CN109593136A - 禽副粘病毒融合蛋白及其制备方法、应用和用于鸽子的apmv疫苗 - Google Patents
禽副粘病毒融合蛋白及其制备方法、应用和用于鸽子的apmv疫苗 Download PDFInfo
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- CN109593136A CN109593136A CN201811604172.4A CN201811604172A CN109593136A CN 109593136 A CN109593136 A CN 109593136A CN 201811604172 A CN201811604172 A CN 201811604172A CN 109593136 A CN109593136 A CN 109593136A
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Abstract
本发明提供了一种禽副粘病毒融合蛋白及其制备方法、应用和用于鸽子的APMV疫苗,涉及生物技术领域,该禽副粘病毒融合蛋白将鸽1型副粘病毒的HN基因和禽副粘病毒7型的HN基因进行串联,包含NDV‑HN区段和AMPV‑7‑HN区段;其中NDV‑HN区段包括如SEQ ID NO.1所示的氨基酸序列,AMPV‑7‑HN区段包括如SEQ ID NO.2所示的氨基酸序列,该禽副粘病毒融合蛋白具有抗原性好,表达量高,应用广泛的优点。
Description
技术领域
本发明涉及生物技术领域,尤其是涉及一种禽副粘病毒融合蛋白及其制备方法、应用和用于鸽子的APMV疫苗。
背景技术
近来,全国各地出现了许多上万对、几万对,个别达10-20万对的大型鸽场,仅广东省鸽子年存栏量达400万对以上,生产乳鸽4500-5000万只以上,是国内养鸽的大省,其他省市的饲养量约为广东的3-4倍,其中以江苏、山东、河北、湖南、广西以及新疆的南疆等地饲养较多。基于如此规模的养殖环境,给养鸽业带来丰厚的利润。
然而,在肉鸽养殖快速发展的过程中,鸽病的发生和流行是妨碍肉鸽养殖快速发展的主要问题之一。随着养鸽业的快速发展和禽病科学的进步,病种较20多年前明显增多,己达60-70种之多,而且还不断有新的疾病出现。在各类疾病中,报道最多的依次是病毒性传染病、细菌性传染病、体内外寄生虫病。
鸽病毒性传染病一直是各类鸽病中发生频率最多、危害性最大的一类传染病,其中鸽1型副粘病毒病从80年代初开始亦在我国各地先后出现,并逐年增多,成为鸽死亡的主要疫病,占各种病毒性疾病的86.47%(147/170)。
禽副粘病毒1型(APMV-1)也被称为新城疫病毒(NDV),新城疫病毒常常感染家禽,也感染家鸽和其他野鸟,其中,来源并感染鸽子的新城疫病毒也被称为鸽1型副粘病毒(Pigeon Paramyxovirus-1,PPMV-1)。NDV主要传播途径是呼吸道和消化道。预防接种是目前大多数国家控制新城疫的主要措施。历史上曾发生过的3次新城疫大流行中,其中上世纪70年代末发生的第三次大流行,鸽子作为传播宿主扮演重要角色。
禽副粘病毒7型(APMV-7)是禽副粘病毒9个血清型中之一,英国曾经从隔离检疫时死亡的鸽中分离获得。该病毒的分离株均来自于野鸟,鸽是易感宿主之一。其余引起家禽发病的重要血清型还有APMV-2,APMV-3,APMV-6和APMV-7。因此对于禽副粘病毒的预防和控制对于禽类养殖业的发展和人类生命财产的安全至关重要。
目前鸽类没有特定的商品化疫苗,市面上其他禽类商品化的疫苗主要为灭活苗和弱毒苗。灭活苗虽然安全稳定,但需要大剂量接种或应用浓缩抗原;免疫期短,常需强化接种;产生完全免疫力需要2周,不利于紧急预防接种与降低疫苗费用,保护效果不佳。而弱毒苗由于存在着成本高、易返祖、有潜在感染危险等缺陷,很难在实践中推广应用。
有鉴于此,特提出本发明。
发明内容
本发明的第一目的在于提供一种禽副粘病毒融合蛋白,该禽副粘病毒融合蛋白将APMV-1型的HN基因和APMV-7型的HN基因进行串联表达。
本发明的第二目的在于提供一种上述禽副粘病毒融合蛋白的制备方法,该制备方法可以制备出上述禽副粘病毒融合蛋白。
本发明的第三目的在于提供一种上述禽副粘病毒融合蛋白、上述禽副粘病毒融合蛋白的制备方法、或由上述制备方法制备得到的禽副粘病毒融合蛋白的应用。
本发明的第四目的在于提供一种用于鸽子的APMV疫苗,该疫苗包含上述禽副粘病毒融合蛋白、表达上述禽副粘病毒融合蛋白的基因或上述禽副粘病毒融合蛋白的制备方法制备得到的禽副粘病毒融合蛋白。
为解决上述技术问题,本发明特采用如下技术方案:
一种禽副粘病毒融合蛋白,包含NDV-HN区段和AMPV-7-HN区段;所述NDV-HN区段包括如SEQ ID NO.1所示的氨基酸序列,所述AMPV-7-HN区段包括如SEQ ID NO.2所示的氨基酸序列。
优选地,所述NDV-HN区段和所述AMPV-7-HN区段通过融合蛋白连接肽连接;
优选地,所述融合蛋白连接肽为柔性连接肽;
优选地,所述融合蛋白连接肽的序列为(PPSPS)n;n为1-3的正整数;n优选为2。
优选地,所述禽副粘病毒融合蛋白具有如SEQ ID NO.3所示的氨基酸序列。
优选地,所述禽副粘病毒融合蛋白的N端和/或C端连接有标签。
优选地,所述禽副粘病毒融合蛋白为乳酸杆菌表达系统表达的蛋白。
