CN116284272B - 一种广谱抗牛科病毒性腹泻病毒的mRNA疫苗及其应用 - Google Patents
一种广谱抗牛科病毒性腹泻病毒的mRNA疫苗及其应用 Download PDFInfo
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Abstract
本发明涉及一种广谱抗牛科病毒性腹泻病毒(BVDV)的mRNA疫苗、制备方法及其应用等。具体提供一种针对多种基因型BVDV毒株的三价BVDV mRNA,从N端到C端依次包含牛IgG1分泌信号肽、牛IgG1 Fc片段、BVDV1a E2蛋白结构域I‑II、BVDV1b E2蛋白结构域I‑II、BVDV2 E2蛋白结构域I‑II。本发明通过使用一条mRNA能够同时表达不同基因型BVDV的抗原,具有强免疫原性,在动物体内有效且高水平的分泌,从而诱导产生针对BVDV1a、BVDV 1b和BVDV 2的更高水平的特异性中和抗体,能够针对多种BVDV基因型毒株实现理想的免疫效果。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及动物疫苗领域,特别是一种广谱抗牛科病毒性腹泻病毒的mRNA疫苗及其应用。
背景技术
牛科病毒性腹泻病毒(Bovine viral diarrhea virus, BVDV,又称牛病毒性腹泻病毒)是引起牛病毒性腹泻的主要病原体,其广泛存在于野生和家养的反刍动物身体中。BVDV归属于瘟病毒属,黄病毒科。临床上BVDV主要引起腹泻、繁殖障碍、免疫抑制和粘膜糜烂坏死等症状。该病毒在世界范围内广泛存在,给养殖业带来了较大的损失。尤其是BVDV可在牛群中广泛持续的存在和传播,严重影响牛的生产能力,导致产奶量下降、乳品质下降、生长迟缓和发育不良等。
BVDV主要有两种基因型,分别是BVDV1和BVDV2。其中BVDV1是经典毒株,包含BVDV1a、BVDV 1b、BVDV 1e、BVDV 1g、BVDV 1h等基因亚型。BVDV1主要引起腹泻,在临床上检出率明显高于BVDV2。BVDV2同样能够引起腹泻,但BVDV2的毒力更强,可引起成年牛急性发病,血小板减少,发生出血综合症等症状。
当前,我国分离到的毒株主要为BVDV1a基因亚型、BVDV1b基因亚型和BVDV2基因型。目前市场上疫苗大多数针对BVDV1a和BVDV1b,但总体效价偏低且保护范围有限,另外尚没有针对BVDV2基因型效果较好的疫苗,且1型对2型交叉保护较弱,无法抵抗强毒力病毒的感染。BVDV为单股正链RNA,共编码4种结构蛋白(分别为C、E0、E1和E2)和8种非结构蛋白(分别为Npro、P7、NS2、NS3、NS4A、NS4B、NS5A和NS5B)。E2蛋白为BVDV重要的结构蛋白,含有主要的抗原决定簇,免疫原性强,能诱导中和抗体产生,可有效预防BVDV的感染。研究发现,E2蛋白在BVDV1和BVDV2之间的同源性偏低,导致单价E2疫苗只能预防同型的BVDV毒株。E2蛋白胞外域部分主要划分为三个结构域,其中结构域I-II暴露于病毒表面。因此,开发能同时预防BVDV1型和2型的多价疫苗对于防控牛病毒性腹泻疾病显得尤为重要。
现有技术中已公开了多种BVDV疫苗。专利文献CN115381935A(公开日期2022年11月25日)公开了制备BVDV灭活疫苗的方法,包括如下步骤:将BVDV毒株病毒液与过氧化氢溶液混合至过氧化氢的浓度为1%-3%,灭活,得BVDV毒株灭活病毒液,将BVDV毒株灭活病毒液与弗氏佐剂混合,乳化得BVDV灭活疫苗。专利文献CN110331155B(公开日期2022年5月13日)公开了一种携带2型BVDV-Erns基因的高繁殖力猪瘟弱毒标记疫苗的构建方法。利用分子克隆及反向遗传方法,将BVDV2-890#毒株的Erns基因和CSFV流行毒株QZ14的E2基因VR1区置换CSFV-C株相应的基因片段,并引入7个特定的突变位点,得到了重组病毒rC-Marker2。获得的重组病毒带有分子标记(BVDV2 Erns),猪瘟病毒2型流行毒株的VR1以及特定的突变位点。然而,灭活疫苗的缺点主要包括免疫效果持续时间短、接种时间相对较长且需要多次接种,以及存在一定的不良反应。重组基因疫苗的缺点是存在安全性问题,不能完全排除少数质粒DNA***到染色体上引起突变的可能性,此外会有免疫耐受问题,基因疫苗体内持续产生抗原蛋白,可能打破机体本身的免疫平衡,引发免疫耐受。
灭活疫苗、重组蛋白疫苗生产周期长,工艺复杂无法应对突发性大规模流行病的大规模接种需要。mRNA则是一种应对爆发性疫情的快速反应疫苗开发平台。近年来,国内外一些公司开发的mRNA(信使核糖核酸)疫苗启动临床试验,提供了mRNA疫苗的安全性和有效性的前期数据。mRNA疫苗是利用线性化的质粒DNA为模板,在体外进行酶转录反应合成的,该合成策略避免了活细胞培养生产方式,需要考虑的安全性以及复杂的生产工艺等问题。mRNA疫苗平台具有安全,有效,生产周期短,工艺简洁的特点,因而特别适合应对爆发性疫情。本领域已知,mRNA翻译快速,起效快,本身具有激活免疫反应的作用;同时,mRNA药物生产简单、易于改造、合成快速、成本较低;更重要的是,mRNA药物不局限于***细胞,没有整合到宿主基因组中的风险,且会在体内自动降解。所以,mRNA疫苗的开发具有很大的优势。然而,未检索到有效用于BVDV防治的多价mRNA疫苗。本领域亟需一种可同时表达多种BVDV基因型的融合蛋白多价抗原BVDV mRNA疫苗,具有强免疫原性,且能够预防或治疗由多种BVDV基因型毒株所造成的感染,从而有效防控牛病毒性腹泻疾病,降低农牧业的损失。
发明内容
为了克服现有技术BVDV灭活疫苗、重组蛋白疫苗、重组基因疫苗等的缺陷,本发明提供一种同时表达BVDV1a、BVDV1b和BVDV2三种BVDV基因型的E2蛋白结构域I-II的串联抗原融合蛋白、编码其的核酸和mRNA、包含所述mRNA的三价BVDV疫苗,以及相关制备方法和应用等。相对于单独表达一种BVDV基因型的E2蛋白的策略,本发明提供更有效的针对多种基因型BVDV毒株的三价BVDV mRNA疫苗,是更有利于产业应用的抗原选择策略。此外本发明的抗原融合蛋白融合了助溶助分泌的结构域IgG1Fc,因此显著地增强了E2蛋白的免疫原性,增加了施用对象体内产生的针对BVDV的中和抗体。
本发明的一个方面提供一种BVDV抗原片段,其特征在于,包含BVDV E2蛋白靠近N端的2个免疫球蛋白样(Ig-like)结构域,即BVDV E2蛋白结构域I-II,图4中给出了BVDV E2结构域I-II空间结构位置示意图,所参考的BVDV E2蛋白结构(PDB: 4ILD),其中所述2个免疫球蛋白样(Ig-like)结构域对应图4中的Dom.I和Dom.II。免疫球蛋白样结构域具备自稳定特征,其形成的空间结构不受上下游序列的影响;所述免疫球蛋白样结构域内部包含Greek key拓扑构象的β-sheet结构。
进一步地,所述的BVDV抗原片段,其特征在于,包含BVDV1a E2蛋白结构域I-II,BVDV1b E2蛋白结构域I-II,和/或BVDV2 E2蛋白结构域I-II中的一种或多种。
本发明的一个方面提供一种BVDV抗原融合片段,其特征在于,包含IgG1 Fc片段与BVDV1a E2蛋白结构域I-II、BVDV1b E2蛋白结构域I-II和/或BVDV2 E2蛋白结构域I-II中的一种或多种融合。
进一步地,所述的抗原融合片段,其特征在于,所述IgG1 Fc片段为牛IgG1 Fc片段。
本发明的一个方面提供一种三价BVDV抗原融合片段,其特征在于,包含IgG1 Fc片段,BVDV1a E2蛋白结构域I-II,BVDV1b E2蛋白结构域I-II,和BVDV2 E2蛋白结构域I-II。
进一步地,所述的三价BVDV抗原融合片段,其特征在于,所述三价BVDV抗原融合片段从N端到C端,依次包含牛IgG1 Fc片段,BVDV1a E2蛋白结构域I-II,BVDV1b E2蛋白结构域I-II,和BVDV2 E2蛋白结构域I-II。
进一步地,所述的三价BVDV抗原融合片段,其特征在于,所述三价BVDV抗原融合片段还包含分泌信号肽。
本发明的一个方面提供一种BVDV抗原融合蛋白,其特征在于,包含如下任一序列:
(I)如SEQ ID NO.1所示的氨基酸序列;
(II)如SEQ ID NO.1所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸残基获得的氨基酸序列,且与(I)所示的氨基酸序列具有相同的功能;或者,
(III)与(I)或(II)所述序列具有至少90%序列一致性的氨基酸序列,且与(I)所示的氨基酸序列具有相同的功能。
本发明的一个方面提供一种重组核酸,其特征在于,包含编码所述BVDV抗原片段、或所述BVDV抗原融合片段、或所述三价BVDV抗原融合片段、或所述BVDV抗原融合蛋白的核酸。
进一步地,所述的重组核酸,其特征在于,包含如下任一序列:
(I)如SEQ ID NO.2所示的核酸序列;
(II)如SEQ ID NO.2所示的核酸序列经取代、缺失或添加一个或多个核苷酸获得的核酸序列,且与(I)所示的核酸序列具有相同的功能;或者,
(III)与(I)或(II)所述序列具有至少90%序列一致性的核酸序列,且与(I)所示的核酸序列具有相同的功能。
本发明的一个方面提供一种重组mRNA,其特征在于,包含编码所述BVDV抗原片段、或所述BVDV抗原融合片段、或所述三价BVDV抗原融合片段、或所述BVDV抗原融合蛋白的mRNA。
进一步地,所述的重组mRNA,其特征在于,所述mRNA还包含5`-UTR,3`-UTR,polyA序列,IRES序列中的一种或多种。
进一步地,所述的重组mRNA,其特征在于,所述5`-UTR的序列如SEQ ID NO.3所示,所述3`-UTR的序列如SEQ ID NO.4所示,所述polyA的序列如SEQ ID NO.5所示,所述IRES序列如SEQ ID NO.8所示。
本发明的一个方面提供一种表达盒,其特征在于,包含所述重组核酸、或所述重组mRNA。
本发明的一个方面提供一种载体,其特征在于,包含所述重组核酸、或所述重组mRNA、或所述表达盒;优选地,所述载体包含自复制载体;更优选地,所述自复制载体包含VEE自复制载体;最优选地,所述载体的序列包含SEQ ID NO.7、SEQ ID NO.9、或SEQ IDNO.11所示的序列。
本发明的一个方面提供一种细胞,其特征在于,包含所述重组核酸、或所述重组mRNA、或所述表达盒、或所述载体。
本发明的一个方面提供一种重组菌,其特征在于,包含所述重组核酸、或所述重组mRNA、或所述表达盒、或所述载体;优选地,所述菌为大肠杆菌;更优选地,所述大肠杆菌为E. coli BL21(DE3)、E. coli Origami B(DE3)或E. coli Rosetta Blue(DE3)中的一种;最优选地,所述大肠杆菌为E. coli BL21(DE3)。
本发明的一个方面提供一种组合物,其特征在于,包含所述BVDV抗原片段、或所述BVDV抗原融合片段、或所述三价BVDV抗原融合片段、或所述BVDV抗原融合蛋白、或所述重组核酸、或所述重组mRNA、或所述表达盒、或所述载体、或所述细胞、或重组菌。
本发明的一个方面提供一种试剂盒,其特征在于,包含所述BVDV抗原片段、或所述BVDV抗原融合片段、或所述三价BVDV抗原融合片段、或所述BVDV抗原融合蛋白、或所述重组核酸、或所述重组mRNA、或所述表达盒、或所述载体、或所述细胞、或所述重组菌、或所述组合物。
本发明的一个方面提供一种三价BVDV mRNA疫苗,包含:
(a) 所述重组mRNA;和
(b) 药学上可接受的载体;优选地,所述载体包含脂类;更优选地,所述脂类包括阳离子脂质、可电离脂质、辅助脂质、胆固醇、DMG-PEG2000中的一种或多种。
