CN114149492A - 一种用于预防甲型流感病毒感染的HA-mRNA疫苗 - Google Patents
一种用于预防甲型流感病毒感染的HA-mRNA疫苗 Download PDFInfo
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Abstract
本发明公开了一种用于预防甲型流感病毒感染的HA‑mRNA疫苗。本发明提供了HA‑mRNA,其核苷酸序列为序列表中序列1编码的RNA。本发明提供了一种用于预防甲型流感病毒感染的HA‑mRNA疫苗,并采用ELISA法检测了接种有HA‑mRNA疫苗的小鼠体内血清抗体的表达水平,并在最后一次免疫后,用致死剂量的病毒攻击小鼠,观察记录小鼠形态、体重,存活率情况。结果体现出HA‑mRNA疫苗能够在小鼠体内引起良好的体液免疫,并且有很好的保护作用。本发明对于甲型流感病毒感染疫苗的研究有重要意义。
Description
技术领域
本发明涉及药物领域,具体涉及一种用于预防甲型流感病毒感染的HA-mRNA疫苗。
背景技术
mRNA疫苗是一种新型亚型疫苗,发展速度很快。目前应用的领域主要有:①感染性疾病的防治研究,如Pollard等使用阳离子脂质DOTAP/DOPE包裹Ⅰ型人类免疫缺陷病毒抗原Gag的mRNA,皮下免疫小鼠,诱导出了Ⅰ型干扰素的分泌,及抗原特异性的、功能性的T细胞反应感染性疾病的防治研究。②肿瘤的防治研究,如Zhou等使用人工合成的人类黑色素瘤相关抗原gp100的mRNA与日本仙台病毒(Hemagglutinating virus of Japan,HVJ)脂质体包裹,然后直接注射至小鼠脾脏,诱导出了靶向gp100的抗体及细胞毒T淋巴细胞的活性。③其它疾病的防治研究,如Weiss等使用mRNA疫苗在小鼠模型上针对Ⅰ型过敏反应的预防,能够有效阻止IgE引起的过敏性表型诱导,如过敏反应相关细胞因子的产生、嗜酸性粒细胞肺浸润、呼吸道高反应性等。
常用于预防甲流的疫苗主要有全病毒灭活疫苗、裂解疫苗和减毒活疫苗。全病毒灭活疫苗是最早应用于预防流感的一类疫苗,制备简单,易于储存,但可能因灭活不完全产生严重毒副作用且不能诱导粘膜免疫和细胞免疫。裂解疫苗的制备基于全病毒活疫苗,仅保留病毒表面HA、NA等抗原蛋白,制备复杂,生产时间长且诱导细胞免疫效果较差。减毒活疫苗免疫原性强,可以诱导细胞免疫和体液免疫且作用时间长,但存在潜在致病危险。
相比现有常用的甲流疫苗,mRNA甲流疫苗具备独特优势:1)研发制备速度快,有利于突发性甲流的应急防控;2)mRNA自身无感染性,相对安全,且不存在基因整合的风险;3)免疫原性强,可以诱导体液免疫和细胞免疫。
现有针对FM1病毒疫苗多为传统灭活疫苗,其安全性、有效性优异;但开发时间相对漫长,疫苗生产需要使用活毒操作且要求相对严苛,不适于流感突发应急的使用。
发明内容
本发明一个目的是提供一种HA-mRNA。
本发明提供的HA-mRNA,其核苷酸序列为序列表中序列1编码的RNA。
编码上述mRNA的核酸分子也是本发明保护的范围。
上述核酸分子的核苷酸序列为序列表中序列1。
上述的HA-mRNA在制备预防或治疗流感病毒产品中的应用也是本发明保护的范围。
上述应用中,所述流感病毒为甲型流感病毒;在本发明的实施例中以FM1病毒为例。
本发明另一个目的是提供一种预防或治疗流感病毒产品。
本发明提供的产品,其包括上述HA-mRNA。
上述还包括佐剂,在本发明的实施例中,所述佐剂为鱼精蛋白。
上述产品为疫苗,包括HA-mRNA和鱼精蛋白,上述mRNA和所述鱼精蛋白的质量比为2:1。
上述产品也可以为试剂盒或其他可以预防或治疗流感病毒的产品形式。
上述HA-mRNA和鱼精蛋白在预防或治疗流感病毒产品中的应用。
上述中,所述产品为疫苗。
本发明提供了一种用于预防甲型流感病毒感染的HA-mRNA疫苗,并采用ELISA法检测了接种有HA-mRNA疫苗的小鼠体内血清抗体的表达水平,并在最后一次免疫后,用致死剂量的病毒攻击小鼠,观察记录小鼠形态、体重,存活率情况。结果体现出HA-mRNA疫苗能够在小鼠体内引起良好的体液免疫,并且有很好的保护作用。本发明对于甲型流感病毒感染疫苗的研究有重要意义。
附图说明
