缓冲液洗柱一次,10000rpm离心15s;
(5)再加入500μL RPE缓冲液洗柱一次,10000rpm离心2min;
(6)加入30~50μL无RNAase的超纯水,10000rpm离心1min洗脱RNA,-70℃保存备用。
5、RT-PCR
以上述的基因组RNA为样品,分别反转录合成G基因和SH基因的cDNA的第一链。合成反应体系为:mRNA 2.2μL,补水10.8μL,于70℃保温5min,立即冰上冷却5min后;补加15μLSample reaction,5μL First strand 5×buffer,40U/μL的RNasin 1μL,37℃保温3min,然后加入:2.5μL dNTP 10mM和1.5μL AMV 20U/μL。充分混合后,于37℃保温1h,70℃保温15min以失活残留酶活性。然后置于冰上,加入RNAase H 0.3μL(18U左右),37℃保温20min。
PCR扩增体系以50μL计包含:5.0μL 10×buffer,4.0μL 2.5mM dNTP,0.4μL 5U/μLTaqase,3μL cDNA,上游引物各1.5μL。扩增程序:94℃2min,94℃45s,45℃45s,72℃1min,30个循环,72℃整体延伸10min。
其中,G基因的引物:F1:tttttcgactaggatacctatttttGGGGCAAATGCAAACATG,小写序列为同源重组序列,如SEQ ID NO.1所示,R1:GCAAGCTTCTACTGGTTTGTTGATGTT,下划线为HindIII酶切位点,如SEQ ID NO.2所示。
SH基因的引物:F2:GCGAATTCACACGCCAGGCAAAATCAAC,下划线为Eco RI酶切位点,如SEQ ID NO.3所示,R2:tttttatccataggatcagctttttTGGCTTGCATGGTGAGATGT,小写序列为同源重组序列,如SEQ ID NO.4所示。
6、凝胶电泳回收G基因目的片段和SH基因目的片段
将上述G基因和SH基因的PCR产物分别进行电泳,切割目的片段,加入至5倍体积的TE(20mM,lmM EDTA,pH8.0),65℃5min;冷却至室温,Tris饱和酚抽提,20℃下4000rpm离心回收水相;50%酚-氯仿、氯仿各抽提一次,离心收集水相;加入0.3×体积的6.25M的乙酸铵,2×体积的-20℃预冷的无水乙醇,-20℃下沉淀30min;10000rpm离心15min,70%乙醇清洗一次,风干,加适量TE(10mM,lmM EDTA,pH8.0)溶解,即可得到G基因目的片段和SH基因目的片段。扩增产物用0.8%的琼脂糖凝胶电泳检测,确定目的条带,如图1泳道1和泳道2所示,测序可知G基因目的片段和SH基因目的片段如下。
G基因目的片段为:
5’-tttttcgactaggatacctatttttggggcaaatgcaaacatgtccaaaaacaaggaccaacgcaccaccaagacactagaaaagacctgggacactctcaatcatctattattcatatcatcgtgcttatacaagttaaatcttaaatctatagcacaaatcacattatccattctggcaatgataatctcaacttcacttataattgcagccatcatattcatagcctcggcaaaccacaaagtcacactaacaactgcaatcatacaagatgcaacaagccagatcaagaacacaaccccaacatacctcacccagaatccccagcttggaatcagcttctccaatctgtctgaaactacatcacaaaccaccaccatactagcttcaacaacaccaagtgtcaagtcaaccctgcaatccacaacagtcaagaccaaaaacacaacaacaaccaaaatacaacccagcaagcccaccacaaaacaacgccaaaacaaaccaccaaacaaacccaataatgattttcactttgaagtgttcaactttgtaccttgcagcatatgcagcaacaatccaacctgctgggctatctgtaaaagaataccaaacaaaaaacctggaaagaaaaccaccaccaagcccacaaaaaaaccaaccatcaagacaaccaaaaaagatctcaaacctcaaaccacaaaaccaaaggaagtacctaccaccaagcccacagaaaagccaaccatcaacaccaccaaaacaaacatcagaactacactgctcaccaacaataccacaggaaatccagaacacacaagtcaaaagggaaccctccactcaacctcctccgatggcaatccaagcccttcacaagtctatacaacatccgagtacctatcacaacctccatctccatccaacacaacaaaccagtagAAGCTTGC-3’,小写下划线为同源连接序列,大写下划线为Hind III酶切位点,如SEQ ID NO.5所示。
SH基因目的片段为:
5’-GCGAATTCacacgccaggcaaaatcaaccaatggaaaatacatccataacaatagaattctcaagcaaattctggccttactttacactaatacacatgataacaacaataatctctttgctaatcataatctccatcatgattgcaatactgaacaaactctgtgaatataacgtattccataacaaaacctttgagctaccaagagctcgagtcaatacatagcattcaccaatctgatggcacaaaacagtaaccttgcatttgtaagtgaacaaccctcacctctttacaaaaccacatcaacatctcaccatgcaagccatttttcgactaggatacctattttt,大写下划线为EcoRI酶切位点,小写下划线为同源连接序列,如SEQ ID NO.6所示。
7、融合蛋白SH-G的目的片段
测定各回收产物中目的片段浓度,在50μL体系中加入等摩尔的待融合片段(800ng),不添加引物,使用Pfu DNA Polymerase(购自Fermentas公司)进行各片段的互补延伸,以形成全长的融合PCR产物(中间产物)。反应体系在50μL体系中依次加入:5μL10×@Long PCR buffer(MgCl2 15mmol/L),2.5Ll dNTP s 10mM,5μL G蛋白目的片段,5μL SH蛋白目的片段,0.5μL Long PCR EnzymeMix,补水至50μL。反应程序:95℃40s,61℃50s,72℃3min,35cycles。扩增产物用0.8%的琼脂糖凝胶电泳检测,确定目的条带,切胶GelExtractionMiniKit回收。结果如图1泳道3所示。目的条带进行测序结果如下:
SH-G:
5’-GCGAATTCacacgccaggcaaaatcaaccaatggaaaatacatccataacaatagaattctcaagcaaattctggccttactttacactaatacacatgataacaacaataatctctttgctaatcataatctccatcatgattgcaatactgaacaaactctgtgaatataacgtattccataacaaaacctttgagctaccaagagctcgagtcaatacatagcattcaccaatctgatggcacaaaacagtaaccttgcatttgtaagtgaacaaccctcacctctttacaaaaccacatcaacatctcaccatgcaagccatttttcgactaggatacctatttttggggcaaatgcaaacatgtccaaaaacaaggaccaacgcaccaccaagacactagaaaagacctgggacactctcaatcatctattattcatatcatcgtgcttatacaagttaaatcttaaatctatagcacaaatcacattatccattctggcaatgataatctcaacttcacttataattgcagccatcatattcatagcctcggcaaaccacaaagtcacactaacaactgcaatcatacaagatgcaacaagccagatcaagaacacaaccccaacatacctcacccagaatccccagcttggaatcagcttctccaatctgtctgaaactacatcacaaaccaccaccatactagcttcaacaacaccaagtgtcaagtcaaccctgcaatccacaacagtcaagaccaaaaacacaacaacaaccaaaatacaacccagcaagcccaccacaaaacaacgccaaaacaaaccaccaaacaaacccaataatgattttcactttgaagtgttcaactttgtaccttgcagcatatgcagcaacaatccaacctgctgggctatctgtaaaagaataccaaacaaaaaacctggaaagaaaaccaccaccaagcccacaaaaaaaccaaccatcaagacaaccaaaaaagatctcaaacctcaaaccacaaaaccaaaggaagtacctaccaccaagcccacagaaaagccaaccatcaacaccaccaaaacaaacatcagaactacactgctcaccaacaataccacaggaaatccagaacacacaagtcaaaagggaaccctccactcaacctcctccgatggcaatccaagcccttcacaagtctatacaacatccgagtacctatcacaacctccatctccatccaacacaacaaaccagtagAAGCTTGC-3’,大写下划线依次为Eco RI和Hind III酶切位点,小写下划线为同源连接序列,如SEQ ID NO.7所示。
7、pUC18-SH-G阳性质粒的筛选
将SH-G目的片段,分别采用Eco RI和Hind III进行酶切,酶切反应体系包含:EcoRI和Hind III各1μL,2.0μL 10×buffer,1μg回收的DNA,补水至20μL,37℃保温1h。
将上述酶切的目的片段分别与pUC18进行连接,得到质粒pUC18-SH-G。连接反应体系为:100ng pUC18,20ng SH-G目的片段,10×Ligation buffer 1μL,0.3μL(1U)T4 DNALigase,补水至10μL;16℃保温1h。
取40μL E.coli 5α感受态细胞,加入50ng的上述pUC18-SH-G的连接产物,冰浴40min后,42℃保温120s,冰浴1min,加入200μLSOC,37℃培养45min,离心后涂布,于37℃保温16h,置于4℃放置3~4h后,挑取白色颗粒,用于质粒提取。
提取阳性pUC18-SH-G质粒:向2mLLB中接种上述转入了重组质粒的E.coli 5a,37℃培养12~15h;10000rpm离心30s收集菌泥,去除培养液,用溶液I重悬,加入200μL溶液II(0.2M氢氧化钠,SDS 1%),室温裂解后;加入150μL预冷的3M乙酸钾(pH4.8),混匀,12000rpm离心5min;上清液转入另一1.5mL EP管中,依次加入等量酚和氯仿液体,各抽提一次;加入900μL-20℃预冷的无水乙醇,-20℃沉淀10min;12000rpm离心5min,收集沉淀,37℃干燥;沉淀重新溶解于含0.1μg RNase A的TE溶液(10mM Tris-Hcl,pH7.5,1mM EDTA)中,37℃反应30min消解RNA;采用50μL的Tris饱和酚和50μL的氯仿各抽提一次,再采用100μL氯仿抽提一次;然后用-20℃预冷的无水乙醇,-20℃沉淀10min,12000rpm离心5min,收集沉淀,37℃干燥,溶解于适量无菌水中,-20℃保存,即为阳性pUC18-SH-G质粒。
阳性pUC18-SH-G质粒经过1.0%的琼脂糖凝胶电泳检测,结果如图2(泳道1、2)所示。
实施例2:表达载体的构建、重组呼吸道合胞病毒蛋白的表达及纯化
1、表达载体pcDNA3.1(+)-SH-G的构建
将以上述得到的pUC18-SH-G质粒为模板,采用F3(GCGGATCCACACGCCAGGCAAAATCAAC,下划线为BamH I酶切位点,如SEQ ID NO.8所示)和R3(GCGCGGCTCTACTGGTTTGTTGATGTT,下划线为Nde I酶切位点,如SEQ ID NO.9所示)作为引物对,进行PCR扩增。
扩增反应体系为:0.1μg(3μL)pUC18-SH-G质粒,3μL(20pM)F2,3μL(20pM)R1,5.0μL10×PCR buffer,4μL(2.5mM)dNTP,0.4μL(5U/μL)Taqase,补水至50μL。扩增程序为:94℃2min,94℃45s,45℃45s,72℃lmin,30循环,72℃延伸10min。
