CN114573666A - 一种猫细小病毒样颗粒及其制备方法与应用 - Google Patents
一种猫细小病毒样颗粒及其制备方法与应用 Download PDFInfo
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- CN114573666A CN114573666A CN202210285881.0A CN202210285881A CN114573666A CN 114573666 A CN114573666 A CN 114573666A CN 202210285881 A CN202210285881 A CN 202210285881A CN 114573666 A CN114573666 A CN 114573666A
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Abstract
本发明公开了一种猫细小病毒样颗粒及其制备方法与应用,选用自主分离的FPV JL‑125毒株的VP2序列进行重组杆状病毒FPV‑VP2株的制备,其中FPV JL‑125毒株和当前中国流行毒株有很高同源性,其VP2序列根据昆虫细胞密码子优化后,制备的重组杆状病毒FPV‑VP2株可正确表达FPVVP2蛋白。本发明采用全悬浮培养工艺培养,表达量大幅度提高同时,HA效价能达到220‑221,是野生病毒培养效价的256‑512倍。表达的蛋白能自主装配成完整的FPV病毒样颗粒,与原病毒具有相似的空间结构,而病毒样颗粒有效价高、安全性更高,同时具有激发体液免疫和细胞免疫的优点。本发明使用基因工程手段制备病毒样颗粒抗原,进行新型猫细小病毒样颗粒疫苗的制备,具有更高的安全性和有效性。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及一种猫细小病毒样颗粒及其制备方法与应用。
背景技术
猫细小病毒病(Feline panleukopenia,FP)又称猫泛白细胞减少症、猫瘟、猫传染性肠炎,是由猫细小病毒(Feline panleukopenia Virus,FPV)引起的一种急性高度接触性传染病,临床表现以患病猫突发高热、呕吐、腹泻、脱水及循环血流中白细胞数量急剧减少为特征。FPV主要感染猫科与鼬科等多种动物,尤其以6月龄以下幼小动物的发病率和死亡率更高,1岁以内的幼猫发病率高达83.5%,而且全窝幼猫会陆续发病,是猫科动物最重要的传染病,对猫科动物生命造成了严重威胁。
为了猫免遭传染病的威胁,保持其身心健康,对其接种疫苗是关键。目前,市售疫苗均为进口疫苗,主要为猫三联疫苗,包括FPV、FCV、FHV。这些疫苗均为弱毒疫苗或灭活疫苗,而弱毒疫苗有潜在的安全隐患,灭活疫苗不能有效刺激机体产生细胞免疫等缺点。此外,病毒变异是导致当前疫苗免疫失败的主要原因。
病毒样颗粒(virus-like particles,VLPs)是含有一种或多种病毒结构蛋白的空心颗粒,形态上类似于天然病毒,没有病毒核酸,不能自主复制,与天然病毒相比较其不具有致病性但不影响其免疫原性,且具有较高的安全性。VLPs结构稳定,颗粒均一性好,具有广泛的应用前景。VLPs常用表达系统包括大肠杆菌表达系统、酵母表达系统、杆状病毒/昆虫细胞表达系统、植物细胞表达系统、哺乳动物细胞表达系统、无细胞表达系统等。本发明的猫细小病毒样颗粒疫苗采用的是昆虫细胞-杆状病毒表达系统,该系统所需要的周期远比动物或植物系统短,可以利用昆虫个体或其培养细胞进行大规模的表达生产,生产的重组蛋白产量高,蛋白翻译后加工比细菌、酵母生产系统完善,而且由于昆虫杆状病毒具有限制性的宿主范围,只对特定种属的昆虫及其细胞进行感染,对人畜等脊椎动物没有感染能力,因此具有比在哺乳动物及其培养细胞生产系统更为安全等优点而成为目前最有效的真核表达系统之一。
FPV VLPs的制备通常是利用病毒结构蛋白VP2表达后组装而获得的。目前常用的表达体系主要分为真核表达体系和原核表达体系,真核包含昆虫细胞表达系统和酵母表达系统,而原核主要是大肠杆菌表达系统。目前比较成熟的CPV VLPs利用真核表达系统的为多,主要原因是真核系统有利于实现外源蛋白翻译后的修饰和折叠,表达的重组VP2结构蛋白具有天然的分子构象,利于VLPs的形成。
目前,采用全悬浮培养工艺培养昆虫杆状病毒VP2的表达量有大幅度提高,然而HA效价不够高,同时,VLPs的纯度也是病毒样颗粒疫苗的重要质量指标之一,目前的VLPs的纯度一般在80%左右,纯度不高的VLPs在临床应用上还存在一些过敏反应,因此开发一种效价、纯度更高的猫细小病毒样颗粒对于生产更加安全、有效的猫泛白细胞减少症疫苗具有重大意义和价值。
发明内容
本发明的目的之一是,提供一种猫细小病毒样颗粒,其氨基酸序列如SEQ ID NO.1所示。
上述的猫细小病毒样颗粒,表达其氨基酸序列的核酸序列如SEQ ID NO.2所示。
上述的猫细小病毒样颗粒,利用昆虫-杆状病毒表达系统表达。
