CN112011572B - 柯萨奇病毒a7的病毒样颗粒及其制备方法和应用 - Google Patents

柯萨奇病毒a7的病毒样颗粒及其制备方法和应用 Download PDF

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CN112011572B
CN112011572B CN202010690365.7A CN202010690365A CN112011572B CN 112011572 B CN112011572 B CN 112011572B CN 202010690365 A CN202010690365 A CN 202010690365A CN 112011572 B CN112011572 B CN 112011572B
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朱朗
李雅静
阴彦辉
马铭江
吕哲
高强
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Abstract

本发明公开了具有免疫原性的柯萨奇病毒A7的病毒样颗粒，所述病毒样颗粒具有SEQ ID NO.3所示的氨基酸序列的P1蛋白和SEQ ID NO.4所示的氨基酸序列的3CD蛋白。本发明还公开了所述病毒样颗粒的制备方法。本发明的病毒样颗粒具有高的免疫原性，可用于制备预防手足口病的病毒感染的疫苗，还可作为抗原用于制备免疫检测试剂盒。

Description

柯萨奇病毒A7的病毒样颗粒及其制备方法和应用
技术领域
本发明属于生物技术领域，具体涉及柯萨奇病毒A7的病毒样颗粒及其制备方法和应用。
背景技术
手足口病是由肠道病毒引起的传染病，在亚太地区呈周期性爆发，多数手足口病的患儿一周左右自愈，少数患儿可引起肺部并发症，心肌炎、无菌性脑膜脑炎等并发症，重症患儿病情发展快，短时间内可导致死亡。目前对手足口病缺乏有效治疗药物对症治疗，造成我国及周边国家严重的公共卫生问题。
EV，Enterovirus，肠道病毒，肠道病毒又称肠病毒，是一种主要寄生于肠道的单链正义RNA病毒，属于小RNA病毒科，包括脊髓灰质炎病毒、柯萨奇病毒和埃可病毒等，与人类及哺乳动物的传染性疾病有关。肠道病毒在人群中感染主要表现为类流感症状、疱疹性咽峡炎及手足口病等症状。
CA，Coxsackievirus groups A，柯萨奇病毒A组，属于肠道病毒属，根据乳鼠感染的临床症状与组织病理特性分成A、B两组。A组目前包含24种血清型，与人类的手足口疾病主要相关。
目前已报道的可引起手足口病的肠道病毒有20多种(型)，包括肠道病毒71型(Enterovirus 71，EV71)、柯萨奇病毒A组16型(CA16)、CA10、CA6、CA4、CA7和CA14等，以EV71引起的重症居多。目前针对EV71引起的手足口病的疫苗已成功上市销售，对由EV71病毒引起的手足口病起到积极的预防作用，但肠道病毒A组病毒抗体之间没有交叉保护作用。CA7是除EV71和脊髓灰质炎病毒以外与弛缓性麻痹爆发相关的少数肠道病毒之一。
肠道病毒颗粒直径约为20～30nm，呈现20面体对称球形，无包膜。病毒衣壳蛋白内部包裹的一条单链正义RNA全长约7500bp，由一个开放阅读框(open reading frame，ORF)和两端的5’-和3’-非编码区(untranslated region，UTR)组成。ORF编码一个较大的多聚前体蛋白，由P1、P2、P3三个前体蛋白组成，P1编码病毒衣壳的结构蛋白VP1、VP2、VP3和VP4，其中VP1包含主要的中和抗原表位所在的区域，P2、P3编码非结构蛋白，包括2A、2B、2C、3A、3B、3C和3D。肠道病毒家族的前体蛋白的剪切一般为ORF表达的多聚前体蛋白首先裂解为P1、P2、P3，P1经3CD蛋白等的作用继而裂解为各种衣壳蛋白VP0(约35kDa)、VP1(约32kDa)和VP3(约28kDa)，病毒的衣壳蛋白一般具有天然的自我装配能力，组装成病毒颗粒，VP0在核酸存在的情况下进一步裂解为VP2和VP4。肠道病毒一般采用适宜的载体序列同时表达P1和3CD蛋白，不需经过筛选纯培养病毒即可获得具有免疫原性的病毒样颗粒(virus-likeparticles,VLPs)。VLPs不含有病毒的核酸，因此不能自主复制，没有感染性，但其形态与天然状态下的病毒颗粒相同或相似，免疫后可通过和病毒感染相同的途径呈递给免疫细胞，有效地诱导机体的免疫系统产生免疫保护反应。
然而，目前本领域还没有一种有效的针对柯萨奇病毒的理想疫苗。
发明内容
为了解决上述技术问题，本发明提供如下技术方案。
第一方面，本发明提供一种重组杆状病毒表达载体。
作为本发明的一个具体实施方式，本发明的重组杆状病毒表达载体具有SEQ IDNO.1所示的核苷酸序列和SEQ ID NO.2所示的核苷酸序列，或具有分别与SEQ ID NO.1所示的核苷酸序列和SEQ ID NO.2所示的核苷酸序列互补的序列，或具有分别与SEQ ID NO.1所示的核苷酸序列和SEQ ID NO.2所示的核苷酸序列具有95％以上同源性的序列。
作为本发明的一个具体实施方式，所述表达载体进一步包含AcMNPV p10启动子和/或polyhedrin启动子。
第二方面，本发明提供一种柯萨奇病毒A7的病毒样颗粒的表达系统。
作为本发明的一个具体实施方式，本发明的柯萨奇病毒A7的病毒样颗粒的表达系统包含所述表达载体。
作为本发明的一个具体实施方式，所述表达系统为大肠杆菌或昆虫细胞，优选昆虫细胞。
作为本发明的一个优选的具体实施方式，所述昆虫细胞是Sf9昆虫细胞。
第三方面，本发明提供一种具有免疫原性的柯萨奇病毒A7的病毒样颗粒(CA7VLP)。
作为本发明的一个具体实施方式，本发明的具有免疫原性的柯萨奇病毒A7的病毒样颗粒具有：(1)：SEQ ID NO.3所示的氨基酸序列的P1蛋白和SEQ ID NO.4所示的氨基酸序列的3CD蛋白，或(2)：在(1)中的SEQ ID NO.3所示的氨基酸序列和SEQ ID NO.4所示的氨基酸序列分别经过取代、缺失或添加一个或几个氨基酸且具有所述病毒样颗粒的免疫原性的由(1)衍生的P1蛋白和3CD蛋白。
第四方面，本发明提供一种制备所述柯萨奇病毒A7的病毒样颗粒的方法。
作为本发明的一个具体实施方式，本发明的制备所述柯萨奇病毒A7的病毒样颗粒的方法包括如下步骤：
(1)构建包含SEQ ID NO.1所示的核苷酸序列和SEQ ID NO.2所示的核苷酸序列的表达载体；
(2)利用所述表达载体转化具有杆状病毒骨架的大肠杆菌，获得杆粒；
(3)用步骤(2)获得的杆粒感染昆虫细胞进行表达，得到所述柯萨奇病毒A7的病毒样颗粒。
作为本发明的一个具体实施方式，所述表达载体进一步包含AcMNPV p10启动子和/或polyhedrin启动子。
第五方面，本发明还提供具有免疫原性的柯萨奇病毒A7的病毒样颗粒或由上述制备方法制备的病毒样颗粒在制备预防手足口病的病毒感染的疫苗中的应用。
第六方面，本发明还提供具有免疫原性的柯萨奇病毒A7的病毒样颗粒或由上述制备方法制备的病毒样颗粒作为抗原在制备免疫检测试剂盒中的应用。
第七方面，本发明还提供预防手足口病的病毒感染的疫苗，所述疫苗通过所述的病毒样颗粒或所述的制备方法制备的病毒样颗粒来制备。
第八方面，本发明还提供免疫检测试剂盒，所述试剂盒通过所述的病毒样颗粒或所述的制备方法制备的病毒样颗粒作为抗原来制备。
名词解释：
杆粒是指带有杆状病毒基因组的质粒，可在细菌和昆虫细胞之间穿梭。
有益效果
本申请通过构建重组杆状病毒表达载体，在适合的条件下可以在表达系统优选昆虫细胞Sf9中表达，形成CA7 VLPs。经过纯化后获得的CA7 VLPs可以与佐剂吸附，制备重组CA7 VLPs疫苗。
本申请的CA7 VLPs病毒样颗粒具有高的免疫原性，可用于制备预防手足口病的病毒感染的疫苗，还可作为抗原用于制备免疫检测试剂盒。
