CN109180821A - 鸡新城疫病毒的融合蛋白及其制备方法、应用和疫苗 - Google Patents
鸡新城疫病毒的融合蛋白及其制备方法、应用和疫苗 Download PDFInfo
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- CN109180821A CN109180821A CN201811097527.5A CN201811097527A CN109180821A CN 109180821 A CN109180821 A CN 109180821A CN 201811097527 A CN201811097527 A CN 201811097527A CN 109180821 A CN109180821 A CN 109180821A
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Abstract
本发明提供了一种鸡新城疫病毒的融合蛋白及其制备方法、应用和疫苗，涉及生物技术领域。该融合蛋白将鸡新城疫病毒F基因和HN基因序列进行串联，表达的融合蛋白具有抗原性好，表达量高的优点。该融合蛋白可应用于制备疫苗、抗体和用于检测鸡新城疫病毒的试剂盒。包含该融合蛋白的疫苗具有较好的免疫原性，该疫苗14日即可测定HI抗体几何平均滴度(GMT)均不低于1:64，而常规的灭活苗免疫后21日HI抗体几何平均滴度(GMT)不低于1:64，缩短断了疫苗免疫的空窗期，降低了生产成本低。
Description
技术领域
本发明涉及生物技术领域，尤其是涉及一种鸡新城疫病毒的融合蛋白及其制备方法、应用和疫苗。
背景技术
鸡新城疫(new castle disease，ND)是鸡新城疫病毒导致的禽类急性、接触性传染病，鸡新城疫病毒(Newcastle disease virus，NDV)属于副黏病毒科(Paramyxoviridae)禽腮腺炎病毒属(Avulavirus)，基因组为单股负链不分节的RNA。鸡新城疫病毒主要能导致鸡败血症，容易出现下痢、呼吸困难、神经紊乱、浆膜与粘膜出血。该病的致死率高，严重威胁禽类养殖，属烈性传染病。在鸡新城疫的防治方面，已经投入了大量的人力、物力与财力来研究鸡新城疫的防止措施。
目前鸡新城疫疫苗主要采用的是灭活疫苗、弱毒疫苗、基因工程苗等，其中灭活疫苗和弱毒疫苗具有生产工艺复杂、免疫保护力弱和生产成本高的缺点，逐步被基因工程疫苗所替代。现在常用的鸡新城疫基因工程苗多采用杆状病毒表达HN蛋白或F蛋白制备疫苗，虽然该疫苗安全稳定，但表达产物的抗体滴度低，影响了其实际应用。
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鸡新城疫病毒的融合蛋白，该融合蛋白将及鸡新城疫病毒的F蛋白和HN蛋白进行串联表达。
本发明的第二目的在于提供一种上述融合蛋白的制备方法，该制备方可以表达上述融合蛋白。
本发明的第三目的在于提供一种上述融合蛋白或上述制备方法制备得到的蛋白的应用，可应用于制备疫苗、抗体和病毒检测等多个方面。
本发明的第四目的在于提供一种包含上述融合蛋白的疫苗，该疫苗免疫原性好，保护效力高。
为解决上述技术问题，本发明特采用如下技术方案：
一种鸡新城疫病毒的融合蛋白，包括F区段和HN区段；所述F区段主要由SEQ IDNO.1所示的核苷酸序列表达，所述HN区段主要由SEQ ID NO.2所示的核苷酸序列表达。
优选地，F区段和HN区段的排列顺序为F-HN，通过Linker连接；
优选地，所述Linker具有如SEQ ID NO.3所示的核苷酸序列。
优选地，所述融合蛋白具有如SEQ ID NO.4所示的氨基酸序列。
本发明还提供了一种上述融合蛋白的制备方法，将所述融合蛋白的基因在宿主中表达；
优选地，使用哺乳动物表达系统表达所述融合蛋白的基因；
优选地，使用CHO细胞表达系统表达所述融合蛋白的基因。
优选地，提供包含表达所述融合蛋白的基因的表达载体，将所述表达载体导入CHO细胞中，然后对CHO细胞进行加压筛选，再将加压筛选出的CHO细胞驯化成悬浮培养的细胞株，使所述悬浮培养的细胞株表达所述融合蛋白。
优选地，使用谷氨酰胺合成酶筛选扩增系统加压筛选表达所述融合蛋白的CHO细胞株；
优选地，所述CHO细胞表达系统使用的表达载体包括pcDNA3、pEE6.4、pEE12.4或pGL4.13。
优选地，采用逐渐驯化的方法传代至少6代获得悬浮培养的表达所述融合蛋白的CHO细胞株。
本发明还提供了一种上述融合蛋白或上述制备方法制备得到的蛋白的应用，包括如下(a)-(d)中的至少一种：
(a)制备鸡新城疫病毒疫苗；
(b)制备鸡新城疫病毒抗体；
(c)制备鸡新城疫病毒抗体的试剂盒；
(d)制备鸡新城疫病毒诊断抗原。
本发明还提供了一种包含上述融合蛋白的鸡新城疫疫苗。
优选地，所述疫苗中所述融合蛋白的浓度为20-100μg/ml，优选30-80μg/ml，更优选50μg/ml；
优选地，所述疫苗还包括辅料，所述辅料包括疫苗佐剂、稳定剂和抗生素中的一种或多种；
优选地，所述疫苗佐剂包括氢氧化铝胶、弗氏完全佐剂、弗氏不完全佐剂、白油佐剂或MF59佐剂，优选使用白油佐剂。
与现有技术相比，本发明具有如下有益效果：
本发明提供的鸡新城疫病毒的融合蛋白，将鸡新城疫病毒F基因和HN基因序列进行串联，本发明通过对F基因和HN基因进行分析，选取了抗原性好，易高表达的区域进行串联，得到的融合蛋白具有抗原性好，表达量高的优点。
本发明提供的上述融合蛋白的制备方法，通过将上述融合蛋白的基因在宿主中表达即可。
本发明提供的上述融合蛋白和上述制备方法制备得到的蛋白的应用范围十分广泛，可以用以制备疫苗和抗体。抗体和上述融合蛋白可以应用于制备多种检测试剂和试剂盒，以用于检测鸡新城疫病毒抗体，例如使用含有鸡新城疫病毒抗体的ELISA试剂盒以检测鸡新城疫病毒，或者使用含有该融合蛋白的胶体金免疫层析试纸检测待测血清样品中的鸡新城疫病毒抗体含量。
本发明提供的包含上述融合蛋白的疫苗，具有较好的免疫原性，免疫动物后可以产生较高滴度的抗体滴度，使动物获得较好的保护效果。通过免疫鸡新城疫融合蛋白，家禽获得良好的保护。同时使生产工艺简便，节省人力物力。该疫苗14日即可测定HI抗体几何平均滴度(GMT)均不低于1:64，而常规的灭活苗免疫后21日HI抗体几何平均滴度(GMT)不低于1:64，缩短断了疫苗免疫的空窗期，降低了生产成本低。
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2提供的pcDNA3-F-HN质粒图谱；
图2为本发明实施例2提供的PCR鉴定结果；
