CN113559254B - 一种狂犬病毒疫苗及其制备方法 - Google Patents
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Abstract
本发明公开了一种狂犬病毒疫苗及其制备方法。本发明的狂犬病毒疫苗是将水疱性口炎病毒的囊膜蛋白G替换为狂犬病毒囊膜蛋白RABVG。本发明通过分子生物学技术，在编码水疱性口炎病毒基因组质粒上，将VSV囊膜蛋白替换为狂犬病毒囊膜蛋白RABVG，利用VSV反向遗传学系统在293T细胞中包装成表达RABVG的以VSV为载体的疫苗，将上述疫苗滴鼻免疫小鼠，证实可有效诱导小鼠针对狂犬病毒的中和抗体，显著提高免疫小鼠抵御病毒感染的能力，具备作为新型狂犬疫苗的应用价值和潜力。
Description
技术领域
本发明涉及一种狂犬病毒疫苗及其制备方法，属于生物医药技术领域。
背景技术
狂犬病病毒(Rabies virus，RABV)属于弹状病毒科，是典型的嗜神经性病毒，病毒粒子呈子弹状，是有囊膜不分节段的单股负链RNA病毒，可经中枢神经系统逆行侵入宿主大脑，引起温血动物的脑脊髓炎，一旦发病，死亡率100％。目前,尚无有效治疗方法，暴露前或暴露后接种狂犬疫苗是唯一有效的预防手段。
目前，由于我国居民生活方式和观念的转变，中国市场宠物行业市场规模不断扩大，2019年增长至2024亿元，宠物猫、犬数量年均增长10％，推动人用狂犬疫苗需求攀升。2020年上半年，人用狂犬疫苗共批签发4795万支，同比增长36％。其中，成大生物占39％，宁波荣安生物药业有限公司占30％，复星医药控股子公司大连雅立峰生物制药有限公司占11％。现售疫苗主要为灭活病毒疫苗，病毒灭活通常采用β-丙内酯作为灭活剂，从培养细胞来源可分为Vero细胞苗、人二倍体细胞苗(HDCV)、原代地鼠肾(PHK)细胞苗、鸡胚细胞苗。Vero细胞狂苗产量大、质量可控，是我国狂苗的主要产品，占到每年狂苗批签发的90％左右，市场占比前四的企业均为生产Vero细胞狂苗的企业，目前只有成都康华一家生产HDCV。因此无论是人用还是兽用狂犬疫苗，每年都在快速递增，市场需求十分巨大。
水泡性口炎病毒(VSV)可作为一种疫苗载体，利用VSV做为疫苗载体的埃博拉疫苗，也于2019年被美国FDA和欧洲药品管理局EMA授予上市许可，充分体现了VSV在人体的安全性可以得到保证。但是，目前VSV作为病毒载体也具有一定的局限性，首先病毒载体容量有限，通常插入的外源基因在1-2K左右大小，而且作为疫苗载体诱导的特异性针对机体的抗体反应也是不一，我们以VSV为载体的柯萨奇病毒B3的心肌炎疫苗能够诱导一个有效的免疫保护反应，但是以新型冠状病毒受体结合区RBD为抗原，VSV作为载体不能诱导一个明显的抗体保护反应。由于外源插入的其他病毒的囊膜蛋白和VSV病毒原有囊膜蛋白存在差异，替换了VSV囊膜蛋白的重组VSV可能不能复制，所以囊膜替换型的重组VSV的拯救是一个大的技术挑战。还没有采用水泡性口炎病毒作为狂犬病毒疫苗载体的报道。
发明内容
为解决现有技术中狂犬疫苗的空缺，本发明利用VSV反向遗传系统，把水疱性口炎病毒的囊膜蛋白G替换为狂犬病毒的囊膜蛋白RABVG，包装制备成表达有狂犬病毒囊膜蛋白的重组VSV-RABVG病毒，并在小鼠模型中检测其免疫应答和保护效果。
本发明的第一个目的是提供一种狂犬病毒疫苗，所述狂犬病毒疫苗是将水疱性口炎病毒的囊膜蛋白G替换为狂犬病毒囊膜蛋白RABVG。
进一步地，所述的囊膜蛋白RABVG为：编号为GenBank:AAA47218.1的囊膜蛋白RABVG，或在编号为GenBank:AAA47218.1的囊膜蛋白RABVG的基础上，进行一个或几个氨基酸的插入、缺失或突变后具有囊膜蛋白RABVG活性的蛋白。
本发明中，狂犬病毒基囊膜蛋白RABVG不限定为编号为GenBank:AAA47218.1的囊膜蛋白RABVG，还包括相类似的其他株型狂犬病毒基囊膜蛋白RABVG。
本发明的第二个目的是提供所述的狂犬病毒疫苗的制备方法，包括如下步骤：
S1、构建质粒pXN2-GRABV：将pXN2载体上囊膜蛋白G的基因序列剪切掉，替换为狂犬病毒囊膜蛋白RABVG，得到质粒pXN2-GRABV；