所得PCR扩增产物和pcDNA3.1(+)(优宝生物)分别采用Nde I和BamH I进行双酶切,酶切反应体系包含:Nde I和BamH I各1μL,2.0μL 10×buffer,1μg回收的DNA,补水至20μL,37℃保温2h。将酶切产物进行连接,连接反应体系为:100ng pUC18,20ng SH-G,10×Ligation buffer 1μL,0.3μL(1U)T4 DNALigase,补水至10μL;16℃保温1h。
取40μL E.coli 5α感受态细胞,加入50ng的上述pUC18-SH-G的连接产物,冰浴40min后,42℃保温120s,冰浴1min,加入200μLSOC,37℃培养45min,离心后涂布,于37℃保温16h,置于4℃放置3~4h后,挑取白色颗粒,用于质粒提取。质粒提取步骤同时。
阳性pcDNA3.1(+)-SH-G表达载体经过1.0%的琼脂糖凝胶电泳检测,结果如图2(泳道3、4)所示。
2、转染
取500μL无血清DMEM稀释8μg阳性pcDNA3.1(+)-SH-G表达载体和20μL的转染试剂Lipgfectamin2000,混合保温20min后,加入至Hep-2细胞(上海歌凡生物细胞库)的孔板中,37℃、5%CO2保温转染4h后,期间更换24h生长培养液,72h后收集培养液上清细胞,采用Western-blot鉴定(方法如下)。
3、表达及目的蛋白分离纯化
收集冰上预冷培养液,8000rpm、4℃离心10min,弃去上清,沉淀物于-70℃反复冻融三次,12000rpm、4℃离心10min,分别收集上清和沉淀进行SDS-PAGE。
按照50μL/mL培养物的比例加入上样缓冲液(0.01M二水磷酸氢二钠,0.01M一水磷酸二氢钠,0.5M氯化钠,20mM咪唑,pH7.5),重悬后,于-70℃反复冻融3次破菌,并用注射器反复抽吸,12000g、4℃离心15min,取上清用0.45μm滤膜过滤,进行亲和层析纯化。
将其上样至由亲和层析凝胶ChelatingFast Flow装填并暗盒镍离子的层析床体积为5mL的亲和层析柱,上样提交为15mL,以洗脱液(0.01M二水磷酸氢二钠,0.01M一水磷酸二氢钠,0.5M氯化钠,60mM咪唑,pH7.5)洗脱,流速1mL/min,收集洗脱液。洗脱液减压浓缩,冷冻干燥后溶于PBS,按10U/mg的比例加入凝血酶,于室温切割16h后,SDS-PAGE检测。
结果如图3所示,融合蛋白SH-G主要处于诱导表达物经冷冻破碎和离心处理的上清液中,沉淀物几乎无融合蛋白,说明该融合蛋白SH-G具有可溶性,为可溶性表达。
4、Western-blot鉴定
将上述纯化的目的蛋白经SDS-PAGE后电转移至硝酸纤维素膜上。将膜置于5%的牛血清白蛋白溶液中37℃封闭2h,然后依次加抗呼吸道合胞病毒抗体(AB1128,Sigma-Aldrich)和羊抗兔HRP-IgG(自中山生物技术公司),分别在37℃孵育2h,最后用二氨基联苯胺显色液显色。
免疫印迹显示,含融合基因SH-G表达物在40kD处出现了抗原抗体结合条带(图4),G基因表达物在32kD处出现结合条带,SH基因表达物在7KD出现结合条带,说明表达的融合蛋白SH-G、G蛋白和SH蛋白均具有RSV特异反应性。而融合基因SH-G的表达沉淀物无结合条带(泳道2),融合蛋白主要处于经冷冻破碎和离心处理的上清液中,再次说明融合基因为可溶性诱导表达。
实施例3:融合蛋白SH-G的免疫活性检测
1、材料与方法
(1)实验动物
SPF级雌性BALB/c小鼠,4~6周龄,体重16~18g,购自北京维通利华实验动物技术有限公司,动物合格证号:SCXK(京)2016-0006。
(2)供试蛋白
上述实施例提供的融合蛋白SH-G,以及SH蛋白及G蛋白。
参照上述方式合成G’基因目的片段为:
