CN116497049A - 一种表达犬疱疹病毒gD糖基化蛋白的重组菌及其应用 - Google Patents
一种表达犬疱疹病毒gD糖基化蛋白的重组菌及其应用 Download PDFInfo
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Abstract
本发明涉及一种表达犬疱疹病毒gD糖基化蛋白的重组菌及其应用,本发明首先提供一种重组表达载体,其表达重组犬疱疹病毒gD糖基化蛋白,该重组蛋白是相应的野生型蛋白的截短体。本发明还构建了表达该重组犬疱疹病毒gD糖基化蛋白的重组乳酸乳球菌,因乳酸乳球菌的保护,可以递送犬疱疹病毒gD蛋白进入犬肠道,从而可以提高犬肠道黏膜的sIgA含量,并且产生特异性免疫抗体,为犬疱疹病毒口服疫苗的研制提供了重要基础。
Description
技术领域
本发明涉及生物工程技术领域,具体涉及一种表达犬疱疹病毒gD糖基化蛋白的重组菌及其应用。
背景技术
犬是人类的重要伙伴,然而由于生理上、先天或遗传、母犬行为、环境状况或细菌性败血症等因素的影响,新生仔犬的死亡率高达15%-25%。在死亡的新生仔犬中,多数仔犬出生一周死亡,其中超过75%的仔犬出生3天即死亡。已有研究证明,犬疱疹病毒感染是1-2周新生仔犬死亡的重要原因之一。
犬疱疹病毒是典型的α-疱疹病毒,为双链DNA病毒,病毒基因组US区的US6基因能够编码gD蛋白,具有高度保守的性质。该蛋白是疱疹病毒囊膜上的重要组成部分,其在病毒穿透和进入宿主细胞的过程中起着重要作用,且能引起比其他蛋白更强更持久的细胞免疫应答。目前该病没有相应的特效药及疫苗,研究预防和治疗犬疱疹病毒病的药物或食品十分迫切,gD蛋白成为防治犬疱疹病毒病的关键因子。
发明内容
本发明的目的在于针对上述现有技术的不足,提供一种表达犬疱疹病毒gD糖基化蛋白的重组菌及其应用,该重组菌能够短期粘附定植、繁殖于肠道内,表达重组gD蛋白,诱导机体产生免疫反应,从而产生特异性抗体。
为此,本发明的第一方面提供了一种重组表达载体,所述重组表达载体表达重组犬疱疹病毒gD糖基化蛋白;所述重组犬疱疹病毒gD糖基化蛋白是野生型犬疱疹病毒gD糖基化蛋白的截短体。
进一步,所述重组表达载体是在表达载体中插入编码重组犬疱疹病毒gD糖基化蛋白的核苷酸序列。所述重组表达载体包含所述重组犬疱疹病毒gD糖基化蛋白的基因表达框。
进一步,所述重组犬疱疹病毒gD糖基化蛋白的氨基酸序列如SEQIDNO:1或SEQIDNO:3所示。
进一步,所述重组犬疱疹病毒gD糖基化蛋白的核苷酸序列如SEQIDNO:2或SEQIDNO:4所示。
进一步,所述重组表达载体适于在乳酸菌中复制并且表达所述重组犬疱疹病毒gD糖基化蛋白。
进一步,所述重组表达载体的构建方法包括:将氨苄抗性基因连入pMG36e质粒中,获得具有氨苄抗性的pLL32a质粒;将所述重组犬疱疹病毒gD糖基化蛋白的基因序列可操作性地连接至pLL32a质粒中,即构建得到所述重组表达载体。
在一些实施方式中,所述pLL32a质粒与专利文献CN113845584A所记载的相同,所述质粒pLL32a的序列如SEQIDNO:5所示。
在一些实施方式中,所述重组表达载体的构建方法包括:
(1)人工合成两端具有酶切位点的氨苄抗性基因序列,通过酶切连接的方法将其连入pMG36e质粒中,然后转化至MC1061感受态细胞中,利用氨苄青霉素进行筛选,获得pLL32a质粒;
(2)人工合成两端具有酶切位点的重组犬疱疹病毒gD糖基化蛋白的基因序列,通过酶切连接的方法将其可操作性地连入pLL32a质粒中的P32启动子下游,然后转化至MC1061感受态细胞中,利用氨苄青霉素进行筛选,获得所述重组表达载体。
本发明的第二方面提供一种重组菌,所述重组菌含有本发明第一方面所述的重组表达质粒。