本申请建立了CA7 VLPs制备的相关方法，初步在动物上验证了其作为基因工程疫苗的有效性，为CA7新型疫苗的研发奠定了基础。
根据本发明的技术方案，在不改变蛋白的活性或功能的情况下，可以对氨基酸序列中的某些氨基酸进行保守取代，参见下面表1：
表1氨基酸保守性替换表
残基 保守性替换 残基 保守性替换
Ala Ser Leu Ile；Val
Arg Lys Lys Arg；Gln
Asn Gln；His Met Leu；Ile
Asp Glu Phe Met；Leu；Tyr
Gln Asn Ser Thr；Gly
Cys Ser Thr Ser；Val
Glu Asp Trp Tyr
Gly Pro Tyr Trp；Phe
His Asn；Gln Val Ile；Leu
Ile Leu；Val
此外，因为碱基的简并性，在不改变多核苷酸序列的活性或功能的情况下，可以对多核苷酸序列的碱基进行取代，参见下面表2：
表2核苷酸简并密码子表
Figure BDA0002589143770000051
Figure BDA0002589143770000061
附图说明
图1为SDS-PAGE分析CA7 VLPs的表达。
图2为CA7 VLPs的电镜照片(68000×)。
具体实施方式
下面通过实施例对本发明作进一步说明，应该理解的是，本发明实施例仅仅是用于说明本发明，而不是本发明的限制，在本发明的构思前提下对本发明的简单改进都属于本发明要求保护的范围。
实施例1.重组杆状病毒表达载体的构建
核苷酸序列优化：优化后的核苷酸序列更适合在昆虫细胞表达系统中表达而氨基酸序列不变。
P1核苷酸序列(SEQ ID NO.1)：
ATGGGTGCTCAGATCAGCACTCAGAAGTCAGGATCCCACGAGACCGGTAACATCGCCACTGAAGGCAGCACCATCAACTTCACTAACATCAACTACTACAAGGACTCTTACGCTGCCAGCGCTTCTAAGCAGGACTTCACTCAGGACCCCGGCAAGTTCACCAGCCCAGTGCTGGACGTCCTGTCTGAGATGGCTGCTCCTCTGCAGAGCCCAACCGCTGAAGCCTGCGGCTACTCTGACCGTGTGGCTCAGCTGACCGTCGGAAACTCCACTATCACCACTCAGGAGGCTGCCAACGTGATCGTCGCCTACGGTGAATGGCCTCAGTACTGCCCCGACACCGACGCTACTGCCGTGGACAAGCCAACCCGCCCTGACGTGAGCGTCAACCGTTTCTACACCCTGGACACTAAGGACTGGTCCAGCTCTTCAAAGGGCTGGTACTGGAAGTTCCCTGACATCCTGGCTGAGACCGGAGTGTTCGGTCAGAACGCCCAGTTCCACTTCCTGTACCGCTCAGGATTCTGCATCCACGTCCAGTGCAACGCTTCCAAGTTCCACCAAGGTGCTCTGCTGGTGGCTGTCCTGCCAGAGTACGTGACCGGAACTGTCTCTGGCAACACCGGACTGGAAAACACTCACCCTCCCTACGCTGCTACCCAGCCTGGTGCTACTGGCTTCGAACTGACCAACCCTTACATCCTGGACGCCGGAATCCCCCTGTCACAGCTGCTGGTGTGCCCACACCAGTGGATCAACCTGCGTACTAACAACTGCGCTACCATCGTGGTCCCTTACATCAACAGCGTGCCCTTCGACTCTGCCCTGAACCACTGCAACTTCGGTCTGGTGGTCATCCCCGTCTCTCCACTGGGTTTCCTGCAGGGCGCTACCCCTACTATCCCCATCACCATCACTGTGGCCCCAATGAACTCAGAGTTCTCCGGACTGCGCCAGGCTGTCACTCAGGGTCTGCCTATGGAACTGAAGCCCGGCACCAACCAGTTCCTGACCACTGACGACGGAGTGTCCGCCCCAATCCTGCCTGGTTTCCACCCAACCCCTGTGATCCACATCCCTGGAGAGGTCCGTAACCTGCTGGAACTGTGCCAGATCGACACTATCATGGAGGTCAACAACCTGACCACTAACGAGGCTACCCCCATGGAACGCCTGCGTATCCCAGTGCAGGTCCAGACTCAGAGCGGCGAACTGTGCGCTGCCTTCAAGGCCGACCCAGGACTGGACGGTCCTTGGCAGTCTACTATGGTGGGTCAGCTGTGCCGCTACTACACCCAGTGGTCAGGCTCCCTGAAGATCACCTTCATGTTCACTGGTTCATTCATGGCTACTGGCAAGATGCTGATCGCTTACACCCCACCTGGTGGCTCCCTGCCCGCTAACAGGATGCAGGCCATGCTGGGCACCCACGTGATCTGGGACTTCGGACTGCAGTCCAGCGTCACTCTGGTGGTCCCATGGATCTCAAACACTCACTACAGATCACAGGCTACCGGATCCTTCTTCGACTACTACGCCACTGGTATCGTGTCCCTGTGGTATCAGACCAACTTCGTGGTCCCAATCGGAGCTCCTACCACTGCCTACATCGTGGTCCTGGGTTCAGCTCAGAAGAACTTCACCATGAGGCTGTGCAGAGACACCTCCGAGCTGACTCAGGCTGCCGAGTACCAGGGTGACGAAATCGTGGACCTGATCGAATCAGCTGTCCAGAACACCACTAAGGCCATCACTTCTTCAATCGACACCAAGACTGGAGCTAACACCCAGGCCTCCCAACACCGTATCGGTCTGGGTGAGGTGCCCGCTCTGCAGGCTGCTGAAACCGGTTCCAGCTCTCTGGTCAGCGACAAGAACATGATCGAGACTCGCTGCGTGGTCAACAAGCACAGCACCGAGGAAACTTCTATCACCAACTTCTACAGCCGTGCTGGCCTGGTGGGAGTGGTCAACATGCCAGTCCAGGGCACTTCTAACACCAAGGGCTTCGCCAAGTGGGGAATCGACATCATGGGTTTCGTGCAGATGCGCCGTAAGCTGGAGCTGATGACCTACATGCGCTTCTCAGCTGAGTTCACTTTCGTCGCCTCCACCCCTGGAGGTGAGACCACTAACCTGATCCTGCAGTACATGTACGCTCCCCCAGGTGCTCCTCTGCCAACCAGGAGAGACTCATACGAATGGCAGACCTCCACTAACCCCAGCATCATCTCTAAGATGGCTGACCCTCCCGCTCAGGTGTCCGTCCCCTTCCTGTCACCAGCTTCCGCCTACCAGTGGTTCTACGACGGCTACCCAACCTTCGGAAAGCACCCTATCGACCAGGACTTCCAGTACGGAATGTGCCCTAACAACATGATGGGCACTTTCTGCGTGAGGATGATCGGTGGTGGCAAGCCCACCCAGAGCGTCACTATCAGGATCTACATGAGACTGAAGCACATCCGTGCTTGGGTGCCTAGGCCACTGAGGTCTCAGAACTACACCATGAGAAACTACCCCAACTACAACGGCGGAGCTATCAAGTGCACTAGCAAGTCTAGAGCCACCATCACCACTCTGTAA
3CD核苷酸序列(SEQ ID NO.2)：