本发明还提供了一种上述禽副粘病毒融合蛋白的制备方法,包括:将所述禽副粘病毒融合蛋白的基因在宿主中表达。
优选地,将所述禽副粘病毒融合蛋白的基因在乳酸杆菌中表达。
本发明还提供了一种上述禽副粘病毒融合蛋白、上述的制备方法或由上述制备方法制备得到的禽副粘病毒融合蛋白的应用,包括如下(x1)-(x6)中的至少一种:(x1)制备鸽亚单位二联疫苗;(x2)制备禽类病毒性疾病的疫苗;(x3)制备禽副粘病毒的抗体;(x4)制备禽副粘病毒的抗原;(x5)制备检测禽副粘病毒的试剂和/或试剂盒;(x6)制备检测禽副粘病毒抗体的试剂和/或试剂盒。
本发明还提供了一种用于鸽子的APMV疫苗,所述APMV疫苗包含上述的禽副粘病毒融合蛋白、表达所述禽副粘病毒融合蛋白的基因或上述制备方法表达的禽副粘病毒融合蛋白。
优选地,所述APMV疫苗以表达上述禽副粘病毒融合蛋白的乳酸杆菌为活性成分。
与现有技术相比,本发明具有如下有益效果:
本发明提供的禽副粘病毒融合蛋白,将鸽1型副粘病毒的HN基因和禽副粘病毒7型的HN基因进行串联,得到的融合蛋白具有抗原性好,表达量高,应用广泛的优点。本发明提供的禽副粘病毒融合蛋白将鸽1型副粘病毒和禽7型副粘病毒的HN基因串联表达,该禽副粘病毒融合蛋白中不仅含有鸽1型副粘病毒病的抗原表位,还含有禽7型副粘病毒的抗原表位,具有较好的免疫原性,因此可作为禽类病毒性疾病的疫苗,既可以作为疫苗中的主要抗原,也可以作为疫苗的免疫原性的增强剂。并且,本发明提供的禽副粘病毒融合蛋白还可以用来制备禽副粘病毒的抗体等,以进一步应用于制备检测鸽1型副粘病毒、其他来源的新城疫病毒、禽7型副粘病毒以及上述病毒的抗原或者抗体的试剂盒。
本发明提供的禽副粘病毒融合蛋白,通过将所述禽副粘病毒融合蛋白的基因在宿主中表达即可,方法简便,适合广泛的应用。
本发明提供的用于鸽子的APMV疫苗,能提供有效地同源攻毒保护,免疫后能够产生较强免疫力,接种的发病率和死亡率均明显减少,能够有效预防鸡新城疫和禽7型副粘病毒引起的感染流行和传播,降低本病对养鸽业造成的经济损失,应用前景广阔。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1提供的重组质粒pMG-NDAP-HN图谱;
图2-A为本发明实施例1提供的pMG-NDAP-HN重组菌NDV HN基因的PCR鉴定结果图;
图2-B为本发明实施例1提供的pMG-NDAP-HN重组菌APMV HN基因的PCR鉴定结果图;
图3为本发明实施例1提供的pMG-NDAP-HN重组菌的双酶切鉴定结果图;
图4为本发明实施例1提供的pMG-NDAP-HN重组抗原的western-blotting结果图;
图5为本发明对比例1提供的重组质粒pMG-NDV-HN图谱;
图6为本发明对比例2提供的重组质粒pMG-APMV-HN图谱;
图7为本发明实施例1提供的红细胞凝集试验;
图8-A为本发明效果例2直肠棉拭子PCR检测结果;
图8-B为本发明效果例2直肠棉拭子PCR检测结果;
图8-C为本发明效果例2直肠棉拭子PCR检测结果。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
本发明提供了一种禽副粘病毒融合蛋白,禽副粘病毒(Avian paramyxovirus,APMV)在分类上属于副粘病毒科(Paramyxoviridae),副粘病毒亚科(Paramyxivirinae),禽副粘病毒属(Avulavirus),多数呈球形,有囊膜病毒。基因组结构为单股、负链、不分段的RNA,整个基因组长度约为15-16kb,基因结构为:3’-NP-P-M-F-HN-L5’,依次编码核衣壳蛋白、磷蛋白、基质蛋白、融合蛋白、血凝素-神经氨酸酶蛋白。根据血凝抑制试验(HI)和神经氨酸酶抑制试验(NI)可以将APMV分为9个血清型。
本发明提供的禽副粘病毒融合蛋白,将NDV-1型的HN基因和APMV-7型的HN基因进行串联。其中,NDV-HN区段包括如SEQ ID NO.1所示的氨基酸序列,该氨基酸序列来源于鸽的新城疫病毒,即鸽1型副粘病毒(Pigeon Paramyxovirus-1,PPMV-1);AMPV-7-HN区段包括如SEQ ID NO.2所示的氨基酸序列。该氨基酸序列来源于禽副粘病毒7型(APMV-7),APMV-7是禽副粘病毒9个血清型中之一,该病毒的分离株均来自于野鸟,鸽是易感宿主之一。
血凝素-神经氨酸酶(Hemagglutinin-Neuraminidase protein,HN)是一种由HN基因编码的膜结合糖蛋白。血凝神经氨酸酶介导副粘病毒属的病毒与细胞膜的附着,主要是由于HN蛋白具有NA与HA的双功能作用。血凝素(hemagglutinin,HA)负责病毒粒子、宿主细胞的连接;神经氨酸酶(Neuraminidase,NA)具有抗原性,能够协助病毒脱离宿主细胞感染新的细胞。HN使病毒与有含唾液酸受体的细胞结合并且两者之间的较高亲和性。病毒通过HN蛋白与宿主细胞受体的结合是触发的F蛋白和靶细胞之间融合的一个重要因素。
本发明提供的禽副粘病毒融合蛋白,将NDV的HN基因和APMV-7型的HN基因进行串联,得到的融合蛋白具有抗原性好,表达量高的优点。
