CN115010814A - 一种诺如病毒p颗粒嵌合棘球蚴eg95蛋白的重组蛋白及其应用 - Google Patents
一种诺如病毒p颗粒嵌合棘球蚴eg95蛋白的重组蛋白及其应用 Download PDFInfo
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Abstract
本发明提供了一种诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白及其应用，所述重组蛋白的氨基酸序列如SEQ ID NO:1所示。所述诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白可作为棘球蚴疫苗用于防治棘球蚴病，所述棘球蚴疫苗结合诺如病毒P颗粒和棘球蚴EG95抗原两者的优势，P颗粒具有高度免疫原性和稳定性，诺如病毒P颗粒作为抗原递呈载体平台，EG95蛋白能够被充分呈递，具有广阔的前景。
Description
技术领域
本发明属于生物技术领域，具体涉及一种诺如病毒P颗粒嵌合棘球蚴EG95 蛋白的重组蛋白及其应用。
背景技术
棘球蚴病(Echinococcosis)又称包虫病(Hydatid disease)，是由棘球属(Echinococcus)绦虫的中绦期幼虫寄生于人体及某些动物体内引起的一种严重的人畜共患寄生虫病。我国流行的以细粒棘球绦虫引起的囊型包虫病(cystic echinococcosis，CE)为主，细粒棘球蚴病对人畜危害极其严重，人畜感染后可在肝脏、肺脏等器官发现占位性病灶(囊泡)，各种动物都可因囊泡破裂产生严重过敏反应而死亡。
CN112250748A公开了一种细粒棘球蚴免疫原性蛋白EG95基因组成型表达载体，且提供含有该表达载体的毕赤酵母基因工程菌。表达的重组蛋白与现有羊棘球蚴病EG95亚单位疫苗免疫阳性血清具有良好的反应原性和特异性，与其他重组蛋白抗体无反应。
CN110016457A公开了一株重组细粒棘球蚴Eg95基因的粗糙型布鲁氏菌的构建及其疫苗。以粗糙型牛种布鲁氏菌弱毒株RA343株为亲本株，以蔗糖自杀质粒为载体，将含有特定启动子序列的细粒棘球蚴Eg95基因经密码子优化后无痕插入到了布鲁氏菌基因组中，成功构建了能够高效表达型重组细粒棘球蚴 Eg95基因的重组布鲁氏菌RA343-Eg95株。该重组菌株不仅保留了原始亲本株 RA343的粗糙型特性，对布鲁氏菌病良好的免疫保护性，而且该重组菌免疫动物后，能产生针对细粒棘球蚴Eg95抗体，从而实现对细粒棘球蚴病的免疫保护。
目前包虫病疫苗研究火热，国内外学者们针对包虫众多候选疫苗位点展开了大量研究，EG95是提取细粒棘球蚴六钩蚴RNA后扩增出相应的cDNA，在细粒棘球蚴原头节、六钩蚴和成虫的生长周期中，EG95基因均有不同程度的表达。目前中国国内仅有一款商品化基因工程疫苗用于棘球蚴病的控制。但在西藏等地区的牛羊免疫EG95基因工程亚单位疫苗实验中发现，免疫组动物剖检时仍有部分动物肺脏和肝脏存在包囊，无法完全抵抗包虫的侵害，并且此疫苗组分仅为EG95蛋白混合Quil A，组分较为简陋，无法充分呈现EG95蛋白的免疫原性。
因此，提供已一种能够充分呈现EG95蛋白的重组疫苗，对棘球蚴病的防治具有重要应用价值。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种诺如病毒P颗粒嵌合棘球蚴蛋白的EG95重组蛋白及其应用。所述诺如病毒P颗粒嵌合棘球蚴EG95 蛋白的重组蛋白可作为棘球蚴疫苗用于防治棘球蚴病，所述棘球蚴疫苗结合诺如病毒P颗粒和棘球蚴EG95抗原两者的优势，P颗粒具有高度免疫原性和稳定性，P颗粒在处于低温、冷冻、冻干和环境温度状态下都很稳定，并且由于每个 P单体具有三个表面环，将外源抗原插入这些环中会使P颗粒表面有24至72 个抗原拷贝，这可以大大增强抗原的抗原性和免疫原性。本发明所述诺如病毒P 颗粒嵌合棘球蚴EG95蛋白的重组蛋白免疫原性好，EG95蛋白能够被充分呈现，所述诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白具有广阔的前景。
为达到此发明目的，本发明采用以下技术方案：
第一方面，本发明提供一种诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白，所述重组蛋白的氨基酸序列如SEQ ID NO:1所示。
诺如病毒P颗粒蛋白就是一个良好的免疫载体，诺如病毒最早是在1929年的俄州诺沃克发生胃肠炎暴发期间从粪便样本中采集到的，整个病毒由具明显杯状凹陷的无包膜蛋白外壳组成，故将其命名为杯状病毒科中的一个新属-诺如病毒(Norwalk Viruses，NoV)。
NoV主要结构蛋白分为S结构域和P结构域，P结构域为突出部分，包含受体结合位点，且单独的P结构域体外能自我组装为结构稳定功能完全的VLP 颗粒，在体外表达用不同末端修饰的P区域蛋白，可形成三种复合物形式：二聚体、12聚体P颗粒、和24聚体P颗粒，电镜显示12聚体和24聚体分别具有正四面体和正八面体对称结构，P颗粒二聚体的每个P蛋白末端均有三个向外突出的环状结构，loop1、loop2和loop3，通过向P颗粒环状结构的基因序列中插入外源抗原序列，可通过原核表达的形式形成带有此抗原的P颗粒嵌合蛋白，并且此抗原蛋白会展示在P颗粒的表面。
原核表达生产的P颗粒具有高度免疫原性和稳定性，P颗粒在处于低温、冷冻、冻干和环境温度状态下都很稳定，由于每个P单体具有三个表面环，将外源抗原插入这些环中会导致P颗粒表面有24至72个抗原拷贝，这可以大大增强抗原的抗原性和免疫原性。因此，本发明结合诺如病毒P颗粒和棘球蚴EG95 抗原两者的优势，制备出诺如病毒P颗粒嵌合EG95蛋白，为棘球蚴病疫苗的制备提供理论基础，为棘球蚴病的防控提供新的思路。
第二方面，本发明提供一种核酸分子，所述核酸分子包括编码第一方面所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的核苷酸序列。
优选地，所述核酸分子包括SEQ ID NO:2所示的核苷酸序列。
本发明从NCBI获取棘球蚴EG95抗原序列，利用Signal P-5.0和TMHMM 分析序列信息以去除信号肽和跨膜结构域序列，并根据大肠杆菌密码子偏好优化序列。利用大肠杆菌异源表达EG95蛋白时，不需要信号肽的定位作用，多余的信号肽和跨膜结构域也无法被大肠杆菌识别切割，有可能影响EG95蛋白的正确折叠和免疫原性，通过去除信号肽和跨膜结构域，能够降低异源表达的EG95 蛋白的错误折叠的概率。
本发明中所述EG95经大肠杆菌密码子优化后的核苷酸序列如SEQ ID NO:5 所示，所述EG95经大肠杆菌密码子优化后的氨基酸序列如SEQ ID NO:6所示；所述NoV P颗粒经大肠杆菌密码子优化后的核苷酸序列如SEQ ID NO:7所示；所述NoV P颗粒经大肠杆菌密码子优化后的氨基酸序列如SEQ ID NO:8所示。
第三方面，本发明提供一种重组载体，所述重组载体含有至少一个拷贝的第二方面所述的核酸分子。
第四方面，本发明提供一种重组菌，所述重组菌含有第三方面所述的重组载体，或所述重组菌的基因组中整合有第二方面所述的核酸分子。