ATGGGTCCTAGCCTGGACTTCGCTCTGTCTCTGCTGCGCCGTAACATCAAGCAGGCCCAGACTGACCAGGGCCACTTCACCATGCTGGGAATCCGCGACCGTCTGGCTATCCTCCCTAGGCACGCTCAGCCAGGAAAGACTATCTGGGTGGAGCACAAGCTGGTGAACGTCCTGGACGCTGTGGAGCTGGTCGACGAACAGGGTGTGAACCTGGAGCTGACCCTGGTCACTCTGGACACCAACGAAAAGTTCCGTGACATCACCAAGTTCATCCCTGAGGTCATCTCTGGTGCTTCCGACGCCACTCTGGTCATCAACACCGAACACATGCCATCTATGTTCGTGCCTGTCGGTGACGTGGTCCAGTACGGCTTCCTGAACCTGTCAGGAAAGCCAACTCACAGGACCATGATGTACAACTTCCCTACTAAGGCTGGTCAATGCGGTGGCGTGGTCACCTCTGTGGGCAAGATCATCGGAATCCACATCGGAGGTAACGGTCGCCAGGGCTTCTGCGCCGGTCTGAAGCGTGGCTACTTCGTCAGCGAGCAGGGCGAAATCCAGTGGATGAAGCCCAACAAGGAGACCGGTCGCCTGAACATCAACGGCCCAACCCGTACTAAGCTGGAACCTTCTGTGTTCTACGACGTCTTCGAGGGCAACAAGGAACCCGCTGTGCTGACTTCTAAGGACCCACGCCTGGAGGTCGACTTCGAACAGGCCCTGTTCTCAAAGTACGTGGGAAACGTCCTGCACGAGCCAGACGAATACGTGACCCAGGCTGCCCTGCACTACGCTAACCAGCTGAAGCAGCTGGACATCAACACTAACAAGATGTCTATGGAGGAAGCCTGCTACGGAACCGAGAACCTGGAAGCTATCGACCTGCACACTTCAGCCGGATACCCTTACTCCACCCTGGGTATCAAGAAGAGGGACATCCTGGACCCCACCACTAGAGACGTGTCAAAGATGAAGCTGTACCTGGACAAGTACGGCCTGGACCTGCCTTACAGCACTTACGTCAAGGACGAGCTGAGGTCTCTGGACAAGATCAAGAAGGGCAAGTCAAGACTGATCGAGGCTTCCTCCCTGAACGACTCCGTGTACCTGCGTATGACCTTCGGCCACCTGTACGAGGTCTTCCACGCTAACCCTGGAACCGTGACTGGTTCTGCTGTCGGCTGCAACCCAGACGTGTTCTGGAGCAAGCTGCCCATCCTGCTGCCAGGCAGCCTGTTCGCTTTCGACTACTCTGGATACGACGCCTCTCTGTCACCAGTGTGGTTCAGGGCTCTGGAGATCGTGCTGAGAGAAGTCGGTTACTCAGAGGAAGCCGTCTCCCTGATCGAGGGCATCAACCACACTCACCACGTGTACAGGAACAAGACCTACTGCGTCCTGGGCGGAATGCCTTCTGGATGCTCAGGTACTTCCATCTTCAACTCCATGATCAACAACATCATCATCAGAACTCTGCTGATCAAGACCTTCAAGGGAATCGACCTGGACGAACTGAACATGGTGGCTTACGGTGACGACGTCCTGGCCTCATACCCTTTCCCCATCGACTGCTCCGAGCTGGCTAAGACCGGAAAGGAATACGGTCTGACCATGACTCCCGCCGACAAGAGCCCATGCTTCAACGAAGTGACTTGGGAAAACGCCACCTTCCTGAAGCGCGGTTTCCTGCCTGACCACCAGTTCCCCTTCCTGATCCACCCAACTATGCCTATGCGTGAGATCCACGAATCCATCAGGTGGACTAAGGACGCTAGAAACACTCAAGACCACGTCCGCAGCCTGTGCCTGCTGGCCTGGCACAACGGCAAGGACGAGTACGAACGCTTCGTGAGCACCATCCGTTCTGTCCCTATCGGAAAGGCTCTGGCCATCCCCAACTTCGAGAACCTGAGGAGAAACTGGCTGGAACTGTTCTAA
P1氨基酸序列(SEQ ID NO.3)：
MGAQISTQKSGSHETGNIATEGSTINFTNINYYKDSYAASASKQDFTQDPGKFTSPVLDVLSEMAAPLQSPTAEACGYSDRVAQLTVGNSTITTQEAANVIVAYGEWPQYCPDTDATAVDKPTRPDVSVNRFYTLDTKDWSSSSKGWYWKFPDILAETGVFGQNAQFHFLYRSGFCIHVQCNASKFHQGALLVAVLPEYVTGTVSGNTGLENTHPPYAATQPGATGFELTNPYILDAGIPLSQLLVCPHQWINLRTNNCATIVVPYINSVPFDSALNHCNFGLVVIPVSPLGFLQGATPTIPITITVAPMNSEFSGLRQAVTQGLPMELKPGTNQFLTTDDGVSAPILPGFHPTPVIHIPGEVRNLLELCQIDTIMEVNNLTTNEATPMERLRIPVQVQTQSGELCAAFKADPGLDGPWQSTMVGQLCRYYTQWSGSLKITFMFTGSFMATGKMLIAYTPPGGSLPANRMQAMLGTHVIWDFGLQSSVTLVVPWISNTHYRSQATGSFFDYYATGIVSLWYQTNFVVPIGAPTTAYIVVLGSAQKNFTMRLCRDTSELTQAAEYQGDEIVDLIESAVQNTTKAITSSIDTKTGANTQASQHRIGLGEVPALQAAETGSSSLVSDKNMIETRCVVNKHSTEETSITNFYSRAGLVGVVNMPVQGTSNTKGFAKWGIDIMGFVQMRRKLELMTYMRFSAEFTFVASTPGGETTNLILQYMYAPPGAPLPTRRDSYEWQTSTNPSIISKMADPPAQVSVPFLSPASAYQWFYDGYPTFGKHPIDQDFQYGMCPNNMMGTFCVRMIGGGKPTQSVTIRIYMRLKHIRAWVPRPLRSQNYTMRNYPNYNGGAIKCTSKSRATITTL*
3CD氨基酸序列(SEQ ID NO.4)：
MGPSLDFALSLLRRNIKQAQTDQGHFTMLGIRDRLAILPRHAQPGKTIWVEHKLVNVLDAVELVDEQGVNLELTLVTLDTNEKFRDITKFIPEVISGASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKIIGIHIGGNGRQGFCAGLKRGYFVSEQGEIQWMKPNKETGRLNINGPTRTKLEPSVFYDVFEGNKEPAVLTSKDPRLEVDFEQALFSKYVGNVLHEPDEYVTQAALHYANQLKQLDINTNKMSMEEACYGTENLEAIDLHTSAGYPYSTLGIKKRDILDPTTRDVSKMKLYLDKYGLDLPYSTYVKDELRSLDKIKKGKSRLIEASSLNDSVYLRMTFGHLYEVFHANPGTVTGSAVGCNPDVFWSKLPILLPGSLFAFDYSGYDASLSPVWFRALEIVLREVGYSEEAVSLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNSMINNIIIRTLLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCSELAKTGKEYGLTMTPADKSPCFNEVTWENATFLKRGFLPDHQFPFLIHPTMPMREIHESIRWTKDARNTQDHVRSLCLLAWHNGKDEYERFVSTIRSVPIGKALAIPNFENLRRNWLELF*