在一些可选地实施方式中,NDV-HN区段和AMPV-7-HN区段通过融合蛋白连接肽连接;融合蛋白连接肽可以将融合蛋白彼此分开,不影响NDV-HN区段和AMPV-7-HN区段各自高级结构的形成。在一些优选地实施方式中,采用柔性连接肽有利于禽副粘病毒融合蛋白中,各区段的高级结构的形成,其中柔性连接肽优选具有(PPSPS)重复序列的连接肽,重复序列的重复数以1个重复至3个重复为佳,例如可以为但不限于为(PPSPS)、(PPSPS)2或(PPSPS)3;更优选为(PPSPS)2。在一些优选的实施方式中,所述禽副粘病毒融合蛋白具有如SEQ IDNO.3所示的氨基酸序列。
在一些可选地实施方式中,所述禽副粘病毒融合蛋的N端和/或C端连接有标签,以便于后续蛋白的分离、纯化和鉴定,所述标签例如可以为但不限于为His标签、Flag标签、GST标签、MBP标签、NusA标签或SUMO标签。
在一些优选地实施方式中,所述禽副粘病毒融合蛋白为乳酸杆菌表达系统表达的蛋白。乳酸杆菌是一种易通过劲膜吸收的常见菌,因其在食品工业各个领域中的长期应用已证明不具有致病性,被公认为安全级微生物;乳酸杆菌还具有在胃肠道、泌尿、生殖系统中或粘膜部位粘附存活且无病原性等优点。利用乳酸杆菌作为宿主菌,可以利用乳酸杆菌作为禽副黏病毒的传递载体进行粘膜疫苗免疫的研究。
本发明还提供了一种上述禽副粘病毒融合蛋白的制备方法,该制备方法通过将所述禽副粘病毒融合蛋白的基因在宿主中表达即可,例如可以为但不限于为在乳酸杆菌表达系统、大肠杆菌表达系统、酵母表达系统、昆虫表达系统、植物表达系统或者哺乳动物表达系统,优选使用乳酸杆菌表达系统,乳酸杆菌表达系统具有上述不具有致病性和乳酸杆菌表达系统可定植于粘膜的优点。
本发明还提供了一种上述禽副粘病毒融合蛋白、上述禽副粘病毒融合蛋白的制备方法或由该制备方法制备得到的禽副粘病毒融合蛋白的应用,包括如下(x1)-(x6)中的至少一种:(x1)制备鸽亚单位二联疫苗;(x2)制备禽类病毒性疾病的疫苗;(x3)制备禽副粘病毒的抗体;(x4)制备禽副粘病毒的抗原;(x5)制备检测禽副粘病毒的试剂和/或试剂盒;(x6)制备检测禽副粘病毒抗体的试剂和/或试剂盒。
本发明提供的禽副粘病毒融合蛋白将鸽1型副粘病毒,即新城疫病毒和禽7型副粘病毒的HN基因串联表达,该禽副粘病毒融合蛋白中不仅含有鸽1型副粘病毒的抗原表位,还含有鸽源的病毒APMV-7的抗原表位,具有较好的免疫原性,因此可作为禽类病毒性疾病的疫苗,既可以作为疫苗中的主要抗原,也可以作为疫苗的免疫原性的增强剂。由于本发明中串联表达的HN基因均源自鸽源的病毒,因此优选用于制备鸽亚单位二联疫苗。并且,本发明提供的禽副粘病毒融合蛋白还可以用来制备禽副粘病毒的抗体等,以进一步应用于制备检测鸽1型副粘病毒、其他来源的新城疫病毒、禽7型副粘病毒以及上述病毒的抗原或者抗体的试剂盒。
本发明还提供了一种用于鸽子的APMV疫苗,该疫苗可以以上述禽副粘病毒融合蛋白或由上述禽副粘病毒融合蛋白的制备方法制备得到的蛋白作为主要抗原或免疫增强剂,还可以包含表达上述禽副粘病毒融合蛋白的基因以制备DNA疫苗。
在一个优选的实施方式中,所述APMV疫苗为基因工程亚单位二联口服疫苗,该疫苗以表达上述禽副粘病毒融合蛋白的乳酸杆菌为活性成分。即该疫苗是一种鸽1型副粘病毒和禽7型副粘病毒基因工程亚单位二联口服疫苗。
机体外周粘膜免疫系统是机体最大的免疫系统。肠道和呼吸道粘膜免疫所产生的分泌型抗体(sIgA)是抵抗NDV和APMV感染的有效抗体。新城疫和禽7型副粘病毒病的免疫机制中,粘膜免疫具有非常重要的作用。传统的非肠道输入的灭活疫苗不产生sIgA抗体,细胞介导免疫反应弱而且维持时间短。
以表达上述禽副粘病毒融合蛋白的乳酸杆菌作为疫苗的活性成分制成的二联口服疫苗,可有效地刺激肠道局部免疫细胞产生分泌型IgA,这尤其适应于肠道粘膜传染病,并且避免了常规注射所引起的应激及疫苗吸收问题,解决疫苗免疫应激性强,免疫原性不强、有毒性的问题。
下面结合优选实施例进一步说明本发明。
实施例1禽副粘病毒融合蛋白的表达
1.1合成和扩增目的基因
通过GenBank获取NDV和APMV-7的全病毒RNA序列,寻找编码HA基因的DNA序列,其中编码NDV的HN基因的氨基酸序列如SEQ ID NO.1所示,核苷酸序列如SEQ ID NO.4所示;编码AMPV-7的HN基因的氨基酸序列如SEQ ID NO.2所示,核苷酸序列如SEQ ID NO.5所示。
根据表达宿主对基因序列进行密码子优化后,选用柔性的融合蛋白连接肽(PPSPS)2将NDV的HN基因和APMV-7的HN基因连接起来,把该序列命名为NDAP-HN,其氨基酸序列如SEQ ID NO.3所示,并送往生物公司合成。
1.2表达载体构建
将合成的NDAP-HN基因与pMG36e表达载体用限制性内切酶XbaI和HindIII酶切,并进行胶回收纯化。将两者16℃过夜连接,转化入大肠杆菌BL21中培养16小时。挑取单克隆菌落进行PCR鉴定、质粒双酶切鉴定以及测序鉴定正确,将构建的表达载体命名为pMG-NDAP-HN,重组质粒pMG-NDAP-HN图谱如图1所示。
1.3重组乳酸杆菌的转化与鉴定
将鉴定正确的阳性质粒0.35-0.5μg分别与感受态细胞(干酪样乳酸杆菌)80μL轻柔混匀后,冰上放置5min,将其转入2mm的预冷电转化杯中,迅速电击,电击参数为电压2.5kV,电击时间为5ms,电击后加入900μL冰预冷的MRS恢复培养基,混匀,将菌液转移至1.5m1离心管中,冰上放置10min,28℃复苏培养2h,取适量菌液涂布于含有5μg/ml红霉素的MRS琼脂培养基上,37℃培养2-3d。于平板上挑取单个菌落,分别接种于含有5μg/ml红霉素的MRS液体培养基中,37℃培养过夜后,从乳酸乳杆菌中分别提取质粒,并分别对质粒进行PCR鉴定、双酶切鉴定和测序鉴定正确,PCR鉴定结果如图2-A和图2-B所示,重组菌的双酶切鉴定结果如图3所示。