上述的猫细小病毒样颗粒,其培养工艺为:将生长良好的Sf9细胞调整至1.2×106~1.5×106个/ml的细胞数量接种于生物反应器,在27℃、100~110r/min、DO 40%~60%的条件下悬浮发酵培养;生物反应器中Sf9细胞数量达到6.0×106~8.0×106个/ml时,补充等体积的新鲜培养基,按MOI为0.1~1.0接种,在27℃、100~110r/min、DO 40~60%的条件下继续培养;接毒后96~120小时收获细胞培养物。
上述的猫细小病毒样颗粒,其纯化工艺为:将收获的含有重组杆状病毒FPV-VP2的细胞培养物经细胞富集、裂解、去除细胞碎片、灭活后,使用100KDa膜包以Tris 20mmol/LpH值8.0、NaCl 0.05mol/L为缓冲液A对灭活原液进行换洗,然后采用Q Focurose HPL与缓冲液A进行线性洗脱,收集洗脱峰1,使用100KDa膜包进行10~50倍超滤浓缩,以4%柱床体积上样,采用以Focudex 200PG为介质的分子筛层析纯化,以PBS 10mmol/LpH值7.0为流动相进行洗脱,收集目标峰1,得到纯化后的猫细小病毒样颗粒。
本发明的目的之二是,提供一种上述的猫细小病毒样颗粒的制备方法,将生长良好的Sf9细胞调整至1.2×106~1.5×106个/ml的细胞数量接种于生物反应器,在27℃、100~110r/min、DO 40%~60%的条件下悬浮发酵培养;生物反应器中Sf9细胞数量达到6.0×106~8.0×106个/ml时,补充等体积的新鲜培养基,按MOI为0.1~1.0接种利用昆虫-杆状病毒表达系统表达重组杆状病毒FPV-VP2,在27℃、100~110r/min、DO 40~60%的条件下继续培养。接毒后96~120小时收获细胞培养物,将收获的细胞培养物经细胞富集、裂解、去除细胞碎片、灭活后,使用100KDa膜包以Tris20mmol/L pH值8.0、NaCl 0.05mol/L为缓冲液A对灭活原液进行换洗,然后采用Q Focurose HPL与缓冲液A进行线性洗脱,收集洗脱峰1,使用100KDa膜包进行10~50倍超滤浓缩,以4%柱床体积上样,采用以Focudex200PG为介质的分子筛层析纯化,以PBS 10mmol/L pH值7.0为流动相进行洗脱,收集目标峰1,得到纯化后的猫细小病毒样颗粒。
本发明的目的之三是,提供上述的猫细小病毒样颗粒在制备猫泛白细胞减少症疫苗中的应用。
具体应用可为,一种猫泛白细胞减少症疫苗,包括上述的猫细小病毒样颗粒。
或者一种多联疫苗,包括上述的猫细小病毒样颗粒。
或者一种多价疫苗,括上述的猫细小病毒样颗粒。
本发明选用自主分离的FPV JL-125毒株的VP2序列进行重组杆状病毒FPV-VP2株的制备。其中FPV JL-125毒株和当前中国流行毒株有很高同源性,其VP2序列根据昆虫细胞密码子优化后,制备的重组杆状病毒FPV-VP2株可正确表达FPV VP2蛋白。本发明采用全悬浮培养工艺培养,表达量大幅度提高同时,HA效价能达到220-221,是野生病毒(能达到212)培养效价的256-512倍。表达的蛋白能自主装配成完整的FPV病毒样颗粒,与原病毒具有相似的空间结构,而病毒样颗粒有效价、安全性更高、可同时激发体液免疫和细胞免疫的优点。本发明使用基因工程手段制备病毒样颗粒抗原(分子量65KD,颗粒直径22nm左右,60个蛋白组装成20面体空间结构),可进行新型猫细小病毒样颗粒疫苗的制备,具有更高的安全性和有效性。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明中记载的一些实施例,对于本领域普通技术人员来讲,还可以根据这些附图获得其他的附图。
图1为本发明实施例1提供的载体构建模式图。
图2为本发明实施例1提供的重组转移载体的酶切鉴定结果,其中1为pFD-FPV-dVP2经Not I+Hind III双酶切鉴定;2为pFD-FPV-dVP2经Xho I+Nhe I双酶切鉴定;M为Marker15000。
图3为本发明实施例1提供的重组杆状病毒PCR鉴定结果,图3A为pH和p10表达盒特异性引物扩增图,图中,1为pH引物PCR扩增产物;2为p10引物PCR扩增产物;M为Marker5000;图3B为VP2引物扩增图,M为Marker2000,1为PCR扩增产物。
图4为本发明实施例3提供的离子交换层析图谱。
图5为本发明实施例3提供的离子交换层析过程样品电泳图。
图6为本发明实施例3提供的离子交换洗脱峰1粒径检测结果。
图7为本发明实施例3提供的分子筛层析图谱。
图8为本发明实施例3提供的分子筛纯化样品电泳图。
图9为本发明实施例3提供的分子筛收获峰1电镜图。
