CN115044562A - 一种嵌合表达分子佐剂的重组狂犬病病毒及其制备方法与应用 - Google Patents
一种嵌合表达分子佐剂的重组狂犬病病毒及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开一种嵌合表达分子佐剂的重组狂犬病病毒及其制备方法与应用,属于生物医学技术领域。为了提供一种诱导机体产生长期、高效的特异性免疫反应的狂犬病疫苗,同时提高疫苗保护力,同时又能减少免疫原的用量,降低疫苗生产成本。本发明提供一种嵌合表达分子佐剂的重组狂犬病病毒是狂犬病病毒CVS11株为出发菌株,在出发菌株的G基因和L基因之间同时表达MAB2560蛋白、狂犬病病毒的G蛋白信号肽和狂犬病病毒的G蛋白TMCD区连接的融合蛋白。MAB2560蛋白的嵌合表达提高了灭活狂犬病病毒的免疫原性,可诱导小鼠产生特异性免疫应答。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种嵌合表达分子佐剂的重组狂犬病病毒及其制备方法与应用。
背景技术
狂犬病是由狂犬病病毒感染引起的一种人兽共患病,一旦出现临床症状,致死率接近100%,对全球公共卫生安全和人类健康构成重要威胁。据WHO统计,每年全球约59000人死于狂犬病。狂犬病病毒属于弹状病毒科狂犬病毒属,外形呈子弹状,为单股负链RNA病毒。狂犬病病毒基因组编码五种结构蛋白:核蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)和RNA依赖的RNA聚合酶蛋白(L),这五种结构蛋白共同调节病毒复制、转录、组装和出芽等过程。
目前,我国批准使用的狂犬病疫苗为灭活疫苗,具有安全性高的优点,但往往需要多次、高剂量免疫才能刺激机体产生有效保护。亟需一种疫苗能够诱导机体产生长期、高效的特异性免疫反应,提高疫苗保护力,同时又能减少免疫原的用量,降低疫苗生产成本。
发明内容
本发明的目的是为了提供一种诱导机体产生长期、高效的特异性免疫反应的狂犬病疫苗,同时提高疫苗保护力,同时又能减少免疫原的用量,降低疫苗生产成本。
本发明提供一种嵌合表达分子佐剂的重组狂犬病病毒,所述重组狂犬病病毒是狂犬病病毒CVS11株为出发菌株,在出发菌株的G基因和L基因之间同时表达MAB2560蛋白、狂犬病病毒的G蛋白信号肽和狂犬病病毒的G蛋白TMCD区连接的融合蛋白。
进一步地限定,所述MAB2560蛋白的氨基酸序列如SEQ ID NO.10所示;所述狂犬病病毒的G蛋白信号肽的氨基酸序列如SEQ ID NO.11所示;所述狂犬病病毒的G蛋白的氨基酸序列如SEQ ID NO.12所示。
进一步地限定,编码MAB2560蛋白的基因序列如SEQ ID NO.1所示;编码狂犬病病毒的G蛋白信号肽的基因序列如SEQ ID NO.2所示;编码狂犬病病毒的G蛋白的基因序列如SEQ ID NO.3所示。
进一步地限定,编码融合蛋白的基因序列的获得方法如下:编码MAB2560蛋白的基因序列的5’端连接编码狂犬病病毒G蛋白信号肽的基因序列,编码MAB2560蛋白的基因序列的3’端连接编码狂犬病病毒G蛋白TMCD区的基因序列。
本发明提供一种上述的重组狂犬病病毒的制备方法,其特征在于,所述制备方法的步骤如下:
步骤1:获得SP-MAB2560-TMCD基因序列如SEQ ID NO.4所示,将SEQ ID NO.4所示的序列插入到狂犬病病毒CVS11全基因组的G基因和L基因之间获得重组基因序列;
步骤2:将步骤1获得的重组基因序列转入到pcDNA3.1载体、pcDNA3.0载体或pCAGGS载体、中获得重组载体;
步骤3:将步骤3获得的重组载体与辅助质粒N、P、L、G共转染BSR细胞,获得重组狂犬病病毒。
本发明提供一种上述的重组狂犬病病毒在制备预防或治疗狂犬病疫苗中的应用。
本发明提供一种嵌合表达分子佐剂的重组狂犬病病毒的融合蛋白,所述融合蛋白是由狂犬病病毒的G蛋白信号肽、MAB2560蛋白和狂犬病病毒的G蛋白TMCD区依次连接获得的融合蛋白。
进一步地限定,所述融合蛋白的氨基酸序列如SEQ ID NO.5所示。
本发明提供一种编码上述的融合蛋白的基因。
本发明提供上述的融合蛋白或上述的编码基因在制备预防或治疗狂犬病疫苗或药物中的应用。
有益效果:本发明提供了一种嵌合表达分子佐剂重组狂犬病病毒主要是通过将RABV-G蛋白的信号肽与TMCD区分别连接至分子佐剂MAB2560两端,并将其连接至狂犬病病毒CVS11株基因组G-L之间,构建嵌合表达MAB2560的重组全长质粒。将全长质粒与辅助质粒共同转染BSR细胞,拯救获得了表达MAB2560分子的重组狂犬病病毒rCVS11-MAB2560,分子佐剂MAB2560的嵌合表达提高了灭活狂犬病病毒的免疫原性,可诱导小鼠产生特异性免疫应答。
附图说明
图1为实施例1中的重组质粒CVS11-MAB2560示意图。
图2为实施例1中的SP-MAB2560-TMCD基因的PCR扩增结果。
图3为实施例1中的重组质粒CVS11-MAB2560的PCR鉴定。
图4为实施例1中重组病毒rCVS11-MAB2560的DFA鉴定;其中,A:重组病毒rCVS11-MAB2560;B:NA细胞。
图5为实施例1中重组病毒rCVS11-MAB2560的IFA鉴定;其中,A:重组病毒rCVS11-MAB2560;B:CVS11;C:NA细胞。
图6为实施例1中重组病毒rCVS11-MAB2560的Western Blot鉴定。
图7为实施例1中重组病毒rCVS11-MAB2560的生长曲线测定;其中,A:MOI=0.接种NA细胞,B;MOI=1接种NA细胞,C:MOI=0.1接种BSR细胞,D:MOI=1接种BSR细胞。
图8为实施例2中重组狂犬病病毒rCVS11-MAB2560的安全性评价;其中,A:体重变化,B:临床症状评分,C:存活率。
图9为实施例2中重组狂犬病病毒免疫小鼠中和抗体检测。
图10为实施例2中小鼠淋巴结DC细胞的活化分析;其中,A:CD11c+CD80+双阳性细胞比例,B:CD11c+MHC-I+双阳性细胞比例,C:CD11c+MHC-II+双阳性细胞比例。
图11为实施例2中小鼠腹股沟淋巴结B细胞活化情况。
图12为实施例2中小鼠腹股沟淋巴结T细胞活化情况;其中A:CD4+CD69+双阳性细胞比例,B:CD8+CD69+双阳性细胞比例。
图13为实施例2中小鼠IL-4和IFN-γ酶联免疫斑点实验;其中A:小鼠IL-4酶联免疫斑点实验,B:小鼠IFN-γ酶联免疫斑点实验。
图14为实施例2中小鼠脾脏B细胞活化情况。
图15为实施例2中小鼠脾脏T细胞活化情况。
图16为实施例2中小鼠脾脏记忆T细胞产生情况;其中A:CD4+CD44+CD62L+阳性细胞比例,B:CD8+CD44+CD62L+阳性细胞比例。
具体实施方式
狂犬病病毒CVS-11株记载在张传明,冷雪梅,蒋雯雯,等.狂犬病病毒CVS-11株的培养及其效价测定[J].现代畜牧兽医,2014(7):3.