第五方面，本发明提供一种棘球蚴疫苗，所述棘球蚴疫苗包括第一方面所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白。
第六方面，本发明提供一种药物组合物，所述药物组合物包括第五方面所述的棘球蚴疫苗。
优选地，所述药物组合物还包括药学上可接受的载体。
第七方面，本发明提供一种第一方面所述的诺如病毒P颗粒嵌合棘球蚴 EG95蛋白的重组蛋白的制备方法，所述制备方法包括如下步骤：
(1)构建含有编码第一方面所述诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的核酸分子的重组载体；
(2)将步骤(1)所述的重组载体转入感受态细胞中，筛选阳性克隆后进行培养，裂解细胞，得到裂解液；
(3)从步骤(2)所述的裂解液中提取和纯化蛋白质，得到所述诺如病毒P 颗粒嵌合棘球蚴EG95蛋白的重组蛋白。
优选地，步骤(1)中，所述重组载体的构建方法包括如下步骤：
将编码诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的序列插入 pET28a-MBP载体的Hind III和Xho I酶切位点之间，构建重组质粒 pET28a-MBP-P-EG95。
优选地，步骤(2)中，所述感受态细胞包括BL21感受态细胞。
优选地，步骤(3)中，所述提取和纯化采用如下步骤进行：
将所述裂解液离心收集上清液，并对所述上清液进行过滤，将过滤后的上清液加入树脂中孵育，再洗脱蛋白，收集目标蛋白。
第八方面，本发明提供第一方面所述的诺如病毒P颗粒嵌合棘球蚴EG95 蛋白的重组蛋白、第二方面所述的核酸分子、第三方面所述的重组载体、第四方面所述的重组菌、第五方面所述的棘球蚴疫苗、第六方面所述的药物组合物或第七方面所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的制备方法中任意一种或至少两种的组合在制备棘球蚴病治疗药物和/或疫苗中的应用。
相对于现有技术，本发明具有以下有益效果：
本发明选用了棘球蚴EG95蛋白作为制备亚单位疫苗的候选抗原，所述抗原具有良好的免疫原性，并且选择NoV P颗粒蛋白作为免疫载体，用于递呈EG95 抗原并提高其免疫原性，NoV P颗粒本身即可充当佐剂加强免疫反应，使机体产生高滴度的特异性抗体。所述诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白可作为棘球蚴疫苗用于防治棘球蚴病，所述棘球蚴疫苗结合诺如病毒P颗粒和棘球蚴EG95抗原两者的优势，P颗粒具有高度免疫原性和稳定性，诺如病毒 P颗粒作为抗原递呈载体平台，EG95蛋白能够被充分呈现，具有广阔的前景。
附图说明
图1是实施例2中重组载体pET-28a-P-EG95的PCR鉴定结果图。
图2是实施例3中P-EG95蛋白的SDS-PAGE电泳结果图。
图3是实施例3中P-EG95蛋白的Western blot结果图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例提供一种诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白，所述诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的氨基酸序列如SEQ ID NO:1 所示，编码所述诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的核酸分子的核苷酸序列如SEQ ID NO:2所示。所述核苷酸序列为根据大肠杆菌的密码子偏好优化之后序列。
SEQ ID NO:1：
CNGRCSRTKPFTVPILTVEEMSNSRFPIPLEKLYTGPSSAFVVQPQNGRCT TDGVLLGTTQLSAVNICTFRGDVTHDYIMNLASQNWNNYDPTEEIPAPLGTP DFVGKIQGMLTQTTREDGSTRAHKATVSTGSVHFTPKLGSVQYTTDQEYKG MGVETRTTETPLRKHFNLTPVGSQGIRLSWEVQHLSDLKGTDISLKAVNPSDP LVYKRQTAKFSDGQLTIGELKPSTLYKMTVEAVKAKKTILGFTVDIETPRAGK KESTVLQTGQNTKFTPVGVIQHQNEPQQWVLPNYSGRTGHNVHLAPAVAPTF PGEQLLFFRSTMPGCSGYPNMNLDCLLPQEWVQHFYQEAAPAQSDVALLRF VNPDTGRVLFECKLHKSGYVTVAHTGPHDLVIPPNGYFRFDSWVNQFYTLAP MGNGAGRRRALCNGRC。
SEQ ID NO:2：
tgcaacggccgttgctcaagaactaaaccattcaccgtcccgatcttaactgttgaggaaatgtccaactcaagattc cccattcctttggaaaagttgtacacgggtcccagcagtgcttttgttgtccaaccacaaaatggcaggtgcacgactgatg gcgtgctcttaggcactacccagctgtctgctgtcaatatctgcaccttcagaggggatgtcacccacgactatataatgaat ttggcttctcaaaattggaacaattatgacccaacagaagaaatcccagcccctctgggaactccagatttcgtgggaaaga tccaaggcatgctcacccaaaccacaagagaggatggctcgacccgcgcccacaaagctacagtgagcactgggagc gtccacttcactccaaagttgggcagtgttcaatacaccactgaccaggaatataaaggcatgggcgtggaaacccgcac caccgaaaccccgttacgcaaacattttaacctgaccccggtgggcagccagggcattcgcctgagctgggaagttcagc atctgagcgatctgaaaggcaccgatattagcctgaaagcggtgaacccgagcgatccgctggtgtataaacgccagac cgcgaaatttagcgatggccagctgaccattggcgaactgaaaccgagcaccctgtataaaatgaccgtggaagcggtg aaagcgaagaaaaccattctgggctttaccgtggatattgaaacccctcgcgcgggcaaaaaggaaagcaccgtgcttca aactggccaaaacacgaaattcaccccagtcggcgtcatccagcaccaaaatgaaccccagcaatgggtactcccaaatt actcaggtagaactggtcataatgtgcacctagctcctgccgttgcccccactttcccaggcgagcaacttctcttctttaggt ccactatgcccgggtgtagcgggtatcccaacatgaatctggattgcctactcccccaggaatgggtgcagcacttctaccaagaagcagctccagcacaatctgatgtggctctgctgagatttgtgaatccagacacaggtagggttctgtttgagtgcaa gctccataaatcaggctatgtcacagtggctcacactggcccgcatgatttggttatcccccccaatggttattttagatttgat tcctgggtcaaccagttctacacacttgcccccatgggaaatggagcggggcgcagacgtgcattatgcaacggccgttg ctaa。
