CN113881617B - 靶向树突状细胞表达h7n9禽流感ha1抗原的重组乳酸菌及其应用 - Google Patents
靶向树突状细胞表达h7n9禽流感ha1抗原的重组乳酸菌及其应用 Download PDFInfo
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Abstract
本申请提供一种靶向树突状细胞表达H7N9禽流感HA1抗原的重组乳酸菌及其应用,所述重组植物乳杆菌中含有HA1‑DCpep融合基因,所述HA1‑DCpep融合基因由HA1基因片段与3个DCpep基因片段串联组成。该重组植物乳杆菌所表达的融合蛋白具有良好的免疫原性,且重组植物乳杆菌能够将禽流感病毒融合抗原锚定表达在植物乳杆菌表面。此外,本发明的个的重组植物乳杆菌为非抗生素乳酸菌对环境而言更为友好,对动物而言避免了抗生素耐受,以其对动物进行免疫,能诱导派氏淋巴结DC的活化,诱导产生特异性T细胞反应,并对B细胞反应产生影响。
Description
技术领域
本申请涉及基因工程技术领域,具体涉及靶向树突状细胞表达H7N9禽流感HA1抗原的重组乳酸菌及其应用。
背景技术
本发明背景技术中公开的信息旨在增加对本发明总体背景的理解,而该公开不应必然被视为承认或以任何形式暗示该信息已经成为本领域一般技术人员所公知的现有技术。
禽流感(AI)是一种严重的家禽疾病。禽流感病毒(avian influenza virus,AIV)属于正黏病毒科流感病毒属甲型流感病毒(国际病毒分类委员会[ICTV],2018)。禽流感病毒根据两种主要表面糖蛋白、血凝素(HA)和神经氨酸酶(NA)的抗原差异分为亚型。目前,在鸟类种群中发现了16种HA亚型(H1-H16)和11种NA亚型(N1-N11)的不同组合。禽流感病毒分为高致病性禽流感(HPAI)和低致病性禽流感(LPAI)病毒。家禽中的高致病性禽流感病毒有H5和H7亚型。(Palese,P.,and Shaw,M.L.(2013).“Orthomyxoviridae:the viruses andtheir replication,”in Fields Virology,eds D.M.Knipe and P.M.Holey(Philadelphia:Lippincott Williams&Wilkins),1151–1186.)。此外,近年来,AIV的H5、H7、H9和H10亚型已引起人类感染,其中H5和H7可引起人类严重疾病。
H7N9是禽流感病毒中的一个亚型,很多研究显示H7N9 AIV是一个新型重组AIV,其六个内部基因来源于H9N2 AIV,而表面的2个基因节段来自野禽。H7N9对禽类具有低致病性,但对于人类是高致病性的,致死率高达41%,对于人类健康具有很大威胁。相较于其他亚型比如H9N2,H7N9更容易引起公众威胁,原因可能在于:H7N9对禽类具有低致病力,因此对鸟和禽类不表现致病力或仅表现温和的致病力,因而基本在家畜的流行中表现不明显,不易被察觉而给防控带来很大难度。但是H7N9相比其他亚型的流感病毒表现出更容易适应哺乳动物的特性。以及,H7N9病毒的绝大多数表现出金刚烷胺的耐药性和神经氨酸酶抑制性。并且,值得注意的是,H7N9在能够被检测到病毒之前,该病毒可能已经在动物体内传播数月之久,加之活禽市场交易的活跃度,也更是加大了H7N9的防控难度。
血凝素(hemagglutinin,HA)是流感病毒中一种重要的多功能蛋白。HA蛋白介导病毒与宿主细胞受体的结合。它是感染期间诱导中和抗体的主要抗原。HA是一种三聚体,具有一个大的球状免疫显性头部。每个HA单体都被合成为无活性的前体蛋白HA0。HA0蛋白被细胞蛋白酶切割成HA1和HA2亚单位。形成球状头部的HA1亚单位在亚型间高度可变,并包含主要的中和表位。HA2亚单位形成大部分柄结构域,在亚型间相当保守,包含少量但交叉反应的中和表位。现有的研究在鸡中评估了重组新城疫病毒表达H5N1高致病性禽流感病毒HA蛋白、HA1和HA2亚单位在中和抗体的诱导和保护中的贡献情况,结果表明,当HA1和HA2亚单位分离时,既不能提供保护,也不能诱导中和抗体,而表达完整HA蛋白时可提供完全保护,因而,在重组病毒载体中使用HA蛋白进行免疫是完全防止高致病性禽流感病毒所必需的(Rong L,Park J K,Zou W,et al.Contributions of HA1 and HA2 Subunits of HighlyPathogenic Avian Influenza Virus in Induction of Neutralizing Antibodies andProtection in Chickens[J].Frontiers in Microbiology,2020,11)。这些结果对流感病毒疫苗的开发有着重要的影响。
在流行国家,接种疫苗被认为是控制AI的实用方法。然而,不建议在家禽中使用针对AIV的活疫苗,因为疫苗和野毒株之间的基因组片段重配存在潜在风险,这可能导致出现具有不同抗原特性的病毒变体。目前该领域使用的绝大多数AIV疫苗(95.5%)是灭活疫苗。目前H7N9禽流感也主要靠灭活苗免疫来达到预防目的,缺乏口服疫苗。灭活疫苗需要通过肌肉注射的方式来免疫动物,既是对操作技术的考验,同时也增加了动物应激的几率。然而,灭活疫苗不会引起强烈的免疫反应,且其生产和施用过程昂贵、劳动密集且耗时。随着免疫学研究的迅速发展,使用基因工程技术研发的新型疫苗不断出现,但依然普遍存在免疫原性弱,不能诱导机体产生有效的免疫应答等缺点。
发明内容
为了改善现有技术的不足,本发明提供了靶向树突状细胞表达H7N9禽流感HA1抗原的重组乳酸菌及其应用,该菌中含有HA1-DCpep融合基因,所述HA1-DCpep融合基因由HA1基因片段与3个DCpep基因片段串联组成。该重组植物乳杆菌所表达的融合蛋白具有良好的免疫原性,且重组植物乳杆菌能够将禽流感病毒融合抗原锚定表达在植物乳杆菌表面。此外,本发明的重组植物乳杆菌为非抗生素乳酸菌对环境而言更为友好,对动物而言避免了抗生素耐受,以其对动物进行免疫,具有更优的免疫效果,能诱导派氏淋巴结DC的活化,诱导产生特异性T细胞反应,并对B细胞反应产生影响。
具体地,本发明提供了下述的技术特征,以下技术特征的一个或多个的结合构成本发明的技术方案。
在本发明的第一方面,本发明提供了一种表达H7N9禽流感抗原的重组植物乳杆菌,其含有HA1-DCpep融合基因,所述HA1-DCpep融合基因由HA1基因片段与3个DCpep基因片段串联组成。
在本发明的实施方式中,发明人在构建融合抗原时发现仅使用HA1时相较于HA表达得到的抗原具有更好的免疫原性,在重组乳酸菌中更容易表达,并且在相同条件下含有HA1-DCpep融合基因的重组植物乳杆菌相较于含有HA-DCpep的重组乳酸菌具有更高的抗原表达量。在本发明的一些实施方式中,所述HA1基因为Influenza A virus(A/chicken/China/WYG1/2019(H7N9))序列,其GenBank登录号为MN700034.1。
在本发明的一些实施方式中,所述DCpep基因片段如SEQ ID NO:1中所示的核苷酸序列,3个DCpep基因片段串联组成的序列表示为3×DCpep,其核苷酸序列如SEQ ID NO:2中所示。
树突状细胞(dendritic cell,DC)的重要作用是摄取、加工处理和递呈抗原,刺激机体产生免疫应答反应。DC是一类成熟时具有许多树突状突起的细胞,因此而命名。这类细胞成熟时能够识别、摄取和加工外源性抗原并将抗原肽提呈给初始T细胞进而诱导T细胞活化增殖的、功能最强的抗原提呈细胞。DC是机体适应性免疫应答的始动者,也是连接固有免疫应答和适应性免疫应答的“桥梁”。
发明人在研究中发现,DCpep(树突状细胞诱导肽)能很好地靶向树突状细胞,从而引起机体的免疫应答。用DCpep作为佐剂,有着序列短的优势,可以解决标签表达的难题,同时也为抗原制备提供了方便。但是,DCpep仅由12个氨基酸组成,DCpep由12个氨基酸组成,能够特异性靶向结合树突状细胞(DCs),有助于DCs识别抗原,诱发免疫反应,且序列短,对免疫稳态起重要作用,然而,在本发明的的一些实施方式中,发明人发现由12个氨基酸组成的DCpep,其组成使其空间构象容易被改变,DCpep的惯常优势难以稳定发挥,发明人对此进行了很多研究来改善这种新发现的不利的情况,最终在研究过程中发现将DCpep做串联使用时能够改善上述问题,尤其是将3个DCpep做串联时使用既能避免空间构象的改变又能使其充分暴露,并且对DC细胞的作用更好,在发挥其惯常优势的前提下,能够更好的协助激发机体更强的免疫应答。
具体地,在发明人的上述构思下,发明人展开了研究,在研究过程中,发明人构建了以1-5个DCpep串联为目的片段(分别标示为1×DCpep、2×DCpep、3×DCpep、4×DCpep、5×DCpep),pWCF为载体的5组重组乳酸菌(原始出发菌株为NC8/Δalr)。然后进行了体外DC刺激试验。结果在CD80分子的检测中,3×DCpep组表面的CD80极显著高于1×DCpep组(P<0.001),也高于2×DCpep组(P<0.05)。在CD86分子的检测中,与1×DCpep组相比,3×Dcpep组CD86分子都显著上调(P<0.05)。此外,发明人还发现,序列过长不利于标签表达以及抗原的制备,因此,综合结果,3个DCpep串联能对DC达到更好的作用效果,随DCpep数量的增加对DC表面分子的活化没有显著差异,因此,3个DCpep串联效果最佳。
以及,相较于传统方法中目的片段需要连接表达载体,再转化到菌中扩增表达,蛋白纯化后再免疫白兔获得抗体血清的方式,在本发明的实施方式中,以3个DCpep为目的片段,能通过给动物比如白兔免疫DCpep多肽来获得多克隆抗体血清,进而能够更方便的检验重组菌的表达,从而在达到验证目的的基础上免去了传统方法的复杂步骤。
在本发明的一些实施方式中,所述HA1-DCpep融合基因为如SEQ ID NO:3中所示的核苷酸序列、或与SEQ ID NO:3所示序列具有至少90%序列同源性的且具有相同功能的核苷酸序列、或在高严谨条件下可与SEQ ID NO:3所示序列杂交的核苷酸序列。
在本发明的一些实施方式中,所述HA1-DCpep融合基因中还可以含有启动子、终止子和酶切位点基因序列。优选地,所述酶切位点为Xba I(TCTAGA)和Hind III(AAGCTT)。
在本发明的一些实施方式中,所述重组乳杆菌中还导入了pWCF的基因片段,其包含如SEQ ID NO:4中所示的核苷酸序列。
在本发明的一些实施方式中,所述重组植物乳杆菌以植物乳杆菌NC8为原始出发菌株,优选为丙氨酸消旋酶基因(alr)缺陷型植物乳杆菌NC8/Δalr。
