CN107022530A - 重组腺病毒载体表达体内自组装呼吸道合胞病毒样颗粒及其制备方法和应用 - Google Patents
重组腺病毒载体表达体内自组装呼吸道合胞病毒样颗粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一种重组腺病毒表达自组装呼吸道合胞病毒样颗粒,所述病毒样颗粒包含呼吸道合胞病毒F和M蛋白,所述F和M蛋白是密码子优化的F和M基因进行编码,F和M基因由2A序列连接,所述连接后的核苷酸序列如SEQ ID No.4所示。本发明还公开了上述呼吸道合胞病毒样颗粒的制备方法及在制备用于预防或治疗呼吸道合胞病毒感染疫苗或药物、呼吸道合胞病毒抗体和呼吸道合胞病毒的诊断试剂中的应用。本发明采用单一重组腺病毒为载体,建立了一种体内自组装表达呼吸道合胞病毒样颗粒的方法,重组腺病毒制备相对简单、显著降低成本,获得较好的免疫效果及免疫保护,并且提供了安全性。
Description
技术领域
本发明涉及基因工程领域。更具体地,涉及一种重组腺病毒载体表达体内自组装呼吸道合胞病毒样颗粒及其制备方法和应用。
背景技术
人呼吸道合胞病毒(Human respiratory syncytial virus,RSV)是引起全球5岁以下婴幼儿下呼吸道感染最重要的病毒性病原,常引发肺炎、支气管炎(Hall,C.B.,E.A.Simoes,and L.J.Anderson,Clinical and epidemiologic features ofrespiratory syncytial virus.Curr Top Microbiol Immunol,2013.372:p.39-57.)。老年人和免疫缺陷病人也是RSV的易感人群(Jorquera,P.A.,L.Anderson,and R.A.Tripp,Understanding respiratory syncytial virus(RSV)vaccine development and aspectsof disease pathogenesis.Expert Rev Vaccines,2016.15(2):p.173-87.)。人在一生中可反复感染RSV,自然感染RSV仅能诱导短期的免疫力,产生不完全的保护(Graham,B.S.,K.Modjarrad,and J.S.McLellan,Novel antigens for RSV vaccines.Curr OpinImmunol,2015.35:p.30-8.)。统计数据显示:1岁以内的婴幼儿,有超过60%的急性呼吸道感染、超过80%的下呼吸道感染是由RSV引起(Mazur,N.I.,et al.,Lower respiratorytract infection caused by respiratory syncytial virus:current management andnew therapeutics.Lancet Respir Med,2015.3(11):p.888-900。)。世界卫生组织估计,每年在世界范围内RSV可引起至少6400万人发病和16万人死亡。尽管RSV危害严重,但是尚缺乏可靠、有效的方法来诊断、预防及治疗RSV病毒感染(Girard,M.P.,et al.,A review ofvaccine research and development:human acute respiratory infections.Vaccine,2005.23(50):p.5708-24;Murata,Y.,Respiratory syncytial virus vaccinedevelopment.Clin Lab Med,2009.29(4):p.725-39.)。具有预防RSV病毒感染作用的RSV疫苗亟待研制。世界卫生组织及美国医学研究院有关21世纪疫苗报告中均将开发RSV疫苗列为最优先发展的疫苗项目之一。
RSV属于副黏病毒科,肺炎病毒属,有一个血清型,二个抗原亚型,单股负链RNA病毒,共编码11种蛋白,其中跨膜包膜糖蛋白F为RSV的主要中和抗原,基质蛋白M为RSV的骨架蛋白(McLellan,J.S.,W.C.Ray,and M.E.Peeples,Structure and function ofrespiratory syncytial virus surface glycoproteins.Curr Top Microbiol Immunol,2013.372:p.83-104)。