图1为人工合成HA-mRNA的模板示意图。
图2为HA-mRNA质量控制结果图。
图3为HA-mRNA细胞内表达检测结果。
图4为ELISA方法检测第二次免疫后抗HA抗体产生结果。
图5为ELISA方法检测第三次免疫后抗HA抗体产生结果。
图6为攻毒后小鼠体重情况检测结果。
图7为小鼠经三次免疫后用病毒攻击后存活情况。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
实施例1、HA-mRNA的设计、制备和体外表达检测
一、HA-mRNA的设计和制备
1、人工合成HA-mRNA的模板序列,如序列表的序列1所示。
序列表的序列1中,自5’端第1至72为5’-UTR,第73至1773为CDS,第1774至1990为3’-UTR。
HA-mRNA的的模板示意图如图1所示。
2、将步骤1人工合成的模板序列插入pcDNA3.1(+)载体的NheI和XhoI位点之间,得到重组质粒(已经测序验证)。
3、对步骤2得到的重组质粒进行质粒大提。
4、采用XbaI和PmeI双酶切步骤3大提的重组质粒,回收并纯化大小约7319bp的含有HA的线性化载体片段。
5、配置表1所示的反应体系,37℃孵育30min进行目的片段的体外转录。
表1为反应体系
试剂 | 体积 |
含有HA的线性化载体片段 | 1μg |
10×mScript T7 Transcription Buffer | 2μL |
NTP Solution | 7.2μL |
100mM DTT | 2μL |
ScriptGuard RNase Inhibitor | 0.5μL |
mScript T7 Enzyme Solution | 2μL |
RNase-Free Water | 补足20uL |
上述表1中的试剂均来自T7-mScriptTM Standard mRNA Production System试剂盒(购买于CELLSCRIPT公司)。
6、完成步骤5后,向反应体系中加入1μL RNase-Free DNase I,37℃孵育15min,以去除体外转录产物体系中模板,得到转录产物。
7、对步骤6得到的转录产物进行纯化,纯化方法如下:
(1)向转录产物中加入179μL RNase-Free Water,使管中溶液体积为200uL;
(2)加入200μL水饱和酚氯仿,涡旋10s,4℃、10000×g离心5min,然后将管中上层水相移至新的管中;
(3)向新管中加入200μL 5M ammonium acetate,混匀后冰上放置15min;
(4)4℃、12000×g离心15min;
(5)弃上清,加入70%乙醇轻轻混匀清洗mRNA,弃去70%乙醇;
(6)室温放置数分钟充分弃去残留乙醇,加适量RNase-Free水重悬。
上述试剂RNase-Free Water(购买于Solarbio公司)、5M ammonium acetate(购买于Sigma公司)、水饱和酚(购买于Solarbio公司)
8、完成步骤7后,取纯化产物,进行mRNA加帽处理,具体步骤如下:
(1)取60μg转录纯化产物,65℃孵育10min进行变性,然后移至冰上。
(2)按照表2所示的配置得到混合物。
表2为混合物组分
(3)取步骤(2)的混合物和步骤(1)的变性产物,按照表3配置反应体系,37℃孵育半小时,得到加帽产物。
表3为反应体系
试剂 | 体积 |
步骤(2)的混合物 | 24μL |
ScriptCap Capping Enzyme | 4μL |
步骤(1)的变性RNA | 72μL |
总计 | 100μL |
上述表2和表3的试剂均来自T7-mScriptTM Standard mRNA Production System试剂盒(购买于CELLSCRIPT公司)。
9、取步骤8得到的加帽产物,按照表4配置反应体系,37℃孵育半小时,得到加尾产物。
表4为反应体系
试剂 | 体积 |
加帽产物 | 100μL |
ScriptGuard RNase Inhibitor | 0.5μL |
10×A-Plus Tailing Buffer | 12μL |
20mM ATP | 6μL |
