CN114685676B - 一种重组蛋白及其表达方法、纯化方法及用途 - Google Patents
一种重组蛋白及其表达方法、纯化方法及用途 Download PDFInfo
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Abstract
本发明涉及一种重组蛋白及其表达方法、纯化方法及用途。重组蛋白为RSV的F蛋白,即RSV‑F,所述RSV‑F的核苷酸序列为序列表SEQ ID No.1,所述RSV‑F蛋白的氨基酸序列为序列表SEQ ID No.2。所述重组蛋白的表达方法包括:1)RSV‑F编码片段的扩增;2)根据扩增后的RSV‑F编码片段构建包含F蛋白的亚克隆质粒;3)根据所述亚克隆质粒构建RSV‑F的真核表达质粒;4)转染所述RSV‑F的真核表达质粒于HEK293E细胞中,在培养基中培养。所述重组蛋白的纯化经阴离子交换柱纯化、阳离子交换柱纯化以及凝胶过滤色谱柱纯化。纯化后的RSV‑F重组蛋白,具有免疫原性,可用于RSV亚单位疫苗的研发或者RSV感染的免疫诊断。
Description
技术领域
本发明属于涉及生物技术领域,特别涉及一种重组蛋白及其表达方法、纯化方法及用途。
背景技术
呼吸道合胞病毒(respiratory syncytial virus,RSV)是引起婴幼儿急性下呼吸道疾病的最主要的病原。全世界每年有3400多万5岁以下的儿童遭受RSV的感染,其中约340万住院,16-20万人死亡,且99%RSV感染的致死病例发生在中低收入的发展中国家。24个月以内的婴儿几乎都感染过一次RSV,3岁以下所有儿童被RSV感染,并在一生中多次重复感染。在全球范围内,RSV导致1岁以下婴幼儿死亡率达6.7%,是疟疾外的第二致死病因。此外老年人和免疫功能低下的患者也易遭受RSV的感染,RSV引起的疾病负担与非流行季的流感引起的疾病负担相当。利巴韦林是用于治疗RSV疾病的抗病毒的药物,但是它的疗效不确定性、价格高和难以施用,仅限于症状严重的病人。帕丽珠单抗是被动预防RSV疾病的单克隆抗体,它可有效降低RSV引起的住院率,但其价格昂贵,需每月注射,目前仅施用于发达国家中RSV疾病高风险的婴幼儿。严重RSV疾病负担迫切需要研发RSV的疫苗,为此,RSV疫苗被世界卫生组织列为全球最优先研发的疫苗之一。
F蛋白为呼吸道合胞病毒表面的糖蛋白,属于I型糖蛋白。在细胞内合成无活性的前体F0,在成熟的过程中,F0由弗林酶切割形成F2(aa1-109),P27(aa110-136)和F1(aa137-574),F2与F1以二硫键形成异源二聚体即F蛋白的单体,三个单体组装形成三聚体。呼吸道合胞病毒包膜上的功能F蛋白以不稳定的融合前构象存在,在尚未探明的因素启动下,折叠形成稳定的融合后构象。F蛋白介导病毒和宿主细胞膜的融合,为病毒侵入细胞所必须。RSV两个亚型F蛋白序列高度保守,同源性高达90%以上;F蛋白的单克隆抗体已被证明能降低高风险婴幼儿重度RSV疾病的发病率,已被批准用于婴幼儿RSV疾病的预防,所以F蛋白是研发疫苗主要靶抗原。
由于F蛋白为包膜蛋白,通过体外培养病毒并裂解病毒,可以获得的F蛋白,但其产率较低;且在F蛋白的纯化过程中易丧失其活性。为了研发F蛋白疫苗必须建立F蛋白制备和纯化的新方法。
发明内容
针对上述问题,本发明提供了一种重组蛋白及其表达方法、纯化方法及用途。
一种重组蛋白,
所述重组蛋白为RSV的F蛋白,即RSV-F;
所述RSV-F的核苷酸序列为序列表SEQ ID No.1。
进一步地,
所述RSV-F蛋白的氨基酸序列为序列表SEQ ID No.2。
进一步地,
一种重组蛋白的表达方法,所述方法包括:
(1)RSV-F编码片段的扩增;