SEQ ID NO:5(EG95蛋白经大肠杆菌密码子优化后的核苷酸序列)：
caggaatataaaggcatgggcgtggaaacccgcaccaccgaaaccccgttacgcaaacattttaacctgaccccg gtgggcagccagggcattcgcctgagctgggaagttcagcatctgagcgatctgaaaggcaccgatattagcctgaaag cggtgaacccgagcgatccgctggtgtataaacgccagaccgcgaaatttagcgatggccagctgaccattggcgaact gaaaccgagcaccctgtataaaatgaccgtggaagcggtgaaagcgaagaaaaccattctgggctttaccgtggatattg aaacccctcgcgcgggcaaaaaggaaagcaccgtg。
SEQ ID NO:6(EG95蛋白经大肠杆菌密码子优化后的氨基酸序列)：
QEYKGMGVETRTTETPLRKHFNLTPVGSQGIRLSWEVQHLSDLKGTDIS LKAVNPSDPLVYKRQTAKFSDGQLTIGELKPSTLYKMTVEAVKAKKTILGFTV DIETPRAGKKESTV。
SEQ ID NO:7(NoV P颗粒经大肠杆菌密码子优化后的核苷酸序列)：
tgcaacggccgttgctcaagaactaaaccattcaccgtcccgatcttaactgttgaggaaatgtccaactcaagattc cccattcctttggaaaagttgtacacgggtcccagcagtgcttttgttgtccaaccacaaaatggcaggtgcacgactgatg gcgtgctcttaggcactacccagctgtctgctgtcaatatctgcaccttcagaggggatgtcacccacgactatataatgaat ttggcttctcaaaattggaacaattatgacccaacagaagaaatcccagcccctctgggaactccagatttcgtgggaaaga tccaaggcatgctcacccaaaccacaagagaggatggctcgacccgcgcccacaaagctacagtgagcactgggagc gtccacttcactccaaagttgggcagtgttcaatacaccactgaccttcaaactggccaaaacacgaaattcaccccagtcg gcgtcatccagcaccaaaatgaaccccagcaatgggtactcccaaattactcaggtagaactggtcataatgtgcacctag ctcctgccgttgcccccactttcccaggcgagcaacttctcttctttaggtccactatgcccgggtgtagcgggtatcccaac atgaatctggattgcctactcccccaggaatgggtgcagcacttctaccaagaagcagctccagcacaatctgatgtggct ctgctgagatttgtgaatccagacacaggtagggttctgtttgagtgcaagctccataaatcaggctatgtcacagtggctca cactggcccgcatgatttggttatcccccccaatggttattttagatttgattcctgggtcaaccagttctacacacttgccccc atgggaaatggagcggggcgcagacgtgcattatgcaacggccgttgc。
SEQ ID NO:8(NoV P颗粒经大肠杆菌密码子优化后的氨基酸序列)：
CNGRCSRTKPFTVPILTVEEMSNSRFPIPLEKLYTGPSSAFVVQPQNGRCT TDGVLLGTTQLSAVNICTFRGDVTHDYIMNLASQNWNNYDPTEEIPAPLGTP DFVGKIQGMLTQTTREDGSTRAHKATVSTGSVHFTPKLGSVQYTTDLQTGQN TKFTPVGVIQHQNEPQQWVLPNYSGRTGHNVHLAPAVAPTFPGEQLLFFRST MPGCSGYPNMNLDCLLPQEWVQHFYQEAAPAQSDVALLRFVNPDTGRVLFE CKLHKSGYVTVAHTGPHDLVIPPNGYFRFDSWVNQFYTLAPMGNGAGRRRA LCNGRC。
实施例2
本实施例提供一种重组载体，所述重组载体中含有编码诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的核酸分子。
(1)EG95抗原基因序列寻找及分析
选择EG95作为疫苗候选基因：从NCBI获得EG95 cDNA序列。利用Signal P-5.0和TMHMM分析序列信息，去除信号肽和跨膜结构域的多肽序列，并根据大肠杆菌密码子偏好优化序列(长度：348bp，GC％：54)。
(2)重组载体的构建
从NCBI基因数据库中获得NoV P结构域cDNA序列，将优化的EG95序列整合入NoV P结构域的loop2环中(长度：1296bp，GC％：52)，交由华大基因公司合成，得到编码诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的核酸分子P-EG95，所述核酸分子的核苷酸序列如SEQ ID NO:2所示，然后将 P-EG95序列插入pET28a-MBP载体的上，构建重组载体pET28a-MBP-P-EG95。
构建重组载体pET28a-MBP-P-EG95的步骤如下所示：
对P-EG95和pET28a-MBP质粒(均含有Hind III和Xho I酶切位点)分别进行双酶切，以上各做5管，共100μL，于37℃水浴锅反应4h以上。双酶切体系如下所示。
将P-EG95和pET28a-MBP的酶切产物进行纯化：
先称量1.5mL干净离心管的重量，后将含有片段的凝胶装于其中，确定凝胶的重量。假设凝胶密度为1g/mL，凝胶的体积由下式得出如下：质量0.3g的凝胶片则具有0.3mL的体积；将凝胶片与结合缓冲液(XP2)1:1混合，在50-60℃环境下直到凝胶完全融化，每2-3min摇动一次；将DNA Mini Column置于2mL 收集管中备用。
将完全融化的DNA琼脂糖溶液(不超过700μL)添加到DNA Mini Column 中；在室温下10000g离心1min。丢弃滤液并重新使用收集管；再重复此步骤。
加入300μL结合缓冲液(XP2)，在室温下以最大速度(≥13000g)离心1 min，丢弃滤液并重新使用收集管；加入700μL SPW洗涤缓冲液，室温下以最大速度离心1min，丢弃滤液并重新使用收集管。