将优化后的CA7-P1和CA7-3CD核苷酸序列连接到至pFastBacTMDual(pFBD，Invitrogen)载体上，构建pFBD-CA7-P1/3CD质粒，P1和3CD基因优化和pFBD-CA7-P1/3CD质粒构建合成均在金斯瑞公司(南京，中国)完成。
实施例2.CA7杆粒的构建
获得pFBD-CA7-P1/3CD质粒后，利用昆虫杆状病毒表达系统(Bac-to-Bacexpression system，Invitrogen)将纯化的质粒转化具有杆状病毒骨架的大肠杆菌DH10BacTM感受态细胞(Invitrogen)，并通过蓝白斑筛选，纯化获得杆粒Bac-CA7-P1/3CD用于后续昆虫细胞中的表达。
实施例3.CA7 VLPs的表达和纯化
小量检测验证表达成功后，取P3代CA7杆状病毒进行VLPs的大量表达。首先，用P3代杆状病毒感染昆虫细胞Sf9，72h～96h后收获细胞培养液离心将上清与细胞分离，将上清液采用30kD～100kD的膜包进行超滤浓缩，得到浓缩液经20％蔗糖垫进行超速离心浓缩，条件为36000～40000rpm 3h，所得沉淀用0.01mol/L PBS重悬。再通过10％-60％蔗糖，30000～35000rpm 3h密度梯度离心，蔗糖密度梯度离心后从上至下取20个等体积组分，通过SDS-PAGE、电镜和质谱进行VLPs表达的分析，确定制备的样品已组装形成完整的CA7 VLPs，收集富含VLP的组分混合，再进行上述操作。纯化后的VLPs用BCA或Lowry法进行蛋白定量，确定CA7-VLP的浓度。
实施例4.CA7 VLPs免疫小鼠后的抗体反应
用以上方法对Bac-CA7-P1/3CD转染的Sf9细胞进行表达和纯化，获得CA7 VLPs用于动物免疫实验。为了检测评估制备的CA7 VLPs的免疫原性，将12只Balb/c小鼠分为两组，分别在第0周、第2周和第4周腹腔注射Sf9细胞裂解液或CA7 VLPs，并分别在第6周和第8周进行眼眶采血，分离获得抗Sf9和抗CA7 VLPs的特异性抗体血清，用于ELISA检测分析和动物保护实验。用0.5μg/ml蛋白含量的CA7 VLPs作为抗原包板，10倍梯度稀释至10-8进行ELISA检测。结果显示，CA7-VLP组的血清与CA7 VLPs结合，产生很强的特异性反应可达106，而Sf9组则没有产生明显反应。该实验结果显示，CA7-VLP免疫小鼠后在体内产生了很强特异性免疫反应。
(1)CA7 VLPs血清的体外交叉中和实验：
将获得的抗CA7血清以1:8起始倍比稀释，对CA6、CA16、CA10及EV71四种病毒的交叉中和实验结果证实CA7血清在最低稀释度对上述四种病毒没有中和交叉效果。
(2)CA7 VLPs疫苗抗血清体内被动保护实验：
为了确定抗CA7 VLPs特异性抗体在体内的被动保护效果，7日龄新生小鼠先被动腹腔注射100μl的抗CA7 VLPs或抗Sf9抗血清，并在24h后接种CA7(ATCC VR-319)病毒进行攻击。实验结果表明，注射抗Sf9血清的小鼠，用CA7病毒攻击后逐渐出现严重的临床症状，包括共济失调和瘫痪，并且均在攻毒后8日内达到100％的死亡率。然而，经过抗CA7 VLPs抗血清注射的小鼠，同样攻击CA7病毒后未出现任何临床症状，也未见死亡。以上实验结果证实抗CA7VLPs血清能够很好地保护小鼠免于病毒的攻击。
(3)CA7 VLPs疫苗体内主动保护实验：
为了进一步确定CA7 VLPs疫苗在动物体内的主动保护效果，新生小鼠分别在1日龄和7日龄时免疫接种2μg CA7 VLPs或空白对照Sf9细胞裂解液，并在14日龄时用CA7(ATCCVR-319)病毒进行攻击。实验结果表明，注射Sf9细胞裂解液的小鼠，用CA7病毒攻击后很快出现严重临床症状，包括共济失调和瘫痪，经CA7病毒攻击的小鼠在8日内达到90％～100％死亡率。然而，经过CA7 VLPs免疫的小鼠，同样攻击CA7病毒后仅出现轻微的一级迟缓症状，死亡率均小于20％。以上实验结果证实CA7 VLPs能够保护小鼠免于CA7病毒的攻击。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。
参考文献
1.Guide to Baculovirus Expression Vector Systems(BEVS)and Insect CellCulture Techniques.Invitrogen Instruction Manual.
2.Seitsonen JJ1,Shakeel S,Susi P,Pandurangan AP,Sinkovits RS,
Figure BDA0002589143770000141
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Figure BDA0002589143770000142
P,
Figure BDA0002589143770000143
-Pelto J,Topf M,
Figure BDA0002589143770000144
T,Butcher SJ.Structuralanalysis of coxsackievirus A7 reveals conformational changes associated withuncoating.J Virol.2012Jul；86(13):7207-15.
3.
Figure BDA0002589143770000145
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Figure BDA0002589143770000151
T,Susi P.Complete genome sequences of three strains of coxsackievirus a7.GenomeAnnounc.2013 Apr 11；1(2):e0014613.
序列表
<110> 北京科兴生物制品有限公司 北京科兴中维生物技术有限公司
<120> 柯萨奇病毒A7的病毒样颗粒及其制备方法和应用
<130> RYP1910490.6
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2586
<212> DNA
<213> P1核苷酸序列
<400> 1
atgggtgctc agatcagcac tcagaagtca ggatcccacg agaccggtaa catcgccact 60
gaaggcagca ccatcaactt cactaacatc aactactaca aggactctta cgctgccagc 120
gcttctaagc aggacttcac tcaggacccc ggcaagttca ccagcccagt gctggacgtc 180
ctgtctgaga tggctgctcc tctgcagagc ccaaccgctg aagcctgcgg ctactctgac 240
cgtgtggctc agctgaccgt cggaaactcc actatcacca ctcaggaggc tgccaacgtg 300
atcgtcgcct acggtgaatg gcctcagtac tgccccgaca ccgacgctac tgccgtggac 360
aagccaaccc gccctgacgt gagcgtcaac cgtttctaca ccctggacac taaggactgg 420
tccagctctt caaagggctg gtactggaag ttccctgaca tcctggctga gaccggagtg 480
ttcggtcaga acgcccagtt ccacttcctg taccgctcag gattctgcat ccacgtccag 540
tgcaacgctt ccaagttcca ccaaggtgct ctgctggtgg ctgtcctgcc agagtacgtg 600