1.4重组蛋白的诱导表达
分别取50μL的菌种接种于5mL含有红霉素的新配MRS液体培养基中,于37℃过夜培养,至OD600达到1.0时,加入nisin至终浓度为10ng/ml,继续培养,于10小时后终止诱导。然后进行SDS-PAGE蛋白检测试验;结果显示所表达的目的蛋白大约为62KD和120KD;Westernblot鉴定重组蛋白具有与抗NDV和抗APMV-7的多抗发生反应的能力,证明重组菌表达的外源蛋白具有良好的反应原性,pMG-NDAP-HN重组抗原的Western-blotting结果如图4所示。
1.5凝集试验
1.5.1 1%红细胞悬液:由鸡心脏或翅静脉采血,放入加有抗凝剂的离心管内(按抗凝剂:全血=1:2的比例),迅速混匀。在离心机中以1500转/分离心8分钟,用吸管吸去上清液和红细胞上层的白细胞薄膜,将沉淀的红细胞加生理盐水,慢慢混合均匀,再在离心机中1500转/分离心8分钟,弃去上清液,再加生理盐水混匀,如此反复离心4~5次,最后一次离心后的红细胞,弃去上清液,即为红细胞泥。放入4℃冰箱中可保存2~3天。使用时用1ml吸管吸取0.1ml红细胞泥,然后加入9.9ml的生理盐水,此即1%红细胞悬液。
1.5.2血凝(HA)试验:采用96孔V型反应板,测定抗原效价。首先用微量移液器向反应板的每孔加生理盐水25微升。用微量移液器吸取待测抗原25微升于第一孔中并挤压6次混合,后吸出25微升至第2孔,依次倍比稀释到第11孔,弃去25微升。微量移液器再吸取1%红细胞悬液依次加入各孔,每孔25微升。置于微量振荡器上振荡1分钟,室温静置30分钟后观察结果。
1.5.3结果观察:将反应板倾斜成45度角,沉于孔底的红细胞沿着倾斜面向下呈线状流动者为沉淀,表明红细胞未被或不完全被病毒凝集;如果孔底的红细胞铺平孔底,凝成均匀薄层,倾斜后红细胞不流动,说明红细胞被病毒所凝集。能使鸡红细胞完全凝集的抗原的最高稀释倍数,称为该抗原滴度红细胞凝集效价。如图7所示第1排各孔红细胞为完全凝集,第2排为阴性对照孔红细胞不凝集。其中,第一排:A表示NDV HN抗原;B表示APMV HN抗原;C表示NDAP H抗原;D表示阳性血清;第二排:各孔为阴性对照孔。
对比例1
对比例1与实施例1的区别在于,将NDV HN基因和pMG36e表达载体用限制性内切酶XbaI和HindIII酶切并进行胶回收纯化,其中NDV HN基因由生物公司合成得到。将两者16℃过夜连接,转化入大肠杆菌BL21中培养16小时。挑取单克隆菌落进行PCR鉴定、质粒双酶切鉴定以及测序鉴定正确,将构建的表达载体命名为pMG-NDV-HN,重组质粒pMG-NDV-HN图谱如图5所示,重组乳酸杆菌的转化与鉴定与重组蛋白的诱导表达同实施例1。
对比例2
对比例2与实施例1的区别在于,将PCR扩增得到的APMV-7 HN基因和pMG36e表达载体用限制性内切酶XbaI和SacI酶切并进行胶回收纯化,其中APMV-7 HN基因由生物公司合成得到。将两者16℃过夜连接,转化入大肠杆菌BL21中培养16小时。挑取单克隆菌落进行PCR鉴定、质粒双酶切鉴定以及测序鉴定正确,将构建的表达载体命名为pMG-APMV-7-HN,重组质粒pMG-APMV-7-HN图谱如图6所示。
实施例2疫苗的制备
西林瓶清洗后包好,121℃15min灭菌,烘干后备用。准备灭菌的多瓣的西林瓶瓶塞。将活化的单菌落,接种至含有培养基的试管,培养好后与冻干保护剂(20g奶粉+80g纯水),按1:3比例加入混匀;每瓶分装2ml混匀液盖上瓶塞(从瓶塞瓣之间的缝隙),瓶身上写上菌株编号。将分装好的混匀液放在-20℃冰箱中过夜。次日,再将混匀液放在-80℃冰箱中冷冻2h。然后将含有混匀液的西林瓶一同放入提取预冻干的冻干机中。按照冻干机操作流程进行冻干。最后将西林瓶取出、压盖,并观察冻干效果,进行真空度检测。使用时,取冻干好的菌粉,加入2ml的生理盐水,混合均匀,根据免疫剂量稀释后直接口服。
对比例3
本对比例提供了一种口服含有NDV-HN蛋白的口服疫苗,与实施例2的区别在于,该疫苗的抗原为对比例1提供的重组质粒pMG-NDV-HN表达的蛋白。
对比例4
本对比例提供了一种口服含有NDV-HN蛋白的口服疫苗,与实施例2的区别在于,该疫苗的抗原为对比例1提供的重组质粒pMG-APMV-7-HN表达的蛋白。
效果例1疫苗的安全性试验
选取18-25g小白鼠,分为2组,分别为试验组和对照组,每组10只。试验组每只灌服实施例2提供的疫苗0.2mL,对照组灌服等剂量无菌生理盐水,灌服一次后,连续14天观察小白鼠精神状态,有无腹泻、采食量下降等不良症状出现。
效果例2疫苗的效力性试验
2.1选取18-25g小白鼠,平均分为对照组和免疫组。免疫组每只小白鼠灌服重组乳酸菌0.1ml,对照组灌服等剂量的无菌生理盐水。分别于免疫前及免疫后的7日、14日、21日、28日对每只小白鼠进行采血,测血液中抗体含量。