将空的HiBind DNA Mini Column以最大速度离心2min至干燥。注意：洗脱前将HiBind DNA Mini Column基质干燥很重要，因为残留乙醇可能会干扰下述操作。
将HiBind DNA Mini Column转移到干净的1.5mL微量离心管中。将15-30 μL洗脱缓冲液或去离子水直接加入色谱柱中心膜。注意：从HiBind DNA Mini Column中洗脱DNA的效率是依赖于pH值的。如果用去离子水洗脱DNA，应确保pH值在8.5左右。
在室温下静置2min，以最大速度离心1min。注意：第一次约70％的结合 DNA被洗脱，而第二次洗脱后残留的DNA都会被洗脱下来，但是浓度较低。最后将DNA保存在-20℃。通过Epoch微孔板分光光度计计算回收浓度。
将P-EG和pET28a-MBP的酶切回收产物进行连接，连接体系如表下所示，分别将所述试剂加样后，将反应体系置于基因扩增仪16℃过夜(12-16h)。
连接产物转化：
取-80℃的100μL DH5α感受态细胞，冰上融化后，加入上一步的连接产物 10μL，混匀，冰浴30min。42℃水浴90s，再放冰上2min。加入400μL LB液体培养基，37℃、220rpm振荡培养45min，恢复质粒抗性。4℃、4000rpm离心5min，弃上清400μL，将剩余菌液涂于Amp+平板，37℃培养30min(平皿正放)，菌液吸收完全后将平皿倒置培养约12h，直至肉眼可见单菌落为止。
连接产物鉴定：
根据P-EG95的碱基序列设计引物，扩增长度为1296bp。
上游引物：5’-3’tgcaacggccgttgctcaag(SEQ ID NO:3)；
下游引物：5’-3’ttagcaacggccgttgcataatgc(SEQ ID NO:4)；
从超低温冰箱中取出BL21(DE3)感受态细胞，置于冰上融化，加入质粒 pET28a-MBP-P-EG95(100ng)，充分混匀，冰浴30min，42℃反应45s，冰浴 3min，加入100μL LB培养基，37℃、200rpm摇床培养60min，均匀涂布在 Amp+平板上，37℃倒置培养过夜。第二天进行菌落PCR鉴定：在培养板上挑取单菌落，接种于新的Amp+平板上，然后配制PCR反应体系，鉴定的PCR反应体系如表下所示。
PCR反应条件为：94℃预变性5min；94℃热变性10s、60℃退火10s、72℃延伸80s，共25个循环；72℃终延伸10min。PCR结束后进行琼脂糖凝胶电泳， 120V电泳30min，于凝胶成像仪上进行分析，重组载体pET-28a-P-EG95的PCR 鉴定结果图如图1所示，图1中，泳道M：2000Ladder Marker；泳道1：P颗粒扩增片段；泳道N：阴性对照。提取阳性克隆的质粒送华大基因公司进行测序，测序结果与P-EG95进行比对，结果正确，最终成功构建重组载体pET28a-MBP-P-EG95。
实施例3
本实施例提供一种由实施例2所述的重组载体诱导表达得到的重组蛋白，所述重组蛋白的制备方法包括如下步骤：
(1)重组载体转化：
将实施例2中的阳性克隆扩繁，接种至5mL含有100μg/mL Amp的LB培养基中，37℃、200rpm震荡培养16-20h，进行质粒提取。
(A)将5mL菌液分装于5支离心管中，10000×g离心1min。
(B)倒出培养基，各加入250μL Solution I，吹打混匀。
(C)加250μL Solution II后轻轻晃动管子2-3min；处理过程中避免剧烈混合，因为这会剪切染色体DNA而导致更低的质粒纯度；且不要让裂解反应进行超过5min；SolutionII在不使用时应密封，以避免空气中的二氧化碳的酸化。
(D)加入350μL SolutionⅢ，立即倒置数次，直到絮状白色沉淀形式，避免局部沉。
(E)最大速度(>13000g)离心10min。
(F)将HiBind DNA Mini Column插入2mL收集管中，将步骤(E)中所得的上清液小心地吸入瓶中，避免吸取颗粒和细胞碎片，以最大速度离心1min。
(G)弃去滤液，重新使用收集管，加入500μL HBC Buffer，以最大速度离心1min。
(H)弃去滤液并重新使用收集管，加入700μL DNA Wash Buffer，最高速度离心1min。
(I)弃滤液，以最大速度将空HiBind DNA Mini Column离心2min。在洗脱前干燥。HiBind DNA Mini Column基质干燥很重要，因为残留乙醇可能会干扰后续的操作。
(J)于1.5mL微量离心管中放入HiBind DNA Mini Column。
(K)于柱膜中心加入30μL无菌去离子水。
(L)在室温下静置1min，最高速度离心1min，第一次约70％的结合DNA 被洗脱，而第二次洗脱后残留的DNA都会被洗脱下来，但是浓度较低。
将上述步骤提取的质粒取2μL转化至BL21表达细胞中，用鉴定引物进行鉴定，得到重组菌。
(2)重组蛋白的诱导表达和鉴定：
挑取阳性的重组菌接种到4mL含有100μg/mL Amp的LB培养基中，37℃、 200rpm震荡培养，当OD600nm处于0.6-0.8之间，向2个试管中分别加入终浓度为0.5mM的IPTG，一个16℃培养16h，一个37℃培养4h，另取一支试管为阴性参照，采用SDS-PAGE和Western blot检测蛋白质的表达和溶解度，检测步骤如下所示：
取450μL培养基离心后的沉淀，重悬于300μL裂解缓冲液(50mM Tris-Hcl， 150mMNaCl，5％glycerol，pH 8.0)，超声裂解1min。
全菌样品：取100μL PBS，与50μL loading buffer(5×)混合均匀，100℃加热10min，12000rpm离心5min。
上清和包涵体样品：取剩余200μL裂解液，15000rpm离心10min，分别取上清和沉淀，混合90μL loading buffer(5×)到180μL上清，作为上清样品。用150μL loadingbuffer(5×)重悬沉淀，作为包涵体样品。100℃加热10min 后，15000rpm离心5min，待上样。
(3)重组蛋白SDS-PAGE检测：
按照碧云天公司的BeyoGelTM SDS-PAGE预制胶(Tris-Gly，4-20％，12孔) 的操作说明进行实验，包括预制胶的处理，样品孔的清洗和上样等，而后进行 80V、30min，120V、60min的电泳，撬开胶板，切割目的胶体，备用。按照碧云天公司的考马斯亮蓝染色试剂盒(P0017A)的操作说明进行凝胶染色试验，包括凝胶染色、室温孵育1h、脱色过夜与凝胶成像等。利用化学发光成像系统的SDS灰度值分析重组蛋白的可溶性比例，以及利用BCA蛋白浓度检测试剂盒检测破碎后样品的总蛋白浓度，通过可溶性比例预测目的蛋白的浓度。
(3)重组蛋白Western-blotting分析：
(a)电泳：将重组蛋白作为样品进行SDS-PAGE电泳试验，电泳结束前预先剪取大小合适的PVDF膜1张和同该膜一样大的滤纸6张。PVDF膜先于甲醇中浸泡10min激活，后于电转缓冲液泡10min；滤纸和海绵在电转缓冲液中稍作浸泡。