accggaactg tctctggcaa caccggactg gaaaacactc accctcccta cgctgctacc 660
cagcctggtg ctactggctt cgaactgacc aacccttaca tcctggacgc cggaatcccc 720
ctgtcacagc tgctggtgtg cccacaccag tggatcaacc tgcgtactaa caactgcgct 780
accatcgtgg tcccttacat caacagcgtg cccttcgact ctgccctgaa ccactgcaac 840
ttcggtctgg tggtcatccc cgtctctcca ctgggtttcc tgcagggcgc tacccctact 900
atccccatca ccatcactgt ggccccaatg aactcagagt tctccggact gcgccaggct 960
gtcactcagg gtctgcctat ggaactgaag cccggcacca accagttcct gaccactgac 1020
gacggagtgt ccgccccaat cctgcctggt ttccacccaa cccctgtgat ccacatccct 1080
ggagaggtcc gtaacctgct ggaactgtgc cagatcgaca ctatcatgga ggtcaacaac 1140
ctgaccacta acgaggctac ccccatggaa cgcctgcgta tcccagtgca ggtccagact 1200
cagagcggcg aactgtgcgc tgccttcaag gccgacccag gactggacgg tccttggcag 1260
tctactatgg tgggtcagct gtgccgctac tacacccagt ggtcaggctc cctgaagatc 1320
accttcatgt tcactggttc attcatggct actggcaaga tgctgatcgc ttacacccca 1380
cctggtggct ccctgcccgc taacaggatg caggccatgc tgggcaccca cgtgatctgg 1440
gacttcggac tgcagtccag cgtcactctg gtggtcccat ggatctcaaa cactcactac 1500
agatcacagg ctaccggatc cttcttcgac tactacgcca ctggtatcgt gtccctgtgg 1560
tatcagacca acttcgtggt cccaatcgga gctcctacca ctgcctacat cgtggtcctg 1620
ggttcagctc agaagaactt caccatgagg ctgtgcagag acacctccga gctgactcag 1680
gctgccgagt accagggtga cgaaatcgtg gacctgatcg aatcagctgt ccagaacacc 1740
actaaggcca tcacttcttc aatcgacacc aagactggag ctaacaccca ggcctcccaa 1800
caccgtatcg gtctgggtga ggtgcccgct ctgcaggctg ctgaaaccgg ttccagctct 1860
ctggtcagcg acaagaacat gatcgagact cgctgcgtgg tcaacaagca cagcaccgag 1920
gaaacttcta tcaccaactt ctacagccgt gctggcctgg tgggagtggt caacatgcca 1980
gtccagggca cttctaacac caagggcttc gccaagtggg gaatcgacat catgggtttc 2040
gtgcagatgc gccgtaagct ggagctgatg acctacatgc gcttctcagc tgagttcact 2100
ttcgtcgcct ccacccctgg aggtgagacc actaacctga tcctgcagta catgtacgct 2160
cccccaggtg ctcctctgcc aaccaggaga gactcatacg aatggcagac ctccactaac 2220
cccagcatca tctctaagat ggctgaccct cccgctcagg tgtccgtccc cttcctgtca 2280
ccagcttccg cctaccagtg gttctacgac ggctacccaa ccttcggaaa gcaccctatc 2340
gaccaggact tccagtacgg aatgtgccct aacaacatga tgggcacttt ctgcgtgagg 2400
atgatcggtg gtggcaagcc cacccagagc gtcactatca ggatctacat gagactgaag 2460
cacatccgtg cttgggtgcc taggccactg aggtctcaga actacaccat gagaaactac 2520
cccaactaca acggcggagc tatcaagtgc actagcaagt ctagagccac catcaccact 2580
ctgtaa 2586
<210> 2
<211> 1941
<212> DNA
<213> 3CD核苷酸序列
<400> 2
atgggtccta gcctggactt cgctctgtct ctgctgcgcc gtaacatcaa gcaggcccag 60
actgaccagg gccacttcac catgctggga atccgcgacc gtctggctat cctccctagg 120
cacgctcagc caggaaagac tatctgggtg gagcacaagc tggtgaacgt cctggacgct 180
gtggagctgg tcgacgaaca gggtgtgaac ctggagctga ccctggtcac tctggacacc 240
aacgaaaagt tccgtgacat caccaagttc atccctgagg tcatctctgg tgcttccgac 300
gccactctgg tcatcaacac cgaacacatg ccatctatgt tcgtgcctgt cggtgacgtg 360
gtccagtacg gcttcctgaa cctgtcagga aagccaactc acaggaccat gatgtacaac 420
ttccctacta aggctggtca atgcggtggc gtggtcacct ctgtgggcaa gatcatcgga 480
atccacatcg gaggtaacgg tcgccagggc ttctgcgccg gtctgaagcg tggctacttc 540
gtcagcgagc agggcgaaat ccagtggatg aagcccaaca aggagaccgg tcgcctgaac 600
atcaacggcc caacccgtac taagctggaa ccttctgtgt tctacgacgt cttcgagggc 660
aacaaggaac ccgctgtgct gacttctaag gacccacgcc tggaggtcga cttcgaacag 720
gccctgttct caaagtacgt gggaaacgtc ctgcacgagc cagacgaata cgtgacccag 780
gctgccctgc actacgctaa ccagctgaag cagctggaca tcaacactaa caagatgtct 840