重组的外源蛋白通常可由质粒表达载体定向运输,根据蛋白与宿主菌的相对位置,我们将重组外源蛋白的表达情况分为胞内分泌表达,胞外分泌表达及菌株表面锚定表达。聚谷氨酸合成酶A(pgsA)来自枯草芽孢杆菌,由1143个核苷酸构成,可编码381个氨基酸,是一段膜蛋白锚定序列,其N端存在一个跨膜区,为其表面展示创造了条件。本发明所述的pWCF载体是在pSIP409载体基础上改进而来,以asd-alr为非抗性筛选标记替换原有的erm,消除了抗生素对环境或是人类健康的潜在影响。该诱导型大肠杆菌-乳酸菌穿梭表达载体以天冬氨酸-β半醛脱氢酶(asd)基因和丙氨酸消旋酶基因(alanine racemase gene,alr)作为无抗筛选标记,采用asd基因缺陷大肠杆菌(E.coliχ6212)作为质粒构建中间宿主菌,alr基因缺失株NC8/Δalr为表达菌。并且,其中包含pgsA’(pgsA截短为pgsA’),来作为表面单锚定表达的原件,使抗原能够在受体菌表面锚定并稳定表达。
在本发明的一些实施方式中,所述重组植物乳杆菌表示为NC8Δ-pWCF-HA1-DCpep,其相较于NC8Δ-pWCF、NC8Δ-pWCF-HA1均具有更优异的蛋白表达量,且表达的蛋白具有优异的免疫原性。并且,值得关注的是,重组植物乳杆菌NC8Δ-pWCF-HA1-DCpep相较于其原始出发菌株具备更好的耐酸碱、胆盐的能力,和良好的肠道粘附力,能够更好的适应机体,更好的定植于体内,并且稳定的表达抗原蛋白。
在本发明的第二方面,本发明提供了一种融合基因HA1-DCpep,其由HA1基因片段与3个DCpep基因片段串联组成其中,所述HA1-DCpep融合基因的核苷酸序列如SEQ ID NO:3中所示、或为与SEQ ID NO:3所示序列具有至少90%序列同源性的且具有相同功能的核苷酸序列、或为在高严谨条件下可与SEQ ID NO:3所示序列杂交的核苷酸序列。
此外,在本发明的一些实施方式中,所述HA1-DCpep融合基因中还可以含有启动子、终止子和酶切位点基因序列;比如,所述酶切位点为Xba I和Hind III。
在本发明的第三方面,本发明提供了一种融合蛋白,其由上述第二方面中所述的融合基因HA1-DCpep编码,所述融合蛋白的氨基酸序列中包含与SEQ ID NO:5所示的氨基酸序列(或者如SEQ ID NO:5所示)、或与SEQ ID NO:5所示的核苷酸序列具有至少90%序列同源性的序列;
以及,所述融合蛋白可由上述第一方面中所述的表达H7N9禽流感抗原的重组植物乳杆菌表达产生。载体上的表面锚定元件会和目的片段融合表达,因此,在本发明的一些实施方式中,所述重组植物乳杆菌表达产生的融合蛋白的氨基酸序列中可以包含表面锚定元件pgsA’表达产生的氨基酸,表达产生的融合蛋白的氨基酸序列如SEQ ID NO:6中所示。
在本发明的第四方面,本发明提供了含有上述第二方面中所述的融合基因HA1-DCpep的表达载体。在本发明的一些实施方式中,所述表达载体由融合基因HA1-DCpep与载体pWCF连接得到,所述载体载体pWCF中包含表面锚定元件pgsA’,优选地,所述pWCF的核苷酸序列如SEQ ID NO:4中所示的核苷酸序列。
在本发明的第五方面,本发明提供了一种药物组合物或药物制剂或菌剂或饲料,其包含上述第一方面中所述的表达H7N9禽流感抗原的重组植物乳杆菌或其发酵产物或代谢产物或上述第三方面中所述的融合蛋白。
在本发明的第六方面,本发明提供了上述第一方面中所述的表达H7N9禽流感抗原的重组植物乳杆菌或上述第二方面中所述的融合基因HA1-DCpep或上述第三方面中所述的融合蛋白在制备预防和/或治疗H7N9禽流感的疫苗或药物或菌剂或饲料中的应用。
在本发明的一些实施方式中,本发明所述饲料还可以包含动物生长所需的可食用物质,比如粮食、脂类、氨基酸、微量元素、饲料添加剂、维生素等等。以及,本发明所述药物组合物或药物制剂中可以包含至少一种药物载体或药学上可接受的辅料,或者其他治疗有效的试剂。这类可以包含在内的物质不影响蛋白的表达和功能的发挥,比如虽然本发明的重组植物乳杆菌相较于其原始菌株已经具备了更优的耐酸、耐胆盐的能力和更好的肠粘附力,这些有助于它的定植,这类物质依然可以是更利于菌株在机体内更好生长和存活的,或者是能够使其缓慢释放的、定点释放的,其中,合适的药用辅料可以是本领域已知的种类,比如溶剂、缓冲剂、稀释剂等等,例如可以是作者Paul J Sheskey等人编著的《药用辅料手册》(Handbook of Parmaceutical Excipients)中所记载的。在本发明的第七方面,本发明提供了上述第一方面中所述的表达H7N9禽流感抗原的重组植物乳杆菌或上述第二方面中所述的融合基因HA1-DCpep或上述第三方面所述的融合蛋白或上述第五方面中所述的药物组合物、药物制剂或菌剂或饲料在如下任意一种中的应用:
1)激活机体免疫细胞和/或制备激活机体免疫细胞的产品;
2)激活派氏淋巴结DC和/或制备激活派氏淋巴结DC的产品;
3)刺激脾和/或肠系膜淋巴结中的特异性细胞因子、T细胞、B细胞的产生和/或制备刺激脾、肠系膜淋巴结和/或派氏淋巴结中的特异性细胞因子的产品;所述特异性细胞因子包括CD4+IFN-γ+、CD8+IFN-γ+。
通过上述一个或多个技术手段,可实现以下有益效果:
本发明提供的重组植入物杆菌中含有HA1-DCpep融合基因,所述HA1-DCpep融合基因由HA1基因片段与3个DCpep基因片段串联组成。该重组植物乳杆菌所表达的融合蛋白具有良好的免疫原性,且重组植物乳杆菌能够将禽流感病毒融合抗原锚定表达在植物乳杆菌表面。此外,本发明的个的重组植物乳杆菌为非抗生素乳酸菌对环境而言更为友好,对动物而言避免了抗生素耐受,以其对动物进行免疫,能诱导派氏淋巴结DC的活化,诱导产生特异性T细胞反应,并对B细胞反应产生影响。以及,本发明的目的片段以3个DCpep为佐剂,能通过给白兔免疫DCpep多肽来获得多克隆抗体血清,进而能够更方便的检验重组菌的表达。传统方法中目的片段需要连接表达载体,再转化到菌中扩增表达,蛋白纯化后再免疫白兔获得抗体血清。本发明中以DCpep为佐剂,在达到验证目的的基础上免去了这一复杂步骤。
附图说明
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解,本申请的示意性实施例及其说明用于解释本申请,并不构成对本申请的不当限定。以下,结合附图来详细说明本申请的实施方案,其中:
*,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001:
图1:白兔免疫及采血时间。
图2:合成的目的片段。
图3:重组质粒pEASY-HA1的双酶切验证(A);M:DL10,000DNA Marker;1:重组质粒pEASY-HA1;2:重组质粒的双酶切验证。重组质粒pWCF-HA1的双酶切验证(B);M:DL10,000DNA Marker;1:重组质粒pWCF-HA1;2:重组质粒的双酶切验证。重组质粒pWCF-HA1-DCpep的双酶切验证(C);M:DL10,000DNA Marker;1:重组质粒pWCF-HA1-DCpep;2:重组质粒的双酶切验证。
图4:重组乳酸菌质粒NC8Δ-pWCF-HA1的PCR验证(A);M:DL2,000DNA Marker;1:重组乳酸菌质粒NC8Δ-pWCF-HA1的PCR验证。重组乳酸菌质粒NC8Δ-pWCF-HA1-DCpep的PCR验证(B);M:DL2,000DNA Marker;1:重组乳酸菌质粒NC8Δ-pWCF-HA1-DCpep的PCR验证。
图5:菌株生长曲线图。
图6:超声破碎法处理的蛋白在重组乳酸菌中表达的Western Blot检测(A);M:蛋白marker;1:NC8-pWCF的表达产物;2:NC8Δ-pWCF-HA1的表达产物;3:NC8Δ-pWCF-HA1-DCpep的表达产物。反复冻融法处理的蛋白在重组乳酸菌中表达的Western Blot检测(B);M:蛋白marker;1:NC8-pWCF的表达产物;2:NC8Δ-pWCF-HA1的表达产物;3:NC8Δ-pWCF-HA1-DCpep的表达产物。
图7:流式细胞术检测结果。
图8:免疫荧光;A,B:NC8Δ-pWCF白光,荧光;C,D:NC8Δ-pWCF-HA1白光,荧光;E,F:NC8Δ-pWCF-HA1-DCpep白光,荧光。
图9:骨髓源DC的CD80表达(左),骨髓源DC的CD86表达(右)。
图10:动物免疫程序。
图11:PPs中DC的活化。
图12:MLN中CD4+IFN-γ+的表达。
图13:MLN中CD8+IFN-γ+的表达。
图14:脾脏中CD4+IFN-γ+的表达。
图15:脾脏中CD8+IFN-γ+的表达。
图16:MLN中CD4+T细胞增值结果。
图17:MLN中CD8+T细胞增值结果。
图18:脾脏中CD4+T细胞增值结果。
图19:脾脏中CD8+T细胞增值结果。
图20:PP中B220+IgA+细胞数量变化。
具体实施方式
下面结合具体实施例,进一步阐述本申请。应理解,这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。
除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。本申请所使用的试剂或原料均可通过常规途径购买获得,如无特殊说明,本申请所使用的试剂或原料均按照本领域常规方式使用或者按照产品说明书使用。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本申请方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1靶向树突状细胞表达H7N9禽流感HA1抗原重组乳酸菌的构建及验证
通过对禽流感病毒的研究发现,HA是流感病毒中一种重要的多功能蛋白。本试验从构建NC8Δ-pWCF-HA、和NC8Δ-pWCF-HA-DCpep非抗生素重组乳酸菌。随后制备兔源DCpep多克隆抗体血清,抗体经western blot技术验证。最后,通过流式细胞术验证了目的蛋白的免疫原性,并使用免疫荧光技术检测目的蛋白的锚定表达情况,为进一步的试验奠定了基础。通过以上试验,证明了经诱导后的重组乳酸菌具有免疫原性且能够锚定表达在NC8/Δalr植物乳杆菌的表面。
1.1材料与方法
菌株及质粒材料:E.coliχ6212(asd auxotroph,cloning host)由吉林农业大学微生态制剂工程研究中心保存并提供;NC8/Δalr(L.plantarum NC8 D-alanineauxotroph)由吉林农业大学微生态制剂工程研究中心保存并提供;409ata(无抗性质粒)由吉林农业大学微生态制剂工程研究中心保存并提供。