A-Plus Poly(A)Polymerase(4U/μL) | 5μL |
总计 | 123.5μL |
上述表4的试剂均来自T7-mScriptTM Standard mRNA Production System试剂盒(购买于CELLSCRIPT公司)。
10、取步骤9的加尾产物,按照如下步骤进行纯化,得到纯化产物(即HA-mRNA)。
(1)配置溶液a和溶液b;溶液a:水饱和酚:氯仿:异戊醇=125:24:1;溶液b:氯仿:异戊醇=24:1。
(2)向步骤9得到的产物中加入76.5μL RNase-Free Water,此时管中溶液体积200μL。
(3)加入200μL a液,涡旋70s,13800×g离心90s,将管中上层水相转入新的EP管中;
(4)加入相同体积b溶液,涡旋60s,13800×g离心90s;
(5)上层水相转入新管中,加入0.1倍与1倍体积3mM Sodium Acetate(pH 5.2)和异丙醇,冰上放置5min,13800×g离心10min;
(6)将管中上清液小心移至新的EP管中,加入1ml 70%乙醇混匀,用移液枪轻轻吹打几次后移除乙醇。室温干燥,加适量的RNAase-free水溶mRNA,做好标记,-70℃保存备用,得到HA-mRNA溶液。
HA-mRNA的核苷酸序列为序列1编码的RNA。
上述试剂RNase-Free Water(购买于Solarbio公司)、3mM Sodium Acetate(购买于Sigma公司)、水饱和酚(购买于Solarbio公司)。
二、HA-mRNA质量控制
采用安捷伦2100RNA Nano 6000Assay Kit对RNA进行质量控制分析,具体步骤如下:
1、使用RNA芯片之前,调节芯片制备器夹子到最上方的位置。将上述一制备的HA-mRNA溶液和实验中用到的Ladder放在金属浴中,70℃变性2min,然后立即放到冰上。用RNase Zap洗3min Agilent 2100仪器,再用RNase-free water洗3min仪器。
2、准备凝胶:按说明将RNA gel matrix加入过滤管中,1500×g室温离心10min,将过滤的凝胶以每份65μL分装到0.5mL RNase-free离心管中,4℃保存备用,最多可保存四周。
3、准备凝胶-染料混合物:RNA染料避光平衡30min,然后涡旋数秒,瞬时离心后以65:1比例配制凝胶染料混合物,将混合物涡旋混匀,13000×g室温离心10min,当天使用。
4、加载凝胶-染料混合物:将RNA芯片放入芯片槽,在注明黑色G的孔中加入9μL凝胶-染料混合物(不要有气泡);注射器活塞在1mL位置处时关闭注胶器,按压注射器,用夹子固定30s后再松开,5s后将活塞拉回1mL处的位置;打开注胶器,在其它注明白色G的孔中加入9μL凝胶-染料混合物。
5、加载Marker与Conditioning Solution:在标CS孔中加入9μL Conditioning溶液,然后在所有样品孔与ladder孔中各加入5μL RNA Marker。
6、加载Ladder与HA mRNA:在标有梯子图样的孔中加入1μL ladder,将HA mRNA加入到剩余11个孔中(没有用到的孔可用RNase-free water代替),将芯片放到芯片涡旋振荡器上,2400r/min振荡1min,然后5min内将芯片放到Agilent 2100仪中,操作检测。
结果如图2所示,结果显示获得的HA-mRNA大小正确,HA-mRNA溶液的浓度为17.65μg/μL。
三、HA-mRNA细胞内表达检测
1、将293T细胞(ATCC)接种于6孔板中,每孔5×105个细胞,37℃,5%CO2培养箱中至细胞达到80%融合度即可进行转染。
2、取步骤1的6孔板,弃掉培养液,每孔加入2mL的无血清DMEM培养基,然后加入转染复合物A或转染复合物B,混合后培养6h,6h后去除6孔板中的溶液,加入含10%(体积百分含量)血清的DMEM培养液继续培养48h。
转染复合物A:混合物A+混合物B,室温静置20min。混合物A:2μg HA-mRNA+125μLOpti-MEM,室温静置5min;混合物B:4μL Lipo2000+125μL Opti-MEM,室温静置5min。