5’-GCGAATTCggggcaaatgcaaacatgtccaaaaacaaggaccaacgcaccaccaagacactagaaaagacctgggacactctcaatcatctattattcatatcatcgtgcttatacaagttaaatcttaaatctatagcacaaatcacattatccattctggcaatgataatctcaacttcacttataattgcagccatcatattcatagcctcggcaaaccacaaagtcacactaacaactgcaatcatacaagatgcaacaagccagatcaagaacacaaccccaacatacctcacccagaatccccagcttggaatcagcttctccaatctgtctgaaactacatcacaaaccaccaccatactagcttcaacaacaccaagtgtcaagtcaaccctgcaatccacaacagtcaagaccaaaaacacaacaacaaccaaaatacaacccagcaagcccaccacaaaacaacgccaaaacaaaccaccaaacaaacccaataatgattttcactttgaagtgttcaactttgtaccttgcagcatatgcagcaacaatccaacctgctgggctatctgtaaaagaataccaaacaaaaaacctggaaagaaaaccaccaccaagcccacaaaaaaaccaaccatcaagacaaccaaaaaagatctcaaacctcaaaccacaaaaccaaaggaagtacctaccaccaagcccacagaaaagccaaccatcaacaccaccaaaacaaacatcagaactacactgctcaccaacaataccacaggaaatccagaacacacaagtcaaaagggaaccctccactcaacctcctccgatggcaatccaagcccttcacaagtctatacaacatccgagtacctatcacaacctccatctccatccaacacaacaaaccagtagAAGCTTGC-3’,大写下划线为EcoRI和HindIII酶切位点,如SEQ IDNO.10所示。
合成SH’基因目的片段为:5’-GCGAATTCacacgccaggcaaaatcaaccaatggaaaatacatccataacaatagaattctcaagcaaattctggccttactttacactaatacacatgataacaacaataatctctttgctaatcataatctccatcatgattgcaatactgaacaaactctgtgaatataacgtattccataacaaaacctttgagctaccaagagctcgagtcaatacatagcattcaccaatctgatggcacaaaacagtaaccttgcatttgtaagtgaacaaccctcacctctttacaaaaccacatcaacatctcaccatgcaagccaAAGCTTGC-3’,大写下划线为Eco RI和Hind III酶切位点,如SEQ ID NO.11所示。
分别采用Eco RI和Hind III进行酶切,酶切的目的片段分别与pUC18进行连接,得到表达质粒pUC18-SH和pUC18-G。转入E.coli 5α感受态细胞,挑取阳性颗粒,提取质粒,鉴定。
将pUC18-SH和pUC18-G采用上述相同的方法构建表达质粒pcDNA3.1(+)-SH和pcDNA3.1(+)-G,分别转入E.coli 5α感受态细胞,挑取阳性颗粒,提取质粒,经1.0%的琼脂糖凝胶电泳检测,结果如图2所示(1、2泳道)。
用到的引物分别为:
表达质粒pcDNA3.1(+)-G:F4(GCGGATCCGGGGCAAATGCAAACATG,下划线为BamH I酶切位点,如SEQ ID NO.12所示)和R4(GCGCGGCTCTACTGGTTTGTTGATGTT,下划线为Nde I酶切位点,如SEQ ID NO.13所示)
表达质粒pcDNA3.1(+)-SH:F5(GCGGATCCACACGCCAGGCAAAATCAAC,下划线为BamH I酶切位点,如SEQ ID NO.14所示)和R5(GCGCGGCTTGGCTTGCATGGTGAGATGT,下划线为Nde I酶切位点,如SEQ ID NO.15所示)。
将提取的表达质粒pcDNA3.1(+)-SH和pcDNA3.1(+)-G分别转入Hep-2细胞,收集细胞,冻融破碎,收集上清进行亲和纯化,并进行WB检测鉴定为SH蛋白和G蛋白(图4泳道3和泳道4)。将制得的SH-G融合蛋白、SH蛋白、G蛋白分别取30μg、加入0.4mg佐剂Al(OH)3,加入200μL PBS配制成供试疫苗溶液。
