CN113603754A - 一种水禽h5n8亚型流感病毒ha重组蛋白及其制备方法与应用 - Google Patents
一种水禽h5n8亚型流感病毒ha重组蛋白及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种水禽H5N8亚型流感病毒HA重组蛋白及其制备方法与应用，所述HA重组蛋白的氨基酸序列如SEQ ID NO:2所示，所述HA重组蛋白的编码基因，它的序列如SEQ ID NO:1所示；本发明的HA重组蛋白与通过计算优化得到的广谱反应HA蛋白(PDB ID 6NTF)具有相同或相似的抗原表位，且共有氨基酸序列之间不包含重大结构变化，这为基因工程亚单位疫苗研发提供了可能，经血凝活性试验证实本发明表达的HA重组蛋白生物活性好；可用于制备水禽H5N8亚型流感病毒亚单位疫苗中。
Description
技术领域
本发明属于人用疫苗和生物制品领域，涉及一种基因工程疫苗，具体涉及一种水禽H5N8亚型流感病毒HA重组蛋白、编码基因、重组载体、免疫组合物、 HA重组蛋白的制备方法以及HA重组蛋白的应用。
背景技术
高致病性禽流感(highly pathogenic avian influenza，HPAI)是由H5亚型或其重组毒株(包括H5N2、H5N5、H5N6和H5N8)流感病毒引起的一种禽类的烈性传染病，引起家禽和野生鸟类数千次暴发并造成大量死亡，对世界家禽业造成了巨大经济损失。近年来，高致病性H5N8病毒在全球迅速蔓延，对家禽业和人类健康构成巨大的威胁。高致病性H5N8病毒的血凝素(hemagglutinin， HA)蛋白编码片段是高致病性H5N1病毒(A/Goose/Guangdong/1/1996)的衍生，导致许多家禽和野生鸟类死亡，对我国养禽业存在危害，因此，加大对流感疫苗研究力度对养殖业的可持续发展具有重要的意义。HA蛋白是A型流感病毒主要表面抗原和保护性抗原，是中和性抗体的重要靶点，是国内外禽流感研究的重点和热点。
目前，在我国广泛用于防控禽流感疫苗为H5亚型基因重组灭活疫苗，但在实际应用中鸡的免疫效果优于水禽，且以鸡胚为基础生产的流感疫苗在扩大生产规模和通过鸡胚传递可能对疫苗病毒株的抗原性生产有相当大的局限性，需要大量工作、存在生产工艺繁杂、成本高、免疫应答较迟、免疫次数较多等缺点。因此，高效、安全、低成本的禽流感新型疫苗的研制一直是科研人员关注的焦点。禽流感亚单位疫苗，主要是基于HA保护性抗原制成的亚单位疫苗，亚单位疫苗不存在毒力返强、散毒和环境污染的问题，相对稳定和生物安全性好。嗜热四膜虫表达系统在流感大流行时期，如果流行病学监测过程中发现流感毒株发生明显的抗原变异，而四膜虫工程株可快速制备HA抗原，从而为调整研制新的亚单位疫苗奠定基础，是制备禽流感病毒疫苗的潜在候选者。
发明内容
本发明的目的在于提供一种水禽H5N8亚型流感病毒HA重组蛋白、编码基因、重组载体、免疫组合物、HA重组蛋白的制备方法以及HA重组蛋白的应用。
本发明的目的通过如下技术方案实现：
本发明利用从自然环境中分离得到的H5N8亚型禽流感毒株(FJ20048病毒)，通过HA氨基酸序列同源进化分析，发现从自然环境中分离得到H5N8亚型禽流感毒株HA多肽能引起动物机体广泛免疫应答。然后将密码子优化后HA 基因序列构建到pD5H8表达载体当中，以嗜热四膜虫为表达系统，通过基因枪将阳性重组质粒转入四膜虫工程株并进行同源重组，诱导表达H5亚型禽流感病毒HA蛋白，免疫小鼠对同源流感病毒的攻击具有良好的免疫保护作用。
本发明提供了一种水禽H5N8亚型流感病毒HA重组蛋白，所述HA重组蛋白的氨基酸序列如SEQ ID NO:2所示。
本发明提供了所述的水禽H5N8亚型流感病毒HA重组蛋白的编码基因，它的序列如SEQ ID NO:1所示。
本发明还提供了一种包含所述编码基因的重组载体。
所述重组载体包括pMD18-T、pEASY Blunt Zero Vector、pTIEV4或pD5H8 等。
本发明还提供了一种包含所述编码基因的宿主细胞。
所述宿主细胞包括嗜热四膜虫株、293T细胞、sf9昆虫细胞、DG44、DXB11、 CHO-K1或CHO-S细胞株等。
本发明还提供了一种免疫组合物，它包括所述的HA重组蛋白以及医药学上可接受的佐剂。
所述医药学上可接受的佐剂包括弗氏佐剂、氢氧化铝佐剂、脂类佐剂等任意一种或者两种以上的组合。
本发明还提供了一种所述的水禽H5N8亚型流感病毒HA重组蛋白的制备方法，它包括以下步骤：
将所述的HA重组蛋白的编码基因构建到表达载体上，获得重组表达载体；以嗜热四膜虫为表达系统，通过基因枪将重组表达载体转入四膜虫工程株并进行同源重组，诱导表达水禽H5N8亚型流感病毒HA蛋白，之后回收并纯化所表达的HA重组蛋白。
所述表达载体为pD5H8；
本发明所选的宿主细胞为嗜热四膜虫，当然还可以为其他宿主细胞如293T 细胞、sf9昆虫细胞、DG44、DXB11、CHO-K1或CHO-S细胞株等。本发明以嗜热四膜虫为表达系统，具有生长周期短(2-2.5h传1代)、培养成本低、操作简便等优点，且表达的蛋白由于含大量甘露糖残基因此具有较高的免疫原性。
本发明还提供了所述的HA重组蛋白或所述的编码基因或所述的免疫组合物在制备水禽H5N8亚型流感病毒检测试剂或检测试剂盒中的应用。