转染复合物B:混合物C+混合物D,室温静置20min。混合物C:1μg HA-mRNA+125μLOpti-MEM,室温静置5min;混合物D:2μL Lipo2000+125μL Opti-MEM,室温静置5min。
3、完成步骤2后,提取各孔细胞总蛋白,进行western blot检测,检测步骤如下:
(1)提取总蛋白后,进行10%SDS-聚丙烯酰胺凝胶电泳(80v电泳30min,然后120v电泳大概60min)。
(2)电泳结束后,取凝胶,依据蛋白marker位置,留取所需部位的凝胶,浸泡到转膜液中备用。
(3)对比凝胶,剪裁PVDF膜,甲醇1min活化膜,接着同滤纸在转膜液中放10min。从下往上分别为滤纸、PVDF膜、凝胶和滤纸,依次放在转膜仪上,其中剪PVDF膜的一角做标记,放好后用干净的50mL离心管轻碾以防止气泡存在。15v,45min进行转膜。
(4)转移完毕后将膜放入50mL 5%脱脂奶粉中摇育,40r/min,封闭1h。
(5)封闭结束后将膜移至flu A HA一抗溶液(稀释1000倍)(Influenza A H1N1(Swine Flu 2009)Hemagglutinin Antibody,北京义翘神州,货号11055-T60)中,40r/min,室温摇育3h。
(6)用PBST分别以5min、5min和20min,80r/min洗膜3次。
(7)将洗好的膜移至稀释好的HRF标记的二抗溶液(Goat Anti-Rabbit IgGSecondary Antibody(HRP),北京义翘神州公司,货号SSA004)中,室温摇育1h。
(8)用PBST溶液分别以5min、5min和20min,80r/min洗膜3次。
(9)在暗室准备好曝光所需用品,将保鲜膜平整地贴在曝光板上,有蛋白那面的膜朝上放到曝光板上,用吸水纸吸取膜上液体后,吸取适量的ECL超敏发光混合液到膜上,1min后用滤纸吸去液体,将保鲜膜盖到膜上,没有气泡,无褶皱。
(10)关闭白灯,将胶片拿出并减去左上角做标记,小心覆盖到膜上,盖上曝光板,曝光10min,拿出胶片,放在显影液浸泡2min,在干净水中洗涮一下后放入定影液浸泡2min。取出胶片,观察结果,拍照保存。
(11)将膜放入膜再生液30min,膜再生后用PBST洗膜5min,加入封闭液,室温摇育1h。
(12)封闭结束后PBST分3次洗膜30min,将膜移至HRPβ-actin抗体稀释液(稀释50000倍)(HRP-conjugated ACTB Rabbit mAb,ABclonal公司,货号AC028)中,室温摇育2h。
(13)孵育结束后,PBST分三次洗膜,共30min。
(14)将膜放在暗室中的曝光板上,吸取膜上液体,加适量ECL超敏发光混合液到膜上,1min后吸取液体,盖上保鲜膜并赶去气泡。
(15)在黑暗环境下降胶片取出做好标记,小心放在膜上,盖上板子曝光5min,将胶片在显影液中显影2min,用水洗一下后放入定影液2min,取出胶片,观察结果,晾干胶片后拍照保存。
结果如图3所示,结果显示,HA-mRNA可在细胞内表达出HA蛋白。
实施例2、HAmRNA免疫小鼠后抗体产生情况和对甲型流感病毒感染的预防保护作用
一、溶液配制
1、戊巴比妥钠溶液(以0.18mL/20g的剂量配置)
称量60mg戊巴比妥钠,用生理盐水(浓度为0.9%的NaCl水溶液)配制6mL、浓度为10mg/mL的戊巴比妥钠溶液。
2、鱼精蛋白溶液
称量1mg鱼精蛋白(Sigma公司),用生理盐水配制1mL、浓度为1mg/mL的鱼精蛋白溶液,配好后置于冰上待用。
3、HA-mRNA+鱼精蛋白疫苗复合物
按80μg HA-mRNA每只小鼠配置,在冰上操作;HA-mRNA原液浓度为17.65μg/μL。
HA-mRNA+鱼精蛋白疫苗复合物:从-70℃冰箱取出冻存的HA-mRNA,将15.9μL浓度为17.65μg/μL的HA-mRNA溶液(280μg)转入新EP管,在新EP管中加入200μL生理盐水,再缓慢加入280μL浓度为1mg/mL的鱼精蛋白溶液,涡旋10min,再取15.9μL浓度为17.65μg/μL的HA-mRNA溶液加入上述溶液,加生理盐水188.2μL(总体积700μL)涡旋2min,置冰上待用,得到HA-mRNA+鱼精蛋白疫苗复合物溶液(鱼精蛋白和HA-mRNA的质量比为1:2)。