(3)免疫
将BALB/c小鼠随机分为实验组、对照组I和对照组II。实验组腹腔注射200μL含SH-G蛋白的供试品溶液,对照组I腹腔注射200μL含SH蛋白的供试品溶液,对照组II腹腔注射200μL含G蛋白的供试品溶液。每隔10d加强免疫一次,共免疫3次,PBS作为阴性组。最后一次免疫后10d心脏取血分离血清,取脾制备脾单细胞悬液,取肺组织匀浆收集上清,用1mL PBS冲洗鼻腔,收集鼻腔冲洗物(NTL)。免疫后3周用106TCID50RSV-A/50μL/只经鼻腔攻击鼠,5d后取肺匀浆上清及NTL。(4)血清IgG抗体效价的检测
采用ELISA法。以2μg/mL抗原(SH-G融合蛋白、SH蛋白、G蛋白)包被酶联板,4℃包被过夜;将待检各组小鼠血清以1:10稀释,进行2倍系列稀释于酶联板中,37℃孵育1h;加入HRP标记的山羊抗鼠IgG(1:2000稀释),37℃孵育1h;TMB显色,终止,于450nm波长处检测A值。
(5)血清中和抗体效价的检测
采用中和试验。将待检血清2倍系列稀释接种至Hep-2细胞,加入20~30PFU RSVLong病毒液后,37℃作用1h;加入含甲基纤维素的DMEM培养基,37℃培养7d;弃培养基,0.75%结晶紫染色并计算空斑数,以空斑数减少50%的血清稀释度作为中和效价(50%plaque redu ction neutralization assays,PRNT50)。
(6)小鼠免疫血清对RSV体外感染的抑制
将Hep-2细胞在96孔板中培养形成细胞单层,用1%DMEM将小鼠免疫血清(上述融合蛋白、SH蛋白和G蛋白分别免疫小鼠的血清)稀释成1:1000的3个稀释度,分别预防法、治疗法测定三组小鼠免疫血清的体外抗病毒活性。
预防法:以小鼠免疫血清100μL与细胞作用2h,30 PFU RSV Long病毒液100μL孵育1h,再以1%DMEM培养细胞。
治疗法:30 PFU RSV Long病毒液加入细胞孔,37℃孵育1h,再以含小鼠免疫血清的1%DMEM100μL培养细胞。同时设病毒对照组、空白细胞组和小鼠空白血清对照组。实验中使用的病毒感染剂量为100TCID50。每个浓度设4个复孔。细胞培养板置34℃、5%CO2条件下培养72h,持续观察细胞病变。当病毒对照孔的细胞病变达75%~100%时,进行0.1%结晶紫染色,595nm处测定A值,记录试验结果,按下式计算病毒抑制率=(试验组A值—病毒对照组A值)/(细胞对照组A值—病毒对照组A值)。
(6)统计学分析
采用Graphpad Prism5.0软件进行统计学分析,组间比较采用方差分析和T检验。
2、结果
ELISA结果显示,实验组小鼠产生了较高水平的针对融合蛋白的抗体,与PBS及对照组I和对照组II相比,均差异有统计学意义,见图5,表明融合蛋白能够诱导机体产生较高水平的体液免疫。中和试验结果显示,PBS组均未检测到中和抗体,而实验组检测到高水平中和抗体,且差异有统计学意义,见图5,表明融合蛋白能有效诱导RSV中和抗体的产生。并且,分别利用SH蛋白、G蛋白包被酶联板检测实验组抗血清的IgG抗体水平,结果其分别可产生6.25±0.58log2和4.69±0.65log2的IgG抗体水平(而对照组I和对照组II无检出),说明该融合蛋白能够诱导交叉保护能力,产生具有多表位的抗体,保护性更强。
各组小鼠免疫血清对RSV感染的Hep-2细胞的抑制作用如图6所示。其中,实验组的小鼠免疫血清的抑制率最高,并显著高于对照组I和对照组II,而阴性组未检出具有抑制作用,说明采用融合蛋白SH-G免疫小鼠所得血清能够抑制RSV引起的Hep-2细胞感染。
实施例4:免疫安全性研究
1、实验方法
将昆明种小鼠(雌性6~8周龄小鼠,济南朋悦实验动物繁育有限公司,动物合格证号:SCXK(鲁)2019-0003)随机分为实验组(免疫融合蛋白SH-G的供试品溶液)、对照组I(免疫蛋白SH的供试品溶液)、对照组II(免疫蛋白G的供试品溶液)和阴性组(免疫PBS)。分别在0、2、4周免疫,每次免疫每只小鼠的注射体积为200μL,免疫结束后,通过检测小鼠肺组织匀浆中IFN-γ、IL-4、IgE含量及ELISPOT检测分泌IFN-γ、IL-4的淋巴细胞数量,以评价融合蛋白、G蛋白和SH蛋白的免疫安全性。
