CN117534736A - 一种用于生产vlp重组疫苗的cmv病毒样颗粒及其制备方法 - Google Patents
一种用于生产vlp重组疫苗的cmv病毒样颗粒及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于生产VLP重组疫苗的CMV病毒样颗粒及其制备方法,该CMV病毒样颗粒将C端G4SLPETG修饰的植物病毒黄瓜花叶病毒CMV衣壳基因克隆入原核表达载体得到重组表达载体,将所述重组表达载体转染大肠杆菌BL21(DE3),经所述重组大肠杆菌BL21(DE3)表达获得;所述C端G4SLPETG修饰的CMV的氨基酸序列为SEQ ID NO.1。试验证明,本发明构建的重组菌株对外源蛋白表达稳定。使用本发明表达的重组蛋白制备病毒样颗粒进行抗原偶联,偶联效率高,抗原纯度高,安全性好,且对小鼠等动物没有致病性,容易通过安全性评价。
Description
本申请是2020年12月15日提交国家知识产权局、申请号为202011476397.3、发明名称为“一种用于生产VLP重组疫苗的CMV病毒样颗粒及其制备方法”的专利申请的分案申请。
技术领域
本发明涉及分子生物学、病毒学、免疫学和医学领域。本发明包含了一种用于生产VLP重组疫苗的CMV病毒样颗粒、VLP重组疫苗和用于生产VLP重组疫苗的CMV病毒样颗粒制备方法。
背景技术
植物病毒及其衍生的病毒样颗粒(VLP)备受关注,这主要是由于植物具有提供独特的翻译后修饰能力和成本效益的能力,是一种经济快速的生产VLP疫苗的替代平台,具有高度安全性、生产速度和可扩展性。
目前,虽然在基于VLP的疫苗开发过程中已经取得了进展,但是仍需要进一步的独特VLP系统。特别地,老年人总体上抗体应答较差、疫苗在被免疫的受试者和个体诱导可变的抗体应答变异通常跨越超过100倍的变异范围等情况,都会发生无抗体反应性。无反应性与某些II类MHC分子有关,即,未能诱导良好的T辅助(Th)细胞反应是造成这些个体中不良抗体反应的原因。因此,基本上在所有受试者和个体中诱导良好的Th细胞应答的疫苗是疫苗开发领域中的重要目标。
近来,一个基于CMV VLP的疫苗平台已经能够使用化学接头偶联技术在其表面上呈递不同抗原。CMV VLP衍生自具有插入的T细胞刺激性表位的CMV修饰衣壳蛋白。但化学接头修饰效率仍然较低且不均一,不能做到定点高效率修饰,该方案可以通过sorteaseA酶定点催化做到目的抗原与CMV定点缩合,催化效率高,工艺简单,CMV的定点修饰可以被工程化。目前CMV重组疫苗充当呈递系统并在其外表面表达源自丙型肝炎病毒(HCV)的表位,来自60名慢性丙型肝炎患者的血清样品显示出对被嵌合CMV感染的粗植物提取物具有显着的免疫反应性。并且,基于CMV VLP的表达系统已经被描述为针对可能的阿尔茨海默氏病的潜在疫苗以及猪圆环病毒特异性疫苗。
自身抗原蛋白通常难以诱导针对自身抗原的抗体应答。提高疫苗接种效率的一种方法是提高所应用抗原的重复性。与分离的蛋白质不同,病毒在没有任何佐剂的情况下在有和没有T细胞帮助的情况下都能诱导迅速有效的免疫反应(Bachmann和Zinkernagel,Ann.Rev.Immunol:15:235-270(1991))。与少数几种蛋白质相比,它们能够触发比其分离成分强得多的免疫反应。对于B细胞反应,众所周知,病毒免疫原性的一个关键因素是表面表位的重复性和顺序。许多病毒表现出准晶体表面,该准晶体表面具有规则排列的表位,可以有效地交联B细胞上的表位特异性免疫球蛋白(Bachxnann和Zinkernagel,Immunol。Today17:553-558(1996))。B细胞表面免疫球蛋白的这种交联是一个强激活信号,直接诱导细胞周期进程和IgM的产生抗体。此外,这种触发的B细胞能够激活T辅助细胞,进而诱导B细胞中IgM抗体向IgG抗体的转换以及任何疫苗接种的长寿B细胞记忆目标的产生(Zinkernagel,Ann.Rev.Immunol.15:235-270(1997))。病毒结构甚至与自身免疫性疾病中抗抗体的产生有关,并且是对病原体的自然应答的一部分(参见Fehr,T.,等人,J.Exp.Med.185:1785-1792(1997))。因此,由高度组织化的病毒表面呈递的抗原能够诱导针对抗原的强抗体应答。
