CN116445546B - 一种质粒载体及通过质粒载体制备的新冠病毒核酸检测标准品和制备方法 - Google Patents
一种质粒载体及通过质粒载体制备的新冠病毒核酸检测标准品和制备方法 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域，提供一种质粒载体及通过质粒载体制备的新冠病毒核酸检测标准品及其制备方法。所述质粒载体依次含有阿拉伯糖启动子；MS2CP基因；琥珀密码子；Linker；His标签序列；带有酶切位点的随机序列，基于酶切可插入新冠病毒核酸检测的靶标序列；MS2噬菌体包装序列；锤头酶序列和MS2A蛋白基因。将该质粒转入琥珀密码子抑制宿主进行诱导表达，离心得到诱导表达菌体，破碎菌体，上清经离子亲和层析纯化得到新冠病毒核酸检测标准品。本发明提供的新冠病毒核酸检测标准品稳定且不易降解，制备方法快速。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及一种质粒载体和用于核酸检测新型冠状病毒2019-nCov的核酸检测标准品及其制备方法。
背景技术
新型冠状病毒被世卫组织命名为2019-nCov，是一类具有囊膜、基因组为线性单股正链的RNA病毒，能引起严重急性呼吸感染。目前，对新冠病毒的预防和诊断以核酸检测为主，国家已批准了多款核酸诊断试剂盒上市，用于临床诊断。这些核酸新冠病毒检测试剂盒都是采用RT-PCR的方法，都需要配备阳性对照或标准品。为监控检测过程，新冠病毒标准品最好是RNA，稳定且不易降解。
大肠杆菌MS2噬菌体是二十面体的单链正义RNA病毒，其基因组编码的衣壳蛋白能与外源ssRNA中特定茎环结构(称为pac位点)相互作用，自发包装形成不具有侵染活性的病毒颗粒(virus-like particles，VLPs)，这里称之为MS2-VLPs。MS2-VLPs携带的RNA，能耐核酸酶降解也称之为盔甲RNA。
目前，MS2-VLPs的生产主要基于各种细胞体内质粒驱动的包装系统。质粒的功能有体现在两方面：1.表达MS2噬菌体的外壳蛋白CP和成熟蛋白A；2.利用启动子转录出带pac位点的靶标RNA。在体内，CP蛋白识别携带pac位点的靶标RNA，自发形成二聚体结构，约90个二聚体和1个成熟蛋白A组装形成MS2-VLP。基于质粒制备的体系中，有多种途径生产MS2-VLP并在两个方面进行优化：1.包装效率，2.简化MS2-VLP的纯化。如zhan(Zhan,S.etal.Armored Long RNA Controls or Standards for Branched DNA Assay forDetection of Human Immunodefciency Virus Type 1.Journal of ClinicalMicrobiology 47,2571–2576,(2009).)等通过增加pac位点提高了包装效率和包装靶标RNA的长度。在解决生产MS2-VLPs纯化复杂且耗时方面，Petr Kralik(Mikel,P.,Vasickova,P.&Kralik,P.Author Correction:One-plasmid double-expression His-tagsystem for rapid production and easy purification of MS2 phage-likeparticles.Sci Rep 9,6377(2019))等为简化纯化步骤，开发了单质粒双表达体系：在单个质粒中一个启动子表达将2个CP蛋白基因与His标签序列进行融合表达，生产带有His标签的2聚体的CP蛋白和A蛋白，另一个启动子转录出带pac位点的靶标RNA。该体系自发组装的MS2-VLPs表面带有His标签，利用快速高效液相色谱(FPLC)就能快速简便的得到目的MS2-VLPs。