进一步,所述重组菌通过将所述重组表达载体转化至宿主细胞中制备得到。
进一步,所述宿主细胞为乳酸菌。
在一些实施方式中,所述宿主细胞为乳酸乳球菌MG1363。
本发明的第三方面,提供所述重组表达载体或所述重组菌在制备预防和/或治疗犬疱疹病毒病的产品中的应用。
进一步,所述产品为药物或食品。
本发明的第四方面,提供一种重组犬疱疹病毒gD糖基化蛋白的制备方法,其包括在适于所述重组菌表达所述重组犬疱疹病毒gD糖基化蛋白的条件下培养所述重组菌,并且对培养产物进行分离纯化。
进一步,培养所述重组菌的条件包括:采用MRS液体培养基,在28~30℃条件下进行培养。
本发明的第五方面,提供一种预防和/或治疗犬疱疹病毒病的产品,所述产品包括根据本发明第四方面所述的制备方法制备得到的重组犬疱疹病毒gD糖基化蛋白。
与现有技术相比,本发明的技术方案具有以下有益效果:
本发明制备的重组菌成功表达重组犬疱疹病毒gD蛋白。大量研究表明gD蛋白是疱疹病毒囊膜上的重要组成部分,在病毒穿透和进入宿主细胞的过程中起着重要作用,且能引起比其他蛋白更强更持久的细胞免疫应答,是诱导宿主产生中和抗体的重要病毒蛋白。本发明成功构建表达重组犬疱疹病毒gD蛋白的乳酸乳球菌,因乳酸乳球菌的保护,可以递送重组犬疱疹病毒gD蛋白进入犬肠道,为制备预防和治疗犬疱疹病毒病药物或食品的研制提供重要基础。
附图说明
通过阅读下文优选实施方式的详细描述,各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的,而并不认为是对本发明的限制。在附图中:
图1:犬疱疹病毒gD蛋白的跨膜结构域预测结果;
图2:表达重组犬疱疹病毒gD蛋白的SDS-PAGE检测结果;其中,泳道1:重组乳酸乳球菌pLL32a/US6-2表达0h的样品,泳道3-4:重组乳酸乳球菌pLL32a/US6-2过夜表达的样品;泳道2:重组乳酸乳球菌pLL32a/US6-1表达0h的样品,泳道5-6:重组乳酸乳球菌pLL32a/US6-1过夜表达的样品;
图3:动物实验中,肠道黏膜sIgA含量的检测结果。
具体实施方式
下面将更详细地描述本公开的示例性实施方式。应当理解,可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反,提供这些实施方式是为了能够更透彻地理解本公开,并且能够将本公开的范围完整的传达给本领域的技术人员。
除非另有定义,本文所用的所有术语具有与本领域的普通技术人员通常理解的相同的含义。这里所描述或引用的许多技术和过程是本领域技术人员所熟知的,并且通常使用传统的方法。适当地,除非另有说明,通常根据制造商定义的规程和/或参数来进行涉及使用市售试剂盒和试剂的过程。
如本文使用的,术语“野生型”是指,天然存在的(例如从自然界中发现的)基因、蛋白质、蛋白质、细胞、菌株等。野生型犬疱疹病毒gD糖基化蛋白(envelope glycoprotein D)的氨基酸序列如SEQIDNO:6所示。
如本文中所使用的,术语“载体”是指,可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白质获得表达时,载体称为表达载体。当表达载体插入可表达为蛋白质的多核苷酸后,称其为重组表达载体。载体可以通过转化、转导或者转染导入宿主细胞,使其携带的遗传物质元件在宿主细胞中获得表达。一种载体可以含有多种控制表达的元件,包括但不限于,启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外,载体还可含有复制起始位点。
如本文中所使用的,术语“表达框”是指表达一个基因所需的完整元件,包括可操作性连接的启动子和基因编码序列。
如本文中所使用的,术语“编码序列”指核酸序列中直接确定其蛋白产物的氨基酸序列的部分。