S2、表达重组VSV-RABVG：第一待感染细胞用带有T7 RNA转录酶的痘病毒感染，感染后的细胞用pXN2-GRABV、pP、pL和pN质粒共转染，转染后收集病变细胞的上清液，采用上清液感染第二待感染细胞，扩增病毒后收取病毒感染的细胞培养上清液，得到所述的狂犬病毒疫苗。
进一步地，第一待感染细胞为BHK细胞、293T细胞、BSRT7细胞或Vero细胞。
进一步地，第二待感染细胞为Vero细胞、BHK细胞、293T细胞或BSRT7细胞。
进一步地，所述的BHK细胞为BHK-21细胞。
进一步地，pXN2-GRABV、pP、pL和pN质粒共转染时，pXN2-GRABV、pP、pL、pN质粒的比例为10：5：4：2。
进一步地，痘病毒感染的时间为1～3小时。
进一步地，共转染后2～4天收集病变细胞的上清液。
在本发明中，水疱性口炎病毒的反向遗传重组系统正是建立在以下四个质粒基础上:pXN2、pP、pL、pN。pXN2包含病毒的全长cDNA序列、T7启动子以及δ肝炎核酶，主要负责提供不包含冗余序列的病毒全长RNA基因组。pP、pN和pL质粒能够提供P、N和L蛋白，以组成RNA聚合酶核心。四个质粒都应用了T7启动子，按照10：5：4：2的比例共同转染BHK-21细胞。在真核细胞中不存在T7 RNA聚合酶，因此在共同转染四个质粒以前，要用包含T7 RNA聚合酶基因的重组痘病毒感染细胞，以提供T7 RNA聚合酶蛋白。
本发明的第三个目的是提供一种中和抗体，所述的中和抗体由所述的狂犬病毒疫苗诱导产生。
本发明的有益效果是：
本发明通过分子生物学技术，在编码水疱性口炎病毒基因组质粒上，将VSV囊膜蛋白替换为狂犬病毒囊膜蛋白RABVG，利用VSV反向遗传学系统在293T细胞中包装成表达RABVG的以VSV为载体的疫苗，将上述疫苗滴鼻免疫小鼠，证实可有效诱导小鼠针对狂犬病毒的中和抗体，显著提高免疫小鼠抵御病毒感染的能力，具备作为新型狂犬疫苗的应用价值和潜力。
附图说明：
图1是VSV-RABVG重组病毒反向遗传重组质粒构建示意图。
图2是重组的VSV-RABVG，VSV-GFP感染293T细胞24小时后RABV GP的Western Blot鉴定，Control为未感染的293T细胞。
图3显示了VSV-RABVG疫苗滴鼻免疫诱导小鼠产生的RABV G特异性血清IgG。
图4是VSV-RABVG疫苗滴鼻免疫诱导小鼠攻毒后3周的生存率检测。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例中采用的质粒、菌种、细胞、动物及试剂如下：
质粒pXN2，pP，pL，pN和pVSVG由本实验室保存、宿主细菌DH5α、stable 2(上海唯地生物科技有限公司)。BHK21和Vero E6由本实验室保存。基因合成委托苏州金唯智公司。HRP标记羊抗小鼠IgG多克隆抗体，HRP标记羊抗小鼠IgA多克隆抗体(SouthernBiotech公司)。限制性核酸内切酶XhoI和NheI(TaKaRa公司)、T4 DNA连接酶(TaKaRa)；Taq DNA聚合酶(TaKaRa公司公司)；dNTP(Promega)；LB培养基(英国OXOID公司)，琼脂粉、琼脂糖、EB(上海化学试剂采购供应站)，Tris(USB公司)，琼脂糖胶回收试剂盒(上海华舜公司)，大量质粒抽提试剂盒(Qiagen)，乳酸脱氢酶LDH试剂盒购自Roche。6-8周龄雄性BALB/c小鼠购自上海斯莱克实验动物中心，清洁级饲养。动物饲养及操作符合国家相关规定。
实施例1：重组VSV-RABVG疫苗的构建及体外表达功能鉴定
1.1)质粒pXN2-GRABV构建：在pXN2载体，xbaI和Mlu I酶切位点之间包含了G基因序列，所以我们委托金唯智合成了XbaI-MluI片段，其中的VSV G基因为RABV流行株G基因所替换，并用xbaI和MluI酶切位点连接到pXN2载体，得到pXN2-GRABV质粒，测序验证，图1是构建示意图。