发明内容
基于CP蛋白先形成二聚体再组装成MS2-VLPs的原理，本发明首次利用琥珀密码子与琥珀密码子抑制宿主配套产生野生型CP蛋白和通读琥珀密码子产生的融合型CP蛋白。上述两种CP蛋白组装形成嵌合型二聚体，在此基础上形成嵌合型的MS2-VLPs。嵌合型CP蛋白末端携带His标签蛋白，产生的嵌合MS2-VLPs表面则展示His标签，方便利用离子亲和层析快速简便的得到目的MS2-VLPs。为优化His标签在MS2-VLPs表面的展示，本发明在琥珀密码子之后设计了Linker序列，该序列编码15氨基酸以上长度就能让His标签充分展示在MS2-VLPs表面，使之与离子亲和层析的镍柱结合力最强，方便得到更纯的MS2-VLPs。在MS2-VLPs形成过程中，CP蛋白与A蛋白的比例约为180：1,A蛋白所需量远少于CP蛋白的量，基于上述考虑，本发明采用无启动子表达成熟蛋白A，为确保A蛋白的表达量，在A蛋白基因前加入锤头酶序列。为制备新冠病毒核酸检测标准品，在编码His标签序列与A蛋白基因之间引入酶切位点和pac位点，通过酶切位点克隆检测新冠病毒序列，该序列经转录后携带pac位点的RNA与CP蛋白作用后形成表面携带His标签的MS2-VLPs。本发明的质粒载体具备在MS2-VLPs表面展示His标签的能力。
本发明提供的质粒载体的特征在于：质粒载体中依次含有阿拉伯糖启动子(图1中所示，AraBAD promoter)，MS2CP基因(图1中所示，A)，琥珀密码子(图1中所示，B),Linker(图1中所示，C)，His标签(图1中所示，D)，带有酶切位点的随机序列(图1中所示，E)，基于酶切可插入新冠病毒核酸检测的靶标序列；MS2噬菌体包装序列(图1中所示，F)，锤头酶序列(图1中所示，G)和MS2 A蛋白基因(图1中所示，H)。
具体的，MS2 CP基因，无终止密码子，编码的蛋白为GenBank:AZS06102.1，如SEQID NO:1所示。
具体的，琥珀密码子紧邻MS2 CP基因，序列为TAG。
具体的，所述Linker序列，编码的蛋白序列为以甘氨酸和丝氨酸为主，长度超过15个氨基酸。
具体的，所述His标签编码序列编码连续6个以上组氨酸。
优选的，上述His标签编码序列编码8-10组氨酸。
具体的，所述MS2噬菌体包装序列如SEQ ID NO:2或SEQ ID NO:3所示。
具体的，所述锤头酶序列如SEQ ID NO:4或SEQ ID NO:5所示。
具体的，锤头酶序列与MS2噬菌体包装序列间加入10bp以上随机序列。
优选的，上述随机序列如SEQ ID NO:7所示。
具体的，MS2 A蛋白基因编码的蛋白为GenBank:AZS06103.1，蛋白序列如SEQ IDNO:6所示。
具体的，带有酶切位点的随机序列的酶切位点为KpnI，新冠病毒核酸检测的靶标序列如SEQ ID NO:8所示。
本发明同时提供通过质粒载体制备的新冠病毒核酸检测标准品，其特征在于，通过上述质粒载体转入琥珀密码子抑制宿主XL1blue获得。
本发明同时提供新冠病毒核酸检测标准品的制备方法，包括以下步骤：
1)构建质粒载体，质粒依次含有阿拉伯糖启动子，MS2CP基因、琥珀密码子TAG、Linker序列、8-10*His的纯化标签序列、2019-nCov检测靶标序列、MS2噬菌体包装序列、锤头酶序列和编码MS2噬菌体成熟蛋白序列；
2)将本发明构建的质粒载体转入琥珀密码子抑制宿主诱导表达；
3)离心诱导表达后的菌液，得到菌体，破碎菌体，释放宿主内蛋白，离心取沉淀获得上清；
4)上清经离子亲和层析纯化得到新冠病毒核酸检测标准品。
本法明的有益效果：
本发明为单质粒体系，基于琥珀密码子和琥珀密码子抑制宿主配套应用，在CP蛋白的羧基端融合His纯化标签表达，提供的新冠病毒核酸检测标准品稳定且不易降解，制备方法快速。
附图说明
图1为本发明一具体实施例中质粒载体的示意图；
