CN116376948B - 一种质粒载体及展示外源蛋白的ms2噬菌体类似颗粒的制备方法 - Google Patents
一种质粒载体及展示外源蛋白的ms2噬菌体类似颗粒的制备方法 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域，具体为一种质粒载体及展示外源蛋白的MS2噬菌体类似颗粒的制备方法。本发明提供的质粒载体的特征在于：该质粒载体依次含有***糖启动子，MS2CP基因、琥珀密码子、Linker序列、外源蛋白基因***部分、His标签编码序列、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。通过该质粒载体可以制备表面展示外源蛋白的MS2噬菌体类似颗粒，可应用于MS2噬菌体亚单位疫苗的制备。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及质粒载体及展示外源蛋白的噬菌体类似颗粒的制备方法。
背景技术
病毒样颗粒(Virus like particles，VLPs)是由多个病毒的一种或几种结构蛋白组装而成的大颗粒，不含病毒核酸，不能进行自主复制，具有与病毒颗粒类似的整体结构。VLPs由于具有安全性高，在结构上与病毒粒子相似，可通过与病毒粒子相同的途径激起免疫***的有效应答等优点，近二十年来在人用、兽用疫苗及药物递送***中的应用越来越受到关注。
病毒样颗粒(VLPs)具高安全性和有效性，代表了亚单位疫苗开发的重大进步。病毒样颗粒(VLPs)的颗粒性质和密集的重复亚基组织使它们成为展示疫苗抗原的理想支架，因可以呈现病原体关键抗原特征而具有很好的免疫原性常用作特定类型的亚单位疫苗。但VLP的抗原展示的往往需要精心设计并劳动密集型的试错优化。另外，纯化展示抗原的VLP步骤也比较繁琐沉淀，往往需要沉淀，过滤，或超高速离心。
大肠杆菌MS2噬菌体是二十面体的单链正义RNA病毒，可感染大肠杆菌和肠杆菌科的其他成员，其基因组含有3569个核苷酸，同时作为mRNA编码4中蛋白质，包括成熟酶蛋白(A蛋白)、衣壳蛋白(CP)、复制酶蛋白(Rep)和裂解蛋白(Lys)。成熟酶蛋白和衣壳蛋白能与外源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，这里称之为MS2-VLPs。MS2-VLPs应用广泛，这些颗粒能够模拟ssRNA病毒，在疫苗、药物传递***和免疫活性多肽显示***或纳米机器等多个领域，可以作为分子检测、过程控制以及定量分析的参照物。
发明内容
本发明开发一种质粒载体，通过该质粒载体可以制备表面展示外源蛋白的MS2噬菌体类似颗粒，可应用于MS2噬菌体亚单位疫苗的制备。同时，在外源蛋白的羧基端引入His标签，便于快速简洁纯化表面展示蛋白的VLP。
本发明提供的质粒载体的特征在于：该质粒载体依次含有***糖启动子，MS2CP基因、琥珀密码子、Linker序列、外源蛋白基因***部分、His标签编码序列、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。
具体的，所述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。
具体的，所述琥珀密码子为TAG。
具体的，所述Linker序列，编码的蛋白序列以甘氨酸和丝氨酸为主，长度超过15个氨基酸。
具体的，外源蛋白基因***部分有酶切位点，方便外源序列的克隆到载体上。
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个组氨酸。
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，所述编码MS2噬菌体成熟蛋白序列如SEQ ID NO.6所示。
具体的，所述MS2噬菌体包装序列和所述锤头酶序列之间加入10bp以上随机序列。
优选的，上述随机序列如SEQ ID NO.7所示。
本发明还提供展示外源蛋白的MS2噬菌体类似颗粒的制备方法，具体步骤如下：
(1)采用本发明提供的质粒载体***外源蛋白基因X后，转入琥珀密码子抑制宿主中表达；
(2)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；
(3)上清经离子亲和层析纯化得到表面展示X蛋白的MS2噬菌体样颗粒。
有益效果：
通过本发明提供的质粒载体可以成功制备表面展示外源蛋白的MS2噬菌体类似颗粒，并可利用HIS标签快速纯化出来，可应用于MS2噬菌体亚单位疫苗的制备。
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MS2PLP--EGFP-His透射电镜图；
图3为MS-VLP表面表达标记的鉴定结果图。
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。
具体实施例中采用的材料来源：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
在本实施例中质粒载体的图谱如图1所示，含有***糖启动子(图中所示，AraBAD promoter)，MS2 CP基因(图1中所示，A)，琥珀密码子(图1中所示，B),Linker序列(图1中所示，C)，外源蛋白基因***部分(图1中所示，D)，His标签编码序列(图1中所示，E),MS2噬菌体包装序列(图1中所示，F)和MS2 A蛋白基因(图1中所示，H)，图1中G部分为锤头酶序列。包含上述序列特征的载体命名为pMS2-P-His。在D部分***外源蛋白基因X后获得载体pMS2-XP-His。