图3为本发明实施例2提供的酶切定结果。
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。
本发明提供了一种鸡新城疫病毒的融合蛋白，该融合蛋白包括F区段和HN区段；所述F区段主要由SEQ ID NO.1所示的核苷酸序列表达，所述HN区段主要由SEQ ID NO.2所示的核苷酸序列表达。
融合蛋白F(Fusionglyeoprotein，F)和血凝素-神经氨酸酶蛋白(HaemagglutininNeuraminidaseprotein，HN)是外部糖蛋白，位于囊膜表面并且这两种蛋白分别形成了病毒的大纤突和小纤突，它们是宿主重要的保护性抗原。
F蛋白的基因分子量为59042kDa，编码一个含553个氨基酸的多肽。F基因从转录起始信号到polyA尾约有1792个核苷酸，含有一个开放阅读框。F蛋白含有3个抗原决定簇，在病毒的抗原性及其结构和功能上起着重要作用，F蛋白有两个七肽重复单位，这种单位每隔7个氨基酸就会出现一个疏水性氨基酸。HN蛋白是NDV中一种较大的蛋白，具有血球凝集(HA)和神经氨酸酶(NA)两种活性。HN基因全厂约2031bp，约占基因组的13.5％，含一个开放阅读框，胞外区有比较保守的半胱氨酸残基和糖基化位点。
本发明提供的融合蛋白，将鸡新城疫病毒F基因和HN基因序列进行串联，本发明通过对F基因和HN基因进行分析，选取了抗原性高，并且较易高表达的区域进行串联，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，F区段和HN区段的排列顺序为F-HN，通过Linker连接；Linker可以将融合蛋白彼此分开，不影响F区段和HN区段各自高级结构的形成，本发明优选使用具有如SEQ ID NO.3所示的核苷酸序列的Linker。在一个优选的实施方式中，所述融合蛋白具有如SEQ ID NO.4所示的氨基酸序列和具有如SEQ ID NO.5所示的核苷酸序列。
本发明还提供了一种上述融合蛋白的制备方法，该制备方法通过将上述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均可，由于哺乳动物细胞表达的蛋白经翻译加工后，其结构与生物学特性更接近于天然蛋白，因此本发明优选使用哺乳动物表达系统表达所述融合蛋白的基因，更优选使用CHO细胞表达系统表达所述融合蛋白的基因。CHO细胞即中国仓鼠卵巢细胞(Chinese hamster ovary)，CHO细胞表达系统具有如下优点：
(1)具有准确的翻译后折叠及修饰功能，表达的蛋白在分子结构，理化特性和生物学功能方面最接近于天然蛋白分子。
(2)具有产物胞外分泌功能，便于下游产物分离纯化。
(3)具有重组基因的高效扩增和表达能力。
(4)具有贴壁生长特性，具有较高的耐受剪切力和渗透压能力。也可以进行悬浮培养，表达水平较高。
(5)CHO属于成纤维细胞，很少分泌自身的内源蛋白质，利于外源蛋白的分离。
在一些优选的实施方式中，本发明对CHO表达系统进行加压筛选，以获得高表达融合蛋白的CHO细胞株。可选地，可以选择谷氨酰胺合成酶基因筛选扩增系统或者二氢叶酸还原酶基因筛选系统进行加压筛选，这两种系统可以单独使用，也可以结合使用，本发明对此不做限制。
选择标记和基因扩增CHO细胞表达载体主要有两类选择标记。一类是非扩增基因，它对目的基因的拷贝数没有影响，用于构建瞬时表达载体。另一类具有基因扩增的功能，也称共扩增基因，如二氢叶酸还原酶基因(dihydrofolatereductase，dhfr)，谷氨酰胺合成酶基因(glutaminesynthetase，GS)。当携带GS基因的表达质粒转染CHO细胞后，可以得到在选择培养基生长的细胞克隆，GS可被甲硫氨酸亚枫(MSX)所抑制，在极少数幸存下来的抗性细胞中，GS基因均得以扩增，结果会导致与GS基因串联在一起的外源基因的共扩增，拷贝数可增加几百到几千倍，从而使目的基因高水平表达，从而抵消MSX的抑制效应。
使用谷氨酰胺合成酶基因筛选扩增系统时，先将表达所述融合蛋白的基因克隆到具有GS筛选标记的表达载体上得到重组载体，然后将重组载体导入CHO细胞中，然后通过使用含有MSX的培养基培养CHO细胞对CHO细胞进行加压，以筛选出高表达目的基因。在一些优选的实施方式中，具有GS筛选标记的表达载体使用pcDNA3、pEE6.4、pEE12.4或pGL4.13。
悬浮生长的细胞其培养和传代都十分简便。传代时不需要再分散，只需按比例稀释后即可继续培养。此法细胞增殖快、产量高、培养过程简单，是大规模培养动物细胞的理想模式。由于CHO细胞具有既可以贴壁生长，也可以进行悬浮培养的特性，因此本发明优选将表达所述融合蛋白的CHO细胞进行驯化，使其能够悬浮培养。并且优选采用逐渐驯化的方法传代至少6代获得悬浮培养的表达所述融合蛋白的细胞株，以使悬浮细胞株细胞表达量高，且传代稳定。
本发明还提供了一种上述融合蛋白和上述制备方法制备得到的蛋白的应用，包括如下(a)-(d)中的至少一种：(a)制备鸡新城疫病毒疫苗；(b)制备鸡新城疫病毒抗体；(c)制备鸡新城疫病毒抗体的试剂盒；(d)制备鸡新城疫病毒诊断抗原。
由于本发明提供的鸡新城疫病毒融合蛋白具有较好的免疫原性，因此可以用以制备鸡新城疫病毒疫苗，优选制备F-HN亚单位疫苗，免疫动物后可产生较高的抗体的滴度。使用该融合蛋白制备的鸡新城疫病毒的抗体以及该融合蛋白可以应用于制备多种检测试剂和试剂盒，以用于检测鸡新城疫病毒抗体，例如使用含有鸡新城疫病毒的抗体的ELISA试剂盒以检测鸡新城疫病毒，或者使用含有该融合蛋白的胶体金免疫层析试纸检测待测血清样品中的鸡新城疫病毒抗体含量。
本发明还提供了一种包含上述融合蛋白的鸡新城疫病毒的疫苗，该疫苗具有较好的免疫原性，免疫动物后可以产生较高滴度的抗体滴度，使动物获得较好的保护效果。通过免疫鸡新城疫融合蛋白，家禽获得良好的保护。同时使生产工艺简便，节省人力物力。该疫苗14日即可测定HI抗体几何平均滴度(GMT)均不低于1:64，而常规的灭活苗免疫后21日HI抗体几何平均滴度(GMT)不低于1:64，缩短断了疫苗免疫的空窗期，降低了生产成本低。
在一些优选的实施方式中，疫苗中所述融合蛋白的浓度为20-100μg/ml，优选30-80μg/ml，更优选50μg/ml。优选地，所述疫苗还包括辅料，例如可以为但不限于为疫苗佐剂、稳定剂或抗生素。优选包括疫苗佐剂，所述疫苗佐剂例如可以为但不限于为氢氧化铝胶、弗氏完全佐剂、弗氏不完全佐剂、白油佐剂或MF59佐剂，优选使用白油佐剂。