发明内容
本发明针对现有化学偶联技术存在的不足,提供一种用于生产VLP重组疫苗的CMV病毒样颗粒及其制备方法,易于定向催化欧联,易于通过菌体培养获得,表达产量高,便于工业化生产,提升目标抗原的免疫原性、免疫反应快。
本发明第一方面提供一种用于生产VLP重组疫苗的CMV病毒样颗粒,该CMV病毒样颗粒将C端G4SLPETG修饰的植物病毒黄瓜花叶病毒CMV衣壳基因克隆入原核表达载体得到重组表达载体,将所述重组表达载体转染大肠杆菌BL21(DE3),经所述重组大肠杆菌BL21(DE3)表达获得;所述C端G4SLPETG修饰的CMV的氨基酸序列为SEQ ID NO.1。
本发明第二方面提供一种VLP重组疫苗,由第一方面的CMV病毒样颗粒与目标抗原按照摩尔比1:1进行混合混匀,在SorteaseA催化下制备而成。
本发明第三方面提供一种用于生产VLP重组疫苗的CMV病毒样颗粒制备方法,包括如下步骤:
(1)将his-CMV-G4SLPETG基因合成片段克隆入pET28a原核表达载体中获得阳性重组质粒his-CMV-G4SLPETG-pET28a;
(2)将步骤(1)中经序列测定正确的重组质粒转化大肠杆菌BL21(DE3),得到重组表达菌株his-CMV-G4SLPETG-pET28a-BL21;
(3)培养所述重组表达菌株his-CMV-G4SLPETG-pET28a-BL21,加入IPTG进行诱导表达,获得CMV病毒样颗粒。
优选的,所述阳性重组质粒his-CMV-G4SLPETG-pET28a将SEQ ID NO.2所示的核苷酸通过同源重组方法克隆入原核表达载体pET28a的NdeI位点和BamhI位点所得。
优选的,所述重组表达菌株his-CMV-G4SLPETG-pET28a-BL21含有所述阳性重组质粒his-CMV-G4SLPETG-pET28a。
优选的,所述的重组表达菌株his-CMV-G4SLPETG-pET28a-BL21培养至OD600达0.6-0.8时加入终浓度为1mM的IPTG进行诱导表达。
本发明具有如下有益效果:
第一、CMV来源于一种黄瓜花叶病毒(CMV)衣壳,衣壳蛋白具有自组装成纳米颗粒的功能,不具有感染性,具有强烈抗原免疫性,是申请人的VLP筛选平台独有的技术;
第二、通过与CMV病毒样颗粒定点催化偶联的抗原,显示出高强度的抗原性,可以在小鼠、大鼠、猫、狗和马中诱导高水平的特异性抗体,体液免疫反应快;
第三、本发明中C端G4SLPETG修饰的CMV病毒样颗粒可由大肠杆菌大量表达,生产工艺简单,CMV-G4SLPETG病毒样颗粒纯度高;
第四、本发明适用于产业化,选择的大肠杆菌重组表达菌株具有生长快、易于培养、遗传操作简单、繁殖速度快,对培养条件要求低,培养基廉价,能进行高密度培养,且能耐受较高的液体静压,便于工业化生产等特点。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案,下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图仅仅是示例性的,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图引伸获得其它的实施附图。
图1为本发明实施例中提供的C端G4SLPETG修饰的CMV基因与pET28a质粒连接后重组质粒示意图;