二、疫苗复合物免疫小鼠
将实验动物BALB/C小鼠(雌,12-14g,北京斯贝福)随机分为如下3组(每组5只),于第0天、21天和42天皮内免疫小鼠,每次免疫后第14天尾静脉取血,通过ELISA法检测小鼠血液中抗HA抗体水平,具体免疫方式如下:
1、免疫
空白组:不进行处理。
阴性组(生理盐水组):依0.18mL戊巴比妥钠溶液/20g体重腹腔注射浓度为10mg/mL戊巴比妥钠溶液麻醉小鼠,待小鼠麻醉后,用剃毛器剃光每只小鼠背部毛发。用酒精棉给小鼠背部消毒后,将生理盐水对小鼠进行皮内免疫(100μL/只),分5个点平行注射。
疫苗组(HA-mRNA+鱼精蛋白组):依0.18mL戊巴比妥钠溶液/20g体重腹腔注射浓度为10mg/mL戊巴比妥钠溶液麻醉小鼠,待小鼠麻醉后,用剃毛器剃光每只小鼠背部毛发。用酒精棉给小鼠背部消毒后,进行三次免疫:
首次免疫:将上述一制备的HA-mRNA+鱼精蛋白疫苗复合物溶液对小鼠进行皮内免疫(100μL/只,相当于80μg HA-mRNA/只),分5个点平行注射,以第一次皮内注射记作第一天。
第二次免疫:首次免疫后第21天做第二次免疫,将上述一制备的HA-mRNA+鱼精蛋白疫苗复合物溶液对小鼠进行皮内免疫(100μL/只,相当于80μg HA-mRNA/只);
第三次免疫:第二次免疫后第21天做第三次免疫将上述一制备的HA-mRNA+鱼精蛋白疫苗复合物溶液对小鼠进行皮内免疫(100μL/只,相当于80μg HA-mRNA/只);
2、攻毒
第三次免疫小鼠后21天,先对每组小鼠称重,记录体重,以0.15mL/20g的剂量腹腔注射戊巴比妥钠麻醉小鼠,待小鼠麻醉后,按组以20μL/只的剂量(致死剂量的FM1病毒溶液(该病毒记载在如下文献中:Sodium ferulate protects against influenza virusinfection by activation of the TLR7/9-MyD88-IRF7signaling pathway andinhibition of the NF-kB signaling pathwayZhu et al.2019),DOI:10.1016/j.bbrc.2019.03.113),对小鼠进行流感病毒滴鼻操作,滴鼻后的第二天记为第一天,每天对小鼠进行称重与观察,观察两周,记录小鼠死亡情况。
3、血清抗体的ELISA测定
尾静脉取血方法:将老鼠用固定器固定好,用酒精棉球擦拭小鼠尾部,用手术刀割破尾部血管,用毛细管采取20μL的血液,将血轻轻打入PBS溶液里,注意不能溶血,取得的血液室温静置两个小时,500×g,4℃离心15min,将上清于无菌操作台分装,-20℃保存,得到待测血清。
血清抗体的ELISA测定方法如下:
(1)包被:用冰盒取流感病毒FM1,利用0.05M过滤的NaOH水溶液稀释100倍,每孔包被100μL,用封口膜包封,4℃过夜,16h左右。
(2)清洗:垂直倒去包被液(0.05M NaOH),每孔加入150μL洗涤液(0.05%Tween-20,1×PBS),手轻轻摇动30s后在纸上拍干,清洗3次。
(3)封闭:每孔加入100μL封闭液(2%BSA),37℃温箱孵育2h。
(4)清洗:垂直倒去封闭液,每孔加入150μL洗涤液(0.05%Tween-20,1×PBS),手轻轻摇动30s后在纸上拍干,清洗3次。
(5)一抗孵育:加入稀释的阳性血清(流感病毒FM1免疫小鼠,收集血清作为阳性血清,稀释100倍)与待测血清(稀释100倍,稀释液配方(0.5%BSA)),每孔100μL,空白孔加入抗体稀释液(0.5%BSA),37℃温箱孵育1h。
(6)清洗:垂直倒去弃去上述一抗,每孔加入150μL洗涤液,手轻轻摇动30s后在纸上拍干,清洗5次。
(7)二抗孵育:加入稀释的抗小鼠二抗(Goat Anti-Mouse IgG SecondaryAntibody(HRP),北京义翘神州,货号SSA007)溶液,每孔100μL,放入37℃温箱孵育1h。
(8)清洗:垂直倒去弃去二抗,每孔加入150μL洗涤液,手轻轻摇动30s后在纸上拍干,清洗5次。