设计序列如SEQ ID NO.9所示(1-6位为NcoI酶切位点；3-392位为编码MS2 CP蛋白的序列；393-395位为琥珀密码子TAG；396-452位为Linker序列；453-503位为外源基因***部分，有酶切位点，453-458位为pstI酶切位点，462-467位为SpeI酶切位点，534-565位为编码10个组氨酸序列与MS2噬菌体包装序列之间的随机序列；504-533编码10个组氨酸；566-584位为MS2噬菌体包装序列；585-626位为随机序列，627-676位为锤头酶序列；677-745位为随机序列，746-1927位为编码MS2噬菌体成熟蛋白的序列)，满足图1中A,B,C,D,E,G,H的顺序要求，上海生物生工有限公司合成并委托其通过NcoI和HindIII克隆到pBAD-HisA质粒质粒上，得到的质粒名pMS2-P-His。其中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；琥珀密码子为TAG；Linker序列，编码的蛋白序列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10个组氨酸；MS2噬菌体包装序列SEQ ID NO.3所示；锤头酶序列如SEQ ID NO.4所示；编码MS2噬菌体成熟蛋白序列如SEQ ID NO.6所示；MS2噬菌体包装序列和所述锤头酶序列之间加入10bp以上随机序列，随机序列如SEQ ID NO.7所示。
展示外源蛋白的MS2噬菌体类似颗粒的制备
序列如SEQ ID NO.8所示，是编码绿色荧光蛋白(EGFP)的序列，由上海生物生工有限公司合成并委托其通过pstI位点克隆到pMS2-P-His上，得到载体pMS2-EGFPP-His。
载体转入宿主表达,宿主为琥珀密码子抑制宿主XL1blue。取40ng pMS2-EGFPP-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。
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。
表面展示EGFP的MS2噬菌体颗粒的纯化采用常规实验方法:
1.5000g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。
(1)试剂：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。
(2)纯化：
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)；
柱子平衡好后，用一次性注射器吸取步骤1过滤样品，手动上样；
待UV-280重新回到基线，采用50nm，150nm，250nm和500nm咪唑梯度洗脱蛋白；
纯化结果显示在150nm咪唑洗脱时，出现洗脱峰；
收集洗脱液，设置为每孔收集0.5mL；
根据洗脱峰对应的收集孔，留取纯化后的洗脱样品。
MS-VLP的鉴定
1)电镜观察。取上述收集的洗脱液适当体积送广东省微生物所电镜(TEM)观察，结果如图2所示(比例尺100nm)。结果显示咪唑洗脱成分的结构为噬菌体样颗粒。
2)MS-VLP表面表达标记的鉴定
取适当体积的上述洗脱成分进行SDS-PAGE电泳分离和蛋白质杂交鉴定。SDS-PAGE电泳的分离胶浓度为12％，一抗为Anti-eGFP mouse monoclonal antibody(购自上海生物工程有限公司)，实验过程和操作参照分子克隆实验指南(J.萨姆布鲁克等，2017年科学出版社出版的图书)进行。SDS-PAGE电泳和蛋白质杂交样品，包括质粒转入宿主后诱导前样品，诱导后样品，超声破碎后上清、沉淀以及纯化后的样品，结果如下图3所示，图3中A为SDS-PAGE检测MS-VLP蛋白，其中M：蛋白标准分子量；1：诱导前总蛋白；2：诱导后总蛋白；3：破碎上清；4：破碎沉淀；5：洗脱液。图3中B为WB检测pMS2-EGFPP-His蛋白表达，其中M：蛋白标准分子量；1：洗脱液样品；2：破碎上清；3：诱导后总蛋白；4：诱导后总蛋白；5：诱导前总蛋白。(上方箭头所指处为衣壳蛋白与EGFP融合的蛋白；下方箭头所指处为衣壳蛋白)。结果显示MS-VLP成分中有CP与EGFP蛋白融合蛋白。
Claims (2)
1.一种质粒载体，其特征在于：所述质粒载体为pMS2-EGFPP-His，由如SEQ ID NO.8所示序列通过pstI位点克隆到pMS2-P-His上获得；所述pMS2-P-His由如SEQ ID NO.9所示序列通过NcoI和HindIII克隆到pBAD-HisA质粒上获得。
2.展示外源蛋白的MS2 噬菌体类似颗粒的制备方法，其特征在于：具体步骤如下：
（1）采用权利要求1提供的质粒载体pMS2-EGFPP-His，转入琥珀密码子抑制宿主XL1blue中表达；
（2）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；
（3）上清经离子亲和层析纯化得到表面展示EGFP蛋白的MS2噬菌体样颗粒。
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