实施例1重组狂犬病病毒rCVS11-MAB2560的构建与鉴定
1、目的序列合成
由南京金斯瑞合成目的基因片段,根据GenBank公布的MAB2560基因序列(DI432724.1),SEQ ID NO.1所示,MAB2560基因序列5’端插入狂犬病病毒CVS11株G蛋白信号肽,SEQ ID NO.2所示,MAB2560基因序列的3’端插入狂犬病病毒CVS11株G蛋白TMCD区,SEQ ID NO.3所示,合成目的序列SP-MAB2560-TMCD,得到的基因序列如SEQ ID NO.4所示。
融合蛋白的制备方法:RABV-G蛋白的信号肽与TMCD区分别连接至分子佐剂MAB2560两端,获得融合蛋白,氨基酸序列如SEQ ID NO.5所示。
2、引物设计与合成
设计特异性扩增引物(表1)。参考合成序列SP-MAB2560-TMCD设计上游引物MAB2560-F(5’端引入酶切位点Bsiw I),下游引物MAB2560-R(3’端引入酶切位点Sac II),根据狂犬病病毒CVS11株全长质粒设计鉴定引物JD-F和JD-R。
表1:引物信息
注:下划线为酶切位点
3、重组质粒rCVS11-MAB2560的构建与鉴定
(1)目的基因SP-MAB2560-TMCD的扩增
以合成的SP-MAB2560-TMCD为模板,MAB2560-F、MAB2560-R为引物扩增目的基因SP-MAB2560-TMCD(图2),反应条件如表2所示,经1%琼脂糖凝胶电泳分离回收目的片段,片段大小与预期大小一致,说明目的片段SP-MAB2560-TMCD扩增成功。
表2 PCR反应条件
(2)重组质粒CVS11-MAB2560的鉴定
将切胶回收后的目的片段与RABV-CVS11株全长质粒(RABV-CVS11株全长如SEQ IDNO.13所示,载体为pcDNA3.1)用限制性内切酶Bsiw I与Sac II进行双酶切,将纯化回收后的酶切产物,16℃,T4连接酶连接过夜。将连接产物转化至Stellar感受态细胞中,挑取单克隆用引物鉴定JD-F、JD-R进行PCR鉴定(图3)(反应条件如表3)并送生工生物公司测序,结果显示其序列与合成的SP-MAB2560-TMCD基因序列同源性为100%,说明重组RABV全长质粒CVS11-MAB2560构建成功。实现目的片段SP-MAB2560-TMCD连接至狂犬病病毒CVS11株基因组G-L之间。按照图1的方式进行构建。
表3 PCR反应条件
4、重组狂犬病病毒rCVS11-MAB2560的拯救
将构建成功的全长质粒CVS11-MAB2560与辅助质粒pD-N、pD-P、pD-M、pD-G(由本实验室保存)使用转染试剂LipofectamineTM3000共同转染至BSR细胞对重组病毒进行拯救。具体方法如下:前一天将BSR细胞铺至6孔板中,待细胞生长至90%时进行转染。全长重组质粒CVS11-MAB2560分别与各辅助质粒(见表3)、将8μL P3000加入250μL OPTI-MEM中后轻吹混匀,室温,静置5min,将其作为转染混合液A;将10μL Lip3000加入250μL OPTI-MEM中轻吹混匀,室温,静置5min,将其作为转染混合液B,将转染混合液A与B混合后室温静置15min。在此期间将细胞上清弃置,用2mL OPTI-MEM清洗细胞两次,然后每孔补加1.5mL OPTI-MEM。将AB混合液分散滴加入六孔板一个孔中,于37℃、5%CO2条件下培养5h后弃置转染液,补加含10%胎牛血清的DMEM培养液进行培养,转染后第三天收取上清,并更换新鲜培养液继续培养,于转染后第5天刮取细胞,将上清-细胞混合液共同保存,并留适量上清液进行RABV的DFA检测。
表3转染各个质粒用量/孔
质粒名称 | 用量/孔(6孔板) |
CVS11-MAB2560 | 2.5μg |
N | 0.625μg |
P | 0.3125μg |
L | 0.125μg |
G | 0.1875μg |
5、重组狂犬病病毒rCVS11-MAB2560的鉴定
(1)重组狂犬病病毒rCVS11-MAB2560的DFA鉴定
为了证明所拯救到的病毒是重组狂犬病病毒,首先对NA细胞进行传代,100μL/孔铺至96孔板内。同步接毒,接种50μL待测拯救病毒上清,于37℃,5%CO2培养箱培养48h。弃去上清,每孔加入150μL80%冷丙酮,室温,固定30min。将固定液弃去,加入PBST 150μL/孔,重复洗涤两次。弃去液体后,避光加入200倍稀释的FITC标记的鼠抗狂犬病病毒N蛋白抗体,40μL/孔,置于37℃孵育1h,弃液后用PBST洗涤两次,于荧光显微镜观察结果,结果如图所示(图4A、图4B),与正常细胞相比,感染重组狂犬病病毒rCVS11-MAB2560的NA细胞在荧光显微镜下呈现明显的绿色荧光,表明重组狂犬病病毒rCVS11-MAB2560初步拯救成功。
(2)间接免疫荧光鉴定
将NA细胞正常传代铺至96孔板内,待细胞长至单层,分别接种母本病毒CVS11、重组病毒rCVS11-MAB2560以及10%FBS的DMEM(阴性对照)。37℃、5%CO2培养箱培养48h后,弃上清,每孔加入150μL提前预冷的80%丙酮,室温固定30min。将固定液弃去,加入PBST 150μL/孔,置小型摇床上5min,以此操作洗板3次,最后一次在纸上轻轻拍打,将孔内液体拍出。将CVS11组与rCVS11-MAB2560组以及阴性对照组,加入抗MAB2560蛋白抗体(1:500稀释),每孔50μL,37℃培养箱避光孵育1h后,PBST洗3次。再加入伊文思蓝染色液(1:500稀释)和FITC标记的山羊抗兔二抗(1:500稀释),37℃避光孵育1h后,PBST洗3次,于倒置荧光显微镜观察结果。结果如图所示(图5A、图5B、图5C),与正常细胞和母本病毒CVS11株相比,感染重组狂犬病病毒rCVS11-MAB2560的NA细胞在荧光显微镜下呈现明显的绿色荧光,表明重组狂犬病病毒rCVS11-MAB2560可以成功表达外源蛋白MAB2560。
(4)Western Blot鉴定
将纯化后的重组病毒rCVS11-MAB2560、CVS11与5×Loading Buffer混合至1×,沸水中煮10min,进行制样。将制好的样品进行SDS-PAGE电泳,转印至NC膜。将NC膜用含5%脱脂奶粉的PBS溶液在室温条件下封闭2h,分用免疫获得的MAB2560、MAB2560多克隆抗体血清作为一抗(稀释倍数:1:500)4℃孵育过夜,1xPBST洗涤。用HRP标记的羊抗兔IgG作为二抗(稀释倍数:1:20000)室温孵育1h,洗涤,凝胶成像系统分析结果。结果如图所示(图6),重组病毒rCVS11-MAB2560在21Kd左右出现目的条带,与MAB2560加RABV G基因TM-CD区的目的片段大小相符。
6.重组狂犬病病毒生长曲线的测定
为测定重组病毒在不同细胞上的生长曲线,选取BSR细胞与Na细胞正常传代至新T25细胞瓶,将F5代重组病毒rCVS11-MAB2560与母本病毒CVS11按病毒感染复数MOI=0.1、1分别感染BSR和Na细胞,置于37℃,5%CO2培养箱培养,每24h吸取部分上清进行病毒滴度测定,并绘制病毒生长曲线。结果如图所示(图7A、图7B、图7C、图7D)重组病毒rCVS11-MAB2560与母本病毒CVS-11的体外增殖无显著差异,表明病毒基因组G-L处插入额外的外源基因MAB2560不影响病毒的复制能力。
实施例2.rCVS11-MAB2560免疫效果评价
1.小鼠免疫
(1)重组狂犬病病毒免疫原的制备
将重组病毒rCVS11-MAB2560与母本毒CVS11分别接种Na细胞后收获上清,1:2000加入β-丙内酯混匀后4℃过夜,37℃水解2h灭活病毒。灭活后的病毒液用直接免疫荧光方法进行灭活验证。将已灭活的病毒液于4℃、3000rpm离心30min去除细胞碎片,然后灭活病毒按总体积5:1与Gel02佐剂混匀,以备后续免疫实验使用。
(2)小鼠免疫分组方案如表4所示:
表4
2、重组狂犬病病毒毒的安全性评价
将重组病毒rCVS11-MAB2560与母本毒CVS11株按103TCID50/只通过脑内注射方式感染4-6周龄BALB/c小鼠,每日观察小鼠临床症状和发病情况。结果如图(图8A、图8B、图8C)所示:重组病毒rCVS11-MAB2560感染小鼠的体重变化与临床症状均优于母本病毒组,感染后9天CVS11感染小鼠已全部死亡(致死率100%),重组病毒rCVS11-MAB2560感染小鼠死亡时间明显延后,其存活率分别20%和30%。结果表明,与母本病毒CVS11相比,嵌合表达MAB2560重组病毒的致病性降低。
3、重组狂犬病病毒免疫小鼠中和抗体检测
首免后每周进行眼缘静脉丛采血,分离血清后使用FAVN法测定其血清中狂犬病病毒中和抗体效价。具体步骤如下:
小鼠眼缘静脉丛采血后,于37℃放置1h,室温4000rpm离心10min后,吸取上层血清至1.5mL离心管中,弃去下层血液。将分离得到的血清56℃灭活30min后,于-80℃保存待用。将提前准备好的96孔板加入DMEM培养液,100μL/孔。再将25μL标准血清与待测血清分别加入到200μL DMEM培养液中进行9倍稀释。将稀释好的血清加入到96孔板中,每孔50μL,每个样品做四个复孔。再按照三倍倍比稀释,将待检血清与标准血清从稀释度为3稀释到稀释度为36。将标准攻击毒株CVS11株稀释到30-300TCID50/50μL,加入96孔板中,每孔50μL。同时设立病毒对照,将稀释完成的病毒进行4倍稀释。将加液完成的96孔板放入37℃、5%CO2细胞培养箱中反应1h,然后加入提前准备好的BHK21细胞,每孔50μL,约2x104cell/孔。将其放置于37℃、5%CO2细胞培养箱中培养48h。将上清弃置与10%NaOH中,进行狂犬病病毒DFA检测,使用倒置荧光显微镜观察结果,使用法计算待测血清中和效价,结果如图所示(图9),一免后7d免疫小鼠中和效价均未超过0.5IU/mL,免疫后14d重组病毒rCVS11-MAB2560免疫小鼠血清中和抗体效价超过0.5IU/mL,显著高于母本病毒对照组。加强免疫后,免疫小鼠血清中和抗体效价均升高,且重组病毒rCVS11-MAB2560免疫组仍显著高于母本病毒组。综上所述,重组病毒rCVS11-MAB2560可以更快更好地诱导小鼠产生抗狂犬病病毒中和抗体,且抗体维持时间更长。
3、腹股沟淋巴结中免疫细胞的流式细胞术分析
将小鼠随机分为3组,分别为重组病毒rCVS11-MAB2560组,CVS11对照组和佐剂对照组。每只小鼠免疫107TCID50的病毒,首免后第3,6,9天采取小鼠腹股沟淋巴结,研磨后制成细胞悬液,取5×105个细胞进行细胞染色,通过流式细胞仪进行分析。
(1)小鼠淋巴结DC细胞的活化分析