图2为本发明实施例中提供的CMV-G4SLPETG病毒样颗粒蛋白SDS-PAGE跑胶示意图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似改进,因此本发明不受下面公开的具体实施例的限制。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施方式的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明实施例中所采用的酶切、同源重组连接等分子生物学实验方法可参考《分子克隆》第二版。制备本发明CMV-G4SLPETG病毒样颗粒的基础材料包括:CMV-G4SLPETG蛋白核苷酸序列pET28a质粒、BL21(DE3)大肠杆菌菌株、天根质粒小提中量试剂盒、LB培养基、IPTG、卡那霉素、镍柱和咪唑等。将核苷酸序列发到华大基因公司合成his-TEV-CMV-G4SLPETG-pET28a重组质粒。
实施例1
本发明实施例1提供一种用于生产VLP重组疫苗的CMV病毒样颗粒,该CMV病毒样颗粒将C端G4SLPETG修饰的植物病毒黄瓜花叶病毒CMV衣壳基因克隆入原核表达载体得到重组表达载体,将所述重组表达载体转染大肠杆菌BL21(DE3),经所述重组大肠杆菌BL21(DE3)表达获得;所述C端G4SLPETG修饰的CMV的氨基酸序列为SEQ ID NO.1。
实施例2
参见图1和图2,本发明实施例2提供一种用于生产VLP重组疫苗的CMV病毒样颗粒制备方法,包括如下步骤:
(1)重组质粒的构建:将CMV-G4SLPETG基因直接让华大基因公司合成到pET28a载体NdeI酶切位点及XhoI酶切位点得到重组质粒his-TEV-CMV-G4SLPETG-pET28a;
(2)重组质粒转入表达菌株:将his-TEV-CMV-G4SLPETG-pET28a重组质粒转入大肠杆菌BL21(DE3)表达菌株,得到重组表达菌株his-TEV-CMV-G4SLPETG-pET28a-BL21;
(3)菌体培养及his-TEV-CMV-G4SLPETG病毒样颗粒纯化:培养重组表达菌株his-TEV-CMV-G4SLPETG-pET28a-BL21,IPTG诱导表达获得his-TEV-CMV-G4SLPETG病毒样颗粒。
(4)CMV-G4SLPETG病毒样颗粒制备:将(3)所得his-TEV-CMV-G4SLPETG病毒样颗粒,经his-TEV酶酶切,将his-TEV标签和CMV-G4SLPETG分离,酶切液经镍柱纯化,穿透液为单纯CMV-G4SLPETG病毒样颗粒。
具体的CMV-G4SLPETG病毒样颗粒表达纯化步骤如下:
(a1)his-TEV-CMV-G4SLPETG-pET28a重组质粒制备
将含有质粒的甘油菌用移液枪吸取5ul接种到5mL的2YT培养基(含50ug/ml卡那霉素),37℃震荡培养14-16小时,培养后取1mL菌液送测序。剩余菌液用质粒小提取试剂盒提取质粒,蛋白核酸检测仪检测核酸浓度。
(a2)his-TEV-CMV-G4SLPETG病毒样颗粒表达
将(a1)所述的质粒转化BL21(DE3)表达菌株,摇菌后离心浓缩,涂布卡那霉素抗性平板,37℃过夜培养,刮取菌落,接种10mL含氨苄青霉素的2×YT培养基,37℃180r摇菌至菌液OD值0.8左右。将其转接到500mL含卡那霉素的2×YT培养基中,37℃180r摇菌至菌液OD值0.8左右。加入终浓度为0.8mMIPTG诱导剂,30℃过夜表达。第三天4500rpm,15min离心收集菌体。超声破碎菌体45min,3s开,4s关,功率125w。超声结束后,9500rpm,20min,4℃收集上清液。
(a3)his-TEV-CMV-G4SLPETG病毒样颗粒纯化
离心上清液,过镍柱纯化,镍柱PBS平衡5柱体积。上样,PBS洗杂5柱体积,25Mm咪唑洗2柱体积,后再用25Mm咪唑洗5柱体积。之后连续用50、100、150、200、250Mm咪唑洗脱5柱体积。收集从上样到500mM咪唑的流出液。