图10为本发明实施例3提供的分子筛层析峰1粒径检测结果。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案,下面将结合实施例和附图对本发明作进一步的详细介绍。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。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。
实施例1重组杆状病毒的构建
选用自主分离的FPV JL-125毒株的VP2序列进行重组杆状病毒FPV-VP2株的制备。其中FPV JL-125毒株和当前中国流行毒株有很高同源性,其VP2序列根据昆虫细胞密码子优化后,制备的重组杆状病毒FPV-VP2株可正确表达FPV VP2蛋白。生产用毒种重组杆状病毒FPV-VP2株构建过程为:
1、VP2基因的优化与合成
对FPV JL-125毒株提取基因组进行测序后,获得VP2基因序列,根据Sf9昆虫细胞密码子偏爱性进行优化,将原始序列中串联的稀有密码子、减少翻译序列或者甚至解除翻译的序列替换为常用密码子,调整了GC含量及不宜峰以延长mRNA的半衰期,那些影响mRNA稳定性及其与核糖体结合的茎环结构被破坏。密码子偏爱性指标由0.65调整至0.95,GC含量由35%调整至59%。优化前的VP2基因序列如SEQ ID NO.3所示,优化后的VP2基因序列如SEQ ID NO.2所示,将优化后序列送公司进行基因合成。直接合成优化后的序列VP2-opti并克隆到载体pUC57上,两端分别带有Not I和Hind III酶切位点。
2、重组杆状病毒的构建与鉴定
将优化后的VP2序列分别连接至pFastBacDual转移载体的两个表达盒,见图1。将pFastBacDual和pUC57-VP2(Not I+Hind III)质粒分别进行双酶切处理,然后将纯化后的VP2片段连入双酶切处理的载体中,将连接产物与感受态细胞(E.coli DH5a)采用热休克方法进行转化,获得阳性质粒pFastBacDual-VP2。利用双酶切以及基因测序分析的方法鉴定重组供体质粒pFastBacDual-dVP2是否构建正确。采用核酸内切酶Not I+Hind III或Xho I+Nhe I对pFastBacDual-dVP2双酶切之后,获得约为1755bp和6993bp左右的两个目的片段,符合插入片段大小,见图2所示。最后,利用基因测序分析方法对pFastBacDual-dVP2进行基因序列分析,测得的基因序列与目的基因序列相比同源性为100%。以上鉴定结果充分说明目的基因片段正确插入pFastBacDual载体上,表明pFastBacDual-dVP2构建正确。
最后,将供体质粒pFastBacDual-dVP2转化感受态(E.coli DH10 Bac)获得Bacmid-dVP2重组质粒,经转染昆虫细胞Sf9细胞后,成功拯救获得表达猫泛白细胞减少症病毒衣壳蛋白VP2的重组杆状病毒AcMNPV-VP2,被命名为重组杆状病毒FPV-VP2株。提取重组杆状病毒基因组进行PCR检测,以p10和pH表达盒特异性引物分别进行PCR鉴定,鉴定引物序列如表1。扩增产物使用1%凝胶进行电泳检测。鉴定结果如图3。pH和p10表达盒特异性引物扩增产物与目的大小一致,分别为2578bp和3733bp(图3A);VP2引物扩增产物与目的大小一致,为1755bp(图3B),表明双拷贝的VP2基因重组至杆状病毒病毒基因组中。氨基酸测序显示其氨基酸序列如SEQ ID NO.1所示。利用电镜(JEM 1200EXII)检测,结果显示颗粒直径26nm左右。
表1VP2表达盒特异性引物信息
实施例2关键制备工艺参数设置:细胞接种密度、转速、收获时间
1、不同细胞接种密度对细胞生长的影响
分别以0.5×106个/ml、0.8×106个/ml、1.2×106个/ml、1.5×106个/ml接种至生物反应器罐体内,每日测定细胞密度和细胞活率,接种密度为0.5×106个/ml经过3日的准备期在第4日进入对数生长期,第8日细胞密度达到9.90×106个/ml,细胞活率97.2%;接种密度为0.8×106个/ml在第3日进入对数生长期,第7日达到最高密度,12.1×106个/ml,细胞活率为95.1%,而后细胞进入衰亡期,活细胞密度和活率下降;接种密度为1.2×106个/ml直接进入对数生长期,在第5日达到最高密度,12.3×106个/ml,细胞活率为88.3%,而后细胞进入衰亡期,活细胞密度和活率下降;接种密度为1.5×106个/ml直接进入对数生长期,在第4日达到最高密度,11.9×106个/ml,细胞活率为94.3%,考虑最高细胞密度及细胞活率,选择1.2×106~1.5×106个/ml作为最佳细胞接种密度。
2、不同转速对细胞生长的影响