本发明的一个方面提供一种表达重组三价BVDV mRNA的加帽mRNA重组表达载体、非加帽mRNA重组表达载体或自复制RNA重组表达载体的制备方法,包括如下步骤:
步骤a:合成所述重组核酸的基因片段;
步骤b:构建加帽mRNA架构载体、构建非加帽mRNA架构载体、或构建VEE自复制载体;
所述加帽mRNA架构载体包含5`-UTR,3`-UTR和polyA序列以及固定加帽mRNA架构的载体,序列分别如SEQ ID NO.3-6所示;或者,
所述非加帽mRNA架构载体包含IRES序列,3`-UTR和polyA序列,以及固定非加帽mRNA架构的载体,其中IRES的序列如SEQ ID No.8所示;或者,
构建VEE自复制载体:根据α病毒家族的基因组获得自复制RNA序列;所述自复制RNA序列包含编码α病毒自复制组件的基因,缺乏制造具有传染性的α病毒颗粒的结构蛋白的基因;将自复制RNA序列构建获得的质粒作为模版扩增,通过合成获得所述VEE自复制载体;所述VEE自复制载体的序列如SEQ ID NO.10所示;
步骤c:制备加帽mRNA、非加帽mRNA或自复制RNA重组表达载体;
将步骤a合成的基因***加帽mRNA架构载体中,得到加帽mRNA重组表达载体,优选地,所述加帽mRNA重组表达载体的序列如SEQ ID NO.7所示;或者,
将步骤a合成的基因***非加帽mRNA架构载体中,得到非加帽mRNA重组表达载体,优选地,所述非加帽mRNA重组表达载体的序列如SEQ ID NO.9所示;或者,
将步骤a合成的基因***到VEE自复制载体的指定位置,得到自复制RNA重组表达载体, 优选地,所述自复制RNA重组表达载体的序列如SEQ ID NO.11所示。
本发明的一个方面提供一种三价BVDV mRNA疫苗的制备方法,其特征在于,包括:
(a)提供所述重组mRNA;
(b)将所述mRNA与脂质颗粒进行混合并孵育,从而形成包裹有mRNA的脂质纳米颗粒。
本发明的一个方面提供一种三价BVDV加帽mRNA架构载体疫苗、非加帽mRNA架构载体疫苗、或基于自复制RNA结构载体疫苗的制备方法,其特征在于,包含如下步骤:
步骤a:将所述加帽mRNA重组表达载体、非加帽mRNA重组表达载体或自复制RNA重组表达载体用限制性内切酶酶切线性化;
步骤b:将线性化的所述加帽mRNA重组表达载体进行体外共转录加帽反应,将7-甲基化鸟苷酸帽结构加至转录的mRNA的5`端,并对模板DNA进行降解;或者,
将线性化的所述非加帽mRNA重组表达载体进行体外非加帽的转录反应,并对模板DNA进行降解;或者,
将线性化的所述自复制RNA重组表达载体进行体外共转录加帽反应,将7-甲基化鸟苷酸帽结构加至转录的RNA的5`端,并对模板DNA进行降解;
步骤c:将步骤b中各自制备的加帽、非加帽、自复制mRNA分别与脂类混合,mRNA包裹进入LNP内,得到mRNA-LNP复合物,通过浓缩及换液制得制剂溶液,从而获得所述疫苗。
本发明的一个方面提供所述BVDV抗原片段、或所述BVDV抗原融合片段、或所述三价BVDV抗原融合片段、或所述BVDV抗原融合蛋白、或所述重组mRNA在以下任一项中的用途:(I)制备治疗或预防BVDV感染引起的疾病的药物;或者,(II)制备治疗或预防牛病毒性腹泻的药物;或者(III)制备在施用对象体内诱导产生针对BVDV的特异性免疫反应的药物。
本发明的广谱抗牛科病毒性腹泻病毒的mRNA疫苗具有如下有益技术效果:
1. 本发明通过使用一条mRNA能够同时表达不同基因型BVDV的抗原,具有强免疫原性,在动物体内有效且高水平的分泌,从而诱导产生更高水平的针对BVDV1a、BVDV1b和BVDV2的特异性中和抗体,能够针对BVDV1a、1b和2等BVDV基因型毒株实现理想的免疫效果。由此,本发明三价BVDV mRNA疫苗可有效阻断多种BVDV毒株所造成的交叉感染,降低对养殖业所造成的损失。
2. 本发明的抗原融合蛋白融合了助溶助分泌的结构域IgG1 Fc,因此显著地增强了E2蛋白的免疫原性,增加了体内产生的针对BVDV的中和抗体。因此可以用较少的疫苗剂量达到保护效果。因此减少接种剂量相应的也减少了脂质体使用量,进而减少脂质体所产生的细胞毒性,在保证免疫效果的同时降低了疫苗的毒性。
3. 本发明基于mRNA的疫苗在小鼠实验中有很强的免疫反应,且mRNA自身具有佐剂效应,因此能够诱导产生显著的免疫应答。
4. 本发明涉及三价BVDV mRNA疫苗,其是利用线性化的质粒DNA为模板,在体外进行酶转录反应合成的,该合成策略避免了活细胞培养生产方式,不需要考虑的安全性以及复杂的生产工艺等问题。
5. 本发明的三价BVDV mRNA疫苗涉及mRNA通用性疫苗平台,可以根据毒株的年度或季节性变异选择新的抗原序列,在不改变工艺流程的情况下快速开发新的疫苗。
附图说明
图1为本发明BVDV三价疫苗的结构示意图,BVDV抗原融合蛋白mRNA,即分泌信号肽+牛IgG1 Fc+BVDV1a E2蛋白结构域I-II+BVDV1b E2蛋白结构域I-II+BVDV2 E2蛋白结构域I-II结构图谱的示意图。
图2A-图2D为本发明BVDV抗原融合蛋白mRNA以及对照的Western Blot和Dot blot检测结果图,其中图2A是本发明mRNA,即分泌信号肽+牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II+BVDV2结构域I-II,在体外转染HEK293T细胞后,牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II+BVDV2结构域I-II的Dot blot蛋白表达图;图2B是本发明mRNA在体外转染HEK293T细胞后,牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II+BVDV2结构域I-II的Western Blot蛋白表达图;图2C是本发明mRNA(Fc+)以及对照(不包含牛IgG1 Fc,即Fc-)在体外转染HEK293T细胞后,Western Blot蛋白表达图;图2D是本发明mRNA(Fc+)以及对照(不包含牛IgG1 Fc,即Fc-)在体外转染HEK293T细胞后,Dot blot蛋白表达图。
图3为本发明BVDV抗原融合蛋白mRNA小鼠免疫和采样过程示意图。
图4为本发明BVDV E2结构域I-II空间结构位置示意图,所参考的BVDV E2蛋白结构(PDB: 4ILD)。
具体实施方式
定义和术语
术语“牛”指牛类动物包括但不限于食用牛、公牛、未生育的小母牛、母牛和牛犊。此处所用的牛指妊娠的和授乳的牛类动物。优选地,本发明方法被施用于非人的哺乳动物;优选地,授乳或妊娠母牛及其胎牛或哺乳的牛犊。
术语“BVDV”指牛科病毒性腹泻病毒,Bovine viral diarrhea virus,又称牛病毒性腹泻病毒,BVDV主要有两种基因型,分别是BVDV1和BVDV2。其中BVDV1是经典毒株,主要引起腹泻,在临床上检出率明显高于BVDV2。BVDV2虽同样能够引起腹泻,但BVDV2的毒力更强,可引起成年牛急性发病,血小板减少,发生出血综合症等症状。BVDV1包含BVDV1a、BVDV1b等基因型。
术语“致免疫的”表示BVDV在动物体内针对1型或2型BVDV或针对1型和2型BVDV两者引起的免疫应答的能力。免疫应答能为主要地由细胞毒性的T细胞介导的细胞免疫应答或主要地通过辅助T细胞介导的体液免疫应答,从而活化B细胞导致抗体产生。
术语“给药”或“接种”指,本发明的mRNA、疫苗组合物优选地经肌内的或皮下的途径给予牛,尽管其他的给药途径也能被使用,如例如,口服、鼻内(例如气雾剂或其他非针剂给药)、淋巴节内、真皮内、腹膜内、直肠或***给药,或通过联用的途径。在动物的颈部肌内的给药是优选的。可采用加速方案 (boosting regimens)并能将给药方案调节为提供最佳免疫。
术语“表达”包括涉及多肽产生的任何步骤,包括但不限于:转录、转录后修饰、翻译、翻译后修饰和分泌。
术语“重组核酸分子”指具有在自然界中不连接在一起的序列的多核苷酸。重组多核苷酸可包括在合适的载体中,且该载体可用于转化至合适的宿主细胞。然后多核苷酸在重组宿主细胞中表达以产生例如“重组多肽”“重组蛋白”“融合蛋白”等。
术语“重组表达载体”指用于表达例如编码所需多肽的多核苷酸的DNA结构。重组表达载体可包括,例如包含(1)对基因表达具有调控作用的遗传元素的集合,例如启动子和增强子;(2)转录成mRNA并翻译成蛋白质的结构或编码序列;以及(3)适当的转录和翻译起始和终止序列的转录亚单位。重组表达载体以任何合适的方式构建。载体的性质并不重要,并可以使用任何载体,包括质粒、病毒、噬菌体和转座子。用于本公开的可能载体包括但不限于染色体、非染色体和合成的DNA序列,例如病毒质粒、细菌质粒、噬菌体DNA、酵母质粒以及从质粒和噬菌体DNA的组合中衍生的载体,来自如慢病毒、逆转录病毒、牛痘、腺病毒、鸡痘、杆状病毒、SV40和伪狂犬病等病毒的DNA。包括自复制型载体和非自复制型载体。
术语“mRNA”,指信使RNA、Messenger RNA,中文译名“信使核糖核酸”,是由DNA的一条链作为模板转录而来的、携带遗传信息能指导蛋白质合成的一类单链核糖核酸。
术语“BVDV mRNA疫苗”,包括基于mRNA的针对不同基因型BVDV的疫苗,mRNA包括常规mRNA或自复制mRNA。现有的mRNA疫苗平台技术有两个主要路线,一个是基于常规mRNA,另一个是基于自复制mRNA;这两个路线都可以应用于本发明。其中基于自复制mRNA疫苗在接种到动物体内后,能够以自身为模板复制出自身的序列,因此相比常规mRNA疫苗需要的接种量更少,并且自复制时所诱导的免疫反应形成的佐剂效应,能够诱导出更强的免疫应答,进一步增强体液和细胞免疫反应。
术语“BVDV mRNA疫苗制备方法”,包括mRNA制备利用线性化的质粒DNA为模板,在体外进行酶转录反应完成。线性化后的质粒DNA使用T7 RNA聚合酶开始体外转录反应,使用DNase酶对模板DNA进行降解,限制性内切酶为SapI/BspQI酶。制备的mRNA与脂类(组成包括阳离子脂质/可电离脂质、辅助脂质、胆固醇和DMG-PEG2000)通过混合器进行快速的混合,从而引起脂类的沉淀及在电荷作用下mRNA包裹进入LNP内。mRNA-LNP复合物随后通过浓缩及换液至纸质溶液中。
术语“BVDV mRNA疫苗应用方法”,包括基于mRNA的广谱牛病毒性腹泻病毒疫苗的应用方法采用疫苗的初免-加强给药应用方式,疫苗的初免-加强给药使用mRNA-分泌信号肽+牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II。疫苗的给药方式为首次注射和加强注射,加强注射的次数根据需要确定。
术语“5’-UTR”,指“5’非翻译区”或“5’UTR”,为转录成初级RNA转录本(前体mRNA)并位于编码序列上游的一部分基因。初级转录本是初始RNA产物,包含内含子和外显子,由DNA转录产生。许多初级转录本必须经过RNA加工以形成具有生理活性的RNA。形成成熟mRNA的加工过程包括修饰末端、切除内含子、加帽和/或从前体RNA上剪切出各rRNA分子。因此,mRNA的5’UTR是不会被翻译成蛋白质并位于编码序列上游的一部分mRNA。在基因组序列中,5’UTR通常被定义为位于转录起始点和起始密码子之间的区域。脊椎动物mRNA的5’非翻译区(5’UTR)长度可以是几十个碱基到几百个碱基。