下面结合优选实施例进一步说明本发明的有益效果。本发明试剂及药品来源如下：中华仓鼠卵巢细胞(CHO)购自美国ATCC公司；细胞培养基和血清均购自美国gibcom公司；真核表达载体pCDNA3购自美国ThermoFisher公司；Lipofectamine LTX购自北京索莱宝科技有限公司；氨甲基喋呤(mnethotrexate MTX)购自Sigma公司；甲硫氨酸亚砜亚铵(L-methioninesulfoximine MSX)购于Sigma公司；BCA蛋白质定量试剂盒购自美国ThermoFisher公司；白油购自美国索诺邦公司。
需要说明的是，实施例中的反应体系和反应条件仅是一种举例。实施例中各步骤中的反应体系和反应条件均可以在可接受的范围内进行调整，以优化反应条件，本发明对此不做限制。
实施例1：F-HN基因设计及合成
F-HN基因设计及合成：选用鸡新城疫病毒La Sota毒株的F基因序列及NH基因序列进行分析，对F基因及HN基因的序列进行密码子的优化，保留其保守序列及抗原表位序列，使F基因及HN基因具有广谱的抗原性。F基因(GeneID:912271)经优化后，设计长度1032bp，具有如SEQ ID NO.1所示序列；HN基因(GeneID:912270)经优化后设计长度1229bp，具有如SEQ ID NO.2所示序列。F基因与HN基因间使用linker序列连接，linker序列具有如SEQ IDNO.3所示序列。F-HN基因全长2291bp，具有如SEQ ID NO.4所示氨基酸序列和如SEQ IDNO.5所示核苷酸序列。F-HN基因合成由上海生工生物公司完成。
实施例2：pcDNA3-F-HN重组质粒构建
2.1添加酶切位点：通过PCR扩增在F-HN基因序列的上游和下游分别添加酶切位点：HindⅢ、XbaⅠ，PCR扩增上游引物如SEQ ID NO.6所示，PCR扩增下游引物如SEQ ID NO.7所示。pcDNA3-F-HN质粒图谱如图1所示。
2.2F-HN基因及载体双酶切反应
2.2.1F-HN串联基因及载体双酶切反应
| 10×buffer | 2.5μL |
| DNA样品 | 1μg |
| HindⅢ | 1μL |
| XbaⅠ | 1μL |
补充dd H20至25μL，将各组分混合均匀后37℃水浴2h。
2.2.2酶切目的DNA片段回收：将酶切后产物，进行琼脂糖凝胶电泳，回收其中的DNA片段采用DNA凝胶回收试剂盒(购自北京莱宝科技有限公司)，回收酶切目的片段，步骤如下：
(1)琼脂糖凝胶电泳后，将目的DNA条带从琼脂糖凝胶中用刀片小心切下，放入1.5mL EP管中，称取重量。
(2)向EP管中加入3倍体积溶胶液，50-55℃水浴10min，期间小心翻转离心管，确保胶块充分溶解。
(3)将上一步所得溶液加入吸附柱中(吸附柱放入收集管中)，12000rpm离心30-60s，倒掉收集管中的废液，将吸附柱重新放入收集管中。
(4)向吸附柱中加入600μL漂洗液，12000rpm离心1min，弃废液，将吸附柱放入收集管中。
(5)向吸附柱中加入600μL漂洗液，12000rpm离心1min，弃废液，将吸附柱放入收集管中。
(6)12000rpm离心2min，尽量除去漂洗液。将吸附柱敞口置于室温或50℃温箱放置2min。
(7)将吸附柱放入1.5mL EP管中，向吸附膜中央悬空滴加适量经65℃水浴预热的洗脱液，室温放置2min，12000rpm离心1min。
2.3F-HN基因及载体连接反应，构建10μL反应体系，然后将连接反应体系混合均匀后置于16℃冷水浴中10-16h，后放入65℃水浴15min，最后4℃保存。
| 10×T4buffer | 1μL |
| DNA片段 | 6μL |
| 载体 | 2μL |
| T4连接酶 | 1μL |
2.4转化反应
将10μL连接反应液加入到100μL感受态细胞中，混匀后冰浴30min，42℃水浴100s，再冰浴2min。然后取出EP管，加入600μL LB培养液，置于37℃恒温摇床，240rpm培养1h后取出EP管，室温离心8000rpm，2min，吸除500μl上清液，重悬吹匀菌体，将重悬的菌液滴到转化平板上，用涂菌棒将菌液均匀铺开。然后将转化平板置于恒温培养箱中，37℃培养1h后，将转化平板倒置进行培养15h，培养完毕后观察转化结果。
2.5质粒抽提与双酶切鉴定
2.5.1质粒提取，使用美国OMEGA质粒提取试剂盒，提取方法按照试剂盒使用说明书进行提取。
(1)挑取转化板中单个菌落至5ml含氨苄抗性的LB液体培养基中，37℃，240rpm培养过夜。
(2)取菌液1.5ml至EP管中，室温条件下离心10000rpm1min，弃上清，然后加入250μL溶液I，振荡混匀；再加入250μL溶液II，小心颠倒EP管4-6次，室温静置2min，至澄清，然后加入350μL溶液III，小心颠倒离心管4-6次，直至出现白色絮状沉淀，10000rpm室温离心10min。
(3)小心吸取上清溶液，并移至吸附柱中心，10000rpm室温离心1min，倒掉收集管中液体后加入500μL Buffer HB，10000rpm离心1min，弃滤液，然后加如700μL WashBuffer，10000rpm离心1min，弃滤液；重复1次。
(4)室温离心空吸附柱，13000rpm，2min，将吸附柱放入干净的1.5ml EP管中，加30μL去离子水在滤膜上，室温静置5min，13000rpm，2min。保存管中DNA溶液。
2.5.2双酶切鉴定，构建20μL反应体系：
| 10×buffer | 2μL |
| DNA样品 | 1μg |
| HindⅢ | 1μL |
| XbaⅠ | 1μL |
补ddH20至20μL后混合均匀，37℃水浴2h后进行凝胶电泳检测，并将插入DNA片段送公司测序，PCR及双酶切鉴定结果如图2和图3所示。
2.6去内毒素质粒大提，采用去内毒素质粒大量提取试剂盒(北京索莱宝科技有限公司)
(1)将测序正确的克隆接种至100ml含氨苄抗性的培养基中，220rpm，37℃恒温培养15h后，取50ml细菌培养物至50ml离心管中，11000rpm离心1min，吸除上清。
(2)加4ml溶液P1，振荡器悬浮细菌细胞沉淀，然后加4ml溶液P2，温和颠倒6-8次使菌体充分裂解，最后再加4ml溶液P3，立即温颠倒6-8次，充分混匀，至出现白色絮状沉淀，11000rpm离心10min后，将上清移到另一个干净的离心管中。
(3)加上清1/5体积的冰上预冷的内毒素清除剂，振荡混匀，冰浴2min至溶液变清亮，然后37℃水浴5min，不时振荡。
(4)11000rpm室温离心5min，溶液分为两相，上层水相含质粒DNA，下层油相含内毒素，将含质粒DNA的上层水相转移至新管，弃下层油相；重复三次。