(9)显色:每孔加入100μL TMB底物(天根),37℃温箱孵育20min。
(10)终止:每孔加入50μL终止液(2M H2SO4),在酶标仪上读取A450的值。
第二次免疫后的第14天尾静脉取血后ELISA方法检测第二次免疫后抗HA抗体产生,结果如图4所示,与阴性组(生理盐水组)相比,疫苗组(HA-mRNA+鱼精蛋白组)在第二次免疫后5只小鼠中有4只小鼠产生了抗HA抗体。
第三次免疫后的第14天尾静脉取血后ELISA方法检测第三次免疫后抗HA抗体产生,结果如图5所示,与阴性组(生理盐水组)相比,疫苗组(HA-mRNA+鱼精蛋白组)在第三次免疫后4只小鼠的抗体水平都显著提高。
上述结果表明,提示HA-mRNA+鱼精蛋白免疫小鼠可在小鼠体内产生抗HA抗体。
4、体重情况检测
对攻毒后小鼠进行称重,结果如图6所示,可以看出,阴性组(生理盐水组)小鼠在感染后第3天开始小鼠体重开始出现显著降低,而疫苗组(HA-mRNA+鱼精蛋白组)小鼠在感染后体重变化不显著。
统计小鼠经三次免疫后用病毒攻击后存活情况,结果如图7所示,可以看出,攻毒感染后第6天开始,阴性组(生理盐水组)小鼠开始出现死亡,在感染后第8天全部死亡,而在疫苗组(HA-mRNA+鱼精蛋白组)小鼠在感染后14天未出现死亡情况。
由以上结果可见,HA-mRNA+鱼精蛋白免疫小鼠对甲型流感病毒感染具有预防保护作用。
SEQUENCE LISTING
<110>中国人民解放军军事科学院军事医学研究院
<120> 一种用于预防甲型流感病毒感染的HA-mRNA疫苗
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1990
<212> DNA
<213> Artificial sequence
<400> 1
tagggagaca agcttgcttg ttctttttgc agaagctcag aataaacgct caactttggc 60
ggatccgcca ccatgaaagc aaaactactg atcctgttat gtgcacttac agctacagat 120
gcagacacaa tatgtatagg ctaccatgcg aacaactcaa ccgacactgt tgacacagta 180
ctcgaaaaga atgtgacagt gacacactct gtaaacctac tcgaagacag ccacaacggg 240
aaattatgca gattaaaagg aatagcccca ctacaattgg ggaaatgtaa cattgccgga 300
tggatcttag gaaacccaga atgcgaatca ctgctttcta agagatcatg gtcctacatt 360
gcagaaacac caaactctga gaatggagca tgttacccag gagatttcgc cgactatgag 420
gaactgaggg agcaattgag ctcagtgtca tcattcgaga gattcgaaat attccccaag 480
gaaagatcat ggcccaaaca caacataacc agaggagtaa cggcagcatg ctcccatgcg 540
gggaaaagca gtttttacaa aaatttgctc tggctgacgg agacagatgg ctcataccca 600
aagctgagca agtcctatgt gaacaataaa gagaaagaag tccttgtgct atggggtgtt 660
catcacccgt ctaacataga ggatcaaaag accctctatc ggaaagaaaa tgcttatgtc 720
tctgtagtgt cttcaaatta taacaggaga ttcaccccgg aaatagcaga aagacccaaa 780
gtaagaggtc aagcagggag aattaactat tactggactt tgctagaacc cggagacaca 840