病毒样颗粒(Virus-like particles,VLPs)是利用病毒衣壳或其他结构蛋白的自组装能力形成的一种具有病毒形态结构特征的颗粒状物质,不含病毒核酸,仅包括大量重复的病毒表面蛋白。病毒的糖蛋白像天然病毒一样插入到VLPs的膜上,从而保持了与天然病毒一样的大小、空间构象、重复排列的中和抗原表位和颗粒特征,VLPs的病毒抗原表位不会发生丢失、变性和增加,能诱导产生有效的免疫应答。
VLPs作为RSV候选疫苗的研究取得了初步成果,但临床应用仍存在许多问题有待探讨。例如,RSV的F蛋白有5-6个N糖基化位点,这些糖基化位点对于F蛋白空间构象、融合活性、抗原性等都具有重要作用,但采用昆虫和禽细胞表达系统制备RSV VLPs,可能存在蛋白质翻译后修饰修饰不足,影响VLPs的空间构象和免疫原性(Fang,N.X.,I.H.Frazer,andG.J.Fernando,Differences in the post-translational modifications of humanpapillomavirus type 6b major capsid protein expressed from a baculovirussystem compared with a vaccinia virus system.Biotechnol Appl Biochem,2000.32(Pt 1):p.27-33;Zhou,W.,et al.,Molecular characterization of recombinantHepatitis B surface antigen from Chinese hamster ovary and Hansenulapolymorpha cells by high-performance size exclusion chromatography and multi-angle laser light scattering.J Chromatogr B Analyt Technol Biomed Life Sci,2006.838(2):p.71-7.)。而以天然病毒感染的宿主(或同源宿主)来源的表达系统能产生与天然病毒生物活性高度一致的VLPs,并诱导产生最有效的免疫应答,取得更好的免疫保护效果(Frietze,K.M.,D.S.Peabody,and B.Chackerian,Engineering virus-likeparticles as vaccine platforms.Curr Opin Virol,2016.18:p.44-9.)。另一方面,VLPs的体外制备、表达和纯化成本高昂,纯化过程也可能引发蛋白构象变化而降低免疫原性。这些不足为其临床应用带来较大限制。
腺病毒载体(Adenovirus vector,Adv)对分裂期及非分裂期细胞均具有高感染效率、遗传稳定性好、易制备高滴度病毒等多种优点被广泛用于基因治疗和疫苗载体研究。重组腺病毒具有体外复制滴度高,制备成本低,能高效表达外源蛋白及安全性好等特点。因此,建立一种以腺病毒为载体的VLPs的新方法,既能发挥VLPs的作用,又能适于大规模生产,且能有效降低成本。
发明内容
本发明的第一个目的在于提供一种重组腺病毒表达自组装呼吸道合胞病毒样颗粒。
本发明的第二个目的在于提供上述呼吸道合胞病毒样颗粒的制备方法。
本发明的第三个目的在于提供上述呼吸道合胞病毒样颗粒的应用。
为达到上述目的,本发明采用下述技术方案:
一种重组腺病毒表达自组装呼吸道合胞病毒样颗粒,该病毒样颗粒包含呼吸道合胞病毒F和M蛋白,所述F和M蛋白分别采用哺乳动物细胞偏爱的密码子优化的F和M基因进行编码。
进一步,所述密码子优化的F基因的核苷酸序列如序列表SEQ ID No.1所示;所述密码子优化的M基因的核苷酸序列如序列表SEQ ID No.2所示;
进一步,所述重组腺病毒表达的呼吸道合胞病毒F和M蛋白的基因由如SEQ IDNo.3所示的2A序列连接,连接后得到的核苷酸序列如SEQ ID No.4所示。
进一步,所述重组腺病毒为一种复制缺陷型重组腺病毒Ad5载体系统,包括一个骨架质粒、一个穿梭质粒和一个包装细胞系。
本发明基于腺病毒的生物递送和表达系统,提供了一种重组腺病毒表达呼吸道合胞病毒F和M蛋白自组装成呼吸道合胞病毒样颗粒的制备方法,包括以下步骤:
(1)按照哺乳动物细胞密码子偏好分别优化RSV F、M基因;PCR方法用2A序列连接F和M基因,得到F-M基因,其核苷酸序列如SEQ ID No.4所示;
(2)将F-M基因克隆到穿梭载体中,得到携带F-M基因的穿梭质粒,并进一步将穿梭质粒线性化后转化到携带腺病毒基因组pAdeasy-1的BJ5183的感受态细胞中进行重组,经筛选鉴定获得重组质粒pAdeasy-F-M;