(5)加入12ml的结合液，充分混匀后加入吸附柱中，室温放置2min，11000rpm离心1min，倒掉收集管中的废液，将吸附柱重新放回收集管中。
(6)然后加入8ml漂洗液，11000rpm离心1min，弃废液，将吸附柱放入收集管中，再加入6ml漂洗液，11000rpm离心1min，弃废液，将吸附柱放入收集管中。然后11000rpm离心3min，将吸附柱敞口置于室温或50℃温箱放置4-5min。
(7)最后将吸附柱放入一个干净的离心管中，向吸附膜滴2ml经65℃水浴预热的洗脱液，室温放置5min，11000rpm离心2min，-20℃保存。
实施例3：pcDNA3-F-HN重组质粒转染CHO-K1细胞
(1)取出细胞，弃去上清培养基，用预温的8ml PBS洗一次，弃去PBS，然后每个培养皿加入2ml 0.25％trypsin-EDTA，室温消化2min，镜下观察细胞变圆，呈单个细胞。加入4mlDMEM/F12(含10％血清，1％氨苄-链霉素双抗)终止消化反应，用移液器将细胞吹散后转移至15ml离心管中，200rpm离心5min。
(2)DMEM/F12(含10％血清，1％氨苄-链霉素双抗)重新悬浮细胞，计数后，稀释细胞至2×105个/ml，取2ml混匀的细胞加入到六孔培养皿，置37℃、5％CO2细胞培养箱中孵育过夜。
(3)观察细胞状态：当细胞交汇度达到80％-90％时开始转染，转染前将培养基换DMEM/F12(无血清无双抗)，2mL/孔。
(4)用OPTI-MEM稀释质粒，125μl OPTI-MEM中加入2.5μg质粒，然后加入2.5μlplus，混合均匀后室温静置5min。
(5)稀释Lipofectamine LTX：125μl OPTI-MEM中加入9μl Lipofectamine LTX，然后加入2.5μl plus，轻轻混匀，室温静置5min。
(6)将稀释质粒和稀释Lipofectamine LTX混合混匀，室温放置5min，然后逐滴加入六孔培养皿中均匀分布。
(7)将六孔培养皿置于37℃，5％CO2细胞培养箱中培养4-6h后换液：弃掉上清培养基，加入2ml DMEM/F12(含10％血清，1％氨苄-链霉素双抗)，将六孔板置于37℃，5％CO2细胞培养箱中培养。
实施例4：单克隆细胞株筛选
(1)转染后24h开始加压：从37℃培养箱中取出六孔培养皿，弃去上清，加入2mlDMEM/F12(含10％血清+25μM MSX)，加压7d，期间镜下观察，死细胞多时换液。
(2)加压筛选至阴性对照细胞死亡至少90％以上时，开始单克隆筛选；
(3)取出六孔培养皿，弃培养基，用PBS洗一次，加入300μl 0.25％trypsin-EDTA，室温消化2min，加2ml DMEM/F12(含10％血清+25μM MSX)终止消化反应，用移液器将细胞吹散，然后将细胞转移至15ml离心管中，200rpm离心5min。
(4)DMEM/F12(含10％血清+25μM MSX)重新悬浮细胞，计数，然后将细胞稀释至5个/ml，取200μL加入到96孔板中，放置到37℃，5％CO2细胞培养箱中孵育4-6h后标记出单个细胞的孔。
(5)96孔板中单个细胞的孔长满时，弃培养基，PBS洗一次，加入100μl 0.25％trypsin-EDTA，室温消化2min，加入2ml DMEM/F12(含10％血清+25μM MSX)终止反应，用移液器将细胞吹散，然后将细胞液转移至12孔板，细胞长满时，取上清，ELISA检测，高效表达的继续培养、冻存。
(6)经过筛选，共收获2株细胞株，编号为11株、35株。
实施例5：CHO-K1细胞株驯化成悬浮培养细胞株
(1)从37℃培养箱中取出细胞培养皿，弃去上清，用8ml PBS洗细胞一次，并弃去PBS，然后向培养皿加入2ml 0.25％trypsin-EDTA，室温消化2min，镜下观察细胞由皱变圆，呈单个细胞。然后加入4ml DMEM/F12(含10％血清，25μM MSX)终止消化反应，用移液器将细胞吹散。然后将细胞液转移至15ml离心管中，200rpm离心5min。
(2)使用100％DMEM/F12(含10％血清，25μM MSX)悬浮细胞，计数后稀释细胞至5×105个/ml，接种30ml培养基于125ml摇瓶中，置37℃、5％CO2细胞培养箱中的轨道式振荡器上115rpm孵育过夜，并且每隔24h计数1次，观察细胞密度及活力。
(3)当第一代细胞培养一次后细胞活率达到94％-97％时，进行第二代培养，将第一代细胞转移至50ml离心管中，200rpm离心5min，然后将DMEM/F12(含10％血清，25μM MSX)和EX-CELL302按1：1混合同时加入对应浓度嘌呤霉素后混匀，重新悬浮细胞，计数后稀释细胞至5×105个/ml，接种30ml培养基于一个125ml摇瓶中，置37℃，5％CO2细胞培养箱中的轨道式振荡器上115rpm孵育过夜，并且每隔24h计数1次，观察细胞密度及活力。
(4)当第二代培养两次后得到的细胞存活率大于95％，第三至六代培养三次后得到的细胞存活率大于95％。7周后，细胞接种3天后繁殖三代，密度达到1×106个/ml，同时细胞存活率达到95％，该细胞被认为已经适应悬浮培养，接种密度降低到3×105个/ml。经过驯化，编号11株的细胞系满足要求，表明驯化成功。
实施例6：细胞发酵
(1)配制培养基：60％的CD-CHO+40％的Ex-cell302置于37℃水浴锅中预热；
(2)从CO2恒温摇床取出摇瓶细胞，计数后稀释细胞至3.0×105个/ml，接种30ml培养基于125ml摇瓶中，置37℃、5％CO2恒温摇床中，115rpm过夜培养。
(3)每隔24h细胞计数，观察密度及活力，监测葡萄糖浓度，当葡萄糖浓度低于2g/L的时候，添加葡萄糖到4g/L；每天取1ml样品上清，检测蛋白表达情况。
(4)补料：约第4天，65g/L CB5，添加基础培养基的10％；第5天，将CO2培养箱温度调整至32℃；第9天，补充60g/L CB5，添加基础培养基的10％；第12天，收获细胞。
(5)Werstern-Blotting检测。
实施例7：蛋白纯化
(1)收集细胞培养液，4℃，11000rpm离心25min，取上清后过滤膜(0.45μm)，准备上样。
(2)柱平衡：用超纯水平衡3个柱体积，排出乙醇保存液；加入BufferA(20mMNaH2PO4，500mM NaCl)4-8ml/min，平衡3个柱体积。
(3)上样：用5ml预装柱，1ml/min进行上样(根据预装柱体积调节上样流速，保留时间5min)，收集Flow through(FT)。
