CN117126870A - 一种质粒载体体系及ms2噬菌体类似颗粒的制备方法 - Google Patents
一种质粒载体体系及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,具体涉及一种质粒载体体系及MS2噬菌体类似颗粒的制备方法。本发明提供的质粒载体体系的特征在于:该质粒载体体系含有MS2噬菌体CP基因、包装序列、重组成熟蛋白基因。通过该质粒载体可以快捷制备MS2噬菌体类似颗粒,可应用于MS2噬菌体递送载体、RNA标准品的制备、亚单位疫苗的纯化。
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噬菌体类似颗粒的制备方法。
背景技术
MS2噬菌体类似颗粒(MS2-VLP)是一种由180个拷贝的单个外壳蛋白(CP)和一个成熟蛋白自组装而成的二十面体的病毒样颗粒。成熟酶蛋白和衣壳蛋白能与外源ssRNA中包装序列(特定茎环结构)相互作用,自发包装形成不具有侵染活性的病毒颗粒MS2-VLP。MS2-VLP应用广泛,这些颗粒能够模拟ssRNA病毒,在疫苗、药物传递系统和免疫活性多肽显示系统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。
但MS2-VLP纯化步骤比较繁琐沉淀,往往需要沉淀,过滤,或超高速离心。为简化MS2-VLP的纯化步骤,之前有研究报道在MS2噬菌体CP蛋白的融合His Tag纯化标签,或在CP蛋白二聚体之间融合His Tag纯化标签,利用离子亲和层析方法一步法可得到比较纯的MS2-VLP。本发明基于MS2-VLP的另一成分成熟蛋白,也就是在成熟蛋白的氨基端融合纯化标签,以此简化MS2-VLP的纯化步骤。
发明内容
本发明开发一种质粒载体体系,通过该质粒载体体系可生产成熟蛋白包含纯化标签的MS2-VLP,便于快速简洁纯化MS2-VLP。
本发明提供的质粒载体体系的特征在于:该质粒载体体系含有至少一个质粒载体,该质粒体系含有MS2噬菌体CP基因、包装序列、重组成熟蛋白基因,所述重组成熟蛋白基因包含成熟蛋白基因和纯化标签,所述成熟蛋白基因编码的蛋白如SEQ ID NO.1所示,所述成熟蛋白的氨基端包含纯化标签。
具体的,所述MS2噬菌体CP基因编码的蛋白序列至少包含如SEQ ID NO.2所示的序列,其5’端有启动子。
具体的,所述包装序列如SEQ ID NO.3,SEQ ID NO.4所示。
具体的,重组成熟蛋白的纯化标签为8×His标签,如SEQ ID NO.5所示。
具体的,成熟蛋白基因与纯化标签间含有Linker序列,所述Linker序列的长度超过5个氨基酸。
优选的,上述Linker序列长度为8-10个氨基酸,以甘氨酸和丝氨酸为主要成分。
本发明还提供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体系转入宿主中表达;
(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)利用纯化标签,采用离子亲和层析方法纯化得到MS2噬菌体样颗粒。
有益效果:
通过本发明提供的质粒载体体系可以成功制备MS2噬菌体类似颗粒,并可利用纯化标签快速纯化出来,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重组成熟蛋白示意图;
图2为pcdfbad-3A8His生产的MS2-VLP透射电镜图;
图3为pcdfbad-3A8His生产的MS2-VLP的SDS-PAGE图。
图4为pcdfbad-3A-P1和pET-MS-P2生产的MS2-VLP透射电镜图;
图5为pcdfbad-3A-P1和pET-MS-P2生产的MS2-VLP透射电镜图;
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:pET系列质粒(购自Invitregen公司)。载体设计由云舟生物公司完成,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。
实例1:单质粒体系生产和纯化MS2-VLP。
(一)单质粒体系将MS2噬菌体2个CP基因的融合、含有包装序列、重组成熟蛋白基因。设计并合成载体pcdfba d-3A8His,序列为SEQ ID NO.6所示,该质粒表达的重组成熟蛋白的氨基端有8×His。SEQ ID NO.6序列长度为6005,第53-931位对应于编码蛋白(MAEAQNDPLLPGYSFNAHLVVGLTP IEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYWHEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRRMEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGISVLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCEEKVNDVAVKLS)序列;第958-1242位为启动子“araBAD promoter”序列,第1276-1665位为2个CP基因融合的序列;第1324-1665位,第1708-2052位分别为编码SEQ ID NO.2编码蛋白的序列;第2080-2098为SEQID NO.3所示的包装序列;第2273-2296位编码蛋白“HHHHHHHH”;第2297-2371位编码蛋白“SSRSGGGGGSGGGGSGGGGSGGGGS”;第2372-3550位为成熟蛋白基因,编码SEQ ID NO.1所示的蛋白;第3763-3849位为转录终止子rrnB T1 terminator(transcri ption terminator T1from the E.coli rrnB gene);第4314-5105位提供streptomycin抗性,编码蛋白“MREA VIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK”;第5245-5983位为“CloDF13 ori”复制子。
(二)MS2噬菌体类似颗粒的制备
将pcdfbad-3A8His载体转入大肠杆菌宿主Dh5a表达。取40ng质粒转入Dh5a感受态细胞。在链霉素抗生素的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100uL阿拉伯糖(20%)诱导后继续培养6-16h。
(三)MS2噬菌体颗粒的纯化方法:
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250nm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。
3)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的鉴定