本发明还提供了所述的HA重组蛋白或所述的免疫组合物在制备水禽H5N8 亚型流感病毒亚单位疫苗中的应用。
较之现有技术而言，本发明的优点在于：
1.通过同源序列比对发现，本发明从自然界分离得到的毒株FJ20048的 Tre-HA蛋白(HA重组蛋白)与通过计算优化得到的广谱反应HA蛋白(PDB ID 6NTF)具有相同或相似的抗原表位，且共有氨基酸序列之间不包含重大结构变化，这为基因工程亚单位疫苗研发提供了可能。经血凝活性试验证实本发明表达的Tre-HA重组蛋白生物活性好；经攻毒保护试验可知，嗜热四膜虫表达的 Tre-HA蛋白可作为防控禽流感亚单位疫苗的候选表达载体，可用于制备水禽 H5N8亚型流感病毒亚单位疫苗中。
2.本发明通过基因枪轰击技术将包埋的重组质粒(重组表达载体)转入接合生殖期四膜虫体内，通过同源重组方式整合靶基因序列并在巴龙霉素筛选下产生基因稳定的微核转化子，最终得到可高效表达HA蛋白的重组四膜虫株，诱导表达H5N8亚型禽流感病毒HA蛋白，并对蛋白进行纯化及免疫原性测定与分析，为今后研发禽流感多价基因工程疫苗奠定基础。
3.本发明证实嗜热四膜虫工程株可表达流感HA蛋白，并能够保护免疫后小鼠不受流感病毒的感染。流感亚单位疫苗的一个优点是避免感染性病毒增殖，从而允许使用低级别的生物安全实验室进行培养。在该系统中表达的蛋白在生物学上是安全可靠的，尚未见嗜热四膜虫携带任何对人类致病的病毒的报道。此外，嗜热四膜虫工程株可以在发酵罐中进行大规模培养，这为流感大流行时期快速研制亚单位疫苗奠定了基础。
附图说明
图1是本发明HA基因PCR扩增产物的凝胶电泳图；其中，M为DL5000 marker，1为HA全长基因，2为构建表达的HA基因截短。
图2是接合生殖期四膜虫大核和小核的发育图；其中，a为接合生殖的四膜虫株；b为四膜虫单体。
图3是SDS-PAGE鉴定重组表达HA蛋白凝胶电泳图。其中，1为HA重组蛋白；M为蛋白Marker；
图4是攻毒保护试验结果图。
具体实施方式
下面结合说明书附图和实施例对本发明内容进行详细说明：
1.实验方法
1.1病毒HA基因扩增
将鸭源FJ20048病毒悬液(其中，FJ20048病毒由福建省农业科学院畜牧兽医研究所动物生物安全三级实验室鉴定及保存)，8000rpm离心5min，取250μl 上清与750μlTrizol混合，再加入200μl预冷的氯仿，充分混匀后冰浴5min，随后12000rpm离心15min，取上清加入等体积的异丙醇，混匀，-20℃沉淀1h。 12000rpm离心15min，弃上清，用DEPC处理过的水重悬，-20℃保存。用流感的反转录引物Uni-12，按照AMV反转酶说明书进行反转录合成cDNA。
以cDNA为模板进行PCR扩增，PCR扩增的反应体系为：10x Buffer 5μL， dNTPmixture(2.5nnol/L)4μL，Ex-Taq DNA polymerase 0.5μL，上/下游引物 (20μM)各1μL，模板cDNA 1μL，ddH2O 37.5μL。反应条件如下：94℃预变性5min，按94℃30S，55℃35S，72℃2min，进行35个循环，最后72℃延伸10min。
其中，所述上/下游引物的序列为：
上游引物：5’-CGCGGATCCGATCAGATTTGCATTGGTTACCA-3’(SEQ ID NO:3)；
下游引物：5’-CCGCTCGAGTCCTATTGATTCTAATTTCACTCCG-3’(SEQ ID NO:4)。
之后，将HA基因PCR产物经切胶回收试剂盒纯化后与pMD18-T载体连接获得pMD-18T-HA(其中，连接反应体系如表1所示)，接着，转化DH5α感受态细胞，铺板37℃过夜培养，挑取单克隆提取质粒，PCR法鉴定重组质粒，阳性质粒送大连宝生物公司测序，选择测序正确的菌液进行保存，得到克隆载体 pMD-18T-HA。
表1构建到pMD18-T载体
结果与分析：
从图1所示的HA基因PCR扩增产物的凝胶电泳图可知，HA基因全长为 1704bp；构建表达的HA基因的长度为1533bp，其编码511个氨基酸。
其中，构建表达的HA基因序列如下：
GATCAGATTTGCATTGGTTACCATGCAAACAACTCGACAGAGCAGGTTGAC ACGATAATGGAAAAGAACGTCACTGTTACACATGCCCAAGACATACTGGA AAAGACACACAACGGGAAGCTCTGCGATCTAAATGGGGTGAAGCCTCTGG TTTTAAAGGATTGTAGTGTAGCTGGATGGCTCCTCGGAAACCCAATGTGCG ACGAATTCATCAGGGTGCCGGAATGGTCTTACATAGTGGAGAGGGCTAACC CAGCCAATGACCTCTGTTACCCAGGGAGCCTCAATGACTATGAAGAACTG AAACACCTATTGAGCAGAATAAATCATTTTGAGAAGATTCTGATCATCCCC AAGAGTTCTTGGCTCGATCATGAAACATCATTAGGGGTGAGCGCAGCATGT CCATACCAGGGAACGCCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAA AAGAACGATGCATACCCAACAATAAAGATAAGCTACAATAATACCAATCAG GAAGATCTTTTGATACTGTGGGGGATTCATCATTCCAACAATGCAGCAGAG CAGACAAATCTCTATAAAAACCCAACCACCTATGTTTCCGTTGGGACATCA ACATTAAACCAGAGATTGGTACCAAAAATAGCTACTAGATCCCAAGTAAAC GGGCAACGTGGAAGAATGGATTTCTTCTGGACAATTTTAAAACCGAATGAT GCAATCCACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACA AAATTGTCAAGAAAGGGGACTCAACAATTATGAAAAGTGAAGTGGAATATGGCCACTGCAACACCAAATGTCAAACTCCAGTAGGGGCGATAAACTCTAG TATGCCATTCCACAATATACATCCTCTCACCATCGGGGAATGCCCCAAATAC GTGAAGTCAAACAAATTGGTCCTTGCGACTGGGCTCAGAAATAGTCCTCTA AGAGAAAAGAGAAGAAAAAGAGGGCTGTTTGGGGCTATAGCAGGTTTTAT AGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATA GCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACCCAAAA GGCAATAGATGGAGTTACCAATAAGGTCAACTCGATCATTGACAAAATGAA CACTCAATTTGAGGCCGTTGGAAGGGAATTTAATAACTTAGAAAGGAGGAT AGAGAATTTAAACAAGAAAATGGAAGACGGATTCCTAGATGTCTGGACTTA TAATGCTGAACTTCTAGTTCTCATGGAAAATGAGAGGACTCTAGATTTCCAT GACTCAAATGTCAAGAACCTTTACGACAAAGTCCGACTACAGCTTAGGGAT AATGCAAAGGAGCTGGGTAATGGTTGTTTCGAGTTCTATCACAAATGTGAT AATGAATGTATGGAAAGTGTAAAAAATGGGACGTATGACTACCCTCAGTAT TCAGAAGAAGCAAGATTAAAAAGAGAAGAAATAAGCGGAGTGAAATTAG AATCAATAGGA
构建表达的HA基因编码的三字母氨基酸序列如下：
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val AspThr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys ThrHis Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys Pro Leu Val Leu Lys Asp CysSer Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Arg Val ProGlu Trp Ser Tyr Ile Val Glu Arg Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro GlySer Leu Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe GluLys Ile Leu Ile Ile Pro Lys Ser Ser Trp Leu Asp His Glu Thr Ser Leu Gly ValSer Ala Ala Cys Pro Tyr Gln Gly Thr Pro Ser Phe Phe Arg Asn Val Val Trp LeuIle Lys Lys Asn Asp Ala Tyr Pro Thr Ile Lys Ile Ser Tyr Asn Asn Thr Asn GlnGlu Asp Leu Leu Ile Leu Trp Gly Ile His His Ser Asn Asn Ala Ala Glu Gln ThrAsn Leu Tyr Lys Asn Pro Thr Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn GlnArg Leu Val Pro Lys Ile Ala Thr Arg Ser Gln Val Asn Gly Gln Arg Gly Arg MetAsp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile His Phe Glu Ser Asn GlyAsn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr IleMet Lys Ser Glu Val