术语“3’-UTR”,指“3’-非翻译区” 或“3’UTR”,涉及位于基因的3’端,在蛋白质编码区的终止密码子下游,并且被转录但不翻译成氨基酸序列的区域,或涉及RNA分子中的对应区域。3’-非翻译区通常从翻译产物的终止密码子延伸至通常在转录过程之后附着的多聚(A)序列。哺乳动物mRNA的3’-非翻译区通常具有已知为AAUAAA六核苷酸序列的同源区。该序列可能是多聚(A)附着信号并且经常位于多聚(A)附着位点上游的10至30个碱基处。3’-非翻译区可以包含一个或更多个反向重复,其可以折叠以产生茎环结构,所述结构充当核糖核酸外切酶的屏障或与已知提高RNA稳定性的蛋白质(例如RNA结合蛋白)相互作用。
术语“polyA”,指“多聚腺苷酸序列”、“多聚(A)序列”或“多聚(A)尾”,是指通常位于RNA分子的3’端的腺苷酸残基序列。本发明允许这样的序列通过DNA模板基于在与编码链互补的链中的重复胸苷酸残基来在RNA转录期间附着,然而所述序列在正常情况下不在DNA中编码,而是在细胞核中转录后通过模板独立性RNA聚合酶附着至RNA的游离3’端。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞,包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”,这包括原代转化的细胞和从其衍生的子代。宿主细胞是可以用来产生本发明mRNA的任何类型的细胞***,包括真核细胞,例如,哺乳动物细胞、昆虫细胞、酵母细胞;和原核细胞,例如,大肠杆菌细胞。宿主细胞包括培养的细胞。
术语“重组宿主细胞”涵盖导入重组核酸分子、重组表达载体、mRNA后不同于亲本细胞的宿主细胞,重组宿主细胞具体通过转化来实现。本公开的宿主细胞可以是原核细胞或真核细胞,主要是能够导入本公开的重组核酸分子、重组表达载体、mRNA等的细胞即可。
术语“转化、转染、转导”具有本领域技术人员普遍理解的意思,即将外源性的DNA、RNA导入宿主的过程。
术语“治疗”是指:表示减少或消除病毒BVDV 1型和2型。在罹患疾病之后,使受试者接触(例如给药)本发明的mRNA、组合物等,从而与不接触时相比使该疾病的症状减轻,并不意味着必需完全抑制疾病的症状。罹患疾病是指:身体出现了疾病症状。
术语“预防”是指:表示减少或消除病毒BVDV 1型和2型。在罹患疾病之前,通过使受试者接触(例如给药)本发明的mRNA、组合物等,从而与不接触时相比减轻罹患疾病后的症状,并不意味着必需完全抑制患病。
术语“有效量”指本发明的重组核酸分子、重组表达载体、mRNA、疫苗或组合物的这样的量或剂量,其以单一或多次剂量施用对象后,在需要治疗或预防的患者中产生预期效果。有效量可以由技术人员通过考虑以下多种因素来容易地确定:诸如哺乳动物的物种;大小、年龄和健康状况;涉及的具体疾病;疾病的程度或严重性;个体患者的应答;施用的具体抗体;施用模式;施用制剂的生物利用率特征;选择的给药方案;和任何伴随疗法的使用。
术语“药学上可接受的载体”是指在药物生产领域中广泛采用的辅助物料。使用载体的主要目的在于提供一种使用安全、性质稳定和/或具有特定功能性的药物组合物,还在于提供一种方法,以便在为受试者体内得到有效吸收。药学上可接受的载体可以是具有惰性的填充剂,也可以是为药用组合物提供某种功能(例如稳定组合物的整体pH值或防止组合物中活性成分的降解)的功效成分。药学上可接受的载体的非限制性实例包括但不限于粘合剂、助悬剂、乳化剂、稀释剂(或填充剂)、成粒剂、粘胶剂、崩解剂、润滑剂、抗粘合剂、助流剂、胶凝剂、吸收延迟剂、溶解抑制剂、增强剂、吸附剂、缓冲剂、螯合剂、防腐剂、着色剂、矫味剂和甜味剂等。
术语“可兽用的载体”包括任何和所有的溶剂、分散剂、 包衣剂、佐剂、稳定剂、稀释剂、防腐剂、抗菌剂和抗真菌剂、等渗剂、 吸附延迟剂等等。稀释剂可包括水、盐水、葡萄糖、乙醇、甘油等等。其 中等渗剂可包括氯化钠、葡萄糖、甘露醇、山梨糖醇和乳糖。其中稳定剂 包括白蛋白等。其中,佐剂包括但不限于RIBI佐剂体系(Ribi Inc.)、明矾、 氢氧化铝凝胶、胆固醇、水包油乳剂、油包水乳剂如,例如弗氏完全的和 不完全佐剂、Block Co-polymer(CytRx,AtlantaGA)、SAF-M(Chiron, Emeryville CA)、AMPHIGEN 佐剂、皂角苷、Quil A、QS-21(Cambridge Biotech Inc.,Cambridge MA)、GPI-0100(GalenicaPharmaceuticals,Inc., Birmingham,AL)或其他的皂角苷级份、单磷酸脂A、阿夫立定脂-胺佐剂 (Avridinelipid-amine adjuvant)、大肠杆菌热不稳定毒素(重组体或者其他)、霍乱菌毒素或胞壁酰二肽。致免疫的组合物能更进一步地包括一种或多种 其他免疫调节剂如,例如白细胞介素、干扰素或其他的细胞因子。
除非另外定义或由背景清楚指示,否则在本公开中的全部技术与科学术语具有如本公开所述领域的普通技术人员通常理解的相同含义。
本发明公开了基于mRNA的针对不同基因型BVDV的疫苗及其制备方法,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,他们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明提供的基于mRNA的针对牛病毒性腹泻病毒的疫苗及其制备方法中,所用原料及试剂均可由市售获得。
下面结合实施例,进一步阐明本发明。其中,作为一种优选,选用常规加帽mRNA架构用于制备BVDV疫苗,常规加帽mRNA利用线性化的质粒DNA为模板,在体外进行酶法共转录加帽反应合成。
实施例1分泌信号肽+牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II基因片段合成以及载体构建
从NCBI上查询BVDV 1a、1b和2基因型各突变系列病毒株的E2蛋白基因,对不同基因型的E2蛋白各自做序列比对,找寻差异度最小的E2蛋白序列;根据已报到的BVDVE2蛋白结构,截取E2结构域I-II;选取牛IgG1的分泌信号肽和Fc片段。将上述蛋白结构域序列组合,得到抗原融合蛋白(从N端到C端依次为):牛IgG1分泌信号肽+牛IgG1 Fc片段+BVDV 1aE2蛋白结构域I-II+ BVDV 1b E2蛋白结构域I-II+ BVDV 2 E2蛋白结构域I-II,C端接6His tag用于表达检测。然后基于密码子简并性对序列做优化,最后通过合成直接获得DNA序列(序列如SEQ ID NO.2所示)(委托金斯瑞公司合成),将合成的目的基因最后***至pUC57载体的多克隆位点上,得到质粒CS-BVDV-210。编码获得BVDV抗原融合蛋白多肽的氨基酸序列如SEQ ID NO.1所示。图1给出本发明BVDV三价疫苗的结构示意图,BVDV抗原融合蛋白mRNA,即分泌信号肽+牛IgG1 Fc+BVDV1a E2蛋白结构域I-II+BVDV1bE2蛋白结构域I-II+BVDV2 E2蛋白结构域I-II结构图谱的示意图。
根据编码BVDV抗原融合蛋白的基因片段制备以下(I)-(III)载体。
(I)常规加帽mRNA制备载体
步骤a:合成信号肽-牛IgG1 Fc片段-BVDV1a E2结构域I-II- BVDV1b E2结构域I-II- BVDV2 E2结构域I-II基因片段;
步骤b:构建加帽mRNA架构载体
所述加帽mRNA架构载体包含5`-UTR,3`-UTR和polyA序列以及固定加帽mRNA架构的载体pUC57,序列分别如SEQ ID NO.3-6所示;
步骤c:制备重组质粒CS-BVDV-210;
将步骤a合成的基因***加帽mRNA架构载体pUC57中,得到质粒CS-BVDV-210,所述质粒CS-BVDV-210的序列如SEQ ID NO.7所示。
(II)非加帽mRNA制备载体
步骤a:如上述(I)步骤a,合成目的基因;
步骤b:构建非加帽mRNA架构载体;
所述非加帽mRNA架构载体包含IRES序列,3`-UTR和polyA序列,以及固定非加帽mRNA架构的载体,其中IRES序列如SEQ ID NO.8所示。
步骤c:制备重组质粒CS-BVDV-220;
将步骤a合成的基因***非加帽mRNA架构载体中,得到质粒CS-BVDV-220;所述质粒CS-BVDV-220的序列如SEQ ID NO.9所示。
(III)自复制RNA架构载体
步骤a:如上述(I)步骤a,合成目的基因;
步骤b:构建VEE自复制载体;
根据α病毒家族的基因组获得自复制RNA序列;所述自复制RNA序列包含编码α病毒自复制组件的基因,缺乏制造具有传染性的α病毒颗粒的结构蛋白的基因;将自复制RNA序列构建获得的质粒作为模版扩增,通过合成获得所述VEE自复制载体;所述VEE自复制载体的序列如SEQ ID NO.10所示;
步骤c:制备重组质粒CS-BVDV-230;
将步骤a合成的基因***到VEE自复制载体的指定位置,得到重组质粒CS-BVDV-230;所述质粒CS-BVDV-230的序列如SEQ ID NO.11所示。
对照体系:在质粒CS-BVDV-210基础上去除牛IgG1 Fc序列获得,其中牛IgG1 Fc核酸序列如SEQID No.12所示)。
表1 BVDV抗原融合蛋白多肽及相关序列
名称 | 氨基酸序列 |
BVDV抗原融合蛋白 | MNPLWTLLFVLSAPRGVLSDPRCKTTCDCCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLPAPIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADGSYFLYSRLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTSKSAGKGGSGGSGGSGGSGGSGGSGGHLDCKPEFSYAIAKSDRIGPLGAEGLTTTWKDYSHEMKLEDTMVIAWCKDGKFVYLQRCTRETRYLAILHSRALPTSVVFKKLFDGRRQEDTVEMDDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCMLANRDTLDTAVVRTYRRSKPFPYRQGCGGSGGGGSGGLPDCKPDFSYAIAKNDEIGPLGATGLTTQWYEYSDGMRLQDTEVVVWCKDGEFKYLIRCEREARYLAILHTRALPTSVVFEKILNGKEQEDVVEMDDNFEFGLCPCDAKPLVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTLANKDTLATTVVRTYKRHRPFPYRQGCGGSGGGGSGGFPECKEGFQYAISKDKKMGLLGPESLTTTWHLPTKKIVDSMVQVWCEGKDLKILRTCTKEERYLVAVHERALSTSAEFLQISDGTTGPEVIDMPDDFEFGLCPCDSKPVIKGKFNTSLLNGPAFQMVCPQGWTGTIECILANQDTLDTTVIRTYRRTTPFQRRKWCGSG (SEQ ID NO.1) |