atggaggaag cctgctacgg aaccgagaac ctggaagcta tcgacctgca cacttcagcc 900
ggataccctt actccaccct gggtatcaag aagagggaca tcctggaccc caccactaga 960
gacgtgtcaa agatgaagct gtacctggac aagtacggcc tggacctgcc ttacagcact 1020
tacgtcaagg acgagctgag gtctctggac aagatcaaga agggcaagtc aagactgatc 1080
gaggcttcct ccctgaacga ctccgtgtac ctgcgtatga ccttcggcca cctgtacgag 1140
gtcttccacg ctaaccctgg aaccgtgact ggttctgctg tcggctgcaa cccagacgtg 1200
ttctggagca agctgcccat cctgctgcca ggcagcctgt tcgctttcga ctactctgga 1260
tacgacgcct ctctgtcacc agtgtggttc agggctctgg agatcgtgct gagagaagtc 1320
ggttactcag aggaagccgt ctccctgatc gagggcatca accacactca ccacgtgtac 1380
aggaacaaga cctactgcgt cctgggcgga atgccttctg gatgctcagg tacttccatc 1440
ttcaactcca tgatcaacaa catcatcatc agaactctgc tgatcaagac cttcaaggga 1500
atcgacctgg acgaactgaa catggtggct tacggtgacg acgtcctggc ctcataccct 1560
ttccccatcg actgctccga gctggctaag accggaaagg aatacggtct gaccatgact 1620
cccgccgaca agagcccatg cttcaacgaa gtgacttggg aaaacgccac cttcctgaag 1680
cgcggtttcc tgcctgacca ccagttcccc ttcctgatcc acccaactat gcctatgcgt 1740
gagatccacg aatccatcag gtggactaag gacgctagaa acactcaaga ccacgtccgc 1800
agcctgtgcc tgctggcctg gcacaacggc aaggacgagt acgaacgctt cgtgagcacc 1860
atccgttctg tccctatcgg aaaggctctg gccatcccca acttcgagaa cctgaggaga 1920
aactggctgg aactgttcta a 1941
<210> 3
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Met Gly Ala Gln Ile Ser Thr Gln Lys Ser Gly Ser His Glu Thr Gly
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Asn Ile Ala Thr Glu Gly Ser Thr Ile Asn Phe Thr Asn Ile Asn Tyr
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Tyr Lys Asp Ser Tyr Ala Ala Ser Ala Ser Lys Gln Asp Phe Thr Gln
35 40 45
Asp Pro Gly Lys Phe Thr Ser Pro Val Leu Asp Val Leu Ser Glu Met
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Ala Ala Pro Leu Gln Ser Pro Thr Ala Glu Ala Cys Gly Tyr Ser Asp
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Arg Val Ala Gln Leu Thr Val Gly Asn Ser Thr Ile Thr Thr Gln Glu
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Ala Ala Asn Val Ile Val Ala Tyr Gly Glu Trp Pro Gln Tyr Cys Pro
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Asp Thr Asp Ala Thr Ala Val Asp Lys Pro Thr Arg Pro Asp Val Ser
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Val Asn Arg Phe Tyr Thr Leu Asp Thr Lys Asp Trp Ser Ser Ser Ser
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Lys Gly Trp Tyr Trp Lys Phe Pro Asp Ile Leu Ala Glu Thr Gly Val
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Phe Gly Gln Asn Ala Gln Phe His Phe Leu Tyr Arg Ser Gly Phe Cys
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Ile His Val Gln Cys Asn Ala Ser Lys Phe His Gln Gly Ala Leu Leu
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Val Ala Val Leu Pro Glu Tyr Val Thr Gly Thr Val Ser Gly Asn Thr
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Gly Leu Glu Asn Thr His Pro Pro Tyr Ala Ala Thr Gln Pro Gly Ala
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Asn Asn Cys Ala Thr Ile Val Val Pro Tyr Ile Asn Ser Val Pro Phe
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Ile Thr Val Ala Pro Met Asn Ser Glu Phe Ser Gly Leu Arg Gln Ala
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Val Thr Gln Gly Leu Pro Met Glu Leu Lys Pro Gly Thr Asn Gln Phe
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Leu Thr Thr Asp Asp Gly Val Ser Ala Pro Ile Leu Pro Gly Phe His
340 345 350