以1.2×106个/ml接种到生物反应器罐体内,分别以90r/min、100r/min、110r/min、120r/min作为单因素研究,设置培养条件为27℃、DO值40%~60%,每日取样测定活细胞密度及细胞活率,绘制生长曲线,90r/min组细胞活率在95%以上的最高活细胞密度为4.88×106个/ml,而后细胞出现结团现象;100r/min组细胞活率在95%以上的最高活细胞密度为9.53×106个/ml;110r/min组细胞活率在95%以上的最高活细胞密度为10.52×106个/ml;120r/min组细胞活率在95%以上的最高活细胞密度为1.19×106个/ml,即为接种时,而后镜下观察细胞出现破碎死亡情况,综合考虑选择100~110r/min作为最佳转速。
3、不同接毒细胞密度对收获液血凝素凝集价的影响
分别在细胞密度达到4×106个/ml、6×106个/ml、8×106个/ml、10×106个/ml时补充等体积的新鲜培养基,按MOI=0.1接种,每日取样进行细胞密度及血凝素凝集价检测,以4×106个/ml作为接毒细胞密度,接毒后血凝素凝集价最高为1:218;以6×106个/ml作为接毒细胞密度,接毒后血凝素凝集价最高为1:220;以8×106个/ml作为接毒细胞密度,接毒后血凝素凝集价最高为1:221;以10×106个/ml作为接毒时细胞密度,接毒后血凝素凝集价最高为1:220,考虑VP2蛋白血凝素凝集价及所需细胞密度,选择6~8×106个/ml作为最佳接毒细胞密度。
4、不同收获时间对病毒收获液血凝素凝集价的影响
以细胞接种密度为1.2×106个/ml、100~110r/min搅拌桨转速培养细胞,待细胞增殖6~8×106个/ml,补充等体积的新鲜培养基,按MOI=0.1接种,设置培养参数为27℃、DO值40%~50%,全程不对pH值进行设置和干预,每24小时记录pH电极参数,细胞在培养阶段的72小时pH值仅下降0.08,而在接毒后的96小时仅下降了0.17,接毒后的120小时仅下降了0.19,完全在细胞承受的范围内,故不对细胞培养及接种病毒后的pH进行干预,pH电极只作为培养体系是否污染的监测。接毒后在96~120小时血凝素凝集价最高,因此选择96~120小时为最佳收获时间。
实施例3猫细小病毒样颗粒的制备
病毒培养:生长良好的Sf9细胞调整至1.2×106~1.5×106个/ml的细胞数量接种于生物反应器,在27℃、100~110r/min、DO 40%~60%的条件下悬浮发酵培养。生物反应器中Sf9细胞数量达到6.0×106~8.0×106个/ml时,补充等体积的新鲜培养基,按MOI为0.1~1.0接种实施例1构建的重组杆状病毒FPV-VP2株,在27℃、100~110r/min、DO 40~60%的条件下继续培养。接毒后96~120小时收获细胞培养物,收集细胞置于-20℃以下冻存。
细胞富集、裂解:收获的重组杆状病毒FPV-VP2株的细胞培养物,采用750KD超滤中空纤维柱富集细胞,使用原液4/5比例的25mmol/LNaHCO3裂解细胞,室温裂解1小时。
除细胞碎片:加入原液1/5比例的4.5%NaCl混匀,0.65μm微滤中空纤维滤柱去除细胞碎片,经0.22μm滤芯除菌过滤后收获纯化后的蛋白液即为原液。
灭活:将原液稀释至血凝素凝集价为1:4096,加入终浓度为0.005mol/L BEI,37℃灭活48小时,灭活后加入终浓度为0.005mol/L的硫代硫酸钠中和BEI,灭活检验应合格。
离子交换纯化(去除核酸和杂蛋白):使用100KDa膜包以缓冲液A(Tris 20mmol/LpH值8.0,NaCl 0.05mol/L)(8.0是因为FPV-vlp的稳定pH区间是6.5~8.0,pH越高吸附性越强)对灭活原液进行换洗,然后采用Q Focurose HPL(武汉汇研生物科技股份有限公司)为介质的离子交换层析纯化,使用缓冲液B(Tris 20mmol/L pH值8.0,NaCl 1.0mol/L)与缓冲液A进行线性洗脱,收集洗脱峰1。
离子交换层析:通过使用Q柱进行离子交换层析,采用离子强度线性洗脱,洗脱过程中一共有4个蛋白峰,对样品及吸收峰进行电泳,洗脱峰E1为主要目标峰,经过立径检测,样品中颗粒大小不均匀。所以再进行分子筛层析纯化,详见图4、图5、图6。
分子筛纯化(将vp2蛋白组成的不同聚体分开,留下完整的vlp):将离子交换E1收集的80ml使用100KDa膜包进行10~50倍超滤浓缩至5ml后,以4%柱床体积(CV)上样,采用以Focudex 200PG(武汉汇研生物科技股份有限公司)为介质的分子筛层析纯化,以流动相(PBS 10mmol/L pH值7.0)1.0cm/ml(0.5cm/min)进行洗脱,层析过程中出现两个蛋白峰,对样品及吸收峰进行电泳,峰1为目的峰,纯度为85%,经电镜检测,大小约为20~30nm的20面体对称结构的病毒样粒子,经粒径检测颗粒均匀,结果显示颗粒直径26.05nm。详见图7、图8、图9、图10,收集峰1目标峰。
样品HA价检测及回收率计算
经对实施例3各步骤进行体积记录及留样进行HA价检测,结果如下,超滤和浓缩过程不损失HA;离子交换层析过程中洗脱峰2、峰3中存在部分目的蛋白,洗脱峰1回收率为93.75%;分子筛收获峰HA价提高2个滴度,回收率为91.4%。全程综合回收率85.0%。详见表2。