(4)洗涤：4％bufferB(20mM NaH2PO4、500mM NaCl、100mM imidazole)洗柱，流速为4ml/min，至OD280nm基线平稳为止。
(5)洗脱：50％bufferB(20mM NaH2PO4、500mM NaCl、100mM imidazole)洗脱目的蛋白，至基线洗平，2ml/min，收集5ml/管。
(6)洗涤：100％bufferB(20mM NaH2PO4、500mM NaCl、500mM imidazole)4ml/min，冲洗2-3个柱体积，至UV基线洗平，超纯水平衡3柱体积。
(7)透析：Millipore 10KD PBS(pH7.4)4℃透析，次换液咪唑稀释倍数为2，换液次数为8次。
(8)除菌过滤：用0.22μm滤器过滤，蛋白样品溶液-80℃冰箱保存。
(9)蛋白浓度和纯度测定：采用BCA法测定蛋白浓度，采用HPLC方法检测纯度，纯度都能达到95％以上。
实施例8：疫苗制备与免疫接种
8.1鸡新城疫亚单位疫苗制备将表达的F-HN融合蛋白与白油佐剂按体积比1：3混合，乳化，蛋白终浓度为50μg/ml。
8.2疫苗免疫及抗体检测：将表达F-HN蛋白超滤换液后，与白油佐剂混合乳化成疫苗(5μg/羽份)，免疫3-4周龄SPF鸡后，14日、21日分别采血测HI抗体，结果显示14日检测到HI抗体几何平均滴度(GMT)均不低于1:64，21日HI抗体几何平均滴度(GMT)均不低于1:128。
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。
序列表
<110> 天康生物股份有限公司
<120> 鸡新城疫病毒的融合蛋白及其制备方法、应用和疫苗
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1032
<212> DNA
<213> 鸡新城疫病毒(Newcastle disease virus)
<400> 1
atgggctcca gaccttttac caagaaccca gcacctatga tgctgactat ccgggtcgcg 60
ctggtattga gttgcatctg tccggcaaac tccattgatg gcaggccttt tgcagctgca 120
ggaattgtgg ttacaggaga caaagcagtc aacatataca cctcatccca gacaggatca 180
atcatagtta agctcctccc gaatctgccc aaggataagg aggcatgtgc gaaagccccc 240
ttggatgcat acaacaggac attgaccact ttgctcaccc cccttggtga ctctatccgt 300
aggatacaag agtctgtgac tacatctgga ggggggagac aggggcgcct tataggcgcc 360
attattggcg gtgtggctct tggggttgca actgccgcac aaataacagc ggccgcagct 420
ctgatacaag ccaaacaaaa tgctgccaac atcctccgac ttaaagagag cattgccgca 480
accaatgagg ctgtgcatga ggtcactgac ggattatccc aactagcagt ggcagttggg 540
aagatgcagc agtttgttaa tgaccaattt aataaaacag ctcaggaatt agactgcata 600
aaaattgcac agcaagttgg tgtagagctc aacctgtacc taaccgaatt gactacagta 660
ttcggaccac aaatcacttc acctgcctta aacaagctga ctattcaggc actttacaat 720
ctagctggtg ggaatatgga ttacttattg actaagttag gtatagggaa caatcaactc 780
agctcattaa tcggtagcgg cttaatcacc ggtaacccta ttctatacga ctcacagact 840
caactcttgg gtatacaggt aactctacct tcagtcggga acctaaataa tatgcgtgcc 900
acctacttgg aaaccttatc cgtaagcaca accaggggat ttgcctcggc acttgtccca 960
aaagtggtga cacaggtcgg ttctgtgata gaagaacttg acacctcata ctgtatagaa 1020
actgacttag at 1032
<210> 2
<211> 1229
<212> DNA
<213> 鸡新城疫病毒(Newcastle disease virus)
<400> 2
atggaccgcg ccgttagcca agttgcgtta gagaatgatg aaagagaggc aaaaaataca 60
tggcgcttga tattccggat tgcaatctta ttcttaacag tagtgacctt ggctatatct 120
gtagcctccc ttttatatag catgggggct agcacaccta gcgatcttgt aggcataccg 180
actaggattt ccagggcaga agaaaagatt acatctacac ttggttccaa tcaagatgta 240
gtagatagga tatataagca agtggccctt gagtctccat tggcattgtt aaatactgag 300
accacaatta tgaacgcaat aacatctctc tcttatcaga ttaatggagc tgcaaacaac 360
agcgggtggg gggcacctat tcatgaccca gattatatag gggggatagg caaagaactc 420
attgtagatg atgctagtga tgtcacatca ttctatccct ctgcatttca agaacatctg 480
aattttatcc cggcgcctac tacaggatca ggttgcactc gaataccctc atttgacatg 540
agtgctaccc attactgcta cacccataat gtaatattgt ctggatgcag agatcactca 600