编码BVDV抗原融合蛋白的DNA序列 | ATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCGACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCCGTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCGGAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCTGGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTTTAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGAGGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGACCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCCTCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTATCCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGTACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTTGAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCACGAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCGGATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCATCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCGCTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGGAGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTGCACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTAGTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATAACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAATACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGTACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCTATAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGGTAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGATGAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATGGCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTACCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTTCCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCGAGATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCGGGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATAGGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCGTTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTCGGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATCAGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACCTGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGTCTGGTGCGAGGGGAAGGATCTGAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGTGCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAGTCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGTTATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGC |
CCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATACGACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGGGTCCGGATAATGA (SEQ ID NO.2) | |
5`-UTR | aggactcactatttgttttcgcgcccagttgcaaaaagtgtcgccacc(SEQ IDNO.3) |
3`-UTR | cccgggctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacacc (SEQID NO.4) |
polyA | aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO.5) |
载体pUC57 | ggaagagctcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatg (SEQ ID NO.6) |
CS-BVDV-210 | tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgtaatacgactcactataAggactcactatttgttttcgcgcccagttgcaaaaagtgtcgccaccATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCGACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCCGTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCGGAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCTGGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTTTAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGAGGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGACCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCCTCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTATCCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGTACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTTGAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCACGAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCGGATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCATCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCGCTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGGAGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTGCACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTAGTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATAACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAATACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGTACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCTATAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGGTAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGATGAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATGGCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTACCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTTCCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCGAGATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCGGGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATAGGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCGTTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTCGGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATCAGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACCTGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGT |
CTGGTGCGAGGGGAAGGATCTGAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGTGCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAGTCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGTTATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGCCCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATACGACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGGGTCCGGACATCACCATCACCACCACTAATGAcccgggctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacaccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaagagctcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtac | |
tcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc(SEQ ID NO.7) | |
IRES | TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGACTCACTATTTGTTTTCGCGCCCAGTTGCAAAAAGTGTCGCCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAA (SEQ ID NO.8) |
CS-BVDV-220 | tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgtaatacgactcactataAggTTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGACTCACTATTTGTTTTCGCGCCCAGTTGCAAAAAGTGTCGCCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAAATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCGACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCCGTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCGGAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCTGGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTTTAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGAGGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGACCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCCTCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTATCCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGTACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTTGAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCACGAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCGGATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCATCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCGCTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGGAGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTGCACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTAGTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATAACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAATACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGTACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCTA |
TAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGGTAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGATGAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATGGCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTACCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTTCCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCGAGATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCGGGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATAGGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCGTTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTCGGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATCAGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACCTGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGTCTGGTGCGAGGGGAAGGATCTGAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGTGCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAGTCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGTTATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGCCCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATACGACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGGGTCCGGACATCACCATCACCACCACTAATGAcccgggctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacaccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaagagctcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagga | |
tctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc (SEQ ID NO.9) | |
VEE自复制载体 | atgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttgacatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaactgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcagaatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcctccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggttgacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactgggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattctctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgtatttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgggtacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtctcttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaacagatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagatagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccaggataggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaaggagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgaggagcccactctggaagccgatgtcgacttgatgttacaagaggctggggccggctcagtggagacacctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctgtgctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagtagtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctgtacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcgagctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatc |
tagtggtgagcgccaagaaagaaaactgtgcagaaattataagggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagtgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagtaccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacgtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaaaacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaacgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactgaacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttccgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataaagaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttcattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaataccatcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcacttgaagagacggaagttctgtttgtattcattcggtacgatcgcaaggcccgtacgcacaattcttacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaaagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattccactgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaatgactctcaaggaagcagtggctaggagagaagcagtggaggaga | |
tatgcatatccgacgactcttcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggtatgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcggaagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaagtatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgagaccaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgctgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatacttgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcgcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccccgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcgggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatgagagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttccagc | |
ctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattcagctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctagtccgccaagtctagaacaagtttgtacaaaaaagcaggctgccaccacccagctttcttgtacaaagtggtggcgcgcctatgttacgtgcaaaggtgattgtcaccccccgaaagaccatattgtgacacaccctcagtatcacgcccaaacatttacagccgcggtgtcaaaaaccgcgtggacgtggttaacatccctgctgggaggatcagccgtaattattataattggcttggtgctggctactattgtggccatgtacgtgctgaccaaccagaaacataattgaatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaagagctagggataacagggtaat(SEQ ID NO.10) | |
CS-BVDV-230 | atgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttgacatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaactgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcagaatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcctccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggttgacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactgggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattctctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgtatttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgggtacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtctcttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaacagatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagatagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccaggataggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaaggagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgaggagcccactctggaagccgatgtcgacttgatgttacaagaggctggggccggctcagtggagacacctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctgtgctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagtagtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctgtacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcgagctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatc |
tagtggtgagcgccaagaaagaaaactgtgcagaaattataagggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagtgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagtaccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacgtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaaaacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaacgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactgaacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttccgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataaagaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttcattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaataccatcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcacttgaagagacggaagttctgtttgtattcattcggtacgatcgcaaggcccgtacgcacaattcttacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaaagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattccactgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaatgactctcaaggaagcagtggctaggagagaagcagtggaggaga | |
tatgcatatccgacgactcttcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggtatgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcggaagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaagtatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgagaccaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgctgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatacttgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcgcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccccgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcgggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatgagagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttccagc | |
ctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattcagctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctagtccgccaagtctagaacaagtttgtacaaaaaagcaggctgccaccATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCGACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCCGTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCGGAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCTGGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTTTAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGAGGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGACCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCCTCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTATCCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGTACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTTGAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCACGAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCGGATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCATCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCGCTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGGAGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTGCACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTAGTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATAACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAATACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGTACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCTATAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGGTAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGATGAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATG | |
GCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTACCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTTCCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCGAGATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCGGGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATAGGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCGTTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTCGGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATCAGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACCTGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGTCTGGTGCGAGGGGAAGGATCTGAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGTGCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAGTCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGTTATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGCCCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATACGACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGGGTCCGGACATCACCATCACCACCACTAATGAacccagctttcttgtacaaagtggtggcgcgcctatgttacgtgcaaaggtgattgtcaccccccgaaagaccatattgtgacacaccctcagtatcacgcccaaacatttacagccgcggtgtcaaaaaccgcgtggacgtggttaacatccctgctgggaggatcagccgtaattattataattggcttggtgctggctactattgtggccatgtacgtgctgaccaaccagaaacataattgaatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaagagctagggataacagggtaat (SEQ ID NO.11) | |
编码牛IgG1 Fc的DNA序列 | GACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCCGTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCGGAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCTGGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTTTAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGAGGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGACCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCCTCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTATCCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGTACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTTGAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCACGAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAA(SEQ ID NO.12) |
实施例2不同类型的mRNA疫苗制备
(I)基于加帽mRNA架构载体疫苗的制备
步骤a:将质粒CS-BVDV-210用限制性内切酶酶切线性化;
步骤b:将线性化的CS-BVDV-210质粒进行体外共转录加帽反应,将7-甲基化鸟苷酸帽结构加至转录的mRNA的5`端,并对模板DNA进行降解;
(II) 基于非加帽mRNA架构载体疫苗的制备
步骤a:将质粒CS-BVDV-220用限制性内切酶酶切线性化;
步骤b:将线性化的CS-BVDV-220质粒进行体外非加帽的转录反应,并对模板DNA进行降解;
(III)基于自复制RNA结构载体疫苗的制备
步骤a:将CS-BVDV-230质粒用限制性内切酶酶切线性化;
步骤b:将线性化的CS-BVDV-230质粒进行体外共转录加帽反应,将7-甲基化鸟苷酸帽结构加至转录的RNA的5`端,并对模板DNA进行降解;
将(I-III)中各自制备的mRNA分别与脂类混合,mRNA包裹进入LNP内,得到mRNA-LNP复合物,通过浓缩及换液制得制剂溶液,从而获得所述疫苗。
实施例3质粒CS-BVDV-210及对照体系线性化和体外共转录加帽制备mRNA
实施例1中的质粒CS-BVDV-210及对照体系线性化采用BspQI酶切线性化,并采用磁珠纯化的方式回收线性化的CS-BVDV-210和对照。1%琼脂糖凝胶电泳,检测质粒CS-BVDV-210和对照是否完全线性化。
线形化后的CS-BVDV-210和对照质粒使用T7 RNA聚合酶开始体外转录反应,帽子类似物7-甲基化鸟苷酸帽结构直接加入体外转录反应体系中,所以体外转录制备的mRNA其5`端携带帽子类似物。体外转录反应结束后,采用DNase酶对模板DNA进行降解。
实施例4 mRNA-分泌信号肽+牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II及对照体系与脂类包裹
本发明mRNA以及实施例1中的对照体系,与下列脂类共同混合,即可电离脂质DHA-1(赛诺邦格,货号0604000930)、辅助脂质DSPC(赛诺邦格,货号06030001100)、胆固醇(艾伟拓,57-88-5)、DMG-PEG2000(赛诺邦格,货号06020112402)四种脂类,其中四种脂类的摩尔比为DHA-1:DSPC:胆固醇:DMG-PEG2000为50:10:38.5:1.5,其中mRNA与LNP(四种脂类总和),摩尔比为1:5。各组分溶解在无水乙醇中,通过混合器进行快速的混合,从而引起脂类的沉淀及在电荷作用下mRNA包裹进入LNP内。mRNA-LNP复合物随后通过浓缩及换液至制剂溶液中。
实施例5 Western Blot和Dot Blot检测mRNA在细胞中的表达和分泌情况
将HEK293T细胞传代培养,待细胞数量足够,胰酶消化至细胞培养6孔板的每孔,铺板37℃ CO2培养箱过夜培养。第二天将脂质体包裹的mRNA转染到铺好的HEK293T细胞中,培养48h后,收集细胞裂解液和上清培养基。
1)Western Blot检测:
电泳:聚丙烯酰胺分离胶浓度为12%,蛋白上样量为40ug,浓缩胶电泳条件为90V,分离胶电泳条件为120V。
转膜:使用PVDF膜转膜,在100v电压下转膜1h。
封闭:使用1xTBST配制5%BSA作为封闭液,封闭1h。
一抗孵育:使用封闭液将6His抗体以1:2500进行稀释,在4℃孵育过夜。/>
二抗孵育:使用封闭液稀释对应的二抗,稀释比为1:5000,在室温下孵育1h。
显影:显影液A液:B液=1:1,在成像***中进行显影。
2)Dot blot检测:
回收的上清培养基,取100ul滴加在PVDF膜上;待其干燥后,用1TBST配制的5%BSA作为封闭液,封闭1h。
一抗孵育:使用封闭液将6His抗体以1:2500进行稀释,在4℃孵育过夜。
二抗孵育:使用封闭液稀释对应的二抗,稀释比为1:5000,在室温下孵育1h。
显影:显影液A液:B液=1:1,在成像***中进行显影。