Pro Thr Pro Val Ile His Ile Pro Gly Glu Val Arg Asn Leu Leu Glu
355 360 365
Leu Cys Gln Ile Asp Thr Ile Met Glu Val Asn Asn Leu Thr Thr Asn
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Glu Ala Thr Pro Met Glu Arg Leu Arg Ile Pro Val Gln Val Gln Thr
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Gln Trp Ser Gly Ser Leu Lys Ile Thr Phe Met Phe Thr Gly Ser Phe
435 440 445
Met Ala Thr Gly Lys Met Leu Ile Ala Tyr Thr Pro Pro Gly Gly Ser
450 455 460
Leu Pro Ala Asn Arg Met Gln Ala Met Leu Gly Thr His Val Ile Trp
465 470 475 480
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485 490 495
Asn Thr His Tyr Arg Ser Gln Ala Thr Gly Ser Phe Phe Asp Tyr Tyr
500 505 510
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515 520 525
Ile Gly Ala Pro Thr Thr Ala Tyr Ile Val Val Leu Gly Ser Ala Gln
530 535 540
Lys Asn Phe Thr Met Arg Leu Cys Arg Asp Thr Ser Glu Leu Thr Gln
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Ala Ala Glu Tyr Gln Gly Asp Glu Ile Val Asp Leu Ile Glu Ser Ala
565 570 575
Val Gln Asn Thr Thr Lys Ala Ile Thr Ser Ser Ile Asp Thr Lys Thr
580 585 590
Gly Ala Asn Thr Gln Ala Ser Gln His Arg Ile Gly Leu Gly Glu Val
595 600 605
Pro Ala Leu Gln Ala Ala Glu Thr Gly Ser Ser Ser Leu Val Ser Asp
610 615 620
Lys Asn Met Ile Glu Thr Arg Cys Val Val Asn Lys His Ser Thr Glu
625 630 635 640
Glu Thr Ser Ile Thr Asn Phe Tyr Ser Arg Ala Gly Leu Val Gly Val
645 650 655
Val Asn Met Pro Val Gln Gly Thr Ser Asn Thr Lys Gly Phe Ala Lys
660 665 670
Trp Gly Ile Asp Ile Met Gly Phe Val Gln Met Arg Arg Lys Leu Glu
675 680 685
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690 695 700
Thr Pro Gly Gly Glu Thr Thr Asn Leu Ile Leu Gln Tyr Met Tyr Ala
705 710 715 720
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725 730 735
Thr Ser Thr Asn Pro Ser Ile Ile Ser Lys Met Ala Asp Pro Pro Ala
740 745 750
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755 760 765
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770 775 780
Gln Tyr Gly Met Cys Pro Asn Asn Met Met Gly Thr Phe Cys Val Arg
785 790 795 800
Met Ile Gly Gly Gly Lys Pro Thr Gln Ser Val Thr Ile Arg Ile Tyr
805 810 815
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820 825 830
Gln Asn Tyr Thr Met Arg Asn Tyr Pro Asn Tyr Asn Gly Gly Ala Ile
835 840 845
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850 855 860
<210> 4
<211> 646
<212> PRT
<213> 3CD氨基酸序列
<400> 4
Met Gly Pro Ser Leu Asp Phe Ala Leu Ser Leu Leu Arg Arg Asn Ile
1 5 10 15
Lys Gln Ala Gln Thr Asp Gln Gly His Phe Thr Met Leu Gly Ile Arg
20 25 30
Asp Arg Leu Ala Ile Leu Pro Arg His Ala Gln Pro Gly Lys Thr Ile
35 40 45
Trp Val Glu His Lys Leu Val Asn Val Leu Asp Ala Val Glu Leu Val
50 55 60
Asp Glu Gln Gly Val Asn Leu Glu Leu Thr Leu Val Thr Leu Asp Thr
65 70 75 80
Asn Glu Lys Phe Arg Asp Ile Thr Lys Phe Ile Pro Glu Val Ile Ser
85 90 95
Gly Ala Ser Asp Ala Thr Leu Val Ile Asn Thr Glu His Met Pro Ser
100 105 110
Met Phe Val Pro Val Gly Asp Val Val Gln Tyr Gly Phe Leu Asn Leu
115 120 125
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130 135 140
Ala Gly Gln Cys Gly Gly Val Val Thr Ser Val Gly Lys Ile Ile Gly
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Ile His Ile Gly Gly Asn Gly Arg Gln Gly Phe Cys Ala Gly Leu Lys
165 170 175
Arg Gly Tyr Phe Val Ser Glu Gln Gly Glu Ile Gln Trp Met Lys Pro
180 185 190
Asn Lys Glu Thr Gly Arg Leu Asn Ile Asn Gly Pro Thr Arg Thr Lys
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210 215 220