酶与主要试验试剂:DNA连接酶T4 DNA ligase、pEASY-Blunt-zero、Trans1-T1感受态购自全式金生物有限公司;DNA末端平滑试剂盒购自碧云天生物技术公司;SppIP由吉林省动物微生态制剂工程研究中心保存并提供;HRP标记的山羊抗鼠IgG购自于Bioss公司;anti-H7N9血凝素鼠单抗购自SinoBiological公司;HRP标记的山抗兔IgG购自于CellSignaling Technology公司;试验所需其他试剂为国产分析纯产品或进口。
试验主要仪器设备:梯度PCR仪(Eppendorf AG)(德国徕卡公司);凝胶成像分析系统(Universal HoodⅡ);制冷型金属浴(TANGEN公司);生化培养箱HERACELL 240i(美国Thermo Scientific公司);电泳仪(041BR 02682);分析天平ME204E(瑞士梅特勒公司);电击穿孔仪(Gene Pulser XcellTM System)。
试验方法
重组乳酸菌的构建与鉴定
目的基因HA1-DCpep的合成:
HA1基因序列:Influenza A virus(A/chicken/China/WYG1/2019(H7N9))GenBank登录号:MN700034.1(https://www.ncbi.nlm.nih.gov/nuccore/AF322026)。HA1序列与3个DCpep串联为目的片段,并连接到pUC-GW-Kan载体。
注:Xba I酶切位点基因序列:TCTAGA;Hind III酶切位点基因序列:AAGCTT;DCpep基因序列:TTTTATCCATCATATCATTCAACTCCACAACGTCCA(SEQ ID NO:1)。3×DCpep基因序列如SEQ ID NO:2所示。
引物设计:
为了从pUC-HA1-DCpep质粒中获得HA1片段,设计了引物HF、HR,其序列如下:
HF 5’-TCTAGAATGGACAAAATCTGCCTCG-3’(SEQ ID NO:7)
HR 5’-AAGCTTATCTCGCAGTCCGTTTTCT-3’(SEQ ID NO:8)
为了鉴定重组质粒的正确性,设计了针对pWCF-alr载体的通用引物,能够鉴定pgsA’序列后Xba I和Hind III之间的基因片段,用于PCR鉴定及测序。引物序列如下:
F 5’-AGATATTGTTGGTGCTGG-3’(SEQ ID NO:9)
R 5’-TCAATCAAAGCAACACG-3’(SEQ ID NO:10)
pEASY-HA1重组质粒的构建
HA1目的片段的获取:
使用高纯度质粒小提试剂盒提取pUC-HA1-DCpep质粒,按照试剂盒说明书中记载的方法进行。然后通过PCR从pUC-HA1-DCpep质粒克隆出HA1目的片段。PCR体系如下:
表1-1PCR回收体系
PCR扩增条件如下:
取PCR产物与10×Loading Buffer按比例混合进行琼脂糖凝胶电泳,然后进行胶回收,胶回收方法按照试剂盒说明书执行。
HA1片段与pEASY-Blunt-zero载体连接:
HA1片段胶回收产物通过T4 DNA连接酶与pEASY-Blunt-zero载体连接。连接体系如下:
表1-2连接反应体系
连接条件为25℃2h。
pEASY-HA1重组质粒转化到Trans1-T1感受态:
通过普通转化方法将pEASY-HA1重组质粒转化到Trans1-T1感受态。
a.取50μL冰浴上融化的感受态细胞,加入目的DNA,轻轻混匀,在冰浴中放置30分钟;
b.42℃水浴热激30秒,然后快速将管转移到冰浴中2分钟,该过程不要摇动离心管;
c.向每个离心管中加入500μL无菌的SOC或LB培养基(不含抗生素),混匀后置于37℃,200rpm培养1小时,使细菌复苏。
d.吸取100μL已转化的感受态细胞加到含卡那霉素的LB琼脂培养基上,将细胞均匀涂开。将平板置于37℃至液体被吸收,倒置平板,37C过夜培养。
pEASY-HA1重组质粒酶切鉴定:
挑取单菌落到5mL LB液体培养基中37℃培养过夜。吸取200μL 80%甘油和800μL菌液到冻存管中,混匀,-80℃冰箱保存菌种。剩下新鲜菌液提取质粒,进行双酶切鉴定。酶切体系如下:
表1-3酶切体系
37℃酶切过夜后进行琼脂糖凝胶电泳,鉴定条带大小。
目的片段和载体的获取:
通过双酶切,从实验室保存的409ata中获得pWCF载体(SEQ ID NO:4)备用。同样地,通过双酶切,从pEASY-HA1、pUC-HA1-DCpep上分别切下HA1、HA1-DCpep目的片段备用。酶切体系如下:
表1-4酶切体系
37℃酶切过夜后进行胶回收,胶回收方法按照试剂盒说明书进行。
目的片段和载体连接:
回收产物通过T4 DNA连接酶与载体连接。连接体系如下:
表1-5连接反应体系
连接条件为16℃过夜。
pWCF空载体构建:
在“目的片段和载体的获取”中双酶切得到载体,胶回收后使用DNA末端平滑试剂盒使酶切位点两端平滑,然后用T4 DNA连接酶使切口两端连接,获得pWCF空载体备用。
连接产物转化到E.coliχ6212感受态:
E.coliχ6212在添加DAP(终浓度为50μg/ml)的LB培养基上活化后传代培养,得到OD600在0.8-1.0的菌液,ddH2O洗一次,10%甘油洗两次菌液,然后浓缩后分装,液氮速冻后转移到-80℃冰箱保存。由此制备E.coliχ6212感受态,通过电转化法将连接产物转到E.coliχ6212感受态中,条件为2500V,200Ω,25μF。
E.coliχ6212中重组质粒的鉴定:取菌液进行小量质粒提取,按照试剂盒说明书方法进行。然后进行酶切及PCR鉴定。
酶切鉴定:酶切鉴定体系如下:
表1-6酶切体系
37℃金属浴8h后,通过琼脂糖凝胶电泳鉴定条带大小。
质粒测序:酶切及PCR鉴定正确后对质粒进行测序(吉林省库美生物科技有限公司)。
重组质粒转化到NC8/Δalr
NC8/Δalr感受态制作:
取NC8/Δalr冻存菌活化后传代培养,NC8/Δalr的培养基需要添加D-丙氨酸(终浓度0.2mg/mL)。获得OD600在0.2~0.3之间的菌液,清洗缓冲液(蔗糖34.21g、MgCl20.029g、ddH2O 100mL,pH值调至7.4)洗2遍,4℃,5000rpm离心10min,弃去上清。用电击缓冲液(Na3PO4 0.19g、MgCl2 0.009g、ddH2O 100mL,pH值调至7.4)重悬沉淀,然后以100μL每管分装,-80℃冰箱保存。
重组质粒的转化:通过电转化方法将构建完成的质粒转入NC8/Δalr。条件为:2000V,400Ω,25μF。
NC8/Δalr中重组质粒验证:先提取NC8/Δalr中质粒,按照小量革兰氏阳性菌质粒试剂盒说明书操作。将提取的重组乳酸菌质粒,使用F、R引物进行PCR鉴定。
DCpep多克隆抗体的制备
制备多克隆抗体:DCpep 12个氨基酸的短肽(生工生物工程(上海)股份有限公司),混合完全弗氏佐剂和不完全弗氏佐剂分别进行初免和加强免疫。耳缘静脉采血,离心获得血清。免疫及采血时间见图1。
鉴定多克隆抗体
乳酸菌蛋白样经过处理,获得蛋白,通过Western blot技术,血清作为一抗,山羊抗兔HRP为二抗,孵育DCpep阳性乳酸菌样品验证抗体是否制备成功。
重组乳酸菌的表达鉴定
首先乳酸菌培养条件优化,测定生长曲线:取冻存菌液活化后传代培养,30℃厌氧。隔一个小时取2mL菌液测定OD600,OD600值在0.3左右时加诱导态SPPIP(12.5μL/5mL)。此外,通过滴板菌落计数,来计算两株重组乳酸菌的免疫剂量(小鼠饲喂乳杆菌最佳数量为0.5×109~1×109CFU)。然后用超声破碎和反复冻融两种方法分别处理乳酸菌蛋白样品,来鉴定表达,包括以下三种鉴定方法:
a.Western blot技术:anti-H7N9血凝素鼠单抗为一抗,HRP标记的山羊抗鼠IgG作为二抗;
b.流式细胞术鉴定:一抗用1:800稀释的anti-H7N9血凝素鼠单抗,二抗用1:800稀释的PE标记抗鼠二抗,混匀菌体于1.5mL离心管中;
c.免疫荧光镜检:一抗用1:300稀释的DCpep兔多克隆抗体血清,二抗用1:800稀释的FITC标记抗兔二抗。
1.2结果
目的基因的合成结果:构建得到的的目的基因HA1-DCpep的大小为1099nt,如图2所示。
HA1目的片段获取结果:通过PCR获得HA1目的片段,在991bp处可见目的基因片段。
pEASY-HA1重组质粒的酶切鉴定结果:pEASY-HA1重组质粒进行双酶切鉴定,在991bp处可见目的基因片段,如图3(A)所示。
载体和目的片段的获取结果:载体和目的片段分别进行双酶切,在8121bp处可见载体条带(pWCF),991bp、1099bp处可见目的片段HA1、HA1-DCpep的条带。
E.coliχ6212中重组质粒的鉴定结果:
酶切鉴定结果:将重组质粒pWCF-HA1双酶切验证,在991bp处可见清晰条带(图3B)。将重组质粒pWCF-HA1-DCpep双酶切验证,在1099bp处可见清晰条带(图3C),表明目的片段成功连接到目的载体。
质粒测序:质粒测序结果正确。
NC8/Δalr中重组质粒的鉴定结果:利用PCR技术对NC8/Δalr中重组质粒的目的片段进行扩增,以此鉴定重组质粒是否成功转化到NC8/Δalr中。在991bp(图4A)、1099bp(图4B)处可见清晰条带,表明重组质粒成功转化到NC8/Δalr中。
DCpep多克隆抗体经Western blot技术鉴定能呈现正确大小的条带,表明多克隆抗体血清制备成功。
重组乳酸菌的表达鉴定结果
乳酸菌培养条件优化:经过3次培养及测定OD600,NC8/Δalr、NC8Δ-pWCF、NC8Δ-pWCF-HA1和NC8Δ-pWCF-HA1-DCpep的生长曲线测定结果(24h)用GraphPad Prism软件绘制,见图5。菌液转接后3h为最佳诱导时间。根据三次菌落滴板计数测定结果选择第9h(诱导后6h)为饲喂时间点,0.2mL NC8/Δalr、NC8Δ-pWCF,0.22mL NC8Δ-pWCF-HA1,0.25mL NC8Δ-pWCF-HA1-DCpep菌液中有1×10 9CFU。
Western blot技术鉴定融合蛋白表达结果:为了鉴定重组乳酸菌的免疫原性,做了Western blot检测。根据氨基酸序列预期HA1的蛋白分子量约为36KDa,HA1-DCpep的蛋白分子量约为41KDa,pgsA’表面锚定元件的蛋白分子量约为21KDa。将诱导后的重组乳酸菌超声破碎后进行检测,在58KDa、62KDa处可见清晰的蛋白条带(图6A)。经过反复冻融处理的乳酸菌,在58KDa、62KDa处可见清晰的蛋白条带(图6B)。另外,在小于融合蛋白处有蛋白条带,是融合蛋白断开的结果。因此,蛋白大小完全符合预期,说明两株重组菌都能表达相应蛋白。相较于原始菌株以及其他重组菌株,NC8Δ-pWCF-HA1-DCpep具有更高的蛋白表达量。