表2纯化过程数据表
实施例4工艺稳定性研究
通过使用实施例3制备方法制备的160ml样品进行超滤、离子交换、洗脱E1浓缩、分子筛收获E1,进行3次纯化试验,试验结果如下,3次纯化均使用160ml样品,收获的纯化样品分别为17ml、18ml、17.5ml,HA价均为223,纯度分别为92%、94%、92%,回收率分别为85.0%、90.0%、87.5%。详见表3。
表3纯化工艺稳定性研究数据表
实施例5表达产物VP2蛋白免疫原性研究
1、疫苗制备:
取实施例3制备的样品,测血凝素凝集价,用PBS稀释至血凝素凝集价为1:220、1:216、1:212、1:28,将抗原与Gel02佐剂按照7∶1的比例进行混合,制备成疫苗。
2、试验分组:
用10周龄健康幼猫(FPV HI抗体效价不高于1∶8)25只,随机分为5组,每组5只。根据血凝素凝集价设为Ⅰ组(对照组)、Ⅱ组(1:28)、Ⅲ组(1:212)、Ⅳ组(1:216)、Ⅴ组(1:220)。
3、接种剂量及方式
试验猫根据不同分组分别颈背部皮下注射血凝素凝集价不同的本发明疫苗,对照组颈背部皮下注射Sf-900 TM II SFM培养基,免疫两次,间隔21日,免疫剂量1mL/只。
4、抗体检测
免疫前、加强免疫后21日采血检测FPV HI抗体效价,统计各组试验猫产生的抗体效价情况。
5、攻毒
加强免疫后21日,猫泛白细胞减少症病毒组每只猫口服猫泛白细胞减少症病毒JL-125株(血凝素凝集价为1:64,病毒含量为105.50TCID50/ml)1.5ml,观察14日,攻毒试验猫隔离饲养。
6、观察指标
(1)临床症状
按照表6攻毒后观察指标,分别在攻毒前、攻毒后第2、4、6、8、10、12、14日观察试验动物临床症状,并根据相应的分值进行记录。
(2)白细胞值的变化
分别在攻毒前、攻毒后第2、4、6、8、10、12、14日,于同一时间段对各组试验猫进行采血,采血后4小时内使用动物用多功能血液分析仪测定各组试验动物白细胞值,比较相同组不同时间点白细胞值的变化情况,并通过统计学方法进行比较。
(3)粪便FPV检测
分别在攻毒前、攻毒后第2~14日用猫泛白细胞减少症病毒抗原检测试纸检测粪便中FPV病毒。
(4)猫泛白细胞减少症发病和保护标准判定
(1)发病标准
①FPV抗原检测试纸呈阳性
②精神沉郁、行动迟缓或昏迷
③食欲减退或废绝
④稀便、水样便、血便
⑤呕吐
⑥白细胞值降低至攻毒前50%
⑦死亡
判定标准:对照组具备①项,同时具备第②~⑦项中的任何一项时,即可判为发病。免疫猫不出现以上任何1项,判为保护。
发病率=发病数量/(发病数量+未发病数量)×100%。
(2)保护标准
①FPV抗原检测试纸呈阳性
②精神沉郁、行动迟缓或昏迷
③食欲减退或废绝
④稀便、水样便、血便
⑤呕吐
⑥白细胞值降低至攻毒前50%
⑦死亡判定标准:免疫猫不出现第①~⑦项中的任何一项时,判为保护。保护率=保护数量/(保护数量+未保护数量)×100%。
FPV抗体效价检测结果如表4所示。
表4FPV抗体效价检测结果
猫泛白细胞减少症病毒攻毒效果结果如表5所示。
表5猫泛白细胞减少症病毒攻毒效果结果
以上只通过说明的方式描述了本发明的某些示范性实施例,毋庸置疑,对于本领域的普通技术人员,在不偏离本发明的精神和范围的情况下,可以用各种不同的方式对所描述的实施例进行修正。因此,上述附图和描述在本质上是说明性的,不应理解为对本发明权利要求保护范围的限制。
序列表
<110> 长春西诺生物科技有限公司
<120> 一种猫细小病毒样颗粒及其制备方法与应用
<160> 5
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<213> 猫细小病毒(Feline panleukopenia Virus)(Feline panleukopenia Virus)
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Ile Phe Asn Val Val Leu Lys Thr Val Ser Glu Ser Ala Thr Gln Pro
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Pro Thr Lys Val Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala
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Glu Thr Leu Gly Phe Tyr Pro Trp Lys Pro Thr Ile Pro Thr Pro Trp
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Arg Tyr Tyr Phe Gln Trp Asp Arg Thr Leu Ile Pro Ser His Thr Gly