分别于免疫后3d、6d、9d分离小鼠腹股沟淋巴结,通过流式细胞术分别筛选CD11c+与CD80+、MHC-I+、MHC-II+的双阳性细胞,检测小鼠腹股沟淋巴结内DCs的活化情况,结果如图(图10A、图10B、图10C)所示:重组病毒rCVS11-MAB2560首免后3d、6d淋巴结细胞中CD11c+与CD80+双阳性细胞显著多于母本病毒CVS11组与adjuvant对照组,首免后3d、6d、9d淋巴结细胞中CD11c+与MHC-I+双阳性细胞数量均显著多于母本病毒CVS11对照组与adjuvant对照组,首免后3d、9d淋巴结细胞中CD11c+与MHC-II+双阳性细胞数量均显著多于母本病毒CVS11对照组与adjuvant对照组。综上所述:与母本病毒相比,重组病毒rCVS11-MAB2560可以在首免后显著促进DCs的增殖与活化。
(2)小鼠腹股沟淋巴结B细胞活化情况
分别于免疫后3d、6d、9d分离小鼠腹股沟淋巴结,通过流式细胞术筛选CD40+与CD19+双阳性细胞,检测小鼠腹股沟淋巴结内B细胞的活化情况。结果如图(图11)所示:首免后3d、6d、9d,重组病毒rCVS11-MAB2560均能诱导B细胞的活化,在6d时重组病毒组小鼠淋巴细胞中CD40+与CD19+双阳性细胞数量显著多于母本病毒CVS11组。综上所述:重组病毒rCVS11-MAB2560在首次免疫后可以显著促进小鼠腹股沟淋巴结内B细胞的募集和/或活化。
(3)小鼠腹股沟淋巴结T细胞活化情况
分别于免疫后3d、6d、9d分离小鼠腹股沟淋巴结,通过流式细胞术分别筛选CD4+、CD69+和CD8+、CD69+双阳性细胞,检测小鼠腹股沟淋巴结内T细胞的活化情况。结果如图(图12)所示:重组病毒rCVS11-MAB2560首免后3d淋巴结细胞中CD4+和CD69+双阳性细胞显著多于母本病毒CVS11对照组,首免后6d、9d未见显著差异;CD8+和CD69+双阳性细胞在首免后3d、6d时,显著多于母本病毒CVS11对照组,9d时未见显著差异。综上所述:重组病毒rCVS11-MAB2560在首次免疫后能促进T细胞的募集和/或活化。
4、脾细胞活化分析
分别于首免后1周和5周采取小鼠脾脏,用100目细胞筛网将脾脏研磨至匀浆,将研磨完成的细胞用5mL含10%FBS的1640培养液冲洗下来,转至15mL离心管中,室温,2000rpm,离心10min。弃去上清,加入5mL红细胞裂解液重悬,静置5min。室温,2000rpm,离心10min,弃去上清,重复此过程,共裂解三次。用5mL含10%FBS的1640培养液重复洗涤细胞两次。细胞计数后将细胞稀释到2.5×106cells/mL,以备后续实验使用。
(1)小鼠IL-4和IFN-γ酶联免疫斑点实验
在生物安全柜中取出ELISPOT 96孔板,将待使用孔中每孔加入200μL含10%FBS的1640培养液,室温孵育30min。弃去上清,每孔加入200μL无菌PBS清洗五次,将稀释好的脾细胞悬液加入待测孔中(200μL/孔,5×105cells/孔)。同时加入稀释好的纯化后的狂犬病病毒CVS11(浓度为10μg/mL),每只小鼠设立两个重复。将加样完成的96孔板用锡纸充分包裹,放置于37℃,5%CO2细胞培养箱中培养48h。弃去上清,用无菌PBS清洗5次,200μL/孔,同时,用含0.5%FBS的无菌PBS 1000倍稀释检测用抗体R4-6A2-biotin,检测孔每孔加入100μL,室温,避光孵育2h。弃去上清,用无菌PBS清洗5次,200μL/孔,用含0.5%FBS的无菌PBS 1000倍稀释检测用抗体Streptavidin-ALP,检测孔每孔加入100μL,室温,避光孵育1h。弃去上清,加入无菌PBS清洗五次。加入TMB底物溶液,室温于暗室中显色5-30min,待检测孔中出现针眼状斑点后弃去显色液,用清水冲洗终止显色。室温晾干后将96孔板送于北京行健雅生物技术有限公司进行读数。结果如图所示(图13A、图13B)重组病毒rCVS11-MAB2560与母本病毒CVS11在二免后一周与五周,均可促进小鼠脾脏细胞中INF-γ与IL-4特异性分泌细胞的活化。同时,重组病毒组的INF-γ与IL-4特异性分泌细胞数量显著高于母本病毒CVS11组。由于INF-γ与IL-4分别是Th1型细胞免疫与Th2型体液免疫的代表性细胞因子,结果表明重组病毒rCVS11-MAB2560可以诱导Th1型与Th2型细胞因子的产生。
(2)小鼠脾脏B细胞活化情况
利用流式细胞术筛选小鼠脾脏中CD69+与CD19+双阳性细胞,检测小鼠脾脏内B细胞的活化情况。结果如图(图14)所示:与母本病毒CVS11相比,重组病毒免疫组在二免后一周时小鼠脾脏CD69+CD19+细胞数量显著增加,但在二免后五周并无明显差异。综上所述:重组病毒二次免疫后,在刺激物刺激下可以促进小鼠脾脏中B细胞的活化。
(3)小鼠脾脏T细胞活化情况
对小鼠脾脏细胞用CD4+、CD8+和CD69+荧光抗体进行染色,检测小鼠脾脏内T细胞的活化情况。结果如图(图15)所示:在二免后五周时重组病毒组与母本病毒CVS11对照组均能促进小鼠脾脏T细胞的活化,但重组病毒组与母本病毒组之间没有明显差异。综上所述:重组病毒二次免疫后,在刺激物刺激下可以促进小鼠脾脏中T细胞的活化。
(4)小鼠脾脏记忆T细胞产生情况
利用流式细胞术筛选小鼠脾脏T细胞中CD44+CD62L+阳性细胞,检测小鼠脾脏内中央记忆性T细胞(Central Memory T cell,TCM)的活化情况。结果如图(图16A、图16B)所示:在二免后一周与五周,与母本病毒CVS11相比,重组病毒rCVS11-MAB2560免疫组CD4+/CD44+CD62L+细胞数量均显著上升,CD8+/CD44+CD62L+细胞数量无显著差异。综上所述:重组病毒二次免疫后,可诱导机体产生更高水平的CD4+中央记忆性T细胞。
实施例3.利用重组狂犬病毒rCVS11-MAB2560制备疫苗
1.利用重组狂犬病毒rCVS11-MAB2560制备疫苗的方法:将重组病毒液按照1:2000的比例加入β-丙内酯,置于4℃摇床上灭活24h,再置于37℃水浴锅中水解2h。灭活后的病毒液用直接免疫荧光方法进行灭活验证。将已灭活的病毒液于4℃、3000rpm离心30min去除细胞碎片,将病毒上清液分别与不同的佐剂配伍制备成免疫原。将制备的免疫原以1:5的比例与Gel02佐剂混合后进行免疫。
CVS11株本身为标准攻击毒株,是适应后的固定毒株,毒力中等。改造过后的重组病毒rCVS11-MAB2560致病性有所降低,由图8中的重组病毒安全性评价体现,重组病毒与母本病毒CVS11分别颅内注射小鼠,重组病毒注射后的小鼠其体重变化、临床症状、存活率均优于母本病毒。
2.安全性:对健康幼犬(4~12周龄)进行一次单剂量注射,一次单剂量重复注射和一次超剂量注射,均安全性良好。对部分免疫的幼犬进行解剖学和组织学检查,均未出现肉眼可见的病理变化。
SEQUENCE LISTING
1.MAB2560,DNA,603bp(SEQ ID NO.1所示)
ATGCGAACTATCGCAATCCGACATCGAGCAGTCATCGGACTGTCCGCTGTCGCTCTGATCACCGTGGGCTGCAGCAACGGCACCTCCGTGGACGTGCCTTCTCCTGAAGTGGGCCTGATCGCCACCACCAGCTCTGCCGCTCCTGCTCAGCCTGCCGAGGTGAAGCTGATCGGCGAGCGGGATGTGGAAGTGACCCTGACCGGACCCATTGCTGCCAAGTACTCCAGCGCCTCTGAATCTCAGAAGCAGGCCCTCGGCAAACCCCTGACCGGCGACCACAACGCCGGCACCAGAGAGTCCGGCGCTGTGTTCCAGCAGTTTCAAGGCGGCGCCATCATCGCCAAGAACAATCAGGCTGGCACACCAGCCTTCATCGTGGTCGGCAAGATCAGAGATGCCTGGAACATCCAGCGGGACGCCGACGGCACACCTTCCATCACCGGCAACAATGGCTCTGCTGGACCTCTGGGCCTGCCTACCTCCGACGAGAACACCGAGGGCGACCAGCTGGTGTCCACCTTCGAGCACGGCAAGATCGAGTACAACGCTAAGTCCGGCGAAGTGGCCGTGACAGTGAACGGAAAAGTGGTGCCCTCTGGTCTG
2.MAB2560,氨基酸,201aa(SEQ ID NO.10)
MRTIAIRHRAVIGLSAVALITVGCSNGTSVDVPSPEVGLIATTSSAAPAQPAEVKLIGERDVEVTLTGPIAAKYSSASESQKQALGKPLTGDHNAGTRESGAVFQQFQGGAIIAKNNQAGTPAFIVVGKIRDAWNIQRDADGTPSITGNNGSAGPLGLPTSDENTEGDQLVSTFEHGKIEYNAKSGEVAVTVNGKVVPSGL
3.RABV CVS11-G信号肽,DNA,63bp(SEQ ID NO.2所示)
ATGGTTCCTCAGGTTCTTTTGTTTGTACTCCTTCTGGGTTTTTCGTTGTGTTTCGGGAAATTT
4.RABV CVS11-G信号肽,氨基酸,21aa(SEQ ID NO.11)
MVPQVLLFVLLLGFSLCFGKF
5.RABV CVS11-G TMCD,DNA,198bp(SEQ ID NO.3所示)
TACGTATTGATGACTGCAGGGGCCATGATTGGCCTGGTGTTGATATTTTCCCTAATGACATGGTGCAGAAGAGCCAATCGACCAGAATCGAAACAACGCAGTTTTGGAGGGACAGGGGGGAATGTGTCAGTCACTTCCCAAAGCGGAAAAGTCATACCTTCATGGGAATCATATAGGAGTGGAGGTGAGATCAGACTG
6.RABV CVS11-G TMCD,氨基酸,66aa(SEQ ID NO.12)
YVLMTAGAMIGLVLIFSLMTWCRRANRPESKQRSFGGTGGNVSVTSQSGKVIPSWESYRSGGEIRL
7.SP-MAB2560-TMCD,DNA,864bp(SEQ ID NO.4所示)