图2为本发明具体实施例中梯度稀释的MS-VLP定量PCR结果图；
图3为本发明具体实施例中MS-VLP耐核酸酶的消化实验结果图；
图4为本发明具体实施例中盔甲RNA温度稳定性实验结果图。
具体实施方式
为使本发明的目的、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具体细节以便于充分理解本发明，本领域技术人员可以在不违背本发明构思的情况下做类似改进，因此本发明的保护范围不受下面公开的具体实施例的限制。
具体实施例中以单个质粒制备新冠标准品。
本发明具体实施例中的生物材料来源：pBAD-HisA质粒购自优宝生物公司。基因序列合成由发明人自行设计并委托上海生物生工有限公司合成。所有的试剂如IPTG和Bl21(DE3)，LB培养基、感受态细胞购自Takara大连生物有限公司。
质粒载体的构建
本实施例中构建的质粒载体中依次含有阿拉伯糖启动子(图1中所示，AraBADpromoter)；MS2 CP基因(图1中所示，A)；琥珀密码子(图1中所示，B)；Linker(图1中所示，C)；His标签(图1中所示，D)；E部分带有酶切位点的随机序列，基于酶切可插入新冠病毒核酸检测的靶标序列；MS2噬菌体包装序列(图1中所示，F)；锤头酶序列(图1中所示，G)和MS2A蛋白基因(图1中所示，H)。MS2 CP基因，无终止密码子，编码的蛋白为GenBank:AZS06102.1，如SEQ ID NO:1所示。琥珀密码子紧邻MS2 CP基因，序列为TAG。Linker序列，编码的蛋白序列为以甘氨酸和丝氨酸为主，长度超过15个氨基酸。His标签编码序列编码连续6个以上组氨酸。本实施例中His标签编码序列编码10个组氨酸，MS2噬菌体包装序列SEQ IDNO:3所示。所述锤头酶序列如SEQ ID NO:4所示。
MS2 A蛋白基因编码的蛋白为GenBank:AZS06103.1，蛋白序列如SEQ ID NO:6所示。锤头酶序列与MS2噬菌体包装序列间加入10bp以上随机序列，随机序列如SEQ ID NO:7所示。
将上述序列进行组装，如SEQ ID NO:9所示。该序列由上海生物生工有限公司合成并委托通过NcoI和HindIII克隆到pBAD-HisA质粒上，得到的质粒载体pMS2-His。
SEQ ID NO:9所示的序列中，1-6位为NcoI酶切位点，3-392位编码MS2 CP蛋白，393-395位为琥珀密码子TAG，396-503位为Linker序列，504-533位编码10个组氨酸，534-565位为带有KpnI酶切位点的随机序列，566-584位为包装位点序列，585-626位为锤头酶序列与包装序列之间的随机序列，627-676位为锤头酶序列，677-745位为锤头酶序列与编码MS2 A蛋白序列之间的随机序列，746-1927位编码MS2 A蛋白，1928-1933位为HINDIII酶切位点。
2019-nCov检测靶标序列如SEQ ID NO:8所示(1-6位,160-165位为KpnI酶切位点)，由上海生物生工有限公司合成并委托通过KpnI位点克隆到pMS2-His上(具体插入图1中的E部分)，得到载体pMS2-CON-His。
在具体实施中可以根据检测需要插入不同的2019-nCov检测靶标序列，获得不同的新冠病毒核酸检测标准品。
质粒载体转入宿主表达
本实施例中选用的宿主为琥珀密码子抑制宿主XL1blue。
(1)取40ng pMS2-CON-His质粒转入XL1blue感受态细胞。在氨苄抗生素的LB平板上挑取单个阳性克隆，接种至液体5mL LB培养基中37℃、180r/min恒温摇床培养过夜，
(2)取1mL过夜培养基加入100mL新鲜液体LB培养基中(含氨苄抗生素50ng/mL),37℃、180r/min恒温摇床培养至OD600为0.4-0.6之间，加入1mM的IPTG诱导后继续培养6-16h。