ataatatttg aggcaaatgg aaatctaata gcgccatggt atgctttcgc actgagtaga 900
gactttggat caggaatcat cacctcaaac gcatcaatgg atgaatgtga cacgaagtgt 960
caaacacccc agggagctat aaacagtagt ctcccttttc agaatataca cccagtcaca 1020
ataggagagt gcccaaaata cgtcaagagt accaaattga ggatggttac aggattaagg 1080
aacatcccat ccattcaatc cagaggtctg tttggagcca ttgccggttt cattgagggg 1140
ggatggactg gaatgataga tggatggtat ggttaccatc atcagaatga acagggatct 1200
ggctatgctg cggatcaaaa aagcacacaa aatgcaatta acgggattac aaataaggtg 1260
aactctgtta tcgagaaaat gaacactcaa ttcacagctg tgggtaaaga attcaacaaa 1320
ttagaaaaaa gaatggaaaa cttaaataaa aaagttgatg atgggtttct ggacatttgg 1380
acatataatg cagaattgtt ggttctactg gaaaatgaaa ggactttgga tttccatgac 1440
tcaaatgtga agaatctgta tgagaaagta aaaaaccaat taaggaataa tgcaaaggaa 1500
ataggaaacg ggtgttttga gttctaccac aagtgtaaca atgaatgcat ggaaagtgta 1560
aaaaatggaa cttatgatta cccaaaatat tcagaggaat caaagttaaa cagggaaaaa 1620
attgatggag tgaaattgga atcaatgggg gtctatcaga ttctggcgat ctactcaact 1680
gtcgccagtt cactggtgct tctggtctcc ctgggggcaa tcagcttctg gatgtgttct 1740
aatgggtctt tgcaatgcag aatatgcatc tgaggtaccg actgactagg atctggttac 1800
cactaaacca gcctcaagaa cacccgaatg gagtctctaa gctacataat accaacttac 1860
actttacaaa atgttgtccc ccaaaatgta gccattcgta tctgctccta ataaaaagaa 1920
agtttcttca cattctaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaac cccccccccc 1980
ccccccctgg 1990
Claims (10)
1.HA-mRNA,其核苷酸序列为序列表中序列1编码的RNA。
2.编码权利要求1所述mRNA的核酸分子。
3.根据权利要求2所述的核酸分子,其特征在于:所述核酸分子的核苷酸序列为序列表中序列1。
4.权利要求1所述的HA-mRNA在制备预防或治疗流感病毒产品中的应用。
5.根据权利要求4所述的应用,其特征在于:所述流感病毒为甲型流感病毒。
6.一种预防或治疗流感病毒产品,其包括权利要求1所述的HA-mRNA。
7.根据权利要求6所述的产品,其特征在于:所述产品还包括佐剂。
8.根据权利要求6或7所述的产品,其特征在于:所述佐剂为鱼精蛋白。
9.权利要求1所述的HA-mRNA和鱼精蛋白在预防或治疗流感病毒产品中的应用。
10.根据权利要求4或5所述的应用或权利要求6-8任一所述产品,其特征在于:所述产品为疫苗。
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