流式细胞术鉴定融合蛋白表达结果:NC8Δ-pWCF、NC8Δ-pWCF-HA1和NC8Δ-pWCF-HA1-DCpep经诱导表达蛋白后应用流式细胞仪进行检测,在横坐标轴上,显示图像越靠右说明其检查物质所带的荧光强度越强,也表明结合的被检测物质越多,则结果表明构建的重组质粒在乳酸菌中得到了表达,且重组乳酸菌具有良好的抗原性,如图7。
免疫荧光鉴定融合蛋白表达结果:FITC标记的二抗可与兔源一抗相结合,从而可见植物乳杆菌表面锚定表达的融合蛋白有绿色荧光。结果可见,NC8Δ-pWCF和NC8Δ-pWCF-HA1没有荧光,如图8B,D;NC8Δ-pWCF-HA1-DCpep看到荧光,如图8F。
目的基因HA1-DCpep的核苷酸序列如SEQ ID NO:3中所示;HA1-Dcpep蛋白的氨基酸序列如SEQ ID NO:5所示,其中经NC8Δ-pWCF-HA1-DCpep表达产生的抗原HA1-DCpep蛋白的氨基酸序列如SEQ ID NO:6所示。
1.3 DCpep序列构建优化试验:
构建以1-5个DCpep串联为目的片段,以pWCF为载体的5组重组乳酸菌(原始出发菌株为NC8/Δalr),其中重组乳酸菌的构建方法同上,差异仅在于目的基因中仅含有不同串联数量的DCpep片段。然后进行了体外DC刺激试验。试验具体方法包括:SPF级雌性C57BL/6小鼠脱颈处死;超净台中用无菌RPMI 1640培养基从小鼠股骨中冲出骨髓细胞;用完全RPMI1640培养基洗涤骨髓细胞2次;洗涤完成后,在骨髓细胞中加入完全RPMI 1640培养基,再添加25ng/mL重组GM-CSF,置于37℃恒温箱培养;在第8天时,通过流式细胞仪用CD11c标志来鉴定DC的纯度;96孔板中,DC(2×105个细胞)与各组重组乳酸菌(2×106CFU)按照1:10的比例共培养12h,然后通过流式细胞仪检测DC表面的CD80和CD86分子。
小鼠骨髓源的DC纯度达到80.5%,可以进行下一步的实验。
结果:小鼠骨髓源DC分别与重组乳酸菌NC8Δ-pWCF-1×DCpep、NC8Δ-pWCF-2×Dcpep、NC8Δ-pWCF-3×DCpep、NC8Δ-pWCF-4×DCpep和NC8Δ-pWCF-5×DCpep共培养12h。在CD80分子的检测中,3×DCpep组表面的CD80极显著高于1×DCpep组(P<0.001);4×DCpep组和5×DCpep组的CD80分子极显著高于1×DCpep组(P<0.01);与2×DCpep组相比,3×Dcpep,4×DCpep和5×DCpep组CD80分子都显著上调(P<0.05),结果见图9(左)。在CD86分子的检测中,与1×DCpep组相比,3×Dcpep和5×DCpep组CD86分子都显著上调(P<0.05),结果见图9(右)。此外,发明人发现,序列过长不利于标签表达以及抗原的制备,因此,综合结果,3个DCpep串联能对DC达到更好的作用效果,随DCpep数量的增加对DC表面分子的活化没有显著差异,因此,3个DCpep串联效果最佳。因此,本发明的实施例示例中选择3个DCpep串联作为序列片段。
1.4小结
本实施例成功构建了NC8Δ-pWCF-HA1和NC8Δ-pWCF-HA1-DCpep重组乳酸菌。此外,成功制备了兔源DCpep多克隆抗体血清。验证了非抗生素重组乳酸菌所表达的融合蛋白免疫原性,且重组乳酸菌NC8Δ-pWCF-HA1-DCpep能够将禽流感病毒融合抗原HA1-DCpep锚定表达在植物乳杆菌表面。
实施例2重组乳酸菌的免疫效果研究
乳酸菌疫苗有着成本低廉,便于规模化生产的优势,且能够在机体肠道内长期定植,持续发挥作用,并且乳酸菌自身就是益生菌,能够提高机体免疫力、促进黏膜免疫。口服的免疫方式,又使之更为方便。因此,重组乳酸菌疫苗有着广阔的发展前景。本发明实施例1构建的重组乳酸菌为非抗生素乳酸菌,对环境而言更为友好,对动物而言避免了抗生素耐受。
现有学者进行了诸多对H7N9亚型禽流感的致病力的研究,有研究表明H7N9对小鼠具有更为明显的临床症状和发病率(Belser J A,Gustin K M,Pearee M B,Maines T RZeng H,Pappas C,Sun X,Carney P J,Villanueva J M,Stevens J,Katz J M,Tumpey TM.Pathogenesis and transmission of avian influenza A(H7N9)virus in ferretsand mice.Nature.Sep 26,2013:501(7468):556-559.),为了便于实验观察,本实施例以小鼠作为免疫对象。
本实施例设定初次免疫与加强免疫程序,诱导3组重组乳酸菌表达,并设置对照组,分别对各组小鼠实施口服灌胃免疫。免疫完成后对各组小鼠的免疫指标进行检测,进而对重组乳酸菌的免疫效果进行评价。
2.1材料与方法
试验菌株:NC8Δ-pWCF-HA1重组乳酸菌、NC8Δ-pWCF-HA1-DCpep重组乳酸菌、NC8△-pWCF空载体乳酸菌。重组乳酸菌按照实施例1的方法构建。
试验动物:SPF级6周龄C57BL/6J小鼠50只(购自河南斯克贝斯生物科技股份有限公司)。
试验试剂:PBS磷酸盐缓冲液,尼龙筛网购自Solarbio公司;蛋白酶抑制剂(PMSF),红细胞裂解液购自碧云天生物技术有限公司;4%多聚甲醛通用型组织固定液购自biosharp公司;细胞固定/细胞破模试剂盒购自BD公司;多肽由上海紫域生物科技有限公司合成;重组禽流感病毒(H5+H7)三联灭活疫苗购自哈尔滨维科生物技术有限公司。
试验仪器:主要仪器同实施例1。
实验动物分组及免疫方案
免疫分组:将小鼠随机分为PBS组、NC8△-pWCF、NC8Δ-pWCF-HA1、NC8Δ-pWCF-HA1-DCpep和疫苗组,共5组,每组10只。处理各组重组乳酸菌菌液,每只小鼠喂饲的活菌数为1.0×109CFU。乳酸菌培养方法及需要体积见1.2.3.1。取相应体积诱导后的菌液,无菌PBS洗三次后用200μL PBS重悬,给小鼠灌胃。动物免疫方案及分组如表(表2-1)。
表2-1动物免疫方案及分组
免疫程序:第1、3、5天为初次免疫,第15、17、19天为加强免疫。加强免疫后对小鼠状态进行观察,在第29天进行流式细胞术检测,见图10。
多肽合成:合成2条多肽,作为流式细胞术检测特异性T细胞相关指标时所需要的细胞刺激抗原肽,多肽的氨基酸序列见表2-2。
表2-2多肽序列
流式细胞术:每组3只小鼠脱颈处死。
制备脾脏、肠系膜淋巴结(MLN)、派氏淋巴结(PPs)细胞悬液:
摘取脾、肠系膜淋巴结与派氏淋巴结,组织放在无菌平皿的铜网里,并浸泡于1mL1640完全培养基,用1ml注射器尾部研磨组织。组织磨碎后吸取液体离心弃掉上清,离心条件为4℃,2000rpm离心5min。此外,脾脏细胞需要裂解,使用红细胞裂解液裂解后离心弃去裂解液。细胞用无菌PBS洗1遍后将各组细胞稀释计数,脾脏需要1.0x107个细胞,MLN需要1.0x107个细胞,PPs需要1.0x106个细胞。细胞原液清洗一遍后使用PBS补齐至1mL制成细胞悬液。
检测派氏淋巴结细胞DC表面CD80+与CD86+:
向制备好的100μL细胞悬液(有1×106个细胞)中混入CD16/CD32 Pure每管0.5μ,4℃,5min。再加CD11c-APC(40倍稀释)、CD80-FITC(60倍稀释)、CD86-PE-Cy7(20倍稀释)、MHC-Ⅱ-PerCy-Cy5.5)抗体各10μL,4℃避光孵育20min。用PBS清洗一次,保留沉淀,加入200μL PBS混匀过尼龙筛网,上机。
检测派氏淋巴结B细胞活化水平:
向制备好的100μL派氏淋巴结细胞悬液中混入CD16/CD32 Pure每管0.5μ,4℃,5min。加B220-APC(110倍稀释)抗体10μL,4℃避光20min,PBS洗一次。加入250μL甲醛固定液,4℃避光20min。使用穿膜液进行两次穿膜后离心,保留沉淀与100μL上清液,直接加IgA-FITC(40倍稀释),4℃避光20min,离心去上清。用1mL PBS洗一次,加入200μL PBS混匀,过尼龙筛网,上机。
检测脾脏与肠系膜淋巴结细胞中细胞因子:
铺细胞24孔板:吸取每组制备好的脾脏与肠系膜淋巴结细胞悬液150μL,加入350μL完全培养基、终浓度为40ng/mL的刺激剂PMA、对应的抗原肽每条终浓度为4μg/mL(各组对应的抗原肽序号见表2-3)。37℃孵育8小时。加入阻断剂(protein transport inhibitor)1μL,再孵育4小时。混匀吸出,加入PBS洗一遍。CD16/CD32 Pure,每管0.5μ,4℃,5min。CD3-percpcy5.5(40倍稀释)、CD4-FITC(60倍稀释)、CD8-APCCY7(60倍稀释)各10μL,4℃避光20min。洗涤一次,加入250μL甲醛固定液,4℃避光20min。孵育结束对细胞使用穿膜液进行穿膜,共两次。加IFN-γ-APC(30倍稀释),每管10μL,避光4℃,20min。洗涤一次,200μLPBS混匀过尼龙筛网,上机检测。
表2-3各组对应的抗原肽序号
检测脾脏与肠系膜淋巴结T细胞增殖情况:
取300μL制备好的细胞悬液染色CFSE(终浓度1μL/mL),条件为37℃孵育10min,加入血清FBS 300μL终止反应,使用PBS洗1遍,1640完全培养液洗1遍。沉淀加1ml 1640完全培养液,PMA 1000倍稀释加1.2μL,抗原肽(1mg/mL)每条2.4μL混匀。铺96孔板,每孔200μL。细胞板置于细胞培养箱37℃培养3天。细胞转至1.5mL离心管,加PBS补齐到1mL离心,1mLPBS洗1遍,剩余100μL混匀细胞沉淀。混入CD16/CD32 Pure每管0.5μ,4℃,5min。加CD4-PERCP(60倍稀)、CD8-APC(60倍稀释)各10μL,孵育条件为4℃,避光20min。PBS洗一遍,200μL PBS混匀沉淀过尼龙筛网,上机。
数据处理与分析:流式细胞图采用Flowjo 6.2.1软件分析。应用GraphPad Prism软件作图和统计数据,并用one-way ANOVA统计各组数据之间的差异(*,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001)。
2.2结果
重组乳酸菌对免疫后小鼠派氏淋巴结DC活化的影响结果:
为评价重组乳酸菌NC8Δ-pWCF-HA1和NC8Δ-pWCF-HA1-DCpep对小鼠派氏淋巴结的影响,给小鼠通过口服与免疫方式饲喂重组乳酸菌,在加强免疫一周后,检测了小鼠派氏淋巴结中DC的情况。本试验为对树突状细胞表面的活化标志CD80、CD86和MHC-II的表达情况进行了检测。结果显示,PPs中的MFI(CD11c+CD80+MFI)HA1-DCpep组与PBS组、空载组、HA1组相比差异极显著(P<0.001),与疫苗组相比差异极显著(P<0.01);PPs中的MFI(CD11c+CD86+)HA1-DCpep组,与疫苗组相比差异极显著(P<0.01),与空载组相比差异显著(P<0.05);PPs中的MFI(CD11c+MHC-II+)HA1-DCpep组与PBS相比差异显著(P<0.05),与疫苗组相比差异极显著(P<0.01),HA1组与疫苗组相比差异极显著(P<0.01)。以上结果说明重组乳酸菌NC8Δ-pWCF-HA1-DCpep对小鼠派氏淋巴结DC有明显更优的活化效果,结果见图11。