如本文中所使用的,术语“可操作性连接的”或“可操作性连接”指两个或多个核苷酸区域或核酸序列的功能性的空间排列。例如,在核酸构建物中,启动子被置于感兴趣基因的核酸序列的特定位置,例如启动子位于该基因编码序列的上游位置,使得该基因编码序列的转录受到该启动子区域的引导,从而,启动子区域被“可操作地连接”到该基因的核酸序列上。“可操作性连接”可以通过基因重组的手段实现。
如本文中所使用的,术语“抗生素抗性基因”指编码对特定抗生素具有抗性的蛋白质的基因,并且携带它们的细胞在被一定浓度范围的该特定抗生素处理的环境中能够存活,因此抗生素抗性基因通常可用作选择标记。例如,“氨苄抗性基因”指编码对氨苄青霉素具有抗性的蛋白质的基因。
如本文中所使用的,术语“转化”是指将外源核酸导入生物体中,使得该核酸可作为染色体外元件或通过染色体整合而复制。
实施例1重组表达载体的构建
(1)人工合成两端分别具有MluI和XhoI酶切位点的氨苄青霉素抗性基因,将其与pMG36e质粒(淼灵生物,MLCC1262)分别进行双酶切(MluI和Xho I),将酶切产物经琼脂糖凝胶电泳分离后,切胶回收目的条带和载体条带,用T4连接酶连接,转化至MC1061感受态细胞中,利用100μg/ml氨苄青霉素进行筛选,获得新质粒pLL32a(序列如SEQ ID NO:5所示);
(2)将gD蛋白去除跨膜区域(跨膜结构预测如图1所示),得到氨基酸序列为SEQIDNO:1所示的重组蛋白,并将其优化为适应乳酸乳球菌的序列,得到核苷酸序列为SEQIDNO:2的编码基因US6-1。人工合成两端分别具有EcoRI和HindIII酶切位点的US6-1基因,将其与pLL32a质粒分别进行双酶切(EcoRI和HindIII),将酶切产物经琼脂糖凝胶电泳分离后,切胶回收目的条带和载体条带,用T4连接酶连接,转化至MC1061感受态细胞中,利用100μg/ml氨苄青霉素进行筛选,经测序验证正确后,培养并提取质粒,获得pLL32a/US6-1重组质粒,用于实施例2;
(3)对gD蛋白进行结构模拟,去除两端区域保留核心结构域,得到氨基酸序列为SEQIDNO:3所示的重组蛋白,并将其优化为适应乳酸乳球菌的序列,得到核苷酸序列为SEQIDNO:4的编码基因US6-2。人工合成两端分别具有EcoRI和HindIII酶切位点的US6-2基因,将其与pLL32a质粒分别进行双酶切(EcoRI和HindIII),将酶切产物经琼脂糖凝胶电泳分离后,切胶回收目的条带和载体条带,用T4连接酶连接,转化至MC1061感受态细胞中,利用100μg/ml氨苄青霉素进行筛选,经测序验证正确后,培养并提取质粒,获得pLL32a/US6-2重组质粒,用于实施例2。
实施例2重组乳酸乳球菌的培养
分别将pLL32a/US6-1重组质粒和pLL32a/US6-2重组质粒转化至MG1363乳酸乳球菌中,具体步骤如下:
将10μL待转化的重组质粒与100μL MG1363乳酸乳球菌感受态细胞轻轻混匀后,加入预冷的2mm电转化杯中,置于冰浴中静置5min,之后迅速将电转杯擦拭干净,置于电转仪中电激,电激条件:200Ω、10kV/cm、25μF。电激结束后,立即向电转杯中加入1mL的再生培养基(含0.5M蔗糖、20mM MgCl2、20mM CaCl2的MRS培养基),混合均匀后,转移至1.5mL离心管中,再置于冰浴静置10min,之后于30℃、220rpm调节下震荡孵育2h。取100μL所得孵育产物涂布于含氨苄抗性的MRS培养基平板上,于30℃过夜培养,得到含有pLL32a/US6-1的重组乳酸乳球菌单克隆、含有pLL32a/US6-2的重组乳酸乳球菌单克隆。
实施例3重组蛋白的表达与鉴定