口服免疫后抗体水平结果:选择商品化小鼠新城疫抗体(NDV-Ab)ELISA检测试剂盒和禽副粘病毒ELISA试剂盒进行HN抗体检测。其中,免疫组1免疫实施例2提供的疫苗;免疫组2免疫对比例3提供的疫苗;免疫组3免疫对比例4提供的疫苗。免疫组对HN抗原的sIgA母源抗体水平都较高。免疫组首免14天后,抗体滴度逐渐上升,开始转阳;对照组无变化。免疫28天后,免疫组的抗体水平则继续升高,至免疫后60天,仍能检测到较高水平抗体。结果如表2所示:
表2小鼠血清中针对HN抗原特异性sIgA抗体转阳率情况
2.2重组菌在小白鼠体内的存留及抗体消长规律:重组菌口服免疫组在免疫后14日、28日、42日、56日以及60日分别采集免疫组1和对照组小鼠的直肠棉拭子进行检测,样品处理后涂布于5ug/ml红霉素的MRS平板,经37℃培养24h,挑取单个菌落,经扩大培养后用碱裂解法提取质粒,并进行PCR分析,PCR检测引物如表2所示;PCR产物经琼脂糖凝胶电泳,观察扩增片段大小,验证粪检菌是否为重组菌,结果如图8-A、图8-B、图8-C和表3所示,其中,M为DL2000DNA Marker,5泳道为阳性质粒,6泳道为PCR阴性对照,1~4泳道和7~27泳道为疫苗免疫组,28~52泳道为试验对照组。
表2 PCR鉴定引物
引物 | 序列(5’-3’) | 编号 |
NDV-HN-F | TCTCCAAGGTCGCCTCCGAA | SEQ ID NO.6 |
NDV-HN-R | CGAGGGCGACCTGCTTGTAG | SEQ ID NO.7 |
表3直肠棉拭子PCR检测结果
从上述实验结果可以看出,所有免疫组小鼠的肛拭子样品仅有1只小鼠粪便检测为阳性,其余均为阴性,说明粪便中重组菌含量较少,有利于机体的免疫应答,有利于抗体产生。
此外,融合表达的抗原产生针对HN抗原特异性sIgA抗体的转阳率高于单独表达的抗原。说明,融合表达的抗原增加了抗原的抗原性。
通过对免疫后小白鼠产生的抗体水平的追踪可以看出重组菌有很好的免疫原性,能够刺激机体产生针对HN的特异性sIgA抗体,并能长时间持续较高水平。结果表明本实验室研究的疫苗安全,可有效促进抗体形成,抑制病毒复制,增强鸽机体特异性和非特异性免疫力。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 天康生物股份有限公司
<120> 禽副粘病毒融合蛋白及其制备方法、应用和用于鸽子的APMV疫苗
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<170> PatentIn version 3.5
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Glu Arg Glu Ala Lys Asn Thr Trp Arg Leu Val Phe Arg Ile Ala Val
595 600 605
Leu Leu Leu Thr Val Val Thr Leu Ala Ile Ser Ala Ala Ala Leu Val
610 615 620
Tyr Ser Thr Gly Ala Ser Thr Pro Arg Asp Leu Val Gly Ile Ser Thr
625 630 635 640
Val Val Ser Lys Val Glu Asp Lys Ile Thr Ser Leu Leu Ser Ser Asn
645 650 655
Gln Asp Val Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro
660 665 670
Leu Ala Leu Leu Asn Thr Glu Ser Ile Ile Met Asn Ala Ile Thr Ser
675 680 685
Leu Ser His Gln Ile Asn Gly Ala Ala Asn Asn Ser Arg Cys Gly Ala
690 695 700
Pro Val His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile
705 710 715 720
Val Asp Asp Ile Ser Gly Val Thr Ser Phe Tyr Pro Ser Ala Tyr Gln
725 730 735
Glu His Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr
740 745 750
Arg Ile Pro Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His
755 760 765
Asn Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser His Gln Tyr
770 775 780
Leu Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe
785 790 795 800
Ser Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser
805 810 815
Cys Ser Val Ser Ala Thr Pro Leu Gly Cys Asp Ile Leu Cys Ser Lys
820 825 830