新冠病毒核酸检测标准品的纯化
采用常规实验方法对新冠病毒核酸检测标准品进行纯化，具体如下：
(1)对质粒载体转入宿主表达后的培养液，在5000g离心10min，回收菌体，用5mL无菌PBS重悬上一步骤离心得到的菌体。用超声破碎仪在冰上进行破碎。超声功率设置为30％，每次破碎8s，暂停12s，共破碎10min。超声结束后，向破碎后的菌液中加DNaseⅠ(终浓度1μg/mL)和RNase A(终浓度10μg/mL)于37℃水浴1-2h，以消化掉一部分大肠杆菌破碎后释放的核酸。将上述消化好的破碎菌液于4℃，4000rpm离心15min，取上清并用0.22μM一次性针头式过滤器过滤。
(2)使用AKTA avant蛋白纯化，所用亲和层析柱为HisTrap HP 5ml，
本步骤中采用的试剂为：Binding buffer:25mM Tris-HCl(pH7.5)；0.5M NaCl；10mM咪唑,10％glycerol；Elution buffer:25mM Tris-HCl(pH7.5)；0.5M NaCl；0.5M咪唑,10％glycerol；20％乙醇；去离子水。上述试剂需超声10-15min以除气泡。
a.用Binding buffer以5ml/min的流速，平衡柱子至紫外峰值(UV-280)不再变化(约5-10个柱体积)；
b.柱子平衡好后，用一次性注射器吸取1)过滤样品，手动上样；
c.待UV-280重新回到基线，采用50nm，150nm，250nm和500nm咪唑梯度洗脱蛋白；
d.纯化结果显示在250nm咪唑洗脱时，出现洗脱峰
e.收集洗脱峰，设置为每孔收集0.5mL；
f.根据洗脱峰对应的收集孔，留取纯化后样品。
MS-VLP的鉴定
(1)定量PCR。将纯化后样品按照浓度梯度稀释后提取RNA并逆转录成cDNA。利用定量PCR探针法鉴定新冠标准品核酸序列，上述操作按照试剂盒说明完成。引物和探针如下：
定量PCR的结果如图2所示。结果显示梯度稀释的盔甲RNA其定量PCR有较好的浓度线性。
耐核酸和耐高温检测
取纯化后样品20uL,分别加入DNaseⅠ(终浓度1μg/mL)和RNase A(终浓度10μg/mL)。加酶组放在提前预设到37℃的水浴锅温育1h，之后样品通过1％的琼脂糖凝胶电泳观察耐核酸酶结果。结果如图3所示，泳道1为DNase I样品，泳道2为加入RNase A样品，说明盔甲RNA能保护好包裹着的核酸不被降解掉，抵抗核酸酶的消化。
取纯化后样品20uL，分别在4℃、45℃、55℃、60℃、65℃、66℃、68℃、70℃温育30min，之后样品通过1％的琼脂糖凝胶电泳观察MS2 VLP的温度稳定性。结果如图4所示，说明盔甲RNA在4-60℃温育30min仍比较稳定。
Claims (3)
1.一种质粒载体，其特征在于：所述质粒载体为pMS2-CON-His，由新冠病毒核酸检测的靶标序列SEQ ID NO:8通过KpnI位点克隆到pMS2-His质粒载体上得到；pMS2-His质粒载体由序列SEQ ID NO:9通过NcoI和HindIII克隆到pBAD-HisA质粒得到。
2.通过质粒载体制备的新冠病毒核酸检测标准品，其特征在于，所述标准品通过如权利要求1所述的质粒载体pMS2-CON-His转入琥珀密码子抑制宿主XL1blue获得。
3.新冠病毒核酸检测标准品的制备方法，其特征在于，包括以下步骤：
1）构建如权利要求1所述的pMS2-CON-His质粒载体；
2）将本发明构建的质粒载体pMS2-CON-His转入琥珀密码子抑制宿主诱导表达；
3）离心诱导表达后的菌液，得到菌体，破碎菌体，释放宿主内蛋白，离心取沉淀获得上清；
4）上清经离子亲和层析纯化得到新冠病毒核酸检测标准品。
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