ATGGTTCCTCAGGTTCTTTTGTTTGTACTCCTTCTGGGTTTTTCGTTGTGTTTCGGGAAATTTCGAACTATCGCAATCCGACATCGAGCAGTCATCGGACTGTCCGCTGTCGCTCTGATCACCGTGGGCTGCAGCAACGGCACCTCCGTGGACGTGCCTTCTCCTGAAGTGGGCCTGATCGCCACCACCAGCTCTGCCGCTCCTGCTCAGCCTGCCGAGGTGAAGCTGATCGGCGAGCGGGATGTGGAAGTGACCCTGACCGGACCCATTGCTGCCAAGTACTCCAGCGCCTCTGAATCTCAGAAGCAGGCCCTCGGCAAACCCCTGACCGGCGACCACAACGCCGGCACCAGAGAGTCCGGCGCTGTGTTCCAGCAGTTTCAAGGCGGCGCCATCATCGCCAAGAACAATCAGGCTGGCACACCAGCCTTCATCGTGGTCGGCAAGATCAGAGATGCCTGGAACATCCAGCGGGACGCCGACGGCACACCTTCCATCACCGGCAACAATGGCTCTGCTGGACCTCTGGGCCTGCCTACCTCCGACGAGAACACCGAGGGCGACCAGCTGGTGTCCACCTTCGAGCACGGCAAGATCGAGTACAACGCTAAGTCCGGCGAAGTGGCCGTGACAGTGAACGGAAAAGTGGTGCCCTCTGGTCTGTACGTATTGATGACTGCAGGGGCCATGATTGGCCTGGTGTTGATATTTTCCCTAATGACATGGTGCAGAAGAGCCAATCGACCAGAATCGAAACAACGCAGTTTTGGAGGGACAGGGGGGAATGTGTCAGTCACTTCCCAAAGCGGAAAAGTCATACCTTCATGGGAATCATATAGGAGTGGAGGTGAGATCAGACTGTGA
8.SP-MAB2560-TMCD,氨基酸,287aa(SEQ ID NO.5所示)
MVPQVLLFVLLLGFSLCFGKFRTIAIRHRAVIGLSAVALITVGCSNGTSVDVPSPEVGLIATTSSAAPAQPAEVKLIGERDVEVTLTGPIAAKYSSASESQKQALGKPLTGDHNAGTRESGAVFQQFQGGAIIAKNNQAGTPAFIVVGKIRDAWNIQRDADGTPSITGNNGSAGPLGLPTSDENTEGDQLVSTFEHGKIEYNAKSGEVAVTVNGKVVPSGLYVLMTAGAMIGLVLIFSLMTWCRRANRPESKQRSFGGTGGNVSVTSQSGKVIPSWESYRSGGEIRL.
9.RABV(CVS11),DNA,11583bp(SEQ ID NO.13)
ACGCTTAACAACAAAACCAGAGAAGAAAAAGACAGCGTCAATTGCAAAGCAAAAATGTAACACCCCTACAATGGATGCCGACAAGATTGTGTTCAAAGTCAATAATCAGGTGGTCTCTTTGAAGCCTGAGATTATCGTGGATCAATATGAGTACAAGTACCCTGCCATCAAGGATTTGAAAAAGCCTTGTATCACCCTAGGGAAAGCCCCCGACTTGAACAAAGCATACAAATCAGTTTTATCAGGCATGAATGCCGCCAAACTTGATCCGGATGATGTATGCTCCTACTTGGCAGCAGCAATGCAGTTCTTTGAGGGGACATGTCCGGAAGACTGGACCAGCTATGGAATCCTGATTGCACGAAAAGGAGATAGGATCACCCCAAACTCTCTAGTGGAGATAAAGCGTACTGATGTAGAAGGGAATTGGGCTCTGACAGGAGGCATGGAATTGACAAGGGACCCCACTGTCTCTGAACATGCATCTTTAGTCGGTCTTCTCCTGAGTCTGTACAGGTTGAGCAAAATATCAGGACAGAACACTGGTAACTATAAGACAAACATTGCAGATAGGATAGAGCAGATTTTCGAGACAGCACCTTTTGTTAAGATCGTGGAACACCATACCCTAATGACAACTCACAAGATGTGTGCTAATTGGAGTACTATACCGAACTTCAGATTTTTGGCCGGAACCTACGACATGTTTTTCTCACGGATTGAGCATCTGTATTCGGCAATCAGAGTGGGCACAGTCGTCACCGCTTATGAAGACTGCTCAGGACTGGTATCGTTTACAGGGTTCATAAAGCAGATCAATCTCACCGCAAGGGAAGCAATACTATATTTCTTCCACAAGAACTTTGAGGAAGAGATAAGAAGAATGTTCGAGCCAGGGCAAGAGACAGCTGTTCCTCACTCTTATTTCATCCACTTCCGTTCACTAGGCTTGAGTGGGAAGTCTCCTTATTCATCGAATGCTGTCGGTCATGTGTTCAATCTCATTCACTTTGTTGGATGCTACATGGGTCAAGTCAGATCTCTAAATGCGACGGTTATTGCTGCATGTGCCCCTCATGAGATGTCTGTTCTAGGGGGCTATTTGGGAGAGGAATTCTTCGGAAAAGGGACATTTGAAAGAAGGTTCTTCAGAGACGAGAAAGAACTTCAAGAATATGAGGCGGCTGAACTAACAAAGTCCGACGTGGCACTGGCAGATGACGGAACCGTCAACTCTGATGACGAGGACTATTTCTCTGGTGAAACCAGAAGTCCAGAAGCTGTCTATACTCGAATCATGATGAATGGAGGTCGACTGAAGAGATCTCATATACGGAGATATGTCTCAGTCAGTTCCAATCATCAAGCCCGTCCAAACTCATTCGCCGAATTTTTAAACAAGACGTATTCGAATGACTCATAAGGAGTTGATTGACAGGGTGCCAGAAATCTATAGATTGTATATATCCATCATGAAAAAAACTAACACTCCTCCTTTCAAACCATCCCAAATATGAGCAAGATCTTTGTTAATCCGAGTGCAATCAGAGCCGGTCTGGCCGATCTTGAGATGGCCGAAGAGACTGTTGATCTGATCAACAGAAACATAGAAGACAATCAGGCTCATCTCCAGGGAGAACCCATAGAAGTGGACAACCTCCCTGAGGACATGAAGCGACTTCACCTGGACGATGAAAAATCGTCCAACCTTGGTGAGATGGTTAGGGTGGGAGAAGGCAAGTATCGAGAGGACTTTCAGATGGATGAGGGAGAGGACCCCAACCTCCTGTTCCAATCGTACCTGGATAATGTTGGAGTCCAAATAGTCAGACAAATGAGGTCAGGAGAGAGATTCCTCAAGATATGGTCACAGACCGTAGAGGAAATTGTATCCTATGTCACGGTCAACTTTCCTAACCCTCCAAGAAGGTCTTCGGAGGATAAATCAACCCAGACTACTGGCAGAGAGCTCAAGAAGGAGACAACGTCTGCTTTCTCTCAGAGAGAAAGCCAACCTTCGAAAGCTAGGATGGTGGCTCAAGTTGCCCCTGGTCCTCCAGCCCTTGAATGGTCAGCCACCAATGAAGAAGATGATCTATCAGTAGAGGCTGAGATCGCTCATCAGATTGCTGAAAGCTTTTCCAAGAAGTACAAGTTTCCCTCCCGATCTTCAGGAATATTCTTGTATAATTTTGAGCAACTGAAGATGAACCTTGATGACATAGTTAAAGAGGCAAAAAATGTACCGGGCGTGACCCGTCTGGCCCATGATGGATCCAAAATCCCCCTGAGATGCGTACTGGGATGGGTCGCTTTGGCTAATTCCAAAAAATTCCAATTACTAGTCGAGGCTGACAAGCTAAGCAAAATCATGCAAGATGATTTGAATCGCTATACATCCTGCTAACCGAGTTTTCGAACTCAGTCCCTCCAGATAATGAAAACTGAGATGTTATGGAGTAGACATGAAAAAAACAGGCAACACCACTGATAAAATGAACGTTCTACGCAAGATAGTGAAAAAATGTAGGGATGAGGACACTCAAAAGCCCTCTCCTGTGTCAGCCCCTCCGTATGACGATGACCTGTGGCTTCCACCTCCTGAATATGTCCCGCTGAAAGAACTCACAAGCAAGAAGAACATGAGGAACTTTTGTGTCAACGGGGAGGTTAAAGCGTGTAGCCCAAATGGTTACTCATTCAGGATTTTGCGGCACATTCTGAGATCATTCAACGAGATATACTCTGGGAATCATAGGATGATTGGGTTAGTCAAAGTTGTTGTTGGACTAGCTTTATCAGGAGCTCCAGTACCTGAGGGCATGAACTGGGTATACAAATTGAGGAGAACCCTTATATTCCAGTGGGCTGATTCCAGGGGCCCTCTTGAAGGGGAGGAGTTAGAATACTCTCAAGAGATCACTTGGGATGATGATACTGAATTCGTCGGATTGCAAATAAGAGTGAGCGCAAGACAATGTCATATTCAAGGCAGGATCTGGTGTATCAACACGAACTCGAGGGCATGTCAACTATGGTCTGACATGTCTCTTCAGACACAAAGGTCTGAAGAGGACAAAGACTCTTCTCTGCTTCTAGAATAATCAGATTATATCCTGCAAGTGTATCACTTGTTTACCTCTGGAGGAGAGAGCATACAGGCTTGACTCCGATCCTTGGGAGCAATAGAACAAAAAAACACACGTTATGGTGCCGTTAAATCGCTGCATTTTATCAAAGTCAAGTTGATAACCTTTACATTTTGAGCCTCTTGGATGTGAAAAAAACTATTAACATCCCTCAAAAGACTTAAGGAAAGATGGTTCCTCAGGTTCTTTTGTTTGTACTCCTTCTGGGTTTTTCGTTGTGTTTCGGGAAGTTCCCCATTTACACGATACCAGACGAACTTGGTCCCTGGAGCCCTATTGACATACACCATCTCAGCTGTCCAAATAACCTGGTTGTGGAGGATGAAGGATGTACCAACCTGTCCGAGTTCTCCTACATGGAACTCAAAGTGGGATACATCTCAGCCATCAAAGTGAACGGGTTCACTTGCACAGGTGTTGTGACAGAGGCAGAGACCTACACCAACTTTGTTGGTTATGTCACAACCACATTCAAGAGAAAGCATTTCCGCCCCACCCCAGACGCATGTAGAGCCGCGTATAACTGGAAGATGGCCGGTGACCCCAGATATGAAGAGTCCCTACACAATCCATACCCCGACTACCACTGGCTTCGAACTGTAAGAACCACCAAAGAGTCCCTCATTATCATATCCCCAAGTGTGACAGATTTGGACCCATATGACAAATCCCTTCACTCAAGGGTCTTCCCTGGCGGAAAGTGCTCAGGAATAACGGTGTCCTCTACCTACTGCTCAACTAACCATGATTACACCATTTGGATGCCCGAGAATCCGAGACCAAGGACACCTTGTGACATTTTTACCAATAGCAGAGGGAAGAGAGCATCCAACGGGAACAAGACTTGCGGCTTTGTGGATGAAAGAGGCCTGTATAAGTCTCTAAAAGGAGCATGCAGGCTCAAGTTATGTGGAGTTCTTGGACTTAGACTTATGGATGGAACATGGGTCGCGATGCAAACATCAGATGAGACCAAATGGTGCCCTCCAGATCAGTTGGTGAATTTGCACGACTTTCGCTCAGACGAGATTGAGCATCTCGTTGTGGAGGAGTTAGTCAAGAAAAGAGAGGAATGTCTGGATGCATTAGAGTCCATCATGACCACCAAGTCAGTAAGTTTCAGACGTCTCAGTCACCTGAGAAAACTTGTCCCAGGGTTTGGAAAAGCATATACCATATTCAACAAAACCTTGATGGAGGCTGATGCTCACTACAAGTCAGTCCGGACCTGGAATGAGATCATCCCCTCAAAAGGGTGTTTGAAAGTTGGAGGAAGGTGCCATCCTCATGTGAACGGGGTGTTTTTCAATGGTATAATATTAGGGCCTGACGACCATGTCCTAATCCCAGAGATGCAATCATCCCTCCTCCAGCAACATATGGAGTTGTTGAAATCTTCAGTTATCCCCCTGATGCACCCCCTGGCAGACCCTTCTACAGTTTTCAAAGAAGGTGATGAGGCTGAGGATTTTGTTGAAGTTCACCTCCCCGATGTGTACAAACAGATCTCAGGGGTTGACCTGGGTCTCCCGAACTGGGGAAAGTATGTATTGATGACTGCAGGGGCCATGATTGGCCTGGTGTTGATATTTTCCCTAATGACATGGTGCAGAAGAGCCAATCGACCAGAATCGAAACAACGCAGTTTTGGAGGGACAGGGGGGAATGTGTCAGTCACTTCCCAAAGCGGAAAAGTCATACCTTCATGGGAATCATATAGGAGTGGAGGTGAGATCAGACTGTGAAGGCCGGTCATCCTTTTGACGATTCCAGTCCCGAGGATAACCAAAAAAACTAACACTTGCGCGCTAACGTACGTAAGAACCGCGGGCTGGGTCATCTAAGCATTTCAGTCGAGAAAAAAACTGTAGACCAAAAGAACAACTAGCAACACTTCTCATCCAGAGACCCATATCAAGATGCTAGATCCGGGAGAGGTTTATGATGACCCTATTGATCCAATTGAGTCAGAGGCTGAACCCAGAGGAACCCCCACTGTCCCCAACATCTTGAGGAACTCCGACTACAATCTCAATTCTCCTTTGATAGAGGATCCTGCCAAACTAATGTTAGAATGGTTGAAGACAGGGAACAGACCTTATCGGATGACTTTGACAGACAATTGCTCCAGGTCTTACAAAGTTTTGAAAGATTATTTCAAGAAAGTAGATTTGGGTTCTCTCAAAGTGGGCGGAACTGCTGCACAGTCAATGGTTTCTCTCTGGTTGTGTGGTGCCCACTCTGAATCAAACAGGAGCCGGAGATGTATAACCGACTTGGCCCATTTCTATTCCAAGTCATCCCCCATAGAGAAGCTATTGAATTGTACGCTAGGAAACAGAGGCCTGAGAATCCCACCAGAGGGGGTGTTAAATTGCCTCGAGAGGGTCAATTATGACAAGGCATTTGGGAGGTATCTGGCCAACACGTATTCCTCTTACTTGTTTTTCCATGTAATCACCTTATACATGAATGCCTTAGACTGGGAAGAGGAAAAGACCATCCTGGCATTATGGAAAGATATAACCTCAGTGGATACCGAGAAGGACTTGGTCAAATTCAAAGATCAAATATGGGGACTGTTGATTGTGACAAAGGACTTTGTTTACTCTCAGAGTTCAAACTGTCTTTTTGACAGAAACTACACACTGATGCTAAAGGATCTTTTCTTGTCTCGATTCAACTCCTTAATGATTCTGCTTTCTCCCCCTGAGCCCCGATACTCAGATGACTTAATATCTCAGCTGTGCCAGCTATACATCGCTGGGGATCAAGTCTTGTCCATGTGTGGGAACTCCGGCTATGAAGTCATCAAAATATTGGAGCCATATGTCGTGAACAGTTTGGTCCAGAGGGCAGAGAAGTTTAGGCCTCTCATCCACCCCTTGGGAGACTTTCCTATGTTCATAAAAGACAAGGTGAATCAACTTGAAGGGACTTTTGGTCCCAGTGCAAAGAGGTTTTTTAGGGTTTTAGATCAATTCGACAACATACATGACTTAGTATTTGTGTATGGCTGTTACAGACATTGGGGGCACCCCTATATAGATTATCGGAAGGGTCTGTCGAAACTATATGATCAAGTTCACATTAAGAAAGTAATAGATAAGTCCTACCAGGAGTGTTTAGCAAGTGACTTGGCCAGAAGGATCCTCAGATGGGGATTTGACAAGTACTCCAAATGGTATCTAGATTCGAGATTCCTTGCCCGAGACCACCCCTTGACTCCTTATGTCAAGACCCAAACATGGCCACCCAAACATATAGTAGACTTGGTGGGGGACACATGGCATAAGCTCCCGATCACGCAGATCTTTGAAATTCCTGAATCAATGGACCCGTCAGAGATACTGGATGACAAATCACATTCTTTCACCAGAACAAGGTTAGCTTCTTGGCTGTCAGAGAACCGAGGGGGGCCTGTTCCTAGCGAGAAGGTCATTATCACGGCCCTGTCTAAGCCACCTGTCAATCCCCGAGAGTTTTTGAAATCTATAGACCTCGGAGGATTGCCAGATGAGGATTTGATAATTGGCCTCAAACCAAAGGAACGGGAGTTGAAGATCGAGGGCCGATTCTTTGCTCTAATGTCATGGAATCTAAGATTATATTTTGTCATCACCGAAAAGCTCTTGGCCAACTACATTTTGCCACTTTTTGACGCACTGACTATGACAGACAACCTGAACAAGGTGTTCAAAAAGTTGATCGACAGGGTCACCGGGCAAGGGCTTTTGGACTATTCTAGGGTCACATACGCATTTCACCTGGACTATGAGAAATGGAACAATCATCAAAGATTGGAGTCAACAGAGGATGTATTCTCTGTCCTAGATCAGGTGTTTGGATTGAAGAGGGTGTTTTCTAGAACACACGAGTTTTTTCAGAAGTCCTGGATCTATTATTCAGACAGATCAGACCTCATTGGGTTATGGGAGGACCAAATATATTGCTTGGATATGTCTAACGGCCCAACCTGCTGGAATGGCCAAGATGGCGGGCTAGAGGGCTTGCGGCAGAAGGGCTGGAGTCTAGTCAGTTTATTAATGATAGATAGAGAATCTCAAACCAGGAACACAAGAACCAAGATACTAGCTCAAGGAGACAACCAGGTTCTGTGTCCTACATACATGTTGTCACCGGGATTGTCTCAAGAGGGGCTTCTCTATGAGTTAGAGAGCATATCAAGGAATGCACTCTCAATATACCGAGCTATCGAGGAAGGAGCATCTAAGCTGGGGCTGATCATCAAGAAGGAAGAGACCATGTGTAGTTATGACTTTCTCATATATGGGAAGACCCCCTTATTTCGAGGCAACATATTGGTGCCTGAATCCAAAAGATGGGCCCGAGTCTCTTGCATCTCTAACGACCAAATAGTCAACCTCGCCAATATAATGTCGACAGTATCCACCAATGCGCTGACAGTGGCACAACACTCTCAATCTCTGATCAAACCTATGAGGGATTTTCTGCTCATGTCAGTACAGGCAGTTTTCCACTACCTGTTGTTTAGCCCAATCCTAAAAGGCAGAGTTTATAAGATTCTGAGTGCTGAAGGGGAGAGCTTTCTCCTAGCCATGTCGCGGATAATCTACCTAGATCCTTCTTTGGGAGGGGTGTCTGGAATGTCTCTCGGGAGGTTCCATATACGTCAGTTCTCAGACCCTGTCTCTGAAGGGTTGTCATTCTGGAGAGAGATCTGGTTAGGCTCTCATGAGTCCTGGATTCACGCGTTGTGTCAGGAGGCCGGGAACCCCGATCTTGGAGAGAGAACACTCGAGAGCTTCACTCGCCTTTTAGAAGATCCTACTACCTTAAATATCAAAGGAGGGGCCAGCCCTACCATTCTACTCAAGGATGCTATCAGAAAGGCTCTGTACGACGAGGTGGACAAGGTGGAAAATTCAGAGTTTCGAGAGGCAATCCTGCTGTCCAAGACCCATAGAGATAACTTTATACTCTTTTTAAAATCTGTTGAGCCTCTGTTCCCTCGATTTCTCAGTGAGCTCTTCAGTTCGTCTTTCTTGGGAATACCGGAGTCAATCATTGGACTGATACAAAACTCCCGGACAATAAGAAGGCAGTTTAGAAAGAGTCTCTCAAGAACTTTAGAAGAGTCCTTCTACAACTCAGAGATCCACGGGATTAATCGGATAACCCAGACACCTCAAAGGGTCGGAAGGGTGTGGCCTTGCTCTTCAGAGAGGGCAGATCTACTTAGGGAGATCTCTTGGGGGAGGAAAGTGGTAGGCACGACAGTTCCTCACCCTTCCGAGATGTTGGAGTTGTTTCCCAAATCCTCCATTTCCTGCACTTGTGGAGCAACAGGGGGAGGCAATCCTAGAGTCTCTGTATCAGTACTCCCGTCCTTCGATCAGTCATTTTTCTCACGGGGCCCCCTAAAGGGATACTTGGGCTCGTCCACCTCCATGTCAACCCAGCTATTCCATGCATGGGAAAAAGTCACTAATGTTCATGTGGTGAAAAGGGCTCTATCGTTAAAAGAATCTATAAACTGGTTCATCAATAGGAATTCCAATTTGGCTCAAACTCTAATTGGAAACATCATGTCTCTGACAGGCCCTGATTTCCCTCTAGAAGAGGCCCCTGTTTTCAAACGGACAGGGTCAGCCTTGCATAGGTTCAAGTCTGCCAGATACAGCGAAGGAGGGTATTCTTCTGTTTGCCCTAACCTTCTCTCCCATATCTCTGTTAGTACAGACACTATGTCTGATTTGACCCAAAACGGGAAGAACTATGATTTCATGTTTCAGCCATTGATGCTTTATGCGCAAACATGGACATCGGAACTGGTACAGAGGGATACAAGACTTAGAGACTCCACGTTTCACTGGCACCTTCGGTGCAACAGATGTGTGAGGCCCATTGATGATATAACACTGGAAACTTCTCAGATCTTCGAGTTCCCGGATGTGTCAAAAAGGATATCCAGGATGGTTTCTGGAGCTGTCCCTCAATTTCAGAAGCTTCCTGATATCCGTCTAAGACCAGGTGATTTTGAATCCCTAAGTGGTAGAGAAAAGTCTCGCCATATAGGGTCAGCTCAGGGGCTCTTATACTCAATCTTAGTAGCAATTCACGACTCAGGATACAATGATGGGACCATCTTCCCTGTCAACATATACGGCAAAGTTTCCCCCAGAGACTATTTGAGAGGGCTTGCAAGAGGGATCTTGATAGGGTCCTCGATTTGCTTCTTGACACGAATGACAAATATTAACATTAACAGACCTCTTGAATTGATCTCAGGGGTAATTTCCTATATTCTCCTGAGGCTGGATAATCATCCCTCTCTGTATATAATGCTTAGAGAACCGTCTCTTAGAGGAGAAATATTCTCTATCCCTCAGAAAATCCCCGCCGCTTACCCAACCACTATGAGAGAAGGCAACAGATCGATTTTGTGTTACCTCCAACACGTGCTACGCTATGAGCGAGAGGCAATCACGGCGTCCCCGGAGAATGACTGGCTGTGGATCTTCTCAGACTTCAGAAGTGTGAAAATGACGTACTTGACCCTCATTACCTACCAGTCTCACCTCCTACTCCAGAGGGTTGAGAGAAACTTGTCTAAGAGTATGAGAGCTACTCTGCGACAAATGAGTTCCTTAATGAGGCAAGTGCTGGGTGGGCACGGAGAAGATACCTTGGAGTCAAACGATGACATTCAACGATTACTAAAAGACTCTTTGCGAAAGACAAGGTGGGTGGATCAAGAGGTGCGCCATGCAGCTAGAACCATGAATGGAGATTACAGCCCCGACAAGAAAGTATCCCACAAGGCAGGATGTTCAGAATGGGTCTGCTCTGCTCAACAGATTGCCGTCTCCACCTCAGCCAACCCGGCCCCTGTCTCAGAGCTTGACATTAGGGCCCTCTCTAAGAGGTTTCAAAACCCCTTGATCTCGGGCCTGAGAGTGGTTCAGTGGGCAACCGGTGCCCATTATAAGCTTAAGCCTATTCTAGATGATCTAAATGTTTTCCCATCTCTCTGTCTTGTAATTGGAGACGGGTCAGGGGGAATATCAAGGGCAGTTCTCAACATGTTTCCAGATTCTAAGCTTGTGTTCAACAGCCTATTGGAGGTGAATGATCTGATGGCTTCCGGAACACATCCACTGCCTCCTTCAGCAATCATGAGTGGAGGAGATGACATCATCTCCAGAGTGATAGACTTTGACTCAATATGGGAGAAACCGTCCGACCTGAGGAACTCGGCCACCTGGAGATACTTCCAGTCGGTTCAAAAACAGGTCAACATGTCGTATGACCTCATTATTTGTGATGCAGAAGTTACTGATATTGCATCTATCAACCGGATAACTCTGTTGATGTCTGATTTCGCATTGTCTATAGATGGACCACTTTATCTGGTCTTCAAAACTTACGGGACTATGCTAGTAAACCCGGACTATAAAGCTATTCAACATCTGTCAAGAGCGTTCCCTTCGGTCACAGGGTTTGTAACCCAAGTAACTTCATCCTTTTCTTCTGAGCTATACCTCCGGTTCTCTAAACGAGGAAAGTTTTTCAGGGACGCCGAGTACTTGACCTCTTCCACCCTTCGAGAGATGAGCCTTGTGTTGTTCAATTGCAGCAGCCCCAAAAGTGAGATGCAGAGAGCTCGTTCCTTAAACTATCAAGACCTGGTAAGGGGATTTCCTGAAGAGATCATATCAAATCCTTACAACGAAATGATCATAACTCTGATTGACAATGATGTAGAGTCCTTCCTAGTCCACAAGATGGTGGATGATCTTGAGCTACAGAGGGGAACTCTGTCTAAAGTGGCTATCATTATATCCATCATGATCGTTTTTTCCAATAGAGTCTTCAACATTTCCAAACCTTTGACTGACCCCTTGTTCTACCCCCCATCTGATCCTAAAATCCTGAGGCACTTCAACATATGTTGCAGTACTATGATGTATCTATCTACCGCTTTAGGCGACGTCCCTAACTTCGCAAGACTTCATGACCTGTATAACAGACCTATAACTTGTTACTTCAGAAAACAAGTTATTCGAGGGAATATTTATCTATCTTGGAGTTGGTCCGATGATACCCCAGTGTTCAAGAGAGTAGCCTGTAATTCTAGCTTGAGTCTGTCATCTCACTGGATCAGGTTGATCTACAAGATAGTGAAGACTACCAGACTCATTGGCAGCATAAAAGACCTATCAGGAGAGGTAGAACGACATCTTCATGGGTATAACAGATGGATCACCCTCGAGGATATCCGATCTAGATCATCCCTACTAGATTACAGTTGTTTGTAAGCCGGATATTACCGAAAGCCTGTGCATGCTAAAATTCTTGTATGATGCATCTTGAAAAAAACAAGATCTTGAATCCGGACCTCTGGTTGTTTGATTGTTTTTTCCATCTTTATTGTTTTTTTGTTAAGCGT
10.RABV(CVS11),氨基酸,3861aa(SEQ ID NO.14)
TLNNKTREEKDSVNCKAKM.HPYNGCRQDCVQSQ.SGGLFEA.DYRGSI.VQVPCHQGFEKALYHPRESPRLEQSIQISFIRHECRQT.SG.CMLLLGSSNAVL.GDMSGRLDQLWNPDCTKRR.DHPKLSSGDKAY.CRRELGSDRRHGIDKGPHCL.TCIFSRSSPESVQVEQNIRTEHW.L.DKHCR.DRADFRDSTFC.DRGTPYPNDNSQDVC.LEYYTELQIFGRNLRHVFLTD.ASVFGNQSGHSRHRL.RLLRTGIVYRVHKADQSHRKGSNTIFLPQEL.GRDKKNVRARARDSCSSLLFHPLPFTRLEWEVSLFIECCRSCVQSHSLCWMLHGSSQISKCDGYCCMCPS.DVCSRGLFGRGILRKRDI.KKVLQRRERTSRI.GG.TNKVRRGTGR.RNRQL..RGLFLW.NQKSRSCLYSNHDEWRSTEEISYTEICLSQFQSSSPSKLIRRIFKQDVFE.LIRS.LTGCQKSIDCIYPS.KKLTLLLSNHPKYEQDLC.SECNQSRSGRS.DGRRDC.SDQQKHRRQSGSSPGRTHRSGQPP.GHEATSPGR.KIVQPW.DG.GGRRQVSRGLSDG.GRGPQPPVPIVPG.CWSPNSQTNEVRREIPQDMVTDRRGNCILCHGQLS.PSKKVFGG.INPDYWQRAQEGDNVCFLSERKPTFES.DGGSSCPWSSSP.MVSHQ.RR.SISRG.DRSSDC.KLFQEVQVSLPIFRNILV.F.ATEDEP..HS.RGKKCTGRDPSGP.WIQNPPEMRTGMGRFG.FQKIPITSRG.QAKQNHAR.FESLYILLTEFSNSVPPDNEN.DVME.T.KKQATPLIK.TFYAR..KNVGMRTLKSPLLCQPLRMTMTCGFHLLNMSR.KNSQARRT.GTFVSTGRLKRVAQMVTHSGFCGTF.DHSTRYTLGIIG.LG.SKLLLD.LYQELQYLRA.TGYTN.GEPLYSSGLIPGALLKGRS.NTLKRSLGMMILNSSDCK.E.AQDNVIFKAGSGVSTRTRGHVNYGLTCLFRHKGLKRTKTLLCF.NNQIISCKCITCLPLEERAYRLDSDPWEQ.NKKTHVMVPLNRCILSKSS..PLHFEPLGCEKNY.HPSKDLRKDGSSGSFVCTPSGFFVVFREVPHLHDTRRTWSLEPY.HTPSQLSK.PGCGG.RMYQPVRVLLHGTQSGIHLSHQSERVHLHRCCDRGRDLHQLCWLCHNHIQEKAFPPHPRRM.SRV.LEDGR.PQI.RVPTQSIPRLPLASNCKNHQRVPHYHIPKCDRFGPI.QIPSLKGLPWRKVLRNNGVLYLLLN.P.LHHLDARESETKDTL.HFYQ.QREESIQREQDLRLCG.KRPV.VSKRSMQAQVMWSSWT.TYGWNMGRDANIR.DQMVPSRSVGEFARLSLRRD.ASRCGGVSQEKRGMSGCIRVHHDHQVSKFQTSQSPEKTCPRVWKSIYHIQQNLDGG.CSLQVSPDLE.DHPLKRVFESWRKVPSSCERGVFQWYNIRA.RPCPNPRDAIIPPPATYGVVEIFSYPPDAPPGRPFYSFQRR..G.GFC.SSPPRCVQTDLRG.PGSPELGKVCIDDCRGHDWPGVDIFPNDMVQKSQSTRIETTQFWRDRGECVSHFPKRKSHTFMGII.EWR.DQTVKAGHPFDDSSPEDNQKN.HLRANVRKNRGLGHLSISVEKKTVDQKNN.QHFSSRDPYQDARSGRGL..PY.SN.VRG.TQRNPHCPQHLEELRLQSQFSFDRGSCQTNVRMVEDREQTLSDDFDRQLLQVLQSFERLFQESRFGFSQSGRNCCTVNGFSLVVWCPL.IKQEPEMYNRLGPFLFQVIPHREAIELYARKQRPENPTRGGVKLPREGQL.QGIWEVSGQHVFLLLVFPCNHLIHECLRLGRGKDHPGIMERYNLSGYREGLGQIQRSNMGTVDCDKGLCLLSEFKLSF.QKLHTDAKGSFLVSIQLLNDSAFSP.APILR.LNISAVPAIHRWGSSLVHVWELRL.SHQNIGAICREQFGPEGREV.ASHPPLGRLSYVHKRQGEST.RDFWSQCKEVF.GFRSIRQHT.LSICVWLLQTLGAPLYRLSEGSVETI.SSSH.ESNR.VLPGVFSK.LGQKDPQMGI.QVLQMVSRFEIPCPRPPLDSLCQDPNMATQTYSRLGGGHMA.APDHADL.NS.INGPVRDTG.QITFFHQNKVSFLAVREPRGACS.REGHYHGPV.ATCQSPRVFEIYRPRRIAR.GFDNWPQTKGTGVEDRGPILCSNVMESKIIFCHHRKALGQLHFATF.RTDYDRQPEQGVQKVDRQGHRARAFGLF.GHIRISPGL.EMEQSSKIGVNRGCILCPRSGVWIEEGVF.NTRVFSEVLDLLFRQIRPHWVMGGPNILLGYV.RPNLLEWPRWRARGLAAEGLESSQFINDR.RISNQEHKNQDTSSRRQPGSVSYIHVVTGIVSRGASL.VREHIKECTLNIPSYRGRSI.AGADHQEGRDHV.L.LSHIWEDPLISRQHIGA.IQKMGPSLLHL.RPNSQPRQYNVDSIHQCADSGTTLSISDQTYEGFSAHVSTGSFPLPVV.PNPKRQSL.DSEC.RGELSPSHVADNLPRSFFGRGVWNVSREVPYTSVLRPCL.RVVILERDLVRLS.VLDSRVVSGGREPRSWRENTRELHSPFRRSYYLKYQRRGQPYHSTQGCYQKGSVRRGGQGGKFRVSRGNPAVQDP.R.LYTLFKIC.ASVPSISQ.ALQFVFLGNTGVNHWTDTKLPDNKKAV.KESLKNFRRVLLQLRDPRD.SDNPDTSKGRKGVALLFREGRST.GDLLGEESGRHDSSSPFRDVGVVSQILHFLHLWSNRGRQS.SLCISTPVLRSVIFLTGPPKGILGLVHLHVNPAIPCMGKSH.CSCGEKGSIVKRIYKLVHQ.EFQFGSNSNWKHHVSDRP.FPSRRGPCFQTDRVSLA.VQVCQIQRRRVFFCLP.PSLPYLC.YRHYV.FDPKREEL.FHVSAIDALCANMDIGTGTEGYKT.RLHVSLAPSVQQMCEAH..YNTGNFSDLRVPGCVKKDIQDGFWSCPSISEAS.YPSKTR.F.IPKW.RKVSPYRVSSGALILNLSSNSRLRIQ.WDHLPCQHIRQSFPQRLFERACKRDLDRVLDLLLDTNDKY.H.QTS.IDLRGNFLYSPEAG.SSLSVYNA.RTVS.RRNILYPSENPRRLPNHYERRQQIDFVLPPTRATL.ARGNHGVPGE.LAVDLLRLQKCENDVLDPHYLPVSPPTPEG.EKLV.EYESYSATNEFLNEASAGWARRRYLGVKR.HSTITKRLFAKDKVGGSRGAPCS.NHEWRLQPRQESIPQGRMFRMGLLCSTDCRLHLSQPGPCLRA.H.GPL.EVSKPLDLGPESGSVGNRCPL.A.AYSR.SKCFPISLSCNWRRVRGNIKGSSQHVSRF.ACVQQPIGGE.SDGFRNTSTASFSNHEWRR.HHLQSDRL.LNMGETVRPEELGHLEILPVGSKTGQHVV.PHYL.CRSY.YCIYQPDNSVDV.FRIVYRWTTLSGLQNLRDYASKPGL.SYSTSVKSVPFGHRVCNPSNFILFF.AIPPVL.TRKVFQGRRVLDLFHPSRDEPCVVQLQQPQK.DAESSFLKLSRPGKGIS.RDHIKSLQRNDHNSD.Q.CRVLPSPQDGG.S.ATEGNSV.SGYHYIHHDRFFQ.SLQHFQTFD.PLVLPPI.S.NPEALQHMLQYYDVSIYRFRRRP.LRKTS.PV.QTYNLLLQKTSYSREYLSILELVR.YPSVQESSL.F.LESVISLDQVDLQDSEDYQTHWQHKRPIRRGRTTSSWV.QMDHPRGYPI.IIPTRLQLFVSRILPKACAC.NSCMMHLEKNKILNPDLWLFDCFFHLYCFFVKR
SEQUENCE LISTING
<110> 吉林大学
<120> 一种嵌合表达分子佐剂的重组狂犬病病毒及其制备方法与应用
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 603
<212> DNA
<213> MAB2560
<400> 1
atgcgaacta tcgcaatccg acatcgagca gtcatcggac tgtccgctgt cgctctgatc 60
accgtgggct gcagcaacgg cacctccgtg gacgtgcctt ctcctgaagt gggcctgatc 120
gccaccacca gctctgccgc tcctgctcag cctgccgagg tgaagctgat cggcgagcgg 180
gatgtggaag tgaccctgac cggacccatt gctgccaagt actccagcgc ctctgaatct 240
cagaagcagg ccctcggcaa acccctgacc ggcgaccaca acgccggcac cagagagtcc 300
ggcgctgtgt tccagcagtt tcaaggcggc gccatcatcg ccaagaacaa tcaggctggc 360
acaccagcct tcatcgtggt cggcaagatc agagatgcct ggaacatcca gcgggacgcc 420
gacggcacac cttccatcac cggcaacaat ggctctgctg gacctctggg cctgcctacc 480
tccgacgaga acaccgaggg cgaccagctg gtgtccacct tcgagcacgg caagatcgag 540
tacaacgcta agtccggcga agtggccgtg acagtgaacg gaaaagtggt gccctctggt 600
ctg 603
<210> 2
<211> 63
<212> DNA
<213> RABV CVS11-G
<400> 2
atggttcctc aggttctttt gtttgtactc cttctgggtt tttcgttgtg tttcgggaaa 60
ttt 63
<210> 3
<211> 198
<212> DNA
<213> RABV CVS11-G TMCD
<400> 3
tacgtattga tgactgcagg ggccatgatt ggcctggtgt tgatattttc cctaatgaca 60
tggtgcagaa gagccaatcg accagaatcg aaacaacgca gttttggagg gacagggggg 120
aatgtgtcag tcacttccca aagcggaaaa gtcatacctt catgggaatc atataggagt 180
ggaggtgaga tcagactg 198
<210> 4
<211> 864
<212> DNA
<213> SP-MAB2560-TMCD
<400> 4
atggttcctc aggttctttt gtttgtactc cttctgggtt tttcgttgtg tttcgggaaa 60
tttcgaacta tcgcaatccg acatcgagca gtcatcggac tgtccgctgt cgctctgatc 120
accgtgggct gcagcaacgg cacctccgtg gacgtgcctt ctcctgaagt gggcctgatc 180
gccaccacca gctctgccgc tcctgctcag cctgccgagg tgaagctgat cggcgagcgg 240
gatgtggaag tgaccctgac cggacccatt gctgccaagt actccagcgc ctctgaatct 300
cagaagcagg ccctcggcaa acccctgacc ggcgaccaca acgccggcac cagagagtcc 360
ggcgctgtgt tccagcagtt tcaaggcggc gccatcatcg ccaagaacaa tcaggctggc 420
acaccagcct tcatcgtggt cggcaagatc agagatgcct ggaacatcca gcgggacgcc 480
gacggcacac cttccatcac cggcaacaat ggctctgctg gacctctggg cctgcctacc 540
tccgacgaga acaccgaggg cgaccagctg gtgtccacct tcgagcacgg caagatcgag 600