Glu Tyr Gly His Cys Asn Thr Lys Cys Gln Thr Pro Val GlyAla Ile Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu CysPro Lys Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser ProLeu Arg Glu Lys Arg Arg Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile GluGly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu GlnGly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val ThrAsn Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly ArgGlu Phe Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp GlyPhe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu ArgThr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu GlnLeu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys CysAsp Asn Glu Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr SerGlu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
1.2重组四膜虫株构建及筛选
将克隆载体pMD-18T-HA和pTIEV4载体分别用BamHI和XhoI酶切后16℃连接过夜(连接体系见表2)，获得pTIEV4-HA克隆，提取质粒、双酶切验证并进一步测序验证得到阳性重组质粒pTIEV4-HA。
表2构建到pTIEV4载体
NotI酶切pTIEV4-HA阳性质粒和pD5H8表达载体，并将pD5H8载体去磷酸化，反应体系如下：10×CIAP Buffer 5μL，pD5H8载体片段20μL，CIAP 1μL，补ddH2O到50μL，37℃反应1h。16℃连接过夜。
通过电击法(Bio-Rad，2mm电击杯，300V，25μF，50Ω)将连接产物转到大肠杆菌HST08感受态细胞。电转后涂板过夜培养，将单克隆扩大培养，提取质粒NotI酶切鉴定重组质粒pD5H8-HA，测序验证插入的HA基因，最终获得阳性重组质粒pD5H8-HA(重组表达载体pD5H8-HA)用于基因枪转化。
将用于接合生殖的嗜热四膜虫株CU427和CU428复苏在Neff培养基中培养，培养温度为30℃、转数为80rpm，培养增殖到5×105个细胞/ml，收集虫液计数然后按照1：1比例混匀离心后重悬在pH7.4 Tris-HCl(终浓度为10mM) 缓冲液中。30℃、80rpm饥饿培养24h后，静置10-11h，体视显微镜下观察接合生殖期CU427和CU428虫株的配对率，当配对率大于等于80％时，800g离心2min收集沉淀，重悬于10mM、pH7.4 Tris-HCl中，Biolistic PDS-1000/He 基因枪进行轰击，将包埋阳性重组质粒pD5H8-HA的子弹金颗粒转到接合生殖期的四膜虫体内。巴龙霉素浓度梯度抗性选择条件下进行筛选，巴龙霉素初始浓度为200μg/ml，然后每天浓度增加一倍，直到1000μg/ml。阳性虫株单克隆后培养至5×105个细胞/ml，液氮冻存备用。
结果与分析：
接合生殖期四膜虫观察：
将饥饿后四膜虫混合后，显微镜下可观察到CU427和CU428虫株会在四膜虫的头端配对进行有性生殖(如图2所示)。当虫株配对率达到80％以上且大核原基形成，供体株系和宿主株系正发生同源重组，这是基因枪转化的最佳时期，可将重组质粒转入接合生殖期的四膜虫体内。最后经巴龙霉素梯度筛选和单克隆后得到阳性的四膜虫株Tre-HA，重组四膜虫株传代稳定。
1.3 HA蛋白诱导表达及纯化
将冻存在液氮中的重组四膜虫工程株Tre-HA复苏，30℃、80rpm避光培养， 100mlNeff培养基培养，并加入100μL 100mg/ml巴龙霉素，混匀)，至5×105个细胞/ml。加入终浓度15μg/ml的金属离子诱导剂氯化镉到重组四膜虫株培养液中诱导培养16h，诱导重组蛋白HA表达。10000g离心5min收集虫株，超声破碎虫体，超声功率为300w，超声2s，间隔6s，超声共100次，18000g 离心20min，取上清，Ni-NTA进行亲和纯化，SDS-PAGE验证和质谱鉴定纯化的蛋白。
结果与分析：
SDS-PAGE分析，阳性重组子虫株Tre-HA诱导表达，Ni-NTA纯化后在62 kDa左右有一条丰度很高的条带(图3)。
1.4血凝活性测定