(b)转膜：待电泳结束，切取所需凝胶部分于电转缓冲液泡10min，按照负极板、海绵、3层滤纸、膜、分离胶、3层滤纸、海绵、正极板的顺序放妥，在放置期间应该用玻棒赶尽滤纸、胶和膜之间可能存在的气泡，否则，在显色时可见膜上有气泡印迹。将上述的转膜板放入电转糟，加入电转缓冲液至膜上沿所在的位置，恒流200mA，电转60min。
(c)洗膜：将转膜结束后的PVDF膜用TBST缓冲液洗涤3次，每次5min。
(d)封闭：把洗毕的膜浸泡在5％脱脂奶粉封闭液，室温摇床80rpm振摇 2h或在4℃过夜；洗膜：用TBST缓冲液洗涤PVDF膜3次，每次5min。
(e)一抗温育：将HIS单抗用TBST作1:10000比例稀释(按照Thermo FisherScientific说明书)，与PVDF膜在室温作用1h或4℃过夜。
(f)洗膜：用TBST洗涤PVDF膜4次，每次5min。
(g)二抗温育：用TBST对兔抗鼠IgG多抗作1:40000稀释(按照Thermo FisherScientific说明书)，将PVDF浸泡于其中，室温作用0.5-1h。
(h)洗膜：用TBST洗涤PVDF膜5次，每次5min；
(i)显色：使用ECL显色液进行显色1min，后用TBST终止反应，利用化学发光成像系统观察特异性条带。
P-EG95重组蛋白表达产物经过SDS-PAGE和Western-blotting鉴定，结果如图2和图3所示，图2是P-EG95蛋白的SDS-PAGE电泳结果图，图2中，箭头指向的条带为P-EG95蛋白对应的条带，泳道M1：Protein marker；泳道PC1： BSA(1μg)；泳道PC2：BSA(2μg)；泳道NC：未诱导的全细胞；泳道1： 15℃诱导16h的全细胞(0.5mM IPTG)；泳道2：37℃诱导4h的全细胞(0.5 mM IPTG)；泳道NC1：未诱导的细胞裂解上清；泳道3：15℃诱导16h的细胞裂解上清(0.5mM IPTG)；泳道4：37℃诱导4h的细胞裂解上清(0.5mM IPTG)；泳道NC2：未诱导的细胞裂解沉淀；泳道5：15℃诱导16h的细胞裂解沉淀(0.5mM IPTG)；泳道6：37℃诱导4h的细胞裂解沉淀(0.5mM IPTG)。图3是P-EG95蛋白的Western blot结果图，图3中箭头指向的条带为P-EG95 蛋白对应的条带，泳道M2：Western blot Protein marker；泳道3：15℃诱导16h 的细胞裂解上清(0.5mM IPTG)；泳道4：37℃诱导4h的细胞裂解上清(0.5mM IPTG)。诱导表达后的目的蛋白P-EG95的分子量约为90.4kDa，而未诱导的在此位置无明显条带。
可溶性分析表明，在15℃、16h的诱导情况下，上清和沉淀中均有目的蛋白存在，但沉淀中目的蛋白更多。最佳诱导结果为：15℃诱导16h，表达水平为25mg/L，可溶性比例为20％。
实施例4
本实施例提供一种对重组蛋白大量表达及纯化的方法，所述方法包括如下步骤：
将重组菌大量扩增，扩增后于6000rpm离心10min，收集菌体，用10mL BindingBuffer重悬，超声20min，随后10000rpm离心10min收集上清，用0.22 μm滤膜过滤后用PurKineTM MBP-Tag Purification Nickel Column试剂盒纯化蛋白，纯化的具体步骤如下所示：
(1)固定重力柱，去掉上端塞和下端塞，流干保护液。
(2)向柱中加入2倍柱体积Binding buffer，平衡树脂，流干后，再重复3 次。
(3)将处理好的蛋白提取物样本(蛋白提取物与等体积的Binding buffer 混合制备样本)加入树脂中孵育。注意收集流穿液，可通过SDS-PAGE分析。注意：为了最大限度结合，样本可在4℃或室温与树脂孵育30min，注意不要超过树脂的载量。
(4)向柱中加入2倍柱体积Wash Buffer，去除非特异性吸附蛋白。注意收集洗杂液用于SDS-PAGE检测分析，重复6次。
(5)向柱中加入5-10倍柱体积的Elution Buffer洗脱蛋白，或直至流出物 OD280nm稳定，分段收集，每个柱体积收集一管，分别检测。
(5)检测和鉴定含有目标蛋白的组分，通过紫外分光光度计，SDS-PAGE 或Westernblotting。
实施例5
本实施例对实施例4中制备的重组蛋白(诺如病毒P颗粒嵌合棘球蚴EG95 蛋白的重组蛋白)进行免疫保护效果评估。
评估步骤如下所示：
将上述纯化的重组蛋白(P-EG95嵌合蛋白)用弗氏佐剂等体积乳化，通过肌注免疫SPF兔，两周后加强免疫一次(首次免疫用完全弗氏佐剂乳化，加强免疫用不完全弗氏佐剂乳化)。分别在一免后一周、一免后两周、二免后一周和二免后两周耳缘静脉采血，每次采1mL，37℃放置1h，再转入4℃过夜，待血块充分收缩后，迅速取出血清4℃、3500rpm离心15min，收集血清，随后利用正典公司(佛山市正典生物科技有限公司)已有的间接ELISA方法检测特异性抗体水平，现有的EG95蛋白免疫血清作为阳性血清对照，未免疫血清为阴性对照。
本实施例提供一种对重组蛋白免疫效果评估的间接ELISA检测方法，所述方法包括如下步骤：
(1)包被：取0.5mg/mL EG95蛋白，用1×包被液按1:1000倍稀释，加到酶标板中，100μL/孔，放到37℃孵育2h。
(2)洗涤拍干：倒掉酶标板内液体，拍干，洗板机洗涤3次，再次拍干。
(3)封闭：拍干后，加封闭液封闭，200μL/孔，4℃过夜。
(4)洗涤拍干：同步骤(2)。
(5)加待检血清：待检血清用5％脱脂奶按1:12800稀释，100μL/孔，放到37℃孵育1h。
(6)洗涤拍干：同步骤(2)。
(7)加二抗：取羊抗鸡IgG-HRP，用5％脱脂奶按1:4000稀释，100μL/ 孔。放到37℃孵育0.5小时。
(8)洗涤拍干：同步骤(2)。
(9)加显色液：每孔加100μL显色液，放到37℃孵育15分钟。
(10)加终止液：时间到直接加终止液，50μL/孔。
(11)读取结果：用OD450nm读取结果并保存。
免疫保护效果评估结果如表1所示：
表1
| 检测免疫蛋白OD<sub>450nm</sub>时间 | 一免后一周 | 一免后两周 | 二免后一周 | 二免后两周 |
| P-EG95嵌合蛋白 | 1.883 | 2.747 | 4.437 | 4.504 |
| EG95蛋白 | 1.147 | 2.269 | 4.484 | 4.495 |
| 阴性对照 | 0.189 | 0.164 | 0.161 | 0.192 |
从表1的统计结果可知，P-EG95嵌合蛋白免疫血清能与EG95蛋白结合，并且P-EG95嵌合蛋白组比单独的EG95蛋白免疫后引起的特异性中和抗体水平更高，证明P-EG95嵌合蛋白不仅很好的呈递了EG95蛋白，还能使动物体产生较高的特异性抗体水平，是一个极具潜力的疫苗蛋白选择。
综上，本发明提供了一种诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白，所述重组蛋白可作为棘球蚴疫苗用于防治棘球蚴病，所述棘球蚴疫苗结合诺如病毒P颗粒和棘球蚴EG95抗原两者的优势，P颗粒具有高度免疫原性和稳定性，诺如病毒P颗粒作为抗原递呈载体平台，EG95蛋白能够被充分呈现，具有广阔的前景。
申请人声明，以上所述仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于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，均落在本发明的保护范围和公开范围之内。