重组乳酸菌对免疫后小鼠特异性细胞因子的影响结果:
在加强免疫后,利用流式细胞术对小鼠肠系膜淋巴结与脾脏中的CD4+IFN-γ+和CD8+IFN-γ+细胞进行了检测,结果显示小鼠的MLN与脾脏的细胞免疫反应都可以被锚定表达非洲猪瘟病毒融合抗原重组乳酸菌激活。
在对小鼠MLN中CD4+IFN-γ+细胞因子的检测,结果见图12,经口服NC8Δ-pWCF-HA1-DCpep重组乳酸菌组的CD4+IFN-γ+细胞的比例与PBS组、疫苗组相比,极显著增加(P<0.001),与空载组相比差异显著(P<0.05),并且在各组中,HA1-DCpep组的CD4+IFN-γ+细胞比例最高;HA1组与空载组、疫苗组相比差异极显著(P<0.01)。而在检测CD8+IFN-γ+细胞中,结果见图13,经口服NC8Δ-pWCF-HA1-DCpep重组乳酸菌组的CD8+IFN-γ+细胞比例与PBS对照组相比,极显著增加(P<0.01),与空载组相比,显著增加(P<0.05);HA1组与PBS组相比差异显著(P<0.05)。
在对小鼠脾脏中CD4+IFN-γ+细胞因子的检测,结果见图14,经口服NC8Δ-pWCF-HA1-DCpep重组乳酸菌组的CD4+IFN-γ+的细胞比例与PBS对照组相比,极显著增加(P<0.0001),与空载组相比,极显著增加(P<0.01),与HA1组相比,显著增加(P<0.05),并且在各组中,HA1-DCpep组的CD4+IFN-γ+细胞比例最高;HA1组与PBS组相比差异显著(P<0.05)。而在检测CD8+IFN-γ+细胞中,结果见图15。经口服NC8Δ-pWCF-HA1-DCpep重组乳酸菌组的CD8+IFN-γ+的细胞比例与PBS对照组相比,极显著增加(P<0.0001),与空载组相比,极显著增加(P<0.01),与HA1组相比,显著增加(P<0.05),并且在各组中,HA1-DCpep组的CD8+IFN-γ+细胞比例最高;HA1组与PBS组相比差异极显著(P<0.01)。
重组乳酸菌对免疫后小鼠MLN和脾脏T细胞增殖的影响结果:
在加强免疫后,利用流式细胞术对小鼠肠系膜淋巴结与脾脏中T细胞增殖进行了检测,结果显示小鼠的MLN与脾脏的T细胞增殖都可以被锚定表达非洲猪瘟病毒融合抗原重组乳酸菌激活。
在对小鼠MLN中CD4+T细胞增殖的检测,结果见图16,与疫苗对照组相比,经口服NC8Δ-pWCF-HA1-DCpep重组乳酸菌组的CD4+T细胞的增殖情况显著增加(P<0.05)。而在检测CD8+T细胞增殖中,结果见图17,与疫苗对照组相比,经口服NC8Δ-pWCF-HA1-DCpep重组乳酸菌组的CD8+T细胞的增殖情况显著增加(P<0.05)。
在对小鼠脾脏中CD4+T细胞增殖的检测,结果见图18,经口服NC8Δ-pWCF-HA1-DCpep重组乳酸菌组的CD4+T细胞增值情况与PBS对照组相比,极显著增加(P<0.01),于空载组相比,显著增加(P<0.05),并且在各组中,HA1-DCpep组的CD4+T细胞增殖最高。而在检测CD8+T细胞增殖中,结果见图19,经口服NC8Δ-pWCF-HA1-DCpep重组乳酸菌组的CD8+T细胞增值情况与PBS组、空载组相比,极显著增加(P<0.01);与PBS组相比HA1组的增值情况差异显著(P<0.05)。
重组乳酸菌对免疫后小鼠B细胞的影响结果
重组乳酸菌对免疫后小鼠派氏淋巴结B细胞的影响结果:
本试验对锚定表达非洲猪瘟病毒融合抗原重组乳酸菌是否能诱导小鼠B细胞活化进行了研究,对加强免疫后小鼠PPs中B细胞的情况进行检测。结果见图20,HA1-DCpep组与PBS对照组、空载体组相比,B220+IgA+细胞的百分数极显著增加(P<0.001);HA1组与PBS对照组、空载体组相比,B220+IgA+细胞的百分数极显著增加(P<0.01);疫苗组与PBS对照组、空载体组相比,B220+IgA+细胞的百分数极显著增加(P<0.0001),与HA1组相比显著增加(P<0.05)。
2.3小结
本实施例试验证明了重组乳酸菌NC8Δ-pWCF-HA1和NC8Δ-pWCF-HA1-DCpep均能诱导小鼠派氏淋巴结DC的活化,诱导小鼠产生特异性T细胞反应,其中,NC8Δ-pWCF-HA1-DCpep具有更优的效果。2株重组乳酸菌均对小鼠B细胞反应产生影响,尤以NC8Δ-pWCF-HA1-DCpep效果更好。
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,尽管参照前述实施例对本申请进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
SEQUENCE LISTING
<110> 吉林农业大学
<120> 靶向树突状细胞表达H7N9禽流感HA1抗原的重组乳酸菌及其应用
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ccaaagagaa aacggactgc gagattttat ccatcatatc attcaactcc acaacgtcca 1020
ttttatccat catatcattc aactccacaa cgtccatttt atccatcata tcattcaact 1080
ccacaacgtc cataagctt 1099
<210> 4
<211> 8127
<212> DNA
<213> 人工序列
<400> 4
ccatgggcaa gaaagaatta agtttccacg agaagttatt aaaattgact aaacaacaaa 60
aaaagaagac taacaagcat gtgtttattg ctattccaat tgttttcgtt ttaatgtttg 120
cttttatgtg ggcaggtaaa gctgagactc caaaagttaa gacttatagt gatgacgttt 180
tgagtgcttc atttgtcggc gacattatga tgggtcgtta cgttgagaaa gtcacggaac 240
aaaagggtgc agatagtatt ttccaatatg ttgaaccgat tttccgtgct agtgattatg 300
ttgctggcaa ttttgaaaat cctgttactt atcagaaaaa ctacaaacaa gctgataaag 360
agattcattt acagactaat aaggaaagtg ttaaagtttt aaaggatatg aattttactg 420
tcttaaatag tgctaataat catgctatgg attatggtgt tcaaggtatg aaagatacgt 480
taggtgagtt tgctaaacag aatttagata ttgttggtgc tggttattca ttaagtgacg 540
ctaagaagaa aattagttac cagaaagtgt ctagaaagct tcaaattaca gcacgtgttg 600
ctttgattga tagccaaaaa gcagcagttg ataaagcaat tactgatatt gctgaaaaat 660
tgtaatttat aaataaaaat caccttttag aggtggtttt tttatttata aattattcgt 720
ttgatttcgc tttcgataga acaatcaaag cgagaataag gaagataaat cccataaggg 780
cgggagcaga atgtccgaga ctaattcatg accaaaatcc cttaacgtga gttttcgttc 840
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 900
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 960
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 1020
aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 1080
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 1140
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 1200
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 1260
ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 1320
ccggtaagcg gcagggtcgg aacaggaggc gcacgaggga gcttccaggg ggaaacgcct 1380
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 1440
gctcgtcagg ggggcggagc ctatcgaaaa acgccagcaa cgcggccttt ttacggttcc 1500
tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 1560
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 1620
gcagcgagtc agtgagcgag aaggattatt cggctggttg agacgttaaa atgataaagg 1680
ttgtattaat cttatattac ggttataatg tactcaactt aataaatgaa cgcaaaaaaa 1740
agaaccctca acttagcaga gttaggattc acgacttatc agcacaacct gataagattt 1800
tcgatagcaa gtactaccaa tacaagctat ctaacttggt actattataa catgtaggct 1860
aagtttttca accattgata cttaaagtaa acggttgtta tcgggaatct taacagaaac 1920