通过微量法进行红细胞凝集试验，测定重组Tre-HA蛋白(HA重组蛋白) 血凝活性，即96孔微量反应孔内加入25μl PBS，第一孔加入表达的Tre-HA抗原(即HA重组蛋白)25μl，然后进行倍比稀释，每孔加入25μl PBS，每孔加入1％鸡红细胞悬液，充分混匀并在20℃条件下孵预育40min观察结果。
结果与分析：
血凝活性试验证实，得到的HA重组蛋白血凝价为27，而对照组灭活H5N8 病毒为28，说明表达的HA蛋白(即HA重组蛋白)生物活性好。
1.5攻毒保护试验
将纯化得到的重组蛋白Tre-HA用PBS稀释，与弗氏佐剂等比例混匀乳化，免疫6周龄的Balb/C小鼠，随机分为3组，每组两个重复小组，每组12只，每组间隔3周分别免疫3次，免疫方式为小鼠后腿肌肉注射。第1组为Tre-HA疫苗组，第2组为空白对照组PBS，免疫剂量为100μl/只。一免佐剂为弗氏完全佐剂，二免为弗氏不完全佐剂，二免21天后纯蛋白进行三免，三免后3周，进行动物攻毒保护试验。用亲本H5N8毒株50MLD50经鼻腔感染小鼠。攻毒后连续观察14d，观察小鼠的体重、发病、死亡情况，并记录小鼠的体重变化，小鼠体重损失超过20％可认定为死亡。攻毒前Balb/C小鼠的体重设定为100％，以天数为横坐标，相对体重Weight(％)为纵坐标，通过GraphPad Prism 8软件作图。
结果与分析：
Tre-HA蛋白三免后用50MLD50亲本菌株攻毒后，免疫组存活率为100％，攻毒组小鼠在攻毒后第4天出现消瘦、精神萎靡等症状，攻毒后第9-12天左右体重均下降了20％(判定为对照组死亡)(图4)。通过上述攻毒保护试验，表明嗜热四膜虫表达的Tre-HA蛋白可作为防控禽流感亚单位疫苗的候选表达载体，下一阶段拟对该疫苗的效果和安全性进行评价，以期为流感候选疫苗的研究开拓新的途径。
2结论
通过CASTp软件进行同源序列比对，发现本实验室从自然界分离得到的毒株FJ20048的Tre-HA蛋白(HA重组蛋白)与通过计算优化得到的广谱反应HA 蛋白(PDB ID6NTF)具有相似的抗原表位，且共有氨基酸序列之间不包含重大结构变化，这为基因工程亚单位疫苗研发提供了可能。本发明通过基因枪轰击技术将包埋的重组质粒(重组表达载体)转入接合生殖期四膜虫体内，通过同源重组方式整合靶基因序列并在巴龙霉素筛选下产生基因稳定的微核转化子，最终得到可高效表达HA蛋白的重组四膜虫株，诱导表达H5N8亚型禽流感病毒 HA蛋白，并对蛋白进行纯化及免疫原性测定与分析，为今后研发禽流感多价基因工程疫苗奠定基础。总之，本发明证实嗜热四膜虫工程株可表达流感HA蛋白，并能够保护免疫后小鼠不受流感病毒的感染。流感亚单位疫苗的一个优点是避免感染性病毒增殖，从而允许使用低级别的生物安全实验室进行培养。在该系统中表达的蛋白在生物学上是安全可靠的，尚未见嗜热四膜虫携带任何对人类致病的病毒的报道。此外，嗜热四膜虫工程株可以在发酵罐中进行大规模培养，这为流感大流行时期快速研制亚单位疫苗奠定了基础。
需要说明的是：以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
序列表
<110> 福建省农业科学院畜牧兽医研究所
<120> 一种水禽H5N8亚型流感病毒HA重组蛋白及其制备方法与应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1533
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gatcagattt gcattggtta ccatgcaaac aactcgacag agcaggttga cacgataatg 60
gaaaagaacg tcactgttac acatgcccaa gacatactgg aaaagacaca caacgggaag 120
ctctgcgatc taaatggggt gaagcctctg gttttaaagg attgtagtgt agctggatgg 180
ctcctcggaa acccaatgtg cgacgaattc atcagggtgc cggaatggtc ttacatagtg 240
gagagggcta acccagccaa tgacctctgt tacccaggga gcctcaatga ctatgaagaa 300
ctgaaacacc tattgagcag aataaatcat tttgagaaga ttctgatcat ccccaagagt 360
tcttggctcg atcatgaaac atcattaggg gtgagcgcag catgtccata ccagggaacg 420
ccctcctttt tcagaaatgt ggtatggctt atcaaaaaga acgatgcata cccaacaata 480
aagataagct acaataatac caatcaggaa gatcttttga tactgtgggg gattcatcat 540
tccaacaatg cagcagagca gacaaatctc tataaaaacc caaccaccta tgtttccgtt 600
gggacatcaa cattaaacca gagattggta ccaaaaatag ctactagatc ccaagtaaac 660