(2)根据扩增后的RSV-F编码片段构建包含F蛋白的亚克隆质粒;
(3)根据所述亚克隆质粒构建RSV-F的真核表达质粒;
(4)转染所述RSV-F的真核表达质粒于HEK293E细胞中,在培养基中培养。
进一步地,
所述RSV-F编码片段的扩增是以优化的RSV-F核苷酸序列为模板,PCR扩增RSV-F片段。
进一步地,
所述RSV-F核苷酸序列为序列表SEQ ID No.1第nt94-nt1563。
进一步地,
所述构建包含F蛋白的亚克隆质粒是将F蛋白与pGEM-Teasy载体连接,进而构建包含F蛋白的亚克隆质粒pTe-F。
进一步地,
所述RSV-F的真核表达质粒将所述pTe-F与表达载体pSEC分别酶切纯化后进行连接而构建。
进一步地,
所述RSV-F的真核表达质粒通过Lipofectamine2000或PEI法转染于HEK293E细胞中;
所述培养基为无血清培养基。
一种重组蛋白的纯化方法,所述方法包括:
(1)RSV-F编码片段的扩增;
(2)根据扩增后的RSV-F编码片段构建包含F蛋白的亚克隆质粒;
(3)根据所述亚克隆质粒构建RSV-F的真核表达质粒;
(4)转染所述RSV-F的真核表达质粒于HEK293E细胞中,在培养基中培养;
(5)收集培养液的上清RSV-F;
(6)将所述收集的上清RSV-F经Capto Q阴离子交换柱纯化;
(7)将步骤(6)纯化后的RSV-F进行Sepharose sp hp阳离子交换柱纯化;
(8)将步骤(7)纯化后的RSV-F进行Surperdex 200凝胶过滤色谱柱纯化。
进一步地,
所述阴离子交换柱纯化使用A液平衡层析柱和层析系统,将A液稀释的含目标蛋白的浓缩上清进行上样,收集流穿液;
所述A液为NaH2PO4,Na2HPO4,pH 7.0。
进一步地,
所述阴离子交换柱纯化使用B液平衡层析柱和层析系统,使用C液进行线性梯度洗脱;
所述B液为柠檬酸-柠檬酸钠缓冲液,pH 6.0;C液为NaCl。
进一步地,
所述凝胶过滤色谱柱纯化使用D液平衡层析柱和层析系统,再用D液洗脱,去除痕量的杂蛋白;
所述D液为Tris缓冲液,NaCl,Tween 20,pH 7.4。
进一步地,
所述纯化后的RSV-F重组蛋白具有免疫原性,可用于RSV亚单位疫苗的研发或者RSV感染的免疫诊断。
本发明具有如下优点:
(1)表达RSV-F重组蛋白仅包含F蛋白的胞外区,不包含F蛋白跨膜区和胞外区;
(2)采用分泌信号肽代替了蛋白原信号肽,使表达的蛋白为可溶性蛋白并分泌至培养基中,便于后期的蛋白纯化;
(3)采用无血清悬浮培养系统,方便培养规模的放大和蛋白纯化;
(4)表达的重组蛋白经过三步纯化后,获得了高纯度的RSV-F重组蛋白,具有免疫原性,可用于RSV亚单位疫苗的研发或者RSV感染的免疫诊断;
(5)RSV-F重组蛋白实现了重组蛋白分泌、可溶、高水平表达。
本发明的其它特征和优点将在随后的说明书中阐述,并且,部分地从说明书中变得显而易见,或者通过实施本发明而了解。本发明的目的和其他优点可通过在说明书、权利要求书以及附图中所指出的结构来实现和获得。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例。
图1示出了根据本发明实施例的RSV-F重组蛋白基因扩增产物电泳结果;
图2示出了根据本发明实施例的RSV-F重组蛋白亚克隆质粒酶切鉴定结果(EcoRI);
图3示出了根据本发明实施例的RSV-F重组蛋白的真核表达质粒的酶切鉴定结果(Age I/Kpn I):1,pSEC;2,pSEC-F;
图4示出了根据本发明实施例的RSV-F重组蛋白的SDS-PAGE(左)和Westernblot(右)鉴定结果:1,pSEC-F;2,pSEC;