将实施例2构建好的重组乳酸乳球菌分别以1%的接种量接种于MRS培养基,30℃培养过夜,第二天取培养液按3%接种量再次接种于新鲜的MRS培养基中,30℃培养约4h,待OD 600nm至0.6左右时,分别收集表达0h及过夜菌液,于4℃、12000rpm离心1min,取上清采用SDS-PAGE电泳进行gD蛋白含量的检测。结果如图2所示,其中,泳道1:重组乳酸乳球菌pLL32a/US6-2表达0h的样品,泳道3-4:重组乳酸乳球菌pLL32a/US6-2过夜表达的样品;泳道2:重组乳酸乳球菌pLL32a/US6-1表达0h的样品,泳道5-6:重组乳酸乳球菌pLL32a/US6-1过夜表达的样品。
实施例4 动物实验
取1月龄犬36只,随机分为对照组、试验组1和实验组2,每组12只。对照组用无菌PBS灌胃,试验组用1×1010cfu/mL浓度的重组菌灌胃(试验组1为重组乳酸乳球菌pLL32a/US6-1,试验组2为重组乳酸乳球菌pLL32a/US6-2),每日一次,每1 kg体重灌胃5 mL,每周根据体重调整用药量一次,连续灌胃2周。取第1d、3d、5d、7d、14d的血清和肠道黏膜。血清用于抗犬疱疹病毒的中和抗体效价,肠道黏膜用于检测sIgA含量。根据检测结果,试验组1和试验组2的犬体内产生的中和抗体水平显著高于对照组(P<0.05),在第14d,试验组1的抗体效价均达到1:64以上,试验组2的抗体效价均达到1:128以上,表明重组菌刺激犬产生了特异性免疫反应。sIgA含量的检测结果如图3所示,试验组1和试验组2的犬肠道中sIgA水平均显著高于对照组(P<0.05),说明重组菌刺激机体产生了黏膜免疫。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (10)
1.一种重组表达载体,其特征在于,所述重组表达载体表达重组犬疱疹病毒gD糖基化蛋白;所述重组犬疱疹病毒gD糖基化蛋白是野生型犬疱疹病毒gD糖基化蛋白的截短体。
2.如权利要求1所述的重组表达载体,其特征在于,所述重组犬疱疹病毒gD糖基化蛋白的氨基酸序列如SEQIDNO:1或SEQIDNO:3所示。
3.如权利要求1所述的重组表达载体,其特征在于,所述重组犬疱疹病毒gD糖基化蛋白的核苷酸序列如SEQIDNO:2或SEQIDNO:4所示。
4.如权利要求1所述的重组表达载体,其特征在于,所述重组表达载体适于在乳酸菌中复制并且表达所述重组犬疱疹病毒gD糖基化蛋白。
5.如权利要求1-4任一项所述的重组表达载体,其特征在于,所述重组表达载体的构建方法包括:将氨苄抗性基因连入pMG36e质粒中,获得具有氨苄抗性的pLL32a质粒;将所述重组犬疱疹病毒gD糖基化蛋白的基因序列可操作性地连接至pLL32a质粒中,即构建得到所述重组表达载体。
6.一种重组菌,其特征在于,所述重组菌含有权利要求1-5任一项所述的重组表达质粒。
7.如权利要求6所述的重组菌,其特征在于,所述重组菌通过将所述重组表达载体转化至宿主细胞中制备得到;
优选地,所述宿主细胞为乳酸菌;
优选地,所述宿主细胞为乳酸乳球菌MG1363。
8.权利要求1-5任一项所述的重组表达载体或权利要求6或7所述的重组菌在制备预防和/或治疗犬疱疹病毒病的产品中的应用;
优选地,所述产品为药物或食品。
9.一种重组犬疱疹病毒gD糖基化蛋白的制备方法,其特征在于,所述制备方法包括:在适于权利要求6或7所述的重组菌表达所述重组犬疱疹病毒gD糖基化蛋白的条件下培养所述重组菌,并且对培养产物进行分离纯化;
优选地,培养所述重组菌的条件包括:采用MRS液体培养基,在28~30℃条件下进行培养。
10.一种预防和/或治疗犬疱疹病毒病的产品,其特征在于,所述产品包括根据权利要求9所述的制备方法制备得到的重组犬疱疹病毒gD糖基化蛋白。
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