cactcatatc agtatttagc acttggtgtg ctccggacat ctgcaacagg gagggtattc 660
ttttctactc tgcgttccat caacctggac gacacccaaa atcggaagtc ttgcagtgtg 720
agtgcaactc ccctgggttg tgatatgctg tgctcgaaag ccacggagac agaggaagaa 780
gattataact cagctgtccc tacgcggatg gtacatggga ggttagggtt cgacggccaa 840
tatcacgaaa aggacctaga tgtcacaaca ttattcgggg actgggtggc caactaccca 900
ggagtagggg gtggatcttt tattgacagc cgcgtatggt tctcagtcta cggagggtta 960
aaacccaatt cacccagtga cactgtacag gaagggaaat atgtgatata caagcgatac 1020
aatgacacat gcccagatga gcaagactac cagattcgaa tggccaagtc ttcgtataag 1080
cctggacggt ttggtgggaa acgcatacag caggctatct tatctatcaa agtgtcaaca 1140
tccttaggcg aagacccggt actgactgta ccgcccaaca cagtcacact catgggggcc 1200
gaaggcagaa ttctcacagt agggacatc 1229
<210> 3
<211> 24
<212> DNA
<213> 人工序列()
<400> 3
ggaggaggat ctggaggagg atct 24
<210> 4
<211> 763
<212> PRT
<213> 人工序列()
<400> 4
Met Gly Ser Arg Pro Phe Thr Lys Asn Pro Ala Pro Met Met Leu Thr
1 5 10 15
Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile
20 25 30
Asp Gly Arg Pro Phe Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys
35 40 45
Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys
50 55 60
Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro
65 70 75 80
Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly
85 90 95
Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly
100 105 110
Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Val Ala Leu Gly
115 120 125
Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala
130 135 140
Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala
145 150 155 160
Thr Asn Glu Ala Val His Glu Val Thr Asp Gly Leu Ser Gln Leu Ala
165 170 175
Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys
180 185 190
Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Ala Gln Gln Val Gly Val
195 200 205
Glu Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gly Pro Gln
210 215 220
Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Ile Gln Ala Leu Tyr Asn
225 230 235 240
Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly
245 250 255
Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn
260 265 270
Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr
275 280 285
Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu
290 295 300
Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Ala Ser Ala Leu Val Pro
305 310 315 320
Lys Val Val Thr Gln Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser
325 330 335
Tyr Cys Ile Glu Thr Asp Leu Asp Gly Gly Gly Gly Ser Gly Gly Gly
340 345 350
Gly Ser Met Asp Arg Ala Val Ser Gln Val Ala Leu Glu Asn Asp Glu
355 360 365
Arg Glu Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu
370 375 380
Phe Leu Thr Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu Tyr
385 390 395 400
Ser Met Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg
405 410 415