实验结果见图2A-图2D是本发明BVDV抗原融合蛋白mRNA以及对照的Western Blot和Dot blot检测结果图,其中图2A是本发明mRNA,即分泌信号肽+牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II,在体外转染HEK293T细胞后,牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II的Dot blot蛋白表达图;图2B是本发明mRNA在体外转染HEK293T细胞后,牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II的Western Blot蛋白表达图;图2C是本发明mRNA(Fc+)以及对照(不包含牛IgG1 Fc,即Fc-)在体外转染HEK293T细胞后,Western Blot蛋白表达图;图2D是本发明mRNA(Fc+)以及对照(不包含牛IgG1 Fc,即Fc-)在体外转染HEK293T细胞后,Dot blot蛋白表达图。
图2A、图2B是mRNA在体外转染HEK293T细胞后,牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II蛋白表达图,WB(图2B)和Dot Blot(图2A)结果表明体外转染细胞后能高效表达和分泌相应的目标蛋白。由图2A、图2B可知,mRNA-牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II在体外转染HEK293T细胞后,牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II蛋白表达和分泌情况较好。
此外,图2C、图2D显示,不包含牛IgG1 Fc的对照,相对于本发明的mRNA,其分泌表达很低。证实本发明mRNA,添加的牛IgG1 Fc结构域,对目的蛋白多肽BVDV抗原融合蛋白的表达和分泌发挥着至关重要的作用。
实施例6 mRNA-牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II疫苗免疫小鼠诱导的抗BVDV中和抗体检测
6~8周龄的BALB/c雌鼠适应性喂养7天后,5只为实施例4制备的mRNA-LNP复合物组(包含mRNA-牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II++BVDV2结构域I-II),5只为对照组(不采取任何给药措施,只用来采血用于对比分析)。给药方式为腹股沟皮下注射给药,每组动物免疫3次,第0天(D0)、第14天(D14)和第28天(D28)给药,给药剂量为10ugmRNA-LNP/只/次。图3给出本发明BVDV抗原融合蛋白mRNA小鼠免疫和采样过程示意图。
图3为本发明BVDV抗原融合蛋白mRNA小鼠免疫和采样过程示意图,其显示,采血时间点为D7/D14/D21/D28/D35/D42,每次取血时每只小鼠全血取100μl以上,以满足血清达到50μl为准。5只小鼠固定为一个生物样品小组,每小组中的5只小鼠提取的血清混合为一个生物样品(250μl)。因此每个样品组每次采样后得到1个样品。
中和抗体滴度检测方法为ELISA,具体操作如下:
将MDBK细胞用胰酶消化后用含10%胎牛血清的DMEM培养基调整为2x105个细胞/mL;均匀的铺到96孔细胞培养板,100ul/孔,37℃ CO2培养箱培养过夜。
待检血清56℃灭火30min,10,000g离心10min,用含2%胎牛血清的DMEM培养基从1:8开始做2倍倍比稀释。将不同亚型的BVDV悬液(BVDV1a:NADL株(GenBank ID:AJ133738.1),BVDV 1b:JS2201株(GenBank ID:OP856581),BVDV2:C201602(GenBankID:MG420995))按照100TCID50/50μl的接种量用含2%胎牛血清的DMEM稀释。将等体积的病毒液与不同稀释度的待检血清混合,37℃孵育1h。孵育完成的混合液加入96孔细胞培养板的MDBK细胞中,每孔100ul,每份样品做4个重复。同时设置阳性、阴性血清对照(1:8稀释)、空白对照和接毒细胞对照。放入细胞培养箱继续培养5天。
通过间接免疫荧光实验进行检测,具体操作如下:
弃培养液,加入免疫染色固定液室温固定细胞20min,PBST洗涤3次,加入免疫染色封闭液37℃封闭1h,PBST洗涤3次,加入BVDV单克隆抗体(AHVLA,1:100稀释),100μl/孔,37℃孵育1h,PBST洗涤3次,加入1:500稀释的FITC标记的羊抗小鼠IgG,100μl/孔,37℃孵育1h,PBST洗涤3次,置荧光显微镜观察。统计阳性和阴性细胞孔数,按照Reed-Muench法计算中和抗体效价。
实验结果:小鼠血清中抗BVDV 1a、1b和2基因型毒株的中和抗体滴度。表2为抗体检测结果(BVDV-1a型);表3为抗体检测结果(BVDV-2型);表4为抗体检测结果(BVDV-1b型)。
表2 抗体检测结果(BVDV-1a型)
表3 抗体检测结果(BVDV-2型)
表4 抗体检测结果(BVDV-1b型)
注:表2-表4中 / 表示未检测
由表2-表4结果可知,mRNA-牛IgG1 Fc+BVDV1a结构域I-II+BVDV1b结构域I-II+BVDV2结构域I-II在动物中能够诱导产生高水平的中和抗体。在本实施例中,所选用的研究对象为小鼠。但是可以推知,该mRNA所表达的Fc片段源自牛IgG1,该Fc所融合的蛋白可提高融合蛋白在牛体内的半衰期,在牛体内进一步放大诱导产生的免疫反应。并且本实施例的实验结果显示,运用本发明的方法得到的三价BVDV mRNA疫苗能够很好的在小鼠体内表达,并诱导产生较为显著的中和抗体效价。本发明通过使用一条mRNA能够同时表达多个BVDV基因型的抗原,具有强免疫原性和显著的免疫效果,因此具有良好的应用前景。
本公开的上述实施例仅是为清楚地说明本公开所作的举例,而并非是对本公开的实施方式的限定。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他不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本公开的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本公开权利要求的保护范围之内。
Claims (17)
1.一种BVDV抗原融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ ID NO.1所示。
2.一种重组核酸,其特征在于,所述重组核酸为编码权利要求1所述BVDV抗原融合蛋白的核酸。
3.根据权利要求2所述的重组核酸,其特征在于,所述重组核酸的核酸序列如SEQ IDNO.2所示。
4.一种重组mRNA,其特征在于,所述重组mRNA为编码权利要求1所述BVDV抗原融合蛋白的mRNA。
5.根据权利要求4所述的重组mRNA,其特征在于,所述mRNA还包含5`-UTR,3`-UTR,polyA序列,IRES序列中的一种或多种。
6.根据权利要求5所述的重组mRNA,其特征在于,所述5`-UTR的序列如SEQ ID NO.3所示,所述3`-UTR的序列如SEQ ID NO.4所示,所述polyA的序列如SEQ ID NO.5所示,所述IRES序列如SEQ ID NO.8所示。
7.一种表达盒,其特征在于,包含权利要求2或3所述重组核酸、或权利要求4-6任一项所述重组mRNA。
8.一种载体,其特征在于,包含权利要求2或3所述重组核酸、或权利要求4-6任一项所述重组mRNA、或权利要求7所述表达盒。
9.一种细胞,其特征在于,包含权利要求2或3所述重组核酸、或权利要求4-6任一项所述重组mRNA、或权利要求7所述表达盒、或权利要求8所述载体。
10.一种重组菌,其特征在于,包含权利要求2或3所述重组核酸、或权利要求4-6任一项所述重组mRNA、或权利要求7所述表达盒、或权利要求8所述载体。
11.一种组合物,其特征在于,包含权利要求1所述BVDV抗原融合蛋白、或权利要求2或3所述重组核酸、或权利要求4-6任一项所述重组mRNA、或权利要求7所述表达盒、或权利要求8所述载体、或权利要求9所述细胞、或权利要求10所述重组菌。
12.一种试剂盒,其特征在于,包含权利要求1所述BVDV抗原融合蛋白、或权利要求2或3所述重组核酸、或权利要求4-6任一项所述重组mRNA、或权利要求7所述表达盒、或权利要求8所述载体、或权利要求9所述细胞、或权利要求10所述重组菌、或权利要求11所述组合物。
13.一种三价BVDV mRNA疫苗,包含:
(a) 权利要求4-6任一项所述重组mRNA;和
(b) 药学上可接受的载体。
14.一种表达重组三价BVDV mRNA的加帽mRNA重组表达载体、非加帽mRNA重组表达载体或自复制RNA重组表达载体的制备方法,其特征在于,包括如下步骤:
步骤a:合成权利要求2或3所述重组核酸的基因片段;
步骤b:构建加帽mRNA架构载体、构建非加帽mRNA架构载体、或构建VEE自复制载体;
所述加帽mRNA架构载体包含5`-UTR,3`-UTR和polyA序列以及固定加帽mRNA架构的载体,序列分别如SEQ ID NO.3-6所示;或者,
所述非加帽mRNA架构载体包含IRES序列,3`-UTR和polyA序列,以及固定非加帽mRNA架构的载体,其中IRES的序列如SEQ ID No.8所示;或者,
构建VEE自复制载体:根据α病毒家族的基因组获得自复制RNA序列;所述自复制RNA序列包含编码α病毒自复制组件的基因,缺乏制造具有传染性的α病毒颗粒的结构蛋白的基因;将自复制RNA序列构建获得的质粒作为模版扩增,通过合成获得所述VEE自复制载体;所述VEE自复制载体的序列如SEQ ID NO.10所示;
步骤c:制备加帽mRNA、非加帽mRNA或自复制RNA重组表达载体;
将步骤a合成的基因***加帽mRNA架构载体中,得到加帽mRNA重组表达载体;或者,
将步骤a合成的基因***非加帽mRNA架构载体中,得到非加帽mRNA重组表达载体;或者,
将步骤a合成的基因***到VEE自复制载体的指定位置,得到自复制RNA重组表达载体。
15.一种三价BVDV mRNA疫苗的制备方法,其特征在于,包括:
(a)提供权利要求4-6任一项所述重组mRNA;
(b)将所述mRNA与脂质颗粒进行混合并孵育,从而形成包裹有mRNA的脂质纳米颗粒。
16.一种三价BVDV加帽mRNA架构载体疫苗、非加帽mRNA架构载体疫苗、或基于自复制RNA结构载体疫苗的制备方法,其特征在于,包含如下步骤:
步骤a:将权利要求14中的所述加帽mRNA重组表达载体、非加帽mRNA重组表达载体或自复制RNA重组表达载体用限制性内切酶酶切线性化;
步骤b:将线性化的所述加帽mRNA重组表达载体进行体外共转录加帽反应,将7-甲基化鸟苷酸帽结构加至转录的mRNA的5`端,并对模板DNA进行降解;或者,
将线性化的所述非加帽mRNA重组表达载体进行体外非加帽的转录反应,并对模板DNA进行降解;或者,
将线性化的所述自复制RNA重组表达载体进行体外共转录加帽反应,将7-甲基化鸟苷酸帽结构加至转录的RNA的5`端,并对模板DNA进行降解;
步骤c:将步骤b中各自制备的加帽、非加帽、自复制mRNA分别与脂类混合,mRNA包裹进入LNP内,得到mRNA-LNP复合物,通过浓缩及换液制得制剂溶液,从而获得所述疫苗。
17.权利要求1所述BVDV抗原融合蛋白、或权利要求4-6任一项所述重组mRNA在以下任一项中的用途:(I)制备治疗或预防BVDV感染引起的疾病的药物;或者,(II)制备治疗或预防牛病毒性腹泻的药物;或者(III)制备在施用对象体内诱导产生针对BVDV的特异性免疫反应的药物。
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