ctgatagcaa ccgttttttt gttattcaat ggttagcaac catcaaagca actaaaggct 1980
ggaaacctgt tcttagctag taaaacctcc cgtgagtgtc gttcgtgacc ccgcttgcag 2040
ttaacaacat aggtatgcta aaccttgtcg agatcaacgc gactaaagac gtggctggaa 2100
gactaggaaa tgatacggac aggctaacta ttaacgcaga ttattcgggt tgctgctaaa 2160
accaactcta ataatagtta gtgcaagggc tggttgagct taaattgtct gataaagagt 2220
tctctcttta tactgcaaaa gaagcgcagt tattcacgat taggataact gtttgagaga 2280
gcctaagggc ttgacccttg atggtttaag caccgctatg cgtgcgggat cctcttccct 2340
aaatttaaat ataaacaacg aattatctcc ttaacgtacg ttttcgttcc attggccctc 2400
aaacccctaa ttaggatcaa taaaacagcg acggaaatga ttcccttcct aacgcaaatt 2460
ccctgataat cgccactgga ctttctgctt gcgcggtaag gcaggataag tcgcattact 2520
gatggcttcg ctatcattga ttaatttcac ttgcgacttt ggctgctttt tgtatggtga 2580
aggatgcgcc acaggatact ggcgcgcata cacagcacat ctctttgcag gaaaaaaacg 2640
ctatgaaaaa tgttggtttt atcggctggc gcggaatggt cggctctgtt ctcatgcaac 2700
gcatggtaga ggagcgcgat ttcgacgcta ttcgccctgt tttcttttct acctcccagt 2760
ttggacaggc ggcgcccacc ttcggcgaca cctccggcac gctacaggac gcttttgacc 2820
tggatgcgct aaaagcgctc gatatcatcg tgacctgcca gggcggcgat tataccaacg 2880
aaatttatcc aaagctgcgc gaaagcggat ggcagggtta ctggattgat gcggcttcta 2940
cgctgcgcat gaaagatgat gccattatta ttctcgaccc ggtcaaccag gacgtgatta 3000
ccgacggcct gaacaatggc gtgaagacct ttgtgggcgg taactgtacc gttagcctga 3060
tgttgatgtc gctgggcggt ctctttgccc ataatctcgt tgactgggta tccgtcgcga 3120
cctatcaggc cgcctccggc ggcggcgcgc gccatatgcg cgagctgtta acccagatgg 3180
gtcagttgta tggccatgtc gccgatgaac tggcgacgcc gtcttccgca attcttgata 3240
ttgaacgcaa agttacggca ttgacccgca gcggcgagct gccggttgat aactttggcg 3300
taccgctggc gggaagcctg atcccctgga tcgacaaaca gctcgataac ggccagagcc 3360
gcgaagagtg gaaaggccag gcggaaacca acaagattct caatactgcc tctgtgattc 3420
cggttgatgg tttgtgtgtg cgcgtcggcg cgctgcgctg tcacagccag gcgttcacca 3480
tcaagctgaa aaaagaggta tccattccga cggtggaaga actgctggcg gcacataatc 3540
cgtgggcgaa agtggtgccg aacgatcgtg atatcactat gcgcgaatta accccggcgg 3600
cggtgaccgg cacgttgact acgccggttg gtcgtctgcg taagctgaac atggggccag 3660
agttcttgtc ggcgtttacc gtaggcgacc agttgttatg gggcgccgcc gagccgctgc 3720
gtcgaatgct gcgccagttg gcgtagtggc tattgcagcg cttatcgggc ctgcgtgtgg 3780
ttctgtaggc cggataaggc gcgtcagcgc cgccatccgg cggggaaatt tgtgttaaac 3840
caggggtgca tcgtcaccct ttttttgcgt aatacaggag taaacgcaga tgtttcattt 3900
ttatcaggag ttaagcagag cattggctat tctttaaggg tagcttaatc ccacgggtat 3960
taagcctaac ctgaaggtag gacgacgcag ataggatgca cagtgtgctg cgccgttcag 4020
gtcaaagaag tgtcactacc tgatgttgtg gacgaaaagc cctgacaacc ctcgttccta 4080
aaaaggaata agcgtttggt cagtaaataa tagaaataaa aaatcagacc taagactgat 4140
gacaaaaaga gcaaattttg ataaaatagt attagaatta aattaaaaag ggaggccaaa 4200
tataatgaaa aatatgaatg acaatgatgt tatggttgta attggggagc accgccacac 4260
acaagtcaca gtggacttgc aggcaattaa gacaaatatt agtaatgaaa tggcgcaaaa 4320
ggatgagttg accgagttat gggcagtcgt taaagcgaat ggttatggac atggaattat 4380
ccaagttgct caggccgcca aagaagccgg ggcgaccggc ttttgtgttg caatcctgga 4440
tgaggcctta gcgttgcggg ccgctggctt tgcggaaccc atcctagtac ttggaattac 4500
ggaaccggaa tacgccccac tggtagctga aaaggatatt tcactagctg ttggaacgca 4560
agattggctg actacggccg cagcaatttt agcggctaat caagtgacga caccacttca 4620
cgttcatctt gcattagata cgggtatggg acgaatcggg tttcagacgc ccgaagaatt 4680
ggcaacggcg gttacgactt tgcgtcaacc gcagtcacca tttgactttg aagggatttt 4740
tacgcatttt gcaacggctg accaggcaga tgatacgtat tttactcatc aattaaataa 4800
ttggaaacac ttgattgcag tggtggatga gctaccacgc tatgtccacg tgtccaattc 4860
ggccaccagt ctctggcatc aaacttgcaa tggcaacatg gtgcgctttg gggttgcact 4920
ctatggtcta aatccttctg gtcgcgaact cagcgcacca taccccttgc aacccgcgtt 4980
gtcgctaacg gcacgcttga cgtttgttaa acgcttggct cggggcaaat cggtcagcta 5040
tggtgccacg tatacggccg cacaggatga atggattggc acggtgccga ttgggtatgc 5100
ggacggctat gaacgccgat tacaaggctt ccatgtactt gttgatggtg agttttgcga 5160
aatcgtcgga cgggtctgca tggaccagct gatggttcgt ctgccacatg aagtaccggt 5220
tggagctaag gtaactttgg ttggcacgga cggtgctcgt accatttcgt tgcaagatat 5280
tgctgactat tgtgggacaa ttcattatga gattgcttgt gggttagcac cacgagtgcc 5340
gagagtttat atagattaat tctatgagtc gcttttttaa atttggaaag ttacacgtta 5400
ctaaagggaa tggagaccgg ggcttcaata gagttcttaa cgttaatccg aaaaaaacta 5460
acgttaatat taaaaaataa gatccgcttg tgaattatgt ataatttgat tagactaaag 5520
aataggagaa agtatgatga tatttaaaaa actttctcgt taagataggt tgttggtgag 5580
catgttatat acggatgtat cggtttcctt aatgcaaaat tttgttgcta tcttattaat 5640
ttttctatta tatagatata ttcaaagaaa gataacattt aaacggatca tattagatat 5700
tttaatagcg attatttttt caatattata tctgtttatt tcagatgcgt cattacttgt 5760
aatggtatta atgcgattag ggtggcattt tcatcaacaa aaagaaaata agataaaaac 5820
gactgataca gctaatttaa ttctaattat cgtgatccag ttattgttag ttgcggttgg 5880