Ile Ser Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn Gln
420 425 430
Asp Val Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu
435 440 445
Ala Leu Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile Thr Ser Leu
450 455 460
Ser Tyr Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala Pro
465 470 475 480
Ile His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val
485 490 495
Asp Asp Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gln Glu
500 505 510
His Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg
515 520 525
Ile Pro Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn
530 535 540
Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser Tyr Gln Tyr Leu
545 550 555 560
Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe Ser
565 570 575
Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser Cys
580 585 590
Ser Val Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys Ala
595 600 605
Thr Glu Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pro Thr Arg Met
610 615 620
Val His Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu
625 630 635 640
Asp Val Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro Gly Val
645 650 655
Gly Gly Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val Tyr Gly
660 665 670
Gly Leu Lys Pro Asn Ser Pro Ser Asp Thr Val Gln Glu Gly Lys Tyr
675 680 685
Val Ile Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln Asp Tyr
690 695 700
Gln Ile Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly Gly
705 710 715 720
Lys Arg Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu
725 730 735
Gly Glu Asp Pro Val Leu Thr Val Pro Pro Asn Thr Val Thr Leu Met
740 745 750
Gly Ala Glu Gly Arg Ile Leu Thr Val Gly Thr
755 760
<210> 5
<211> 2291
<212> DNA
<213> 人工序列()
<400> 5
atgggctcca gaccttttac caagaaccca gcacctatga tgctgactat ccgggtcgcg 60
ctggtattga gttgcatctg tccggcaaac tccattgatg gcaggccttt tgcagctgca 120
ggaattgtgg ttacaggaga caaagcagtc aacatataca cctcatccca gacaggatca 180
atcatagtta agctcctccc gaatctgccc aaggataagg aggcatgtgc gaaagccccc 240
ttggatgcat acaacaggac attgaccact ttgctcaccc cccttggtga ctctatccgt 300
aggatacaag agtctgtgac tacatctgga ggggggagac aggggcgcct tataggcgcc 360
attattggcg gtgtggctct tggggttgca actgccgcac aaataacagc ggccgcagct 420
ctgatacaag ccaaacaaaa tgctgccaac atcctccgac ttaaagagag cattgccgca 480
accaatgagg ctgtgcatga ggtcactgac ggattatccc aactagcagt ggcagttggg 540
aagatgcagc agtttgttaa tgaccaattt aataaaacag ctcaggaatt agactgcata 600
aaaattgcac agcaagttgg tgtagagctc aacctgtacc taaccgaatt gactacagta 660
ttcggaccac aaatcacttc acctgcctta aacaagctga ctattcaggc actttacaat 720
ctagctggtg ggaatatgga ttacttattg actaagttag gtatagggaa caatcaactc 780
agctcattaa tcggtagcgg cttaatcacc ggtaacccta ttctatacga ctcacagact 840
caactcttgg gtatacaggt aactctacct tcagtcggga acctaaataa tatgcgtgcc 900
acctacttgg aaaccttatc cgtaagcaca accaggggat ttgcctcggc acttgtccca 960