(3)将重组质粒pAdeasy-F-M线性化后转染HEK293细胞,获得重组腺病毒rAd-F-M;
(4)用CsCl密度梯度法离心纯化重组腺病毒rAd-F-M;
(5)将纯化后的重组腺病毒rAd-F-M感染Vero细胞,用透射电镜可观察病毒样颗粒的形成。
优选地,所述用透射电镜观察病毒样颗粒的步骤为:
(1)重组腺病毒rAd-F-M感染Vero细胞72h后收取细胞,2.5%戊二醛固定;
(2)经脱水、包埋、固化后做超薄切片;
(3)3%醋酸铀-柠檬酸铅双染色;
(4)透射电镜观察、拍照。
优选地,所述穿梭载体为pShuttle载体。
本发明进一步提供了重组腺病毒表达自组装呼吸道合胞病毒样颗粒在制备用于预防或治疗呼吸道合胞病毒感染疫苗或药物、呼吸道合胞病毒抗体和呼吸道合胞病毒的诊断试剂中的应用。
其中,所述疫苗为注射剂、口服剂、滴鼻剂或透皮剂。
本发明的有益效果如下:
在本发明中,对RSV A亚型Long株F、M蛋白基因进行了密码子优化,有效的增加了蛋白的表达量。本发明以单一腺病毒为载体,通过构建可共表达RSV F-M的重组腺病毒rAd-F-M,感染宿主细胞后可原位产生自组装的内生性RSV VLPs(endogenous RSV VLPs,RSVeVLPs),避免了体外制备、纯化VLP面临的诸多问题,可在最大程度上保持F蛋白天然的构象和免疫原性,诱导机体产生更具保护性的中和抗体。因此,以重组腺病毒载体传递RSV F和M蛋白原位组装VLPs的方法,制备相对简单、显著降低成本,有助于提高RSV疫苗的安全性、免疫效果和免疫保护作用。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出pShuttle-CMV-F-M酶切后琼脂糖凝胶电泳图,1:DL15000 DNA Marker;2:目的基因,F-M,大小是2547bp;
图2示出重组腺病毒质粒pAdeasy-F-M经Pac I酶切后的琼脂糖凝胶电泳图,1:DL15000 DNA Marker;2:目的基因F-M,条带大小是33Kb和4.5Kb的片段;
图3示出为重组腺病毒rAd-F-M感染293细胞,导致293细胞发生病变,A:病变的293细胞,B:正常的293细胞;
图4示出重组腺病毒rAd-F-M感染293细胞72h后细胞上清的Western blot鉴定结果,A为RSV F单抗检测F蛋白的条带,B为RSV M多抗检测M蛋白的条带,位置均在270kDa处;
图5示出氯化铯密度梯度离心法纯化重组腺病毒rAd-F-M形成的条带;
图6示出氯化铯密度梯度离心法纯化重组腺病毒rAd-F-M的电镜照片;
图7示出重组腺病毒rAd-F-M感染Vero细胞形成VLPs的电镜观察结果(3%醋酸铀-柠檬酸铅双染色,JEM-1400,20,000×);
图8示出免疫后小鼠血清抗体分析,A为RSV特异性血清抗体滴度检测结果,B为中和抗体滴度检测结果;
图9示出免疫后小鼠在RSV攻毒后肺病毒滴度分析结果,A为实时定量PCR法检测小鼠肺病毒滴度的结果,B为对肺病毒滴度检测结果的数据分析;
图10示出免疫后小鼠在RSV攻毒后肺病理分析结果,A为肺血管周围炎症评分,B为肺支气管周围炎症评分,C为肺间质炎症评分。
具体实施方式
为了更清楚地说明本发明,下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
本发明所用的呼吸道合胞病毒和重组腺病毒来自北京交通大学生物工程与生命科学研究院基因工程实验室。
实施例1重组穿梭质粒的构建
1.构建:为提高RSV F、M蛋白在哺乳动物细胞中的表达量,采用哺乳动物细胞偏好的密码子对RSV F、M密码子进行优化并全基因合成,PCR方法用2A序列连接F和M,经Xho I、Sac II双酶切后将目的片段连接至pShuttle载体。
2.酶切鉴定及测序:所获得的质粒经Xho I、Sac II双酶切鉴定,琼脂糖凝胶电泳结果在2547bp处和7500bp处出现特异性条带(图1),分别与F-M和pShuttle载体大小一致,酶切鉴定阳性结果的质粒经测序验证为正确质粒,重组质粒pShuttle-F-M构建成功。
实施例2构建含有F-M基因的重组腺病毒质粒
1.线性化穿梭质粒及重组
Pme I线性化所获得的重组质粒pShuttle-F-M,取0.5μg的已线性化的pShuttle-F-M转化至携带腺病毒基因组pAdeasy-1的BJ5183的感受态细胞中进行重组,Kana+抗性LB平板37℃培养16h,挑取单菌落进行培养,并提取质粒。
2.酶切筛选鉴定重组质粒