Val Thr Glu Ala Glu Glu Glu Asp Tyr Lys Ser Val Thr Pro Thr Ser
835 840 845
Met Val His Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp
850 855 860
Leu Asp Ile Thr Val Leu Phe Lys Asp Trp Val Ala Asn Tyr Pro Gly
865 870 875 880
Val Gly Ser Gly Ser Phe Ile Asp Asp Arg Val Trp Phe Pro Val Tyr
885 890 895
Gly Gly Leu Lys Pro Asn Ser Pro Ser Asp Thr Ala Gln Glu Gly Lys
900 905 910
Tyr Val Ile Tyr Lys Arg Tyr Asn Asn Thr Cys Pro Asp Gly Gln Glu
915 920 925
Tyr Gln Ile Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly
930 935 940
Gly Lys Arg Val Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser
945 950 955 960
Leu Gly Glu Asp Pro Val Leu Thr Val Pro Pro Asn Thr Ile Thr Leu
965 970 975
Met Gly Ala Glu Gly Arg Val Leu Thr Val Gly Thr Ser His Phe Leu
980 985 990
Tyr Gln Arg Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met
995 1000 1005
Thr Val Tyr Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe
1010 1015 1020
Asn Ala Phe Thr Arg Pro Gly Ile Val Pro Cys Gln Ala Ser Ala
1025 1030 1035
Arg Cys Pro Asn Pro Cys Ile Thr Gly Val Tyr Thr Asp Pro Tyr
1040 1045 1050
Pro Leu Val Phe Tyr Arg Asn His Thr Leu Arg Gly Val Phe Gly
1055 1060 1065
Thr Met Leu Asp Asp Glu Gln Glu Arg Leu Asn Pro Val Ser Ala
1070 1075 1080
Val Phe Asp Asn Ile Ser Arg Ser Arg Val Thr Arg Val Ser Ser
1085 1090 1095
Ser Ser Thr Lys Ala Ala Tyr Thr Thr Ser Thr Cys Phe Lys Val
1100 1105 1110
Val Lys Thr Asn Lys Val Tyr Cys Leu Ser Ile Ala Glu Ile Ser
1115 1120 1125
Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro Leu Leu Val Glu
1130 1135 1140
Ile Leu Lys Asp Asp Arg Val
1145 1150
<210> 4
<211> 1716
<212> DNA
<213> 鸽1型副粘病毒(Pigeon Paramyxovirus-1)
<400> 4
atggaccacg ccgtctccaa ggtcgcctcc gaaaacgaag aacgcgaagc caagaacacc 60
tggcgcctcg tcttccgcat cgccgtcctc ctcctcaccg tcgtcaccct cgccatctcc 120
gccgccgccc tcgtctactc caccggcgcc tccacccccc gcgacctcgt cggcatctcc 180
accgtcgtct ccaaggtcga agacaagatc acctccctcc tctcctccaa ccaggacgtc 240
gtcgaccgca tctacaagca ggtcgccctc gaatcccccc tcgccctcct caacaccgaa 300
tccatcatca tgaacgccat cacctccctc tcccaccaga tcaacggcgc cgccaacaac 360
tcccgctgcg gcgcccccgt ccacgacccc gactacatcg gcggcatcgg caaggaactc 420
atcgtcgacg acatctccgg cgtcacctcc ttctacccct ccgcctacca ggaacacctc 480
aacttcatcc ccgcccccac caccggctcc ggctgcaccc gcatcccctc cttcgacatg 540