tacaacgcta agtccggcga agtggccgtg acagtgaacg gaaaagtggt gccctctggt 660
ctgtacgtat tgatgactgc aggggccatg attggcctgg tgttgatatt ttccctaatg 720
acatggtgca gaagagccaa tcgaccagaa tcgaaacaac gcagttttgg agggacaggg 780
gggaatgtgt cagtcacttc ccaaagcgga aaagtcatac cttcatggga atcatatagg 840
agtggaggtg agatcagact gtga 864
<210> 5
<211> 287
<212> PRT
<213> SP-MAB2560-TMCD
<400> 5
Met Val Pro Gln Val Leu Leu Phe Val Leu Leu Leu Gly Phe Ser Leu
1 5 10 15
Cys Phe Gly Lys Phe Arg Thr Ile Ala Ile Arg His Arg Ala Val Ile
20 25 30
Gly Leu Ser Ala Val Ala Leu Ile Thr Val Gly Cys Ser Asn Gly Thr
35 40 45
Ser Val Asp Val Pro Ser Pro Glu Val Gly Leu Ile Ala Thr Thr Ser
50 55 60
Ser Ala Ala Pro Ala Gln Pro Ala Glu Val Lys Leu Ile Gly Glu Arg
65 70 75 80
Asp Val Glu Val Thr Leu Thr Gly Pro Ile Ala Ala Lys Tyr Ser Ser
85 90 95
Ala Ser Glu Ser Gln Lys Gln Ala Leu Gly Lys Pro Leu Thr Gly Asp
100 105 110
His Asn Ala Gly Thr Arg Glu Ser Gly Ala Val Phe Gln Gln Phe Gln
115 120 125
Gly Gly Ala Ile Ile Ala Lys Asn Asn Gln Ala Gly Thr Pro Ala Phe
130 135 140
Ile Val Val Gly Lys Ile Arg Asp Ala Trp Asn Ile Gln Arg Asp Ala
145 150 155 160
Asp Gly Thr Pro Ser Ile Thr Gly Asn Asn Gly Ser Ala Gly Pro Leu
165 170 175
Gly Leu Pro Thr Ser Asp Glu Asn Thr Glu Gly Asp Gln Leu Val Ser
180 185 190
Thr Phe Glu His Gly Lys Ile Glu Tyr Asn Ala Lys Ser Gly Glu Val
195 200 205
Ala Val Thr Val Asn Gly Lys Val Val Pro Ser Gly Leu Tyr Val Leu
210 215 220
Met Thr Ala Gly Ala Met Ile Gly Leu Val Leu Ile Phe Ser Leu Met
225 230 235 240
Thr Trp Cys Arg Arg Ala Asn Arg Pro Glu Ser Lys Gln Arg Ser Phe
245 250 255
Gly Gly Thr Gly Gly Asn Val Ser Val Thr Ser Gln Ser Gly Lys Val
260 265 270
Ile Pro Ser Trp Glu Ser Tyr Arg Ser Gly Gly Glu Ile Arg Leu
275 280 285
<210> 6
<211> 33
<212> DNA
<213> MAB2560-F
<400> 6
atacgtacga tggttcctca ggttcttttg ttt 33
<210> 7
<211> 30
<212> DNA
<213> MAB2560-R
<400> 7
ataccgcggt cacagtctga tctcacctcc 30
<210> 8
<211> 18
<212> DNA
<213> JD-F
<400> 8
accagaatcg aaacaacg 18
<210> 9
<211> 19
<212> DNA
<213> JD-R
<400> 9
tgaaatgctt agatgaccc 19
<210> 10
<211> 201
<212> PRT
<213> MAB2560
<400> 10
Met Arg Thr Ile Ala Ile Arg His Arg Ala Val Ile Gly Leu Ser Ala
1 5 10 15
Val Ala Leu Ile Thr Val Gly Cys Ser Asn Gly Thr Ser Val Asp Val
20 25 30
Pro Ser Pro Glu Val Gly Leu Ile Ala Thr Thr Ser Ser Ala Ala Pro
35 40 45
Ala Gln Pro Ala Glu Val Lys Leu Ile Gly Glu Arg Asp Val Glu Val
50 55 60
Thr Leu Thr Gly Pro Ile Ala Ala Lys Tyr Ser Ser Ala Ser Glu Ser
65 70 75 80
Gln Lys Gln Ala Leu Gly Lys Pro Leu Thr Gly Asp His Asn Ala Gly
85 90 95
Thr Arg Glu Ser Gly Ala Val Phe Gln Gln Phe Gln Gly Gly Ala Ile
100 105 110
Ile Ala Lys Asn Asn Gln Ala Gly Thr Pro Ala Phe Ile Val Val Gly
115 120 125
Lys Ile Arg Asp Ala Trp Asn Ile Gln Arg Asp Ala Asp Gly Thr Pro
130 135 140
Ser Ile Thr Gly Asn Asn Gly Ser Ala Gly Pro Leu Gly Leu Pro Thr
145 150 155 160
Ser Asp Glu Asn Thr Glu Gly Asp Gln Leu Val Ser Thr Phe Glu His
165 170 175
Gly Lys Ile Glu Tyr Asn Ala Lys Ser Gly Glu Val Ala Val Thr Val
180 185 190
Asn Gly Lys Val Val Pro Ser Gly Leu
195 200
<210> 11
<211> 21
<212> PRT
<213> RABV CVS11-G
<400> 11
Met Val Pro Gln Val Leu Leu Phe Val Leu Leu Leu Gly Phe Ser Leu
1 5 10 15
Cys Phe Gly Lys Phe
20
<210> 12
<211> 66
<212> PRT
<213> RABV CVS11-G TMCD
<400> 12
Tyr Val Leu Met Thr Ala Gly Ala Met Ile Gly Leu Val Leu Ile Phe
1 5 10 15
Ser Leu Met Thr Trp Cys Arg Arg Ala Asn Arg Pro Glu Ser Lys Gln
20 25 30
Arg Ser Phe Gly Gly Thr Gly Gly Asn Val Ser Val Thr Ser Gln Ser
35 40 45
Gly Lys Val Ile Pro Ser Trp Glu Ser Tyr Arg Ser Gly Gly Glu Ile
50 55 60
Arg Leu
65
Claims (10)
1.一种嵌合表达分子佐剂的重组狂犬病病毒,其特征在于,所述重组狂犬病病毒是狂犬病病毒CVS11株为母本毒株,在母本毒株的G基因和L基因之间同时表达MAB2560蛋白、狂犬病病毒的G蛋白信号肽和狂犬病病毒的G蛋白TMCD区连接的融合蛋白。
2.根据权利要求1所述的重组狂犬病病毒,其特征在于,所述MAB2560蛋白的氨基酸序列如SEQ ID NO.10所示;所述狂犬病病毒的G蛋白信号肽的氨基酸序列如SEQ ID NO.11所示;所述狂犬病病毒的G蛋白的氨基酸序列如SEQ ID NO.12所示。
3.根据权利要求1所述的重组狂犬病病毒,其特征在于,编码MAB2560蛋白的基因序列如SEQ ID NO.1所示;编码狂犬病病毒的G蛋白信号肽的基因序列如SEQ ID NO.2所示;编码狂犬病病毒的G蛋白的基因序列如SEQ ID NO.3所示。
4.根据权利要求1所述的重组狂犬病病毒,其特征在于,编码融合蛋白的基因序列的获得方法如下:编码MAB2560蛋白的基因序列的5’端连接编码狂犬病病毒G蛋白信号肽的基因序列,编码MAB2560蛋白的基因序列的3’端连接编码狂犬病病毒G蛋白TMCD区的基因序列。
5.权利要求1-4任一项所述的重组狂犬病病毒的制备方法,其特征在于,所述制备方法的步骤如下:
步骤1:获得SP-MAB2560-TMCD基因序列如SEQ ID NO.4所示,将SEQ ID NO.4所示的序列插入到狂犬病病毒CVS11全基因组的G基因和L基因之间获得重组基因序列;
步骤2:将步骤1获得的重组基因序列转入到pcDNA3.1载体、pcDNA3.0载体或pCAGGS载体中获得重组载体;
步骤3:将步骤3获得的重组载体与辅助质粒N、P、L、G共转染BSR细胞,获得重组狂犬病病毒。
6.权利要求1-4任一项所述的重组狂犬病病毒在制备预防或治疗狂犬病疫苗中的应用。
7.一种嵌合表达分子佐剂的重组狂犬病病毒的融合蛋白,其特征在于,所述融合蛋白是由狂犬病病毒的G蛋白信号肽、MAB2560蛋白和狂犬病病毒的G蛋白TMCD区依次连接获得的融合蛋白。
8.根据权利要求7所述的融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ IDNO.5所示。
9.编码权利要求7或8所述的融合蛋白的基因。
10.权利要求7或8所述的融合蛋白或权利要求9所述的编码基因在制备预防或治疗狂犬病疫苗或药物中的应用。
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