Pac I酶切所获得的质粒,经琼脂糖凝胶电泳检测重组质粒酶切结果(图2),电泳结果显示获得了4.5Kb和33Kb二个片段。经酶切鉴定获得正确重组质粒pAdeasy-F-M。
3.扩增重组质粒
用获得的正确重组质粒pAdeasy-F-M转化Ecoli.DH10B感受态,扩增并纯化获得较大量的pAdeasy-F-M质粒。具体方法参考AdEasy Adenoviral Vector System第24页:Amplifying recombinant Ad plasmid。
实施例3转染HEK293细胞包装重组腺病毒
1.Pac I线性化重组腺病毒质粒pAdeasy-F-M
用Pac I酶切重组腺病毒质粒pAdeasy-F-M,乙醇沉淀、室温干燥,取20μl灭菌去离子水溶解。
2.转染HEK293细胞
HEK293细胞来自于由中国疾病预防控制中心病毒病预防控制研究所。
HEK293细胞接种细胞培养板(6孔板),转染时细胞丰度约为50%-70%;取10μlPac I线性化处理的重组腺病毒质粒pAdeasy-F-M加入240μl opti-MEM(Gibco)并混匀,10μl Lipofactamine 2000加入240μl opti-MEM并混匀,室温孵育5min;将上述二种溶液混匀,室温孵育20min。将上述500μl的转染复合物加入细胞培养基中,于37℃培养继续培养4h后更换新的培养基。约在转染后10d左右,HEK293细胞出现细胞肿胀、变圆、折光性增强等典型的细胞病变(CPE)(图3A),图3B为正常细胞对照。收集细胞及上清于-80℃、37℃反复冻融3次,取离心后的上清(P0代)感染HEK293细胞,至细胞出现完全CPE,重复上述操作,获得P1、P2、P3代病毒。
实施例4重组腺病毒蛋白表达及鉴定
1.接种重组腺病毒于HEK293细胞,72h后收获上清,上清经冷冻干燥保存于-80℃。
2.取冻干浓缩10倍的细胞上清上样做SDS-PAGE电泳。
3.转膜做Western blot,分别用RSV F单抗(131-2A;Merck Millipore)、RSV M小鼠多抗检测,结果在上清中均可检测到约270kDa的条带,说明RSVF蛋白与M蛋白结合在一起,且均成功表达,结果见图4。
实施例5重组腺病毒rAd-F-M的制备和纯化
1.扩大培养HEK293细胞,约用15个150mm培养皿,细胞传代6h后(丰度为90%)接种重组腺病毒,约3d后细胞呈现完全CPE且约有50%脱落时收获细胞上清,于4℃5000rpm离心10min,收获细胞。
2.用PBS重悬细胞沉淀,冻存于-80℃。
3.氯化铯密度梯度法离心纯化重组腺病毒(图5)。具体方法参考DJPalmer,PNg.Methods for the Production of First Generation Adenoviral Vectors.Methodsin Molecular Biology,2008,433(433):55-78
4.收集纯化后的腺病毒经透析后加入5%甘油保存于-80℃。
5.取10μl纯化样品制备电镜负染样品,并用透射电镜观察拍照(图6)。
实施例6重组腺病毒滴度测定
重组腺病毒滴度测定参考Clontech的Adeno-X Rapid Titer Kit方法检测。以2.5×105个细胞/孔接种HEK293细胞于24孔板,10倍系列稀释病毒,每孔加入100μl病毒稀释液,37℃5%CO2培养箱中继续培养48h,经固定后加入鼠抗-Hexon抗体、相应的二抗、TMB显色,在显微镜下计数,计算病毒滴度(方法参考:Clontech,Adeno-X Rapid Titer KitManual第9页的Adeno-XTM Rapid Titer Procedure),所获得的重组腺病毒滴度为:2.4×1010PFU/mL。
实施例7电镜检测重组腺病毒rAd-F-M感染Vero细胞组装VLPs
为检测所获得的重组腺病毒rAd-F-M是否能在细胞内表达组装VLPs,用30MOI的rAd-F-M感染Vero细胞,72h收集细胞,2.5%戊二醛固定,经脱水、包埋、固化后制备电镜超薄切片,3%醋酸铀-柠檬酸铅双染色,用透射电镜观察拍照,观察到病毒样颗粒形成(图7)。
实施例8重组腺病毒免疫小鼠诱导产生RSV特异性抗体及中和抗体
为评价重组腺病毒免疫小鼠抗体产生情况,通过滴鼻途径免疫小鼠,检测小鼠血清中RSV特异性抗体产生情况(图8A),抗体滴度测定是采用ELISA方法检测。进一步地,分析了所获得的小鼠血清中的RSV特异性中和抗体的滴度(图8B)。从抗体及中和抗体的数据分析,采用重组腺病毒rAd-F-M表达RSV F、M能有效诱导小鼠产生抗体及中和抗体,rAd-F-M与rAd-F两组之间没有统计学差异,这意味着这种方法没有影响RSV F蛋白的功能。