序列表
<110> 佛山市正典生物技术有限公司
<120> 一种诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白及其应用
<130> 2022
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 431
<212> PRT
<213> 人工序列
<400> 1
Cys Asn Gly Arg Cys Ser Arg Thr Lys Pro Phe Thr Val Pro Ile Leu
1 5 10 15
Thr Val Glu Glu Met Ser Asn Ser Arg Phe Pro Ile Pro Leu Glu Lys
20 25 30
Leu Tyr Thr Gly Pro Ser Ser Ala Phe Val Val Gln Pro Gln Asn Gly
35 40 45
Arg Cys Thr Thr Asp Gly Val Leu Leu Gly Thr Thr Gln Leu Ser Ala
50 55 60
Val Asn Ile Cys Thr Phe Arg Gly Asp Val Thr His Asp Tyr Ile Met
65 70 75 80
Asn Leu Ala Ser Gln Asn Trp Asn Asn Tyr Asp Pro Thr Glu Glu Ile
85 90 95
Pro Ala Pro Leu Gly Thr Pro Asp Phe Val Gly Lys Ile Gln Gly Met
100 105 110
Leu Thr Gln Thr Thr Arg Glu Asp Gly Ser Thr Arg Ala His Lys Ala
115 120 125
Thr Val Ser Thr Gly Ser Val His Phe Thr Pro Lys Leu Gly Ser Val
130 135 140
Gln Tyr Thr Thr Asp Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg
145 150 155 160
Thr Thr Glu Thr Pro Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly
165 170 175
Ser Gln Gly Ile Arg Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu
180 185 190
Lys Gly Thr Asp Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu
195 200 205
Val Tyr Lys Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile
210 215 220
Gly Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val
225 230 235 240
Lys Ala Lys Lys Thr Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro
245 250 255
Arg Ala Gly Lys Lys Glu Ser Thr Val Leu Gln Thr Gly Gln Asn Thr
260 265 270
Lys Phe Thr Pro Val Gly Val Ile Gln His Gln Asn Glu Pro Gln Gln
275 280 285
Trp Val Leu Pro Asn Tyr Ser Gly Arg Thr Gly His Asn Val His Leu
290 295 300
Ala Pro Ala Val Ala Pro Thr Phe Pro Gly Glu Gln Leu Leu Phe Phe
305 310 315 320
Arg Ser Thr Met Pro Gly Cys Ser Gly Tyr Pro Asn Met Asn Leu Asp
325 330 335
Cys Leu Leu Pro Gln Glu Trp Val Gln His Phe Tyr Gln Glu Ala Ala
340 345 350
Pro Ala Gln Ser Asp Val Ala Leu Leu Arg Phe Val Asn Pro Asp Thr
355 360 365
Gly Arg Val Leu Phe Glu Cys Lys Leu His Lys Ser Gly Tyr Val Thr
370 375 380
Val Ala His Thr Gly Pro His Asp Leu Val Ile Pro Pro Asn Gly Tyr
385 390 395 400
Phe Arg Phe Asp Ser Trp Val Asn Gln Phe Tyr Thr Leu Ala Pro Met
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Gly Asn Gly Ala Gly Arg Arg Arg Ala Leu Cys Asn Gly Arg Cys
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<210> 2
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<213> 人工序列
<400> 2
tgcaacggcc gttgctcaag aactaaacca ttcaccgtcc cgatcttaac tgttgaggaa 60
atgtccaact caagattccc cattcctttg gaaaagttgt acacgggtcc cagcagtgct 120
tttgttgtcc aaccacaaaa tggcaggtgc acgactgatg gcgtgctctt aggcactacc 180
cagctgtctg ctgtcaatat ctgcaccttc agaggggatg tcacccacga ctatataatg 240
aatttggctt ctcaaaattg gaacaattat gacccaacag aagaaatccc agcccctctg 300
ggaactccag atttcgtggg aaagatccaa ggcatgctca cccaaaccac aagagaggat 360
ggctcgaccc gcgcccacaa agctacagtg agcactggga gcgtccactt cactccaaag 420
ttgggcagtg ttcaatacac cactgaccag gaatataaag gcatgggcgt ggaaacccgc 480
accaccgaaa ccccgttacg caaacatttt aacctgaccc cggtgggcag ccagggcatt 540