从各收集液中吸取10uL加入10uL 2×蛋白电泳loading buffer后100℃加热4min。之后蛋白电泳,染色观察目的条带所在收集液浓度。将目的蛋白使用3kDa超滤管进行超滤,置换成PBS溶剂。
(a4)CMV-G4SLPETG病毒样颗粒纯化
将(a3)得到的his-TEV-CMV-G4SLPETG病毒样颗粒均由PBS溶液(10mM)溶解,加入his-TEV重组酶进行酶切,按照10ug TEV酶酶切1mg重组蛋白的比例进行酶切,30摄氏度震荡酶切3h,然后将酶切液分别过镍柱,穿透液为CMV-G4SLPETG病毒样颗粒,而his-TEV酶及酶切不完全的重组蛋白则会挂在镍柱上,分别用3K浓缩管对穿透液即CMV-G4SLPETG病毒样颗粒进行浓缩。将目的蛋白使用3kDa超滤管进行超滤,置换成PBS溶剂。
实施例3
本发明实施例3提供一种VLP重组疫苗,由实施例1的CMV病毒样颗粒与目标抗原按照摩尔比1:1进行混合混匀,在SorteaseA催化下制备而成,实现了疫苗简单化生产。
具体的,将实施例1中CMV-G4SLPETG病毒样颗粒与N端3个甘氨酸修饰的目标抗原分别用PBS(10mM)配制成1mg/ml,按照1:1进行混合,加入his-Sotase A5酶,按照100ug催化2mg混合蛋白的比例进行37℃震荡催化5h,然后将催化液过镍柱,穿透液为CMV-G4SLPETGGG-目标抗原病毒样颗粒,而his-Sotase A重组酶会挂在镍柱上,用3K浓缩管用对穿透液即CMV-G4SLPETGGG-目标抗原病毒样颗粒进行浓缩,即可得到CMV病毒样颗粒修饰的VLP重组疫苗。
序列:
SEQ ID NO.1:
MHHHHHHENLYFQGMGQYIKANSKFIGITERRRRPRRGSRSAPSSAD
ANFRVLSQQLSRLNKTLA
AGRPTINHPTFVGSERCKPGYTFTSITLKPPKIDRGSYYGKRLLLPDSVTEY
DKKLVSRIQIRVN
PLPKFDSTVWVTVRKVPASSDLSVAAISAMFADGASPVLVYQYAASGVQA
NNKLLYDLSAMRADI
GDMRKYAVLVYSKDDALETDELVLHVDVEHQRIPTSGVLPVGGGGSLPETG*
SEQ ID NO.2:
ATGCACCATCATCATCATCACgagaatctttattttcagggcATGggccagtatattaaggccaa ctccaaatttatcgggattaccgagcgtcgacgtcgtccgcgtcgtggttcccgctccgccccctcctccgcggatgctaactttagagtcttgtcgcagcagctttcgcgacttaataagacgttagcagctggtcgtccaactattaaccacccaacctttgtagggagtgaacgctgtaaacctgggtacacgttcacatctatcaccctaaagccaccaaaaatagaccgtgggtcttattatggtaaaaggttgttattacctgattcagtcacggaatatgataagaaacttgtttcgcgcattcaaattcgagttaatcctttgccgaaatttgattcaaccgtgtgggtgacagtccgtaaagttcctgcctcttcggacttatccgttgccgccatttctgctatgtttgcggacggagcctcaccggtactggtttatcagtacgctgcatctggagtccaagctaacaacaaactgttgtatgatctttcggcgatgcgcgctgatataggcgacatgagaaagtacgccgtcctcgtgtattcaaaagacgatgcactcgagacagacgagttagtacttcatgttgacgtcgagcaccaacgtattcccacatctggggtgctcccagttGGTGGAGGAGGTTCTCTGCCAGAAACCGGTTAG。