aaagtggtga cacaggtcgg ttctgtgata gaagaacttg acacctcata ctgtatagaa 1020
actgacttag atggaggagg aggatctgga ggaggaggat ctatggaccg cgccgttagc 1080
caagttgcgt tagagaatga tgaaagagag gcaaaaaata catggcgctt gatattccgg 1140
attgcaatct tattcttaac agtagtgacc ttggctatat ctgtagcctc ccttttatat 1200
agcatggggg ctagcacacc tagcgatctt gtaggcatac cgactaggat ttccagggca 1260
gaagaaaaga ttacatctac acttggttcc aatcaagatg tagtagatag gatatataag 1320
caagtggccc ttgagtctcc attggcattg ttaaatactg agaccacaat tatgaacgca 1380
ataacatctc tctcttatca gattaatgga gctgcaaaca acagcgggtg gggggcacct 1440
attcatgacc cagattatat aggggggata ggcaaagaac tcattgtaga tgatgctagt 1500
gatgtcacat cattctatcc ctctgcattt caagaacatc tgaattttat cccggcgcct 1560
actacaggat caggttgcac tcgaataccc tcatttgaca tgagtgctac ccattactgc 1620
tacacccata atgtaatatt gtctggatgc agagatcact cacactcata tcagtattta 1680
gcacttggtg tgctccggac atctgcaaca gggagggtat tcttttctac tctgcgttcc 1740
atcaacctgg acgacaccca aaatcggaag tcttgcagtg tgagtgcaac tcccctgggt 1800
tgtgatatgc tgtgctcgaa agccacggag acagaggaag aagattataa ctcagctgtc 1860
cctacgcgga tggtacatgg gaggttaggg ttcgacggcc aatatcacga aaaggaccta 1920
gatgtcacaa cattattcgg ggactgggtg gccaactacc caggagtagg gggtggatct 1980
tttattgaca gccgcgtatg gttctcagtc tacggagggt taaaacccaa ttcacccagt 2040
gacactgtac aggaagggaa atatgtgata tacaagcgat acaatgacac atgcccagat 2100
gagcaagact accagattcg aatggccaag tcttcgtata agcctggacg gtttggtggg 2160
aaacgcatac agcaggctat cttatctatc aaagtgtcaa catccttagg cgaagacccg 2220
gtactgactg taccgcccaa cacagtcaca ctcatggggg ccgaaggcag aattctcaca 2280
gtagggacat c 2291
<210> 6
<211> 30
<212> DNA
<213> 人工序列()
<400> 6
ccaagcttat gggctccaga ccttttacca 30
<210> 7
<211> 28
<212> DNA
<213> 人工序列()
<400> 7
gctctagaga tgtccctact gtgagaat 28
Claims (10)
1.一种鸡新城疫病毒的融合蛋白，其特征在于，包括F区段和HN区段；所述F区段主要由SEQ ID NO.1所示的核苷酸序列表达，所述HN区段主要由SEQ ID NO.2所示的核苷酸序列表达。
2.根据权利要求1所述的融合蛋白，其特征在于，F区段和HN区段的排列顺序为F-HN，通过Linker连接；
优选地，所述Linker具有如SEQ ID NO.3所示的核苷酸序列。
3.根据权利要求1或2所述的融合蛋白，其特征在于，所述融合蛋白具有如SEQ ID NO.4所示的氨基酸序列。
4.一种权利要求1-3中任一项所述的融合蛋白的制备方法，其特征在于，将所述融合蛋白的基因在宿主中表达；
优选地，使用哺乳动物表达系统表达所述融合蛋白的基因；
优选地，使用CHO细胞表达系统表达所述融合蛋白的基因。
5.根据权利要求4所述的制备方法，其特征在于，提供包含表达所述融合蛋白的基因的表达载体，将所述表达载体导入CHO细胞中，然后对CHO细胞进行加压筛选，再将加压筛选出的CHO细胞驯化成悬浮培养的细胞株，使所述悬浮培养的细胞株表达所述融合蛋白。
6.根据权利要求5所述的制备方法，其特征在于，使用谷氨酰胺合成酶筛选扩增系统加压筛选表达所述融合蛋白的CHO细胞株；
优选地，所述CHO细胞表达系统使用的表达载体包括pcDNA3、pEE6.4、pEE12.4或pGL4.13。
7.根据权利要求5所述的制备方法，其特征在于，采用逐渐驯化的方法传代至少6代获得悬浮培养的表达所述融合蛋白的CHO细胞株。
8.权利要求1-3中任一项所述的融合蛋白或权利要求4-7中任一项所述的制备方法制备得到的蛋白的应用，其特征在于，包括如下(a)-(d)中的至少一种：
(a)制备鸡新城疫病毒疫苗；
(b)制备鸡新城疫病毒抗体；
(c)制备鸡新城疫病毒抗体的试剂盒；
(d)制备鸡新城疫病毒诊断抗原。
9.一种包含权利要求1-3中任一项所述的融合蛋白的鸡新城疫疫苗。
10.根据权利要求9所述的疫苗，其特征在于，所述疫苗中所述融合蛋白的浓度为20-100μg/ml，优选30-80μg/ml，更优选50μg/ml；
优选地，所述疫苗还包括辅料，所述辅料包括疫苗佐剂、稳定剂和抗生素中的一种或多种；
优选地，所述疫苗佐剂包括氢氧化铝胶、弗氏完全佐剂、弗氏不完全佐剂、白油佐剂或MF59佐剂，优选使用白油佐剂。
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