小鼠肺组织匀浆中IFN-γ、IL-4及IgE含量的测定:设置标准品孔、样本孔和空白孔,根据IFN-γELISA检测试剂盒(CB10139-Mu,科艾博生物)、IL-4 ELISA检测试剂盒(CB10185-Mu,科艾博生物)和IgEELISA检测试剂盒(CB10432-Mu,科艾博生物)操作说明,分别检测小鼠肺组织匀浆中IFN-γ、IL-4及IgE的含量。
ELISPOT检测分泌IFN-γ和IL-4的淋巴细胞数量:利用单独的融合蛋白、G蛋白和SH蛋白分别免疫小鼠后,获取脾脏,在体外分离出脾淋巴细胞,进行ELISPOT实验(Mabtech公司),检测分泌INF-γ和IL-4脾淋巴细胞的数量,计算脾淋巴细胞的数IFN-γ和IL-4的比值。
采用Graphpad Prism 5.0软件进行统计学分析,组间比较采用方差分析和T检验。
2、实验结果
将所测得各组的每只小鼠肺部匀浆液中的INF-γ含量和IL-4含量进行比值分析。如图7所示,PBS组、对照组I、对照组II这3组间INF-γ/IL-4比值无差异性,而实验组INF-γ/IL-4比值显著高于阴性组(P<0.01)、对照组I(P<0.01)和对照组II(P<0.01),对照组I和对照组II无显著差异。
如图8所示,与阴性组小鼠的肺部相比,实验组和对照组I小鼠的肺部未出现明显的病变,而对照组II的小鼠肺部充血,部分出现了肺部变实以及大量淋巴细胞浸润的情况,但肺泡结构相对完整,说明融合蛋白疫苗具有较高的免疫安全性。
这些结果表明,免疫融合蛋白SH-G,相对于免疫SH或G蛋白,能够促使免疫应答向Th1方向发展,以降低G蛋白的Th2反应,增强Th1型应答,使得Th1/Th2反应平衡,弥补G蛋白疫苗的T细胞表位免疫缺陷,以通过体液免疫有效地产生T淋巴细胞,增加保护模式,保护和预防力度更广,安全性更高。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。
Claims (3)
1.一种重组呼吸道合胞病毒蛋白疫苗的制备方法,其特征在于,包括以下步骤:
(1)获得如SEQ ID NO.7所示的目的片段;
(2)将所述目的片段插入至真核表达载体pcDNA3.1(+)-中,以得到所述重组表达载体;
(3)将所述重组表达载体转染Hep-2细胞;
(4)培养和诱导表达细胞,收获表达细胞,纯化得到所述呼吸道合胞病毒的SH蛋白和G蛋白的融合蛋白;
(5)以所述融合蛋白为抗原成分制备所述重组呼吸道合胞病毒蛋白疫苗;
其中,步骤(1)包括:
(1-1)从呼吸道合胞病毒基因组RNA中反转录和扩增得到G基因目的片段和SH基因目的片段;所述G基因目的片段如SEQ ID NO.5所示,所述SH基因目的片段如SEQ ID NO.6所示,其中,所述G基因目的片段的5’端具有“tttttcgactaggatacctattttt”的同源序列,所述SH基因目的片段的3’端具有“tttttcgactaggatacctattttt”的同源序列;
(1-2)将所述G基因目的片段和所述SH基因目的片段混合进行互补延伸,以得到所述融合蛋白的目的片段。
2.重组表达载体在制备预防呼吸道合胞病毒感染的疫苗中的应用,所述重组表达载体携带如SEQ ID NO.7所示的目的片段;所述重组表达载体的构建方法包括:
(1)获得如SEQ ID NO.7所示的目的片段;
(2)将所述目的片段插入至真核表达载体pcDNA3.1(+)-中,以得到所述重组表达载体;
所述应用包括:
将所述重组表达载体转染Hep-2细胞;
培养和诱导表达细胞,收获表达细胞,纯化得到所述呼吸道合胞病毒的SH蛋白和G蛋白的融合蛋白;
以所述融合蛋白为抗原成分制备所述重组呼吸道合胞病毒蛋白疫苗。
3.表达细胞在制备预防呼吸道合胞病毒感染的疫苗中的应用,所述表达细胞为携带有一重组表达载体,所述重组表达载体携带如SEQ ID NO.7所示的目的片段,所述重组表达载体的构建方法包括:
(1)获得如SEQ ID NO.7所示的目的片段;
(2)将所述目的片段插入至真核表达载体pcDNA3.1(+)-中,以得到所述重组表达载体;
所述应用包括:
将所述重组表达载体转染Hep-2细胞以得到表达细胞;
培养和诱导表达细胞,收获表达细胞,纯化得到所述呼吸道合胞病毒的SH蛋白和G蛋白的融合蛋白;
以所述融合蛋白为抗原成分制备所述重组呼吸道合胞病毒蛋白疫苗。
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