Ala Val Leu Thr Ser Lys Asp Pro Arg Leu Glu Val Asp Phe Glu Gln
225 230 235 240
Ala Leu Phe Ser Lys Tyr Val Gly Asn Val Leu His Glu Pro Asp Glu
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Tyr Val Thr Gln Ala Ala Leu His Tyr Ala Asn Gln Leu Lys Gln Leu
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Asp Ile Asn Thr Asn Lys Met Ser Met Glu Glu Ala Cys Tyr Gly Thr
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Glu Asn Leu Glu Ala Ile Asp Leu His Thr Ser Ala Gly Tyr Pro Tyr
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Ser Thr Leu Gly Ile Lys Lys Arg Asp Ile Leu Asp Pro Thr Thr Arg
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Asp Val Ser Lys Met Lys Leu Tyr Leu Asp Lys Tyr Gly Leu Asp Leu
325 330 335
Pro Tyr Ser Thr Tyr Val Lys Asp Glu Leu Arg Ser Leu Asp Lys Ile
340 345 350
Lys Lys Gly Lys Ser Arg Leu Ile Glu Ala Ser Ser Leu Asn Asp Ser
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Val Tyr Leu Arg Met Thr Phe Gly His Leu Tyr Glu Val Phe His Ala
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Asn Pro Gly Thr Val Thr Gly Ser Ala Val Gly Cys Asn Pro Asp Val
385 390 395 400
Phe Trp Ser Lys Leu Pro Ile Leu Leu Pro Gly Ser Leu Phe Ala Phe
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Asp Tyr Ser Gly Tyr Asp Ala Ser Leu Ser Pro Val Trp Phe Arg Ala
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Leu Glu Ile Val Leu Arg Glu Val Gly Tyr Ser Glu Glu Ala Val Ser
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Leu Ile Glu Gly Ile Asn His Thr His His Val Tyr Arg Asn Lys Thr
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Phe Asn Ser Met Ile Asn Asn Ile Ile Ile Arg Thr Leu Leu Ile Lys
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Thr Phe Lys Gly Ile Asp Leu Asp Glu Leu Asn Met Val Ala Tyr Gly
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Met Pro Met Arg Glu Ile His Glu Ser Ile Arg Trp Thr Lys Asp Ala
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Arg Asn Thr Gln Asp His Val Arg Ser Leu Cys Leu Leu Ala Trp His
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Asn Gly Lys Asp Glu Tyr Glu Arg Phe Val Ser Thr Ile Arg Ser Val
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Pro Ile Gly Lys Ala Leu Ala Ile Pro Asn Phe Glu Asn Leu Arg Arg
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Asn Trp Leu Glu Leu Phe
645

Claims (11)

1.重组杆状病毒表达载体，其特征在于，所述表达载体具有SEQ ID NO.1所示的核苷酸序列和SEQ ID NO.2所示的核苷酸序列。
2.根据权利要求1所述的表达载体，其特征在于，所述表达载体进一步包含AcMNPV p10启动子和/或polyhedrin启动子。
3.柯萨奇病毒A7的病毒样颗粒的表达系统，其特征在于，所述表达系统包含权利要求1或2所述的表达载体，所述表达系统为昆虫细胞。
4.根据权利要求3所述的表达系统，其特征在于，所述昆虫细胞是Sf9昆虫细胞。
5.具有免疫原性的柯萨奇病毒A7的病毒样颗粒，其特征在于，所述病毒样颗粒具有SEQID NO.3所示的氨基酸序列的P1蛋白和SEQ ID NO.4所示的氨基酸序列的3CD蛋白；
所述柯萨奇病毒A7的病毒样颗粒通过如下步骤制备：
(1)构建包含SEQ ID NO.1所示的核苷酸序列和SEQ ID NO.2所示的核苷酸序列的表达载体；
(2)利用所述表达载体转化具有杆状病毒骨架的大肠杆菌，获得杆粒；
(3)用步骤(2)获得的杆粒感染昆虫细胞进行表达，得到所述柯萨奇病毒A7的病毒样颗粒。
6.制备权利要求5所述的柯萨奇病毒A7的病毒样颗粒的方法，其特征在于，所述方法包括如下步骤：
(1)构建包含SEQ ID NO.1所示的核苷酸序列和SEQ ID NO.2所示的核苷酸序列的表达载体；
(2)利用所述表达载体转化具有杆状病毒骨架的大肠杆菌，获得杆粒；
(3)用步骤(2)获得的杆粒感染昆虫细胞进行表达，得到所述柯萨奇病毒A7的病毒样颗粒。
7.根据权利要求6所述的方法，其特征在于，所述表达载体进一步包含AcMNPV p10启动子和/或polyhedrin启动子。
8.权利要求5所述的病毒样颗粒或权利要求6或7所述的制备方法制备的病毒样颗粒在制备预防手足口病的病毒感染的疫苗中的应用。
9.权利要求5所述的病毒样颗粒或权利要求6或7所述的制备方法制备的病毒样颗粒作为抗原在制备手足口病的病毒感染免疫检测试剂盒中的应用。
10.预防手足口病的病毒感染的疫苗，其特征在于，所述疫苗通过权利要求5所述的病毒样颗粒或权利要求6或7所述的制备方法制备的病毒样颗粒制备。
11.手足口病的病毒感染免疫检测试剂盒，其特征在于，所述试剂盒通过权利要求5所述的病毒样颗粒或权利要求6或7所述的制备方法制备的病毒样颗粒作为抗原制备。
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