实施例9重组腺病毒免疫小鼠诱导RSV特异性免疫保护
重组腺病毒rAd-F-M诱导的免疫保护是通过用RSV Long株对免疫后的小鼠进行攻毒来检测。在RSV感染后的第5天取小鼠的肺脏进行肺病毒滴度检测(图9)及肺病理分析(图10)。对数据进行分析的结果显示,rAd-F-M及rAd-F均能有效地降低小鼠肺组织中的RSV病毒滴度,且rAd-F-M免疫组的肺病毒数量仅为rAd-F免疫组的12%(表1)。肺病理分析结果显示:rAd-F-M免疫组较rAd-F免疫组能更有效地减轻小鼠肺血管周围炎病变的严重程度(图10)。
表1对肺病毒滴度检测结果的数据分析
显然,本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定,对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动,这里无法对所有的实施方式予以穷举,凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
SEQUENCE LISTING
<110> 北京交通大学
<120> 重组腺病毒载体表达体内自组装呼吸道合胞病毒样颗粒及其制备方法和应用
<130> JLC17I0053EA
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1725
<212> DNA
<213> 人工合成的密码子优化后的F基因序列
<400> 1
atggagctgc ctatcctgaa ggccaacgcc atcaccacaa ttctggccgc cgtgaccttc 60
tgttttgcca gcagccagaa catcaccgag gagttctacc agagcacctg tagcgccgtg 120
agcaagggct atctgagcgc cctgagaacc ggctggtaca ccagcgtgat caccatcgag 180
ctgagcaaca tcaaggagaa caagtgcaac ggcaccgacg ccaaggtgaa gctgatgaag 240
caggagctgg acaagtacaa gaacgccgtg accgaactgc agctgctgat gcagtctacc 300
cctgccgcca acaacagagc cagacgggag ctgccccggt tcatgaacta caccctgaac 360
aacaccaaga aaaccaacgt gaccctgagc aagaagcgga agcggagatt cctgggcttt 420
ctgctgggag tgggctctgc catcgcctct ggcatcgccg tgtctaaggt gctgcacctg 480
gagggagagg tgaacaagat caagagcgcc ctgctgagca ccaataaggc cgtggtgagc 540
ctgagcaatg gcgtgagcgt gctgacaagc aaggtgctgg acctcaagaa ctacatcgac 600
aagcagctgc tgcccatcgt gaacaagcag agctgccgga tcagcaacat cgagaccgtg 660
atcgagttcc agcagaagaa caaccggctg ctggagatca ccagggagtt cagcgtgaat 720
gtgggcgtga ccacccctgt gagcacctac atgctgacca acagcgagct gctgagcctg 780
atcaacgaca tgcccatcac caacgaccag aagaagctga tgtccaacaa cgtgcagatc 840
gtgcggcagc agagctacag catcatgtcc atcatcaagg aggaggtgct ggcttacgtg 900
gtgcagctgc ctctgtacgg cgtgatcgac accccttgct ggaagctgca caccagccct 960
ctgtgcacca ccaataccaa ggagggcagc aacatctgcc tgaccaggac cgatagaggc 1020
tggtactgcg acaatgccgg cagcgtgagc ttctttccac aggccgagac ctgtaaggtg 1080