取适当体积的上述洗脱成分进行SDS-PAGE电泳和蛋白杂交离鉴定纯化的MS-VLP。SDS-PAGE电泳的分离胶浓度为12%。蛋白杂交的抗体是anti-his HRP标记的抗体。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,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:纯化后MS2-VLP。从第3泳道可以看出有明显的CP-CP(2个CP基因融合表达)条带(箭头所示),同时anti-His标签的蛋白杂交实验显示在40Kd条带作用有条带,与预期的融合His标签的成熟蛋白一致。因此,实验结果说明,通过我们的质粒载体可以成功制备MS2噬菌体类似颗粒,并可利用纯化标签快速纯化出来。
实例2:多质粒体系生产和纯化MS2-VLP。
(一)2个质粒载体系统含有MS2噬菌体CP基因、包装序列、重组成熟蛋白基因。设计并合成载体pcdfbad-3A-P1(序列为SEQ ID NO.7所示)和pET-MS-P2(序列为SEQ ID NO.8所示),该质粒体系表达的重组成熟蛋白的氨基端有8×His。
SEQ ID NO.7所示序列长度为5602,第53-931位编码蛋白“MAEAQNDPLLPGYSFNAHLVVGLTPIEANGYLDFFIDRPLGMKGYILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYWHEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRRMEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGISVLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCEEKVNDVAVKLS”;第958-1242位为启动子“araBADpromoter”;第1324-1665为CP基因,编码SEQ ID NO.2所示的蛋白;第1870-1893位编码“HHHHHHHH”;第1894-1968位编码“SSRSGGGGGSGGGGSGGGGSGGGGS”;第1969-3147位为成熟蛋白基因,编码SEQ ID NO.1所示的蛋白;第3360-3446位为终止子rrnB T1 ter minator;第3911-4702位提供streptomycin抗性,编码蛋白“MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK”;第4842-5580位为CloDF13 ori复制子。
SEQ ID NO.8所示序列长度为5661,第26-73位为终止子T7 terminator;第400-418位、第520-538为SEQ ID NO.3所示包装序列;第660-678为启动子T7 promoter;第987-1064为启动子lacI promoter;第1065-2147位为lac repressor编码蛋白“MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ”;第4287-5102为aph(3')-Ia卡那抗性基因,编码蛋白“MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF”。
(二)MS2噬菌体类似颗粒的制备
将质粒pcdfbad-3A-P1和pET-MS-P2同时转入大肠杆菌BL21(DE3)中。在链霉素和卡那抗生素的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100uL阿拉伯糖(20%)和1mM IPTG诱导后继续培养6-16h。
(三)MS2噬菌体颗粒的纯化方法:
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250nm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。
3)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4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的鉴定
取适当体积的上述洗脱成分进行SDS-PAGE电泳和蛋白杂交离鉴定纯化的MS-VLP。SDS-PAGE电泳的分离胶浓度为12%。蛋白杂交的抗体是anti-his HRP标记的抗体。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,结果如下图5所示,图5中为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:纯化后MS2-VLP。从第3泳道可以看出有明显的CP条带(箭头所示),同时anti-His标签的蛋白杂交实验显示在40Kd条带作用有条带,与预期的融合His标签的成熟蛋白一致。实验结果说明,通过我们的质粒载体可以成功制备MS2噬菌体类似颗粒,并可利用纯化标签快速纯化出来。
Claims (10)
1.一种质粒载体体系,其特征在于:该质粒载体体系含有至少一个质粒载体,该质粒体系含有MS2噬菌体CP基因、包装序列、重组成熟蛋白基因;所述重组成熟蛋白基因包含成熟蛋白基因和纯化标签,所述成熟蛋白基因编码的蛋白如SEQ ID NO.1所示,所述成熟蛋白的氨基端包含纯化标签。
2.如权利要求1所述的质粒载体体系,其特征在于:所述质粒载体体系含单质粒,序列如SEQ ID NO.6所示。
3.如权利要求1所述的质粒载体体系,其特征在于:所述质粒载体体系含两个质粒,序列如SEQ ID NO.7、SEQ ID NO.8所示。
4.如权利要求1所述的质粒载体体系,其特征在于:所述MS2噬菌体CP基因编码的蛋白序列至少包含如SEQ ID NO.2所示的序列,其5’端有启动子。
5.如权利要求1所述的质粒载体体系,其特征在于:所述包装序列如SEQ ID NO.3,SEQID NO.4所示。
6.如权利要求1所述的质粒载体体系,其特征在于:所述重组成熟蛋白的氨基端纯化标签为8×His标签,如SEQ ID NO.5所示。
7.如权利要求1所述的质粒载体体系,其特征在于:所述重组成熟蛋白的纯化标签与所述成熟蛋白之间有Linker序列。
8.如权利要求7所述的的质粒载体体系,其特征在于:所述Linker序列的长度超过5个氨基酸。
9.如权利要求8所述的的质粒载体体系,其特征在于:所述Linker序列长度为8-10个氨基酸,以甘氨酸和丝氨酸为主要成分。
10.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1-9所述的质粒载体体系转入宿主中表达;
(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)利用纯化标签,采用离子亲和层析方法纯化得到MS2噬菌体样颗粒。
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