tccgccaccc actactgcta cacccacaac gtcatcctct ccggctgccg cgaccactcc 600
cactcccacc agtacctcgc cctcggcgtc ctccgcacct ccgccaccgg ccgcgtcttc 660
ttctccaccc tccgctccat caacctcgac gacacccaga accgcaagtc ctgctccgtc 720
tccgccaccc ccctcggctg cgacatcctc tgctccaagg tcaccgaagc cgaagaagaa 780
gactacaagt ccgtcacccc cacctccatg gtccacggcc gcctcggctt cgacggccag 840
taccacgaaa aggacctcga catcaccgtc ctcttcaagg actgggtcgc caactacccc 900
ggcgtcggct ccggctcctt catcgacgac cgcgtctggt tccccgtcta cggcggcctc 960
aagcccaact ccccctccga caccgcccag gaaggcaagt acgtcatcta caagcgctac 1020
aacaacacct gccccgacgg ccaggaatac cagatccgca tggccaagtc ctcctacaag 1080
cccggccgct tcggcggcaa gcgcgtccag caggccatcc tctccatcaa ggtctccacc 1140
tccctcggcg aagaccccgt cctcaccgtc ccccccaaca ccatcaccct catgggcgcc 1200
gaaggccgcg tcctcaccgt cggcacctcc cacttcctct accagcgcgg ctcctcctac 1260
ttctcccccg ccctcctcta ccccatgacc gtctacaaca agaccgccac cctccactcc 1320
ccctacacct tcaacgcctt cacccgcccc ggcatcgtcc cctgccaggc ctccgcccgc 1380
tgccccaacc cctgcatcac cggcgtctac accgacccct accccctcgt cttctaccgc 1440
aaccacaccc tccgcggcgt cttcggcacc atgctcgacg acgaacagga acgcctcaac 1500
cccgtctccg ccgtcttcga caacatctcc cgctcccgcg tcacccgcgt ctcctcctcc 1560
tccaccaagg ccgcctacac cacctccacc tgcttcaagg tcgtcaagac caacaaggtc 1620
tactgcctct ccatcgccga aatctccaac accctcttcg gcgaattccg catcgtcccc 1680
ctcctcgtcg aaatcctcaa ggacgaccgc gtctaa 1716
<210> 5
<211> 1710
<212> DNA
<213> 禽副粘病毒7型(Avian paramyxovirus-7)
<400> 5
atggaatcca tcggcaaggg cacctggcgc accgtctacc gcgtcctcac catcctcctc 60
gacgtcgtca tcatcatcct ctccgtcatc gccctcatct ccctcggcct caagcccggc 120
gaacgcatca tcaacgaagt caacggctcc atccacaacc agctcgtccc cctctccggc 180
atcacctccg acatccaggc caaggtctcc tccatctacc gctccaacct cctctccatc 240
cccctccagc tcgaccagat caaccaggcc atctcctcct ccgcccgcca gatcgccgac 300
accatcaact ccttcctcgc cctcaacggc tccggcacct tcatctacac caactccccc 360
gaattcgcca acggcttcaa ccgcgccatg ttccccaccc tcaaccagtc cctcaacatg 420
ctcacccccg gcaacctcat cgaattcacc aacttcatcc ccacccccac caccaagtcc 480
ggctgcatcc gcatcccctc cttctccatg tcctcctccc actggtgcta cacccacaac 540
atcatcgcct ccggctgcca ggaccactcc acctcctccg aatacatctc catgggcgtc 600
gtcgaagtca ccgaccaggc ctaccccaac ttccgcacca ccctctccat caccctcgcc 660
gacaacctca accgcaagtc ctgctccatc gccgccaccg gcttcggctg cgacatcctc 720