cagagcaacc gggtgttctg cgacaccatg aacagcctga ccctgccttc tgaggtgaac 1140
ctgtgcaacg tggacatctt caaccccaag tacgactgca agatcatgac cagcaagacc 1200
gacgtgagca gcagcgtgat tacaagcctg ggcgccatcg tgagctgtta cggcaagacc 1260
aagtgcaccg ccagcaacaa gaaccgcggc atcatcaaga ccttcagcaa cggctgcgac 1320
tacgtgagca acaagggcgt ggatacagtg agcgtgggca acaccctgta ctacgtcaac 1380
aagcaggagg gcaagagcct gtacgtgaag ggcgagccca tcatcaactt ctacgacccc 1440
ctggtgttcc ctagcgacga gttcgatgcc agcatcagcc aggtgaacga gaagatcaac 1500
cagagcctgg ccttcatcag gaagagcgac gagctgctgc acaatgtgaa cgccggcaag 1560
agcaccacca acatcatgat caccaccatc atcatcgtga tcatcgtcat cctgctgtcc 1620
ctgattgctg tgggcctgct gctgtactgt aaggccagaa gcacccccgt gaccctgtct 1680
aaggatcagc tgagcggcat caacaacatc gccttctcca actga 1725
<210> 2
<211> 771
<212> DNA
<213> 人工合成的密码子优化后的M基因序列
<400> 2
atggaaacct acgtgaacaa gctgcacgag ggcagcacct acacagccgc cgtgcagtac 60
aacgtgctgg aaaaggacga cgaccccgcc agcctgacca tctgggtgcc catgttccag 120
agcagcatgc ccgccgacct gctgatcaaa gaactggcca acgtgaacat cctggtcaag 180
cagatcagca cccccaaggg ccccagcctg agagtgatga tcaacagcag aagcgccctg 240
ctggcccaga tgcccagcaa gttcaccatc tgcgccaacg tgtccctgga cgagcggagc 300
aagctggcct acgacgtgac caccccctgc gagatcaagg cctgcagcct gacctgcctg 360
aagtccaaga acatgctgac caccgtgaag gacctgacca tgaagaccct gaaccccacc 420
cacgacatca ttgccctgtg cgagttcgag aacatcgtga ccagcaagaa agtgatcatc 480
cccacctacc tgcggagcat cagcgtgcgg aacaaggacc tgaacaccct ggaaaacatc 540
accaccaccg agttcaagaa cgccatcacc aacgccaaga tcatccctta cagcggcctg 600
ctgctggtca tcaccgtgat cgacaacaag ggcgccttca agtacatcaa gccccagagc 660
cagttcatcg tggacctggg cgcctacctg gaaaaagaat ccatctacta cgtcaccacc 720
aactggaagc acaccgccac cagattcgcc atcaagccca tggaagattg a 771
<210> 3
<211> 108
<212> DNA
<213> 人工合成的2A序列
<400> 3
gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctgagggc 60
agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccct 108
<210> 4
<211> 2547
<212> DNA
<213> F-M基因序列
<400> 4
atggagctgc ctatcctgaa ggccaacgcc atcaccacaa ttctggccgc cgtgaccttc 60