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Val Gln Phe Tyr Thr Ile Glu Asn Ser Val Pro Val His Leu Leu Arg
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Thr Gly Asp Glu Phe Ala Thr Gly Thr Phe Phe Phe Asp Cys Lys Pro
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Pro Phe Leu Asn Ser Leu Pro Gln Ser Glu Gly Ala Thr Asn Phe Gly
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Asp Ile Gly Val Gln Gln Asp Lys Arg Arg Gly Val Thr Gln Met Gly
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Asn Thr Asp Tyr Ile Thr Glu Ala Thr Ile Met Arg Pro Ala Glu Val
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Gly Tyr Ser Ala Pro Tyr Tyr Ser Phe Glu Ala Ser Thr Gln Gly Pro
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<210> 2
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<213> 猫细小病毒(Feline panleukopenia Virus)(Feline panleukopenia Virus)
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cctaccaagg tctacaacaa cgacctgacc gcttccctga tggtcgccct ggacagcaac 540
aacaccatgc ccttcactcc cgctgccatg cgctccgaga ccctgggttt ctacccctgg 600
aagcccacca tccccactcc ttggcgctac tacttccagt gggaccgcac tctgatccct 660
agccacaccg gtacctccgg tactcccacc aacgtctacc acggcactga ccctgacgac 720
gtccagttct acactatcga gaactccgtc cccgtccacc tgctgcgcac tggcgacgaa 780
ttcgccactg gcactttctt cttcgactgc aagccctgcc gcctgaccca cacctggcag 840
accaaccgtg ccctgggtct gcctcctttc ctgaacagcc tgccccagtc cgagggtgcc 900
actaacttcg gtgacatcgg cgtgcagcag gacaagcgcc gtggcgtcac tcagatgggc 960
aacaccgact acatcactga agctactatc atgcgtcccg ccgaagtggg ctacagcgct 1020
ccctactaca gcttcgaggc cagcacccag ggtcctttca agactcccat cgccgccggt 1080
cgcggcggag ctcagactga tgagaaccag gccgccgacg gcgaccctcg ttacgctttc 1140
ggtcgtcagc acggtcagaa gactactacc accggtgaaa cccccgaacg cttcacctac 1200
atcgcccacc aggacaccgg tcgttaccct gaaggcgact ggatccagaa catcaacttc 1260
aacctgcccg tgactaacga caacgtgctg ctgcctaccg accctatcgg tggcaagacc 1320
ggtatcaact acactaacat cttcaacact tacggccccc tgaccgctct gaacaacgtc 1380
ccccctgtct accccaacgg ccagatctgg gacaaggaat tcgacaccga cctgaagcct 1440