Claims (9)
1.一种用于生产VLP重组疫苗的CMV病毒样颗粒,其特征在于,该CMV病毒样颗粒将C端G4SLPETG修饰的植物病毒黄瓜花叶病毒CMV衣壳基因克隆入原核表达载体得到重组表达载体,将所述重组表达载体转染大肠杆菌BL21(DE3),经所述重组大肠杆菌BL21(DE3)表达获得;所述C端G4SLPETG修饰的CMV的氨基酸序列为SEQ ID NO.1;
生产所述CMV病毒样颗粒制备方法,包括如下步骤:
(1)将his-CMV-G4SLPETG基因合成片段克隆入pET28a原核表达载体中获得阳性重组质粒his-CMV-G4SLPETG-pET28a;
(2)将步骤(1)中经序列测定正确的重组质粒转化大肠杆菌BL21(DE3),得到重组表达菌株his-CMV-G4SLPETG-pET28a-BL21;
(3)培养所述重组表达菌株his-CMV-G4SLPETG-pET28a-BL21,加入IPTG进行诱导表达,获得CMV病毒样颗粒。
2.如权利要求1所述的CMV病毒样颗粒,其特征在于,所述阳性重组质粒his-CMV-G4SLPETG-pET28a将SEQ ID NO.2所示的核苷酸通过同源重组方法克隆入原核表达载体pET28a的NdeI位点和BamhI位点所得。
3.如权利要求2所述的CMV病毒样颗粒,其特征在于,所述重组表达菌株his-CMV-G4SLPETG-pET28a-BL21含有所述阳性重组质粒his-CMV-G4SLPETG-pET28a。
4.如权利要求3所述的CMV病毒样颗粒,其特征在于,所述的重组表达菌株his-CMV-G4SLPETG-pET28a-BL21培养至OD600达0.6-0.8时加入终浓度为1mM的IPTG进行诱导表达。
5.一种VLP重组疫苗,其特征在于,由权利要求1至4任一项所述的CMV病毒样颗粒与目标抗原按照摩尔比1:1进行混合混匀,在SorteaseA催化下制备而成。
6.一种用于生产VLP重组疫苗的CMV病毒样颗粒制备方法,其特征在于,包括如下步骤:
(1)将his-CMV-G4SLPETG基因合成片段克隆入pET28a原核表达载体中获得阳性重组质粒his-CMV-G4SLPETG-pET28a;
(2)将步骤(1)中经序列测定正确的重组质粒转化大肠杆菌BL21(DE3),得到重组表达菌株his-CMV-G4SLPETG-pET28a-BL21;
(3)培养所述重组表达菌株his-CMV-G4SLPETG-pET28a-BL21,加入IPTG进行诱导表达,获得CMV病毒样颗粒。
7.根据权利要求6所述的一种用于生产VLP重组疫苗的CMV病毒样颗粒制备方法,其特征在于,所述阳性重组质粒his-CMV-G4SLPETG-pET28a将SEQ ID NO.2所示的核苷酸通过同源重组方法克隆入原核表达载体pET28a的NdeI位点和BamhI位点所得。
8.根据权利要求7所述的一种用于生产VLP重组疫苗的CMV病毒样颗粒制备方法,其特征在于,所述重组表达菌株
his-CMV-G4SLPETG-pET28a-BL21含有所述阳性重组质粒his-CMV-G4SLPETG-pET28a。
9.根据权利要求8所述的一种用于生产VLP重组疫苗的CMV病毒样颗粒制备方法,其特征在于,所述的重组表达菌株his-CMV-G4SLPETG-pET28a-BL21培养至OD600达0.6-0.8时加入终浓度为1mM的IPTG进行诱导表达。
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