tgctccgtcg tcaccgaaac cgaaaacgac gactaccagt cccccgaacc cacccagatg 780
atctacggcc gcctcttctt caacggcacc tactccgaaa tgtccctcaa cgtcaaccag 840
atgttcgccg actgggtcgc caactacccc gccgtcggct ccggcgtcga actcgccgac 900
ttcgtcatct tccccctcta cggcggcgtc aagatcacct ccaccctcgg cgcctccctc 960
tcccagtact actacatccc caaggtcccc accgtcaact gctccgaaac cgacgcccag 1020
cagatcgaaa aggccaaggc ctcctactcc ccccccaagg tcgcccccaa catctgggcc 1080
caggccgtcg tccgctgcaa caagtccgtc aacctcgcca actcctgcga aatcctcacc 1140
ttcaacacct ccaccatgat gatgggcgcc gaaggccgcc tcctcatgat cggcaagaac 1200
gtctacttct accagcgctc ctcctcctac tggcccgtcg gcatcatcta caagctcgac 1260
ctccaggaac tcaccacctt ctcctccaac cagctcctct ccaccatccc catccccttc 1320
gaaaagttcc cccgccccgc ctccaccgcc ggcgtctgct ccaagcccaa cgtctgcccc 1380
gccgtctgcc agaccggcgt ctaccaggac ctctgggtcc tctacgacct cggcaagctc 1440
gaaaacacca ccgccgtcgg cctctacctc aactccgccg tcggccgcat gaaccccttc 1500
atcggcatcg ccaacaccct ctcctggtac aacaccaccc gcctcttcgc ccagggcacc 1560
cccgcctcct actccaccac cacctgcttc aagaacacca agatcgacac cgcctactgc 1620
ctctccatcc tcgaactctc cgactccctc ctcggctcct ggcgcatcac ccccctcctc 1680
tacaacatca ccctctccat catgtcctaa 1710
<210> 6
<211> 20
<212> DNA
<213> 人工序列
<400> 6
tctccaaggt cgcctccgaa 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列
<400> 7
cgagggcgac ctgcttgtag 20
Claims (10)
1.一种禽副粘病毒融合蛋白,其特征在于,包含NDV-HN区段和AMPV-7-HN区段;所述NDV-HN区段包括如SEQ ID NO.1所示的氨基酸序列,所述AMPV-7-HN区段包括如SEQ IDNO.2所示的氨基酸序列。
2.根据权利要求1所述的禽副粘病毒融合蛋白,其特征在于,所述NDV-HN区段和所述AMPV-7-HN区段通过融合蛋白连接肽连接;
优选地,所述融合蛋白连接肽为柔性连接肽;
优选地,所述融合蛋白连接肽的序列为(PPSPS)n;n为1-3的正整数;n优选为2。
3.根据权利要求2所述的禽副粘病毒融合蛋白,其特征在于,所述禽副粘病毒融合蛋白具有如SEQ ID NO.3所示的氨基酸序列。
4.根据权利要求1-3中任一项所述的禽副粘病毒融合蛋白,其特征在于,所述禽副粘病毒融合蛋白的N端和/或C端连接有标签。
5.根据权利要求1-3中任一项所述的禽副粘病毒融合蛋白,其特征在于,所述禽副粘病毒融合蛋白为乳酸杆菌表达系统表达的蛋白。
6.一种权利要求1-5中任一项所述的禽副粘病毒融合蛋白的制备方法,其特征在于,包括:将所述禽副粘病毒融合蛋白的基因在宿主中表达。
7.根据权利要求6所述的制备方法,其特征在于,将所述禽副粘病毒融合蛋白的基因在乳酸杆菌中表达。
8.权利要求1-5中任一项所述的禽副粘病毒融合蛋白、权利要求6或7所述的制备方法或由所述制备方法制备得到的禽副粘病毒融合蛋白的应用,其特征在于,包括如下(x1)-(x6)中的至少一种:
(x1)制备鸽亚单位二联疫苗;
(x2)制备禽类病毒性疾病的疫苗;
(x3)制备禽副粘病毒的抗体;
(x4)制备禽副粘病毒的抗原;
(x5)制备检测禽副粘病毒的试剂和/或试剂盒;
(x6)制备检测禽副粘病毒抗体的试剂和/或试剂盒。
9.一种用于鸽子的APMV疫苗,其特征在于,所述APMV疫苗包含权利要求1-5中任一项所述的禽副粘病毒融合蛋白、表达所述禽副粘病毒融合蛋白的基因或由权利要求6或7所述的制备方法表达的禽副粘病毒融合蛋白。
10.根据权利要求9所述的APMV疫苗,其特征在于,所述APMV疫苗以表达上述禽副粘病毒融合蛋白的乳酸杆菌为活性成分。
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