cgcctgcacg tcaacgctcc tttcgtctgc cagaacaact gccctggtca gctgttcgtg 1500
aaggtcgctc ccaacctgac taacgaatac gaccccgacg cttccgctaa catgagccgc 1560
atcgtgactt actccgactt ctggtggaag ggtaaactgg tcttcaaggc taagctgcgc 1620
gcttcccaca cctggaaccc tatccagcag atgtccatca acgtcgacaa ccagttcaac 1680
tacgtcccta acaacatcgg tgccatgaag atcgtgtacg agaagagcca gctggcccct 1740
cgtaagctgt actaa 1755
<210> 3
<211> 1755
<212> DNA
<213> 猫细小病毒(Feline panleukopenia Virus)(Feline panleukopenia Virus)
<400> 3
atgagtgatg gagcagttca accagacggt ggtcaacctg ctgtcagaaa tgaaagagct 60
acaggatctg ggaacgggtc tggaggcggg ggtggtggtg gttctggggg tgtggggatt 120
tctacgggta ctttcaataa tcagacggaa tttaaatttt tggaaaacgg gtgggtggaa 180
atcacagcaa actcaagcag acttgtacat ttaaatatgc cagaaagtga aaattataaa 240
agagtagttg taaataatat ggataaaact tcagttaaag gaaacatggc tttagatgat 300
actcatgtac aaattgtaac accttggtca ttggttgatg caaatgcttg gggagtttgg 360
tttaatccag gagattggca actaattgtt aatactatga gtgagttgca tttagttagt 420
tttgaacaag aaatttttaa tgttgtttta aagactgttt cagaatctgc tactcaacca 480
ccaactaaag tttataataa tgatttaact gcatcattga tggttgcatt agatagtaat 540
aatactatgc catttactcc agcagctatg agatctgaga cattgggttt ttatccatgg 600
aaaccaacca taccaactcc atggagatat tattttcaat gggatagaac attaatacca 660
tcccatactg gaactagtgg cacaccaaca aatgtatatc atggtacaga tccagatgat 720
gttcaatttt atactattga aaattctgta ccagtacact tactaagaac aggtgatgaa 780
tttgctacag gaacattttt ttttgattgt aaaccatgta gactaacaca tacatggcaa 840
acaaatagag cattgggctt accaccattt ctaaattctt tgcctcaatc tgaaggagct 900
actaactttg gtgatatagg agttcaacaa gataaaagac gtggtgtaac tcaaatggga 960
aatacagact atattactga agctactatt atgagaccag ctgaggttgg ttatagtgca 1020
ccatattatt cttttgaagc atctacacaa ggaccattta aaacacctat tgcagcagga 1080
cgggggggag cgcaaacaga tgaaaaccaa gcagcagatg gtgatccaag atatgcattt 1140
ggtagacaac atggtcaaaa aactactaca acaggagaaa cacccgagag atttacatat 1200
atagcacatc aagatacagg aagatatcca gaaggagatt ggattcaaaa tattaacttt 1260
aaccttcctg taacaaatga taatgtattg ctaccaacag atccaattgg aggtaaaaca 1320
ggaattaact atactaatat atttaatact tatggtcctt taactgcatt aaataatgta 1380
ccaccagttt atccaaatgg tcaaatttgg gataaagaat ttgatactga cttaaaacca 1440