1.2)重组VSV-RABVG的制备及表达检测：BHK-21细胞首先用带有T7 RNA转录酶的痘病毒感染，感染后2小时细胞用pXN2-GRABV、pP、pL和pN质粒按照比例10：5：4：2共转染。转染后3天看到细胞病变，收集细胞上清再次感染Vero细胞，扩增病毒。我们收取病毒感染的细胞培养上清液，用蛋白印迹的方法检测了VSV-RABVG中RABV囊膜蛋白GRABV的表达，如图2。
实施例2：重组VSV-RABVG疫苗诱导的特异性抗体应答
2.1)小鼠滴鼻免疫方法及血清收集
将6-8周BALB/c雄性小鼠分为3组,2组滴鼻免疫分别为：106PFU VSV-GFP(阴性对照)、106PFU VSV-RABVG、1组肌肉注射106PFU LBNSE(减毒疫苗)，每组6只小鼠。滴鼻免疫程序如下：以0.75％戊巴比妥钠80-120μl经腹腔注射小鼠，使小鼠轻微麻醉。以含有VSV-GFP的PBS病毒疫苗溶液逐滴滴入小鼠两侧鼻腔，VSV-RABVG组采取相同的操作方法。在免疫后的第10天，20天和30天经眼眶后眦静脉采血，37℃静置30min，6000rpm离心10min，收集血清，分装冻存于-80℃。
2.2)RABV G特异性血清IgG的ELISA检测：
ELISA方法：以20ug/ml RABV G全蛋白、包被液(0.1M Na2CO3，pH9.6、0.1％BSA)4℃包板；以5％milk-PBS封闭；依次加1：40稀释血清、HRP-羊抗小鼠IgG、IgA、OPD，显色中止，测OD450nm。
VSV-RABVG免疫组在免疫10天后即诱生了较高水平的特异性IgG，第30天OD值达1.0左右，较减毒活疫苗LBNSE产生的抗体水平低(OD450nm＝1.5)。而单独的空病毒VSV-GFP不能诱生血清IgG，OD450nm值约为0.15，明显低于前者。结果提示：VSV-RABVG和LBNSE免疫小鼠，都能在血清诱导一个较强的中和抗体免疫反应(图3)。
实施例3：在小鼠感染模型中，重组VSV-RABVG疫苗的保护反应检测
将6-8周BALB/c雄性小鼠分为3组，每组10个，小鼠免疫方法如2.1所叙。
在免疫后的第13周，我们用脑内注射感染小鼠，观测感染20内小鼠的存活情况。我们发现VSV-GFP免疫组在感染后8天开始出现死亡，至13天小鼠全部死亡；而LBNSE组小鼠最后存活率为60％(6/10)，VSV-RABVG组小鼠虽然在抗体水平上检测低于LBNSE组，但是在攻毒实验中全部存活下来(n＝10)，可能是诱导了更多的中和性抗体(图4)。因为小鼠攻毒实验是在免疫后第13周，我们的结果也提示VSV-RABVG可以提供一个长期的保护免疫反应。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (5)
1.一种狂犬病毒疫苗的制备方法，其特征在于，所述狂犬病毒疫苗是将水疱性口炎病毒的囊膜蛋白G替换为狂犬病毒囊膜蛋白RABVG，制备方法包括如下步骤：
S1、构建质粒pXN2-GRABV：将pXN2载体上囊膜蛋白G的基因序列剪切掉，替换为狂犬病毒囊膜蛋白RABVG，得到质粒pXN2-GRABV；
S2、表达重组VSV-RABVG：第一待感染细胞用带有T7 RNA转录酶的痘病毒感染，感染后的细胞用pXN2-GRABV、pP、pL和pN质粒共转染，转染后收集病变细胞的上清液，采用上清液感染第二待感染细胞，扩增病毒后收取病毒感染的细胞培养上清液，得到所述的狂犬病毒疫苗；
第一待感染细胞为BHK-21细胞；
第二待感染细胞为Vero细胞；
pXN2-GRABV、pP、pL和pN质粒共转染时，pXN2-GRABV、pP、pL、pN质粒的比例为10：5：4：2。
2.根据权利要求1所述的制备方法，其特征在于，所述的囊膜蛋白RABVG为：编号为GenBank: AAA47218.1的囊膜蛋白RABVG。
3.根据权利要求1所述的制备方法，其特征在于，痘病毒感染的时间为1~3小时。
4.根据权利要求1所述的制备方法，其特征在于，共转染后2~4天收集病变细胞的上清液。
5.一种权利要求1~4任一项所述的制备方法制备得到的狂犬病毒疫苗。
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