gactattatt agtcagttta ccatatcgat tatcaaaagt gatttcagcc aaaatatatt 5940
gaacaatagt gcaacagata taactttatt aggtattttc tttgctgttt tatttgacgg 6000
cttgttcttt atattattga agaataagcg gactgaatta caacatttaa atcaagaaat 6060
cattgaattt tcgttagaaa aacaatattt tatatttata tttattttat ttatagtaat 6120
agaaattatt ttagcagttg ggaatcttca aggagtaaca gccacgatat tattaaccat 6180
tatcattatt ttttgtgtcc ttatcgggat gactttttgg caagtgatgc tttttttgaa 6240
ggcttattcg attcgccaag aagccaatga ccaattggtc cggaatcaac aacttcaaga 6300
ttatctagtc aatatcgaac agcagtacac cgaattacgg cgatttaagc atgattatca 6360
aaacatctta ttatcgttgg agagttttgc cgaaaagggc gatcagcaac agtttaaggc 6420
gtattaccaa gaattattag cacaacggcc aattcaaagt gaaatccaag gggcagtcat 6480
tgcacaactc gactacttga aaaatgatcc tattcgagga ttagtcattc aaaagttttt 6540
ggcagccaaa caggctggtg ttactttaaa attcgaaatg accgaaccaa tcgaattagc 6600
aaccgctaat ctattaacgg ttattcggat tatcggtatt ttattagaca atgcgattga 6660
acaagccgtt caagaaaccg atcaattggt gagttgtgct ttcttacaat ctgatggttt 6720
aatcgaaatt acgattgaaa atacggccag tcaagttaag aatctccaag cattttcaga 6780
gttaggctat tcaacgaaag gcgctggtcg ggggactggt ttagctaatg tgcaggattt 6840
gattgccaaa caaaccaatt tattcttaga aacacagatt gaaaatagaa agttacgaca 6900
gacattgatg attacggagg aaacttaatt tgtatcccgt ttatttatta gaggatgatt 6960
tacagcaaca agcgatttat cagcaaatta tcgcgaatac gattatgatt aacgaatttg 7020
caatgacttt aacatgcgct gccagtgata ctgagacatt gttggcggca attaaggatc 7080
agcaacgagg tttattcttt ttggatatgg aaattgagga taaccgccaa gccggtttag 7140
aagtggcaac taagattcgg cagatgatgc cgtttgcgca aattgtcttc attacaaccc 7200
acgaggaact gacattatta acgttagaac gaaaaatagc gcctttagat tacattctca 7260
aggaccaaac aatggctgaa atcaaaaggc aattgattga tgatctattg ttagctgaga 7320
agcaaaacga ggcggcagcg tatcaccgag aaaatttatt tagttataaa ataggtcctc 7380
gctttttctc attaccatta aaggaagttg tttatttata tactgaaaaa gaaaatccgg 7440
gtcatattaa tttgttagcc gttaccagaa aggttacttt tccaggaaat ttaaatgcgc 7500
tggaagccca atatccaatg ctctttcggt gtgataaaag ttacttagtt aacctatcta 7560
atattgccaa ttatgacagt aaaacacgga gtttaaaatt tgtagatggc agtgaggcaa 7620
aagtctcgtt ccggaaatca cgggaactag tggccaaatt aaaacaaatg atgtagcgcc 7680
tgcaggcacg ccaaatgatc ccagtaaaaa gccacccgca tggcgggtgg ctttttatta 7740
gccctagaag ggcttcccac acgcatttca gcgccttagt gccttagttt gtgaatcata 7800
ggtggtatag tcccgaaata cccgtctaag gaattgtcag ataggcctaa tgactggctt 7860
ttataatatg agataatgcc gactgtactt tttacagtcg gttttctaat gtcactaacc 7920
tgccccgtta gttgaagaag gtttttatat tacagctcca gatctaccgg tttaatttga 7980
aaattgatat tagcgtttaa cagttaaatt aatacgttaa taattttttt gtctttaaat 8040
agggatttga agcataatgg tgttatagcg tacttagctg gccagcatat atgtattcta 8100
taaaatacta ttacaaggag attttag 8127
<210> 5
<211> 364
<212> PRT
<213> 人工序列
<400> 5
Ser Arg Met Asp Lys Ile Cys Leu Gly His His Ser Val Ser Asn Gly
1 5 10 15
Thr Lys Val Asn Thr Leu Thr Glu Lys Gly Val Glu Val Val Asn Ala
20 25 30
Thr Glu Thr Val Glu Arg Thr Asn Thr Pro Arg Ile Cys Ser Lys Gly
35 40 45
Lys Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr
50 55 60
Gly Pro Pro Gln Cys Asp Gln Phe Leu Lys Phe Ser Ala Asp Leu Ile
65 70 75 80
Val Glu Arg Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val
85 90 95
Asn Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp
100 105 110
Lys Glu Pro Met Gly Phe Lys Tyr Asn Gly Ile Arg Thr Asn Gly Thr
115 120 125
Thr Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys
130 135 140
Trp Leu Leu Ser Asn Thr Asp Asn Ala Thr Phe Pro Gln Met Thr Lys
145 150 155 160
Ser Tyr Lys Asn Thr Arg Glu Ser Pro Ala Ile Val Val Trp Gly Ile
165 170 175
His His Ser Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly
180 185 190
Asn Lys Leu Val Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val
195 200 205
Pro Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Gln Ser Gly Arg Ile
210 215 220
Asp Phe His Trp Leu Ile Leu Asn Pro Asn Asp Thr Val Thr Phe Ser
225 230 235 240
Phe Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Met Arg Gly
245 250 255
Lys Ser Met Gly Ile Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu
260 265 270
Gly Asp Cys Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe
275 280 285
Gln Asn Ile Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Arg
290 295 300
Gln Arg Ser Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Val
305 310 315 320
Pro Lys Arg Lys Arg Thr Ala Arg Phe Tyr Pro Ser Tyr His Ser Thr
325 330 335
Pro Gln Arg Pro Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg Pro
340 345 350
Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg Pro
355 360
<210> 6
<211> 553
<212> PRT
<213> 人工序列
<400> 6
Met Gly Lys Lys Glu Leu Ser Phe His Glu Lys Leu Leu Lys Leu Thr