agacttcatg taaatgcacc atttgtttgt caaaataatt gtcctggtca attatttgta 1500
aaagttgcgc ctaatttaac aaatgaatat gatcctgatg catctgctaa tatgtcaaga 1560
attgtgactt actcagattt ttggtggaaa ggtaaattag tttttaaagc taaactaaga 1620
gcatctcata cttggaatcc aattcaacaa atgagtatta atgtagataa ccaatttaac 1680
tatgtaccaa ataatattgg agctatgaaa attgtatatg aaaaatctca actagcacct 1740
agaaaattat attaa 1755
<210> 5
<211> 16
<212> DNA
<213> 人工序列(Feline panleukopenia Virus)(Feline panleukopenia Virus)
<400> 5
atgtccgacg gagccg 16
<210> 4
<211> 17
<212> DNA
<213> 人工序列(Feline panleukopenia Virus)(Feline panleukopenia Virus)
<400> 4
ttagtacagt tgcggga 17
Claims (10)
1.一种猫细小病毒样颗粒,其特征在于,其氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的猫细小病毒样颗粒,其特征在于,表达其氨基酸序列的核酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述的猫细小病毒样颗粒,其特征在于,利用昆虫-杆状病毒表达系统表达。
4.根据权利要求1所述的猫细小病毒样颗粒,其特征在于,其培养工艺为:将生长良好的Sf9细胞调整至1.2×106~1.5×106个/ml的细胞数量接种于生物反应器,在27℃、100~110r/min、DO 40%~60%的条件下悬浮发酵培养;生物反应器中Sf9细胞数量达到6.0×106~8.0×106个/ml时,补充等体积的新鲜培养基,按MOI为0.1~1.0接种,在27℃、100~110r/min、DO 40~60%的条件下继续培养;接毒后96~120小时收获细胞培养物。
5.根据权利要求1所述的猫细小病毒样颗粒,其特征在于,其纯化工艺为:将收获的含有重组杆状病毒FPV-VP2的细胞培养物经细胞富集、裂解、去除细胞碎片、灭活后,使用100KDa膜包以Tris 20mmol/L pH值8.0、NaCl 0.05mol/L为缓冲液A对灭活原液进行换洗,然后采用Q Focurose HPL与缓冲液A进行线性洗脱,收集洗脱峰1,使用100KDa膜包进行10~50倍超滤浓缩,以4%柱床体积上样,采用以Focudex 200PG为介质的分子筛层析纯化,以PBS 10mmol/L pH值7.0为流动相进行洗脱,收集目标峰1,得到纯化后的猫细小病毒样颗粒。
6.根据权利要求1所述的猫细小病毒样颗粒的制备方法,其特征在于,将生长良好的Sf9细胞调整至1.2×106~1.5×106个/ml的细胞数量接种于生物反应器,在27℃、100~110r/min、DO 40%~60%的条件下悬浮发酵培养;生物反应器中Sf9细胞数量达到6.0×106~8.0×106个/ml时,补充等体积的新鲜培养基,按MOI为0.1~1.0接种利用昆虫-杆状病毒表达系统表达重组杆状病毒FPV-VP2,在27℃、100~110r/min、DO 40~60%的条件下继续培养。接毒后96~120小时收获细胞培养物,将收获的细胞培养物经细胞富集、裂解、去除细胞碎片、灭活后,使用100KDa膜包以Tris20mmol/L pH值8.0、NaCl 0.05mol/L为缓冲液A对灭活原液进行换洗,然后采用Q Focurose HPL与缓冲液A进行线性洗脱,收集洗脱峰1,使用100KDa膜包进行10~50倍超滤浓缩,以4%柱床体积上样,采用以Focudex200PG为介质的分子筛层析纯化,以PBS 10mmol/L pH值7.0为流动相进行洗脱,收集目标峰1,得到纯化后的猫细小病毒样颗粒。
7.根据权利要求1-5任一项所述的猫细小病毒样颗粒在制备猫泛白细胞减少症疫苗中的应用。
8.一种猫泛白细胞减少症疫苗,其特征在于,包括权利要求1-5任一项所述的猫细小病毒样颗粒。
9.一种多联疫苗,其特征在于,包括权利要求1-5任一项所述的猫细小病毒样颗粒。
10.一种多价疫苗,其特征在于,包括权利要求1-5任一项所述的猫细小病毒样颗粒。
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