tgttttgcca gcagccagaa catcaccgag gagttctacc agagcacctg tagcgccgtg 120
agcaagggct atctgagcgc cctgagaacc ggctggtaca ccagcgtgat caccatcgag 180
ctgagcaaca tcaaggagaa caagtgcaac ggcaccgacg ccaaggtgaa gctgatgaag 240
caggagctgg acaagtacaa gaacgccgtg accgaactgc agctgctgat gcagtctacc 300
cctgccgcca acaacagagc cagacgggag ctgccccggt tcatgaacta caccctgaac 360
aacaccaaga aaaccaacgt gaccctgagc aagaagcgga agcggagatt cctgggcttt 420
ctgctgggag tgggctctgc catcgcctct ggcatcgccg tgtctaaggt gctgcacctg 480
gagggagagg tgaacaagat caagagcgcc ctgctgagca ccaataaggc cgtggtgagc 540
ctgagcaatg gcgtgagcgt gctgacaagc aaggtgctgg acctcaagaa ctacatcgac 600
aagcagctgc tgcccatcgt gaacaagcag agctgccgga tcagcaacat cgagaccgtg 660
atcgagttcc agcagaagaa caaccggctg ctggagatca ccagggagtt cagcgtgaat 720
gtgggcgtga ccacccctgt gagcacctac atgctgacca acagcgagct gctgagcctg 780
atcaacgaca tgcccatcac caacgaccag aagaagctga tgtccaacaa cgtgcagatc 840
gtgcggcagc agagctacag catcatgtcc atcatcaagg aggaggtgct ggcttacgtg 900
gtgcagctgc ctctgtacgg cgtgatcgac accccttgct ggaagctgca caccagccct 960
ctgtgcacca ccaataccaa ggagggcagc aacatctgcc tgaccaggac cgatagaggc 1020
tggtactgcg acaatgccgg cagcgtgagc ttctttccac aggccgagac ctgtaaggtg 1080
cagagcaacc gggtgttctg cgacaccatg aacagcctga ccctgccttc tgaggtgaac 1140
ctgtgcaacg tggacatctt caaccccaag tacgactgca agatcatgac cagcaagacc 1200
gacgtgagca gcagcgtgat tacaagcctg ggcgccatcg tgagctgtta cggcaagacc 1260
aagtgcaccg ccagcaacaa gaaccgcggc atcatcaaga ccttcagcaa cggctgcgac 1320
tacgtgagca acaagggcgt ggatacagtg agcgtgggca acaccctgta ctacgtcaac 1380
aagcaggagg gcaagagcct gtacgtgaag ggcgagccca tcatcaactt ctacgacccc 1440
ctggtgttcc ctagcgacga gttcgatgcc agcatcagcc aggtgaacga gaagatcaac 1500
cagagcctgg ccttcatcag gaagagcgac gagctgctgc acaatgtgaa cgccggcaag 1560
agcaccacca acatcatgat caccaccatc atcatcgtga tcatcgtcat cctgctgtcc 1620
ctgattgctg tgggcctgct gctgtactgt aaggccagaa gcacccccgt gaccctgtct 1680
aaggatcagc tgagcggcat caacaacatc gccttctcca acgagggcag aggaagtctt 1740