1 5 10 15
Lys Gln Gln Lys Lys Lys Thr Asn Lys His Val Phe Ile Ala Ile Pro
20 25 30
Ile Val Phe Val Leu Met Phe Ala Phe Met Trp Ala Gly Lys Ala Glu
35 40 45
Thr Pro Lys Val Lys Thr Tyr Ser Asp Asp Val Leu Ser Ala Ser Phe
50 55 60
Val Gly Asp Ile Met Met Gly Arg Tyr Val Glu Lys Val Thr Glu Gln
65 70 75 80
Lys Gly Ala Asp Ser Ile Phe Gln Tyr Val Glu Pro Ile Phe Arg Ala
85 90 95
Ser Asp Tyr Val Ala Gly Asn Phe Glu Asn Pro Val Thr Tyr Gln Lys
100 105 110
Asn Tyr Lys Gln Ala Asp Lys Glu Ile His Leu Gln Thr Asn Lys Glu
115 120 125
Ser Val Lys Val Leu Lys Asp Met Asn Phe Thr Val Leu Asn Ser Ala
130 135 140
Asn Asn His Ala Met Asp Tyr Gly Val Gln Gly Met Lys Asp Thr Leu
145 150 155 160
Gly Glu Phe Ala Lys Gln Asn Leu Asp Ile Val Gly Ala Gly Tyr Ser
165 170 175
Leu Ser Asp Ala Lys Lys Lys Ile Ser Tyr Gln Lys Val Ser Arg Met
180 185 190
Asp Lys Ile Cys Leu Gly His His Ser Val Ser Asn Gly Thr Lys Val
195 200 205
Asn Thr Leu Thr Glu Lys Gly Val Glu Val Val Asn Ala Thr Glu Thr
210 215 220
Val Glu Arg Thr Asn Thr Pro Arg Ile Cys Ser Lys Gly Lys Arg Thr
225 230 235 240
Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly Pro Pro
245 250 255
Gln Cys Asp Gln Phe Leu Lys Phe Ser Ala Asp Leu Ile Val Glu Arg
260 265 270
Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn Glu Glu
275 280 285
Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys Glu Pro
290 295 300
Met Gly Phe Lys Tyr Asn Gly Ile Arg Thr Asn Gly Thr Thr Ser Ala
305 310 315 320
Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp Leu Leu
325 330 335
Ser Asn Thr Asp Asn Ala Thr Phe Pro Gln Met Thr Lys Ser Tyr Lys
340 345 350
Asn Thr Arg Glu Ser Pro Ala Ile Val Val Trp Gly Ile His His Ser
355 360 365
Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn Lys Leu
370 375 380
Val Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro Ser Pro
385 390 395 400
Gly Ala Arg Pro Gln Val Asn Gly Gln Ser Gly Arg Ile Asp Phe His
405 410 415
Trp Leu Ile Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe Asn Gly
420 425 430
Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Met Arg Gly Lys Ser Met
435 440 445
Gly Ile Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu Gly Asp Cys
450 455 460
Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln Asn Ile
465 470 475 480
Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Arg Gln Arg Ser
485 490 495
Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Val Pro Lys Arg
500 505 510
Lys Arg Thr Ala Arg Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg
515 520 525
Pro Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg Pro Phe Tyr Pro
530 535 540
Ser Tyr His Ser Thr Pro Gln Arg Pro
545 550
<210> 7
<211> 25
<212> DNA
<213> 人工序列
<400> 7
tctagaatgg acaaaatctg cctcg 25
<210> 8
<211> 25
<212> DNA
<213> 人工序列
<400> 8
aagcttatct cgcagtccgt tttct 25
<210> 9
<211> 18
<212> DNA
<213> 人工序列
<400> 9
agatattgtt ggtgctgg 18
<210> 10
<211> 17
<212> DNA
<213> 人工序列
<400> 10
tcaatcaaag caacacg 17
<210> 11
<211> 12
<212> PRT
<213> 人工序列
<400> 11
Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg Pro
1 5 10
<210> 12
<211> 20
<212> PRT
<213> 人工序列
<400> 12
Lys Ser Tyr Lys Asn Thr Arg Glu Ser Pro Ala Ile Val Val Trp Gly
1 5 10 15
Ile His His Ser
20
Claims (16)
1.一种表达H7N9禽流感抗原的重组植物乳杆菌,其含有HA1-DCpep融合基因,所述HA1-DCpep融合基因由HA1基因片段与3个DCpep基因片段串联组成;
所述HA1-DCpep融合基因的核苷酸序列如SEQ ID NO:3中所示。
2.根据权利要求1所述的表达H7N9禽流感抗原的重组植物乳杆菌,其特征在于,所述HA1-DCpep融合基因中还含有启动子、终止子和酶切位点基因序列。
3.根据权利要求2所述的表达H7N9禽流感抗原的重组植物乳杆菌,其特征在于,所述酶切位点为Xba I和Hind III。
4.根据权利要求1-3任一项所述的表达H7N9禽流感抗原的重组植物乳杆菌,其特征在于,所述重组乳杆菌中还导入了pWCF的基因片段,如SEQ ID NO:4中所示的核苷酸序列。
5.根据权利要求4所述的表达H7N9禽流感抗原的重组植物乳杆菌,其特征在于,所述重组植物乳杆菌以植物乳杆菌NC8为原始出发菌株。
6.根据权利要求5所述的表达H7N9禽流感抗原的重组植物乳杆菌,其特征在于,所述重组植物乳杆菌为丙氨酸消旋酶基因缺陷型植物乳杆菌NC8/Δalr。
7.一种融合基因HA1-DCpep,其由HA1基因片段与3个DCpep基因片段串联组成;
所述HA1-DCpep融合基因的核苷酸序列如SEQ ID NO:3中所示。
8.根据权利要求7所述的融合基因HA1-DCpep,其特征在于,所述HA1-DCpep融合基因中还可以含有启动子、终止子和酶切位点基因序列。
9.根据权利要求8所述的融合基因HA1-DCpep,其特征在于,所述酶切位点为Xba I和Hind III。
10.一种融合蛋白,其由权利要求9所述的融合基因HA1-DCpep编码,所述融合蛋白的氨基酸序列如SEQ ID NO:5所示。
11.根据权利要求10所述的融合蛋白,其特征在于,所述融合蛋白由权利要求1至6中任一项所述的表达H7N9禽流感抗原的重组植物乳杆菌表达产生;所述融合蛋白的氨基酸序列中包含表面锚定元件pgsA’表达产生的氨基酸,所述融合蛋白的氨基酸序列如SEQ ID NO:6中所示。
12.含有权利要求7所述的融合基因HA1-DCpep的表达载体。
13.根据权利要求12所述的融合基因HA1-DCpep的表达载体,其特征在于,所述表达载体由融合基因HA1-DCpep与载体pWCF连接得到,所述pWCF为SEQ ID NO:4中所示的核苷酸序列。
14.一种药物制剂或菌剂或饲料,其包含权利要求1至6中任一项所述的表达H7N9禽流感抗原的重组植物乳杆菌或代谢产物或权利要求10所述的融合蛋白。
15.权利要求1至6中任一项所述的表达H7N9禽流感抗原的重组植物乳杆菌或权利要求7所述的融合基因HA1-DCpep或权利要求10所述的融合蛋白在制备预防和/或治疗H7N9禽流感的药物或菌剂或饲料中的应用。
16.权利要求1至6中任一项所述的表达H7N9禽流感抗原的重组植物乳杆菌或权利要求10所述的融合蛋白或权利要求14所述的药物制剂或菌剂或饲料在如下任意一种中的应用:
1)制备激活机体免疫细胞的产品;
2)制备激活派氏淋巴结DC的产品;
3)制备刺激脾、肠系膜淋巴结和/或派氏淋巴结中的特异性细胞因子的产品;所述特异性细胞因子包括CD4+IFN-γ+、CD8+IFN-γ+。
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