gggcaacgtg gaagaatgga tttcttctgg acaattttaa aaccgaatga tgcaatccac 720
ttcgagagta atggaaattt cattgctcca gaatatgcat acaaaattgt caagaaaggg 780
gactcaacaa ttatgaaaag tgaagtggaa tatggccact gcaacaccaa atgtcaaact 840
ccagtagggg cgataaactc tagtatgcca ttccacaata tacatcctct caccatcggg 900
gaatgcccca aatacgtgaa gtcaaacaaa ttggtccttg cgactgggct cagaaatagt 960
cctctaagag aaaagagaag aaaaagaggg ctgtttgggg ctatagcagg ttttatagag 1020
ggaggatggc agggaatggt agatggttgg tatgggtacc accatagcaa tgagcagggg 1080
agtgggtacg ctgcagacaa agaatccacc caaaaggcaa tagatggagt taccaataag 1140
gtcaactcga tcattgacaa aatgaacact caatttgagg ccgttggaag ggaatttaat 1200
aacttagaaa ggaggataga gaatttaaac aagaaaatgg aagacggatt cctagatgtc 1260
tggacttata atgctgaact tctagttctc atggaaaatg agaggactct agatttccat 1320
gactcaaatg tcaagaacct ttacgacaaa gtccgactac agcttaggga taatgcaaag 1380
gagctgggta atggttgttt cgagttctat cacaaatgtg ataatgaatg tatggaaagt 1440
gtaaaaaatg ggacgtatga ctaccctcag tattcagaag aagcaagatt aaaaagagaa 1500
gaaataagcg gagtgaaatt agaatcaata gga 1533
<210> 2
<211> 511
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
1 5 10 15
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
20 25 30
Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys
35 40 45
Pro Leu Val Leu Lys Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
50 55 60
Pro Met Cys Asp Glu Phe Ile Arg Val Pro Glu Trp Ser Tyr Ile Val
65 70 75 80
Glu Arg Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Ser Leu Asn
85 90 95
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu
100 105 110
Lys Ile Leu Ile Ile Pro Lys Ser Ser Trp Leu Asp His Glu Thr Ser
115 120 125
Leu Gly Val Ser Ala Ala Cys Pro Tyr Gln Gly Thr Pro Ser Phe Phe
130 135 140
Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asp Ala Tyr Pro Thr Ile
145 150 155 160
Lys Ile Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Ile Leu Trp
165 170 175
Gly Ile His His Ser Asn Asn Ala Ala Glu Gln Thr Asn Leu Tyr Lys
180 185 190
Asn Pro Thr Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Gln Arg
195 200 205
Leu Val Pro Lys Ile Ala Thr Arg Ser Gln Val Asn Gly Gln Arg Gly
210 215 220
Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile His
225 230 235 240
Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile
245 250 255
Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Val Glu Tyr Gly
260 265 270
His Cys Asn Thr Lys Cys Gln Thr Pro Val Gly Ala Ile Asn Ser Ser
275 280 285
Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
290 295 300
Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
305 310 315 320
Pro Leu Arg Glu Lys Arg Arg Lys Arg Gly Leu Phe Gly Ala Ile Ala
325 330 335
Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly
340 345 350
Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu
355 360 365
Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile
370 375 380
Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn
385 390 395 400
Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly
405 410 415
Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu
420 425 430
Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr
435 440 445
Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn
450 455 460
Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser
465 470 475 480
Val Lys Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg
485 490 495
Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
500 505 510
<210> 3
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cgcggatccg atcagatttg cattggttac ca 32
<210> 4
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccgctcgagt cctattgatt ctaatttcac tccg 34
Claims (10)
1.一种水禽H5N8亚型流感病毒HA重组蛋白，其特征在于：所述HA重组蛋白的氨基酸序列如SEQ ID NO:2所示。
2.如权利要求1所述的水禽H5N8亚型流感病毒HA重组蛋白的编码基因，其特征在于：所述编码基因的序列如SEQ ID NO:1所示。
3.一种包含权利要求2所述编码基因的重组载体。
4.根据权利要求3所述的重组载体，其特征在于：所述重组载体包括pMD18-T、pEASYBlunt Zero Vector、pTIEV4或pD5H8。
5.一种包含权利要求2所述编码基因的宿主细胞。
6.根据权利要求5所述的宿主细胞，其特征在于：所述宿主细胞包括嗜热四膜虫细胞株、293T细胞、sf9昆虫细胞、DG44、DXB11、CHO-K1或CHO-S细胞株。
7.一种免疫组合物，其特征在于：它包括权利要求1所述的HA重组蛋白以及医药学上可接受的佐剂。
8.一种如权利要求1所述的水禽H5N8亚型流感病毒HA重组蛋白的制备方法，其特征在于：它包括以下步骤：
将权利要求1所述的HA重组蛋白的编码基因构建到表达载体上，获得重组表达载体；以嗜热四膜虫为表达系统，通过基因枪将重组表达载体转入四膜虫工程株并进行同源重组，诱导表达水禽H5N8亚型流感病毒HA蛋白，之后回收并纯化所表达的HA重组蛋白。
9.如权利要求1所述的HA重组蛋白或权利要求2所述的编码基因或权利要求7所述的免疫组合物在制备水禽H5N8亚型流感病毒检测试剂或检测试剂盒中的应用。
10.如权利要求1所述的HA重组蛋白或权利要求7所述的免疫组合物在制备水禽H5N8亚型流感病毒亚单位疫苗中的应用。
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| WO2023236822A1 (zh) * | 2022-06-08 | 2023-12-14 | 中科南京生命健康高等研究院 | H5n6禽流感广谱性疫苗的开发及其应用 |
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