cgcctgagct gggaagttca gcatctgagc gatctgaaag gcaccgatat tagcctgaaa 600
gcggtgaacc cgagcgatcc gctggtgtat aaacgccaga ccgcgaaatt tagcgatggc 660
cagctgacca ttggcgaact gaaaccgagc accctgtata aaatgaccgt ggaagcggtg 720
aaagcgaaga aaaccattct gggctttacc gtggatattg aaacccctcg cgcgggcaaa 780
aaggaaagca ccgtgcttca aactggccaa aacacgaaat tcaccccagt cggcgtcatc 840
cagcaccaaa atgaacccca gcaatgggta ctcccaaatt actcaggtag aactggtcat 900
aatgtgcacc tagctcctgc cgttgccccc actttcccag gcgagcaact tctcttcttt 960
aggtccacta tgcccgggtg tagcgggtat cccaacatga atctggattg cctactcccc 1020
caggaatggg tgcagcactt ctaccaagaa gcagctccag cacaatctga tgtggctctg 1080
ctgagatttg tgaatccaga cacaggtagg gttctgtttg agtgcaagct ccataaatca 1140
ggctatgtca cagtggctca cactggcccg catgatttgg ttatcccccc caatggttat 1200
tttagatttg attcctgggt caaccagttc tacacacttg cccccatggg aaatggagcg 1260
gggcgcagac gtgcattatg caacggccgt tgctaa 1296
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
tgcaacggcc gttgctcaag 20
<210> 4
<211> 24
<212> DNA
<213> 人工序列
<400> 4
ttagcaacgg ccgttgcata atgc 24
<210> 5
<211> 348
<212> DNA
<213> 人工序列
<400> 5
caggaatata aaggcatggg cgtggaaacc cgcaccaccg aaaccccgtt acgcaaacat 60
tttaacctga ccccggtggg cagccagggc attcgcctga gctgggaagt tcagcatctg 120
agcgatctga aaggcaccga tattagcctg aaagcggtga acccgagcga tccgctggtg 180
tataaacgcc agaccgcgaa atttagcgat ggccagctga ccattggcga actgaaaccg 240
agcaccctgt ataaaatgac cgtggaagcg gtgaaagcga agaaaaccat tctgggcttt 300
accgtggata ttgaaacccc tcgcgcgggc aaaaaggaaa gcaccgtg 348
<210> 6
<211> 116
<212> PRT
<213> 人工序列
<400> 6
Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu Thr Pro
1 5 10 15
Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly Ile Arg
20 25 30
Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr Asp Ile
35 40 45
Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln
50 55 60
Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro
65 70 75 80
Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr
85 90 95
Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly Lys Lys
100 105 110
Glu Ser Thr Val
115
<210> 7
<211> 945
<212> DNA
<213> 人工序列
<400> 7
tgcaacggcc gttgctcaag aactaaacca ttcaccgtcc cgatcttaac tgttgaggaa 60
atgtccaact caagattccc cattcctttg gaaaagttgt acacgggtcc cagcagtgct 120
tttgttgtcc aaccacaaaa tggcaggtgc acgactgatg gcgtgctctt aggcactacc 180
cagctgtctg ctgtcaatat ctgcaccttc agaggggatg tcacccacga ctatataatg 240
aatttggctt ctcaaaattg gaacaattat gacccaacag aagaaatccc agcccctctg 300
ggaactccag atttcgtggg aaagatccaa ggcatgctca cccaaaccac aagagaggat 360
ggctcgaccc gcgcccacaa agctacagtg agcactggga gcgtccactt cactccaaag 420
ttgggcagtg ttcaatacac cactgacctt caaactggcc aaaacacgaa attcacccca 480
gtcggcgtca tccagcacca aaatgaaccc cagcaatggg tactcccaaa ttactcaggt 540
agaactggtc ataatgtgca cctagctcct gccgttgccc ccactttccc aggcgagcaa 600
cttctcttct ttaggtccac tatgcccggg tgtagcgggt atcccaacat gaatctggat 660
tgcctactcc cccaggaatg ggtgcagcac ttctaccaag aagcagctcc agcacaatct 720
gatgtggctc tgctgagatt tgtgaatcca gacacaggta gggttctgtt tgagtgcaag 780
ctccataaat caggctatgt cacagtggct cacactggcc cgcatgattt ggttatcccc 840