ctaacatgcg gtgacgtgga ggagaatccc ggccctatgg aaacctacgt gaacaagctg 1800
cacgagggca gcacctacac agccgccgtg cagtacaacg tgctggaaaa ggacgacgac 1860
cccgccagcc tgaccatctg ggtgcccatg ttccagagca gcatgcccgc cgacctgctg 1920
atcaaagaac tggccaacgt gaacatcctg gtcaagcaga tcagcacccc caagggcccc 1980
agcctgagag tgatgatcaa cagcagaagc gccctgctgg cccagatgcc cagcaagttc 2040
accatctgcg ccaacgtgtc cctggacgag cggagcaagc tggcctacga cgtgaccacc 2100
ccctgcgaga tcaaggcctg cagcctgacc tgcctgaagt ccaagaacat gctgaccacc 2160
gtgaaggacc tgaccatgaa gaccctgaac cccacccacg acatcattgc cctgtgcgag 2220
ttcgagaaca tcgtgaccag caagaaagtg atcatcccca cctacctgcg gagcatcagc 2280
gtgcggaaca aggacctgaa caccctggaa aacatcacca ccaccgagtt caagaacgcc 2340
atcaccaacg ccaagatcat cccttacagc ggcctgctgc tggtcatcac cgtgatcgac 2400
aacaagggcg ccttcaagta catcaagccc cagagccagt tcatcgtgga cctgggcgcc 2460
tacctggaaa aagaatccat ctactacgtc accaccaact ggaagcacac cgccaccaga 2520
ttcgccatca agcccatgga agattga 2547
Claims (10)
1.一种重组腺病毒表达自组装呼吸道合胞病毒样颗粒,其特征在于:所述病毒样颗粒包含呼吸道合胞病毒F和M蛋白,所述F和M蛋白是密码子优化的F和M基因进行编码。
2.根据权利要求1所述的呼吸道合胞病毒样颗粒,其特征在于:所述密码子优化的F基因的核苷酸序列如序列表SEQ ID No.1所示;所述密码子优化的M基因的核苷酸序列如序列表SEQ ID No.2所示。
3.根据权利要求1或2所述的呼吸道合胞病毒样颗粒,其特征在于:所述密码子优化的F和M基因由如SEQ ID No.3所示的2A序列连接,所述连接后的核苷酸序列如SEQ ID No.4所示。
4.根据权利要求1或2所述的呼吸道合胞病毒样颗粒,其特征在于:所述重组腺病毒为一种复制缺陷型重组腺病毒Ad5载体系统,包括一个骨架质粒、一个穿梭质粒和一个包装细胞系。
5.一种如权利要求1-4任一所述的呼吸道合胞病毒样颗粒的制备方法,其特征在于,包括以下步骤:
(1)按照密码子偏好分别优化人呼吸道合胞病毒F、M基因;用2A序列连接F和M基因,得到F-M基因,其核苷酸序列如SEQ ID No.4所示;
(2)将F-M基因克隆到穿梭载体中,得到携带F-M基因的穿梭质粒,并进一步将穿梭质粒转化到携带腺病毒基因组pAdeasy-1的BJ5183的感受态细胞中进行重组,经筛选鉴定获得重组质粒pAdeasy-F-M;
(3)重组质粒pAdeasy-F-M转染HEK293细胞,获得重组腺病毒rAd-F-M;
(4)用CsCl密度梯度法离心纯化重组腺病毒rAd-F-M;
(5)将纯化后的重组腺病毒rAd-F-M感染Vero细胞,用透射电镜可观察病毒样颗粒的形成。
6.权利要求1-4任一所述呼吸道合胞病毒样颗粒在制备用于预防或治疗呼吸道合胞病毒感染疫苗中的应用。
7.根据权利要求6所述的应用,其特征在于:所述疫苗为注射剂、口服剂、滴鼻剂或透皮剂。
8.权利要求1-4任一所述呼吸道合胞病毒样颗粒在制备用于预防或治疗呼吸道合胞病毒感染药物中的应用。
9.权利要求1-4任一所述呼吸道合胞病毒样颗粒在制备呼吸道合胞病毒抗体中的应用。
10.权利要求1-4任一所述呼吸道合胞病毒样颗粒在制备呼吸道合胞病毒诊断试剂中的应用。
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