cccaatggtt attttagatt tgattcctgg gtcaaccagt tctacacact tgcccccatg 900
ggaaatggag cggggcgcag acgtgcatta tgcaacggcc gttgc 945
<210> 8
<211> 315
<212> PRT
<213> 人工序列
<400> 8
Cys Asn Gly Arg Cys Ser Arg Thr Lys Pro Phe Thr Val Pro Ile Leu
1 5 10 15
Thr Val Glu Glu Met Ser Asn Ser Arg Phe Pro Ile Pro Leu Glu Lys
20 25 30
Leu Tyr Thr Gly Pro Ser Ser Ala Phe Val Val Gln Pro Gln Asn Gly
35 40 45
Arg Cys Thr Thr Asp Gly Val Leu Leu Gly Thr Thr Gln Leu Ser Ala
50 55 60
Val Asn Ile Cys Thr Phe Arg Gly Asp Val Thr His Asp Tyr Ile Met
65 70 75 80
Asn Leu Ala Ser Gln Asn Trp Asn Asn Tyr Asp Pro Thr Glu Glu Ile
85 90 95
Pro Ala Pro Leu Gly Thr Pro Asp Phe Val Gly Lys Ile Gln Gly Met
100 105 110
Leu Thr Gln Thr Thr Arg Glu Asp Gly Ser Thr Arg Ala His Lys Ala
115 120 125
Thr Val Ser Thr Gly Ser Val His Phe Thr Pro Lys Leu Gly Ser Val
130 135 140
Gln Tyr Thr Thr Asp Leu Gln Thr Gly Gln Asn Thr Lys Phe Thr Pro
145 150 155 160
Val Gly Val Ile Gln His Gln Asn Glu Pro Gln Gln Trp Val Leu Pro
165 170 175
Asn Tyr Ser Gly Arg Thr Gly His Asn Val His Leu Ala Pro Ala Val
180 185 190
Ala Pro Thr Phe Pro Gly Glu Gln Leu Leu Phe Phe Arg Ser Thr Met
195 200 205
Pro Gly Cys Ser Gly Tyr Pro Asn Met Asn Leu Asp Cys Leu Leu Pro
210 215 220
Gln Glu Trp Val Gln His Phe Tyr Gln Glu Ala Ala Pro Ala Gln Ser
225 230 235 240
Asp Val Ala Leu Leu Arg Phe Val Asn Pro Asp Thr Gly Arg Val Leu
245 250 255
Phe Glu Cys Lys Leu His Lys Ser Gly Tyr Val Thr Val Ala His Thr
260 265 270
Gly Pro His Asp Leu Val Ile Pro Pro Asn Gly Tyr Phe Arg Phe Asp
275 280 285
Ser Trp Val Asn Gln Phe Tyr Thr Leu Ala Pro Met Gly Asn Gly Ala
290 295 300
Gly Arg Arg Arg Ala Leu Cys Asn Gly Arg Cys
305 310 315
Claims (10)
1.一种诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白，其特征在于，所述重组蛋白的氨基酸序列如SEQ ID NO:1所示。
2.一种核酸分子，其特征在于，所述核酸分子包括编码权利要求1所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的核苷酸序列；
优选地，所述核酸分子包括SEQ ID NO:2所示的核苷酸序列。
3.一种重组载体，其特征在于，所述重组载体含有至少一个拷贝的权利要求2所述的核酸分子。
4.一种重组菌，其特征在于，所述重组菌含有权利要求3所述的重组载体，或所述重组菌的基因组中整合有权利要求2所述的核酸分子。
5.一种棘球蚴疫苗，其特征在于，所述棘球蚴疫苗包括权利要求1所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白。
6.一种药物组合物，其特征在于，所述药物组合物包括权利要求5所述的棘球蚴疫苗；
优选地，所述药物组合物还包括药学上可接受的载体。
7.一种权利要求1所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的制备方法，其特征在于，所述制备方法包括如下步骤：
(1)构建含有编码权利要求1所述诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的核酸分子的重组载体；
(2)将步骤(1)所述的重组载体转入感受态细胞中，筛选阳性克隆后进行培养，裂解细胞，得到裂解液；
(3)从步骤(2)所述的裂解液中提取和纯化蛋白质，得到所述诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白。
8.根据权利要求7所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的制备方法，其特征在于，步骤(1)中，所述重组载体的构建方法包括如下步骤：
将编码诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的序列插入pET28a-MBP载体的Hind III和Xho I酶切位点之间，构建重组质粒pET28a-MBP-P-EG95。
9.根据权利要求7或8所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的制备方法，其特征在于，步骤(2)中，所述感受态细胞包括BL21感受态细胞；
优选地，步骤(3)中，所述提取和纯化采用如下步骤进行：
将所述裂解液离心收集上清液，并对所述上清液进行过滤，将过滤后的上清液加入树脂中孵育，再洗脱蛋白，收集目标蛋白。
10.权利要求1所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白、权利要求2所述的核酸分子、权利要求3所述的重组载体、权利要求4所述的重组菌、权利要求5所述的棘球蚴疫苗、权利要求6所述的药物组合物或权利要求7-9中任一项所述的诺如病毒P颗粒嵌合棘球蚴EG95蛋白的重组蛋白的制备方法中任意一种或至少两种的组合在制备棘球蚴病治疗药物和/或疫苗中的应用。
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