图5示出了根据本发明实施例的RSV-F重组蛋白阴离子交换层析图;
图6示出了根据本发明实施例的RSV-F重组蛋白阳离子交换层析图;
图7示出了根据本发明实施例的RSV-F重组蛋白凝胶过滤层析图;
图8示出了根据本发明实施例的纯化后的RSV-F重组蛋白的SDS-PAGE(左)和Westernblot(右)鉴定结果;
图9示出了根据本发明实施例的RSV-F重组蛋白的免疫小鼠后诱导的抗血清滴度。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地说明,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
一、材料、试剂、仪器
主要材料:
细胞:人胚肾细胞HEK293E
主要试剂、仪器:
EasyApolymerase购自Agilent公司;
DNA marker、BenchMark Prestained Protein Ladder和BioluninescenceProtein Ladder购自Takara公司;
EcoRI、Age I、Kpn I购自NEB公司;
QIAquick Gel Extraction kit、QIAprep Spin Miniprep Kit和Endo-freePlasmid Maxi Kit购自QIAGEN;
Amp、X-gal、IPTG购自北京鼎国公司;
Polypepton购自日本制药株式会社;
Yeast extract购自OXOID公司;
大肠杆菌E.coli Top10购自Thermofisher公司,由申请人保存;Na2HPO4.12H2O、NaH2PO4.2H2O、NaCl、NaOH、Tween-20等购自国药集团化学试剂有限公司;
Trizma@base、Lumi-LightWestern BlottingSubstrate购自Sigma公司;
Mouse monoclonal[2F7]to RSV(ab43812)、Goat Anti-Mouse IgG H&L(HRP)(ab205719)购自Abcam公司;
FBS购自Excell公司;
FreeStyleTM F17 Expression Medium购自Gibco公司;
Pen Strep、0.05%Trypsin-EDTA、Opti-MEM、DMEM购自Gibco公司Lipofectamine2000购自Invitrogen;
PEI购自Sigma公司;
Capto Q、Sepharose sp hp、Superdex 200购自GE公司;
超滤浓缩柱购自Millipore Ireland Ltd。
本发明实施例涉及到的材料、试剂和实验设备,如无特别说明,均为符合生物工程技术领域的市售产品。
实施例1、RSV-F重组蛋白的表达方法
1、RSV-F编码片段的扩增
RSV-F编码片段的扩增是以优化的RSV-F核苷酸序列为模板,PCR扩增RSV-F片段;
具体地,
所述RSV-F核苷酸序列为序列表SEQ ID No.1第nt94-nt1563;
具体地,
以野生型RSV的Long株的天然融合蛋白基因编码序列的优化序列为模板,根据优化序列设计如下引物:
F-f GAAACCGGTCAGAATATCACCGAGGAGTTC(Age I)
F-r CTTGGTACCTCATTACAGCAGCTCATCGGACTTCC(KpnI)
扩增RSV-F蛋白的不含信号肽的胞外区(AA26-AA512)的基因序列,即基因开放阅读框nt94-nt1557;
具体地,
PCR的反应条件:94℃预变性2min,94℃变性30s,56℃复性30s,72℃延伸1.5min,30个循环,最后72℃延伸10min;
PCR的反应体系为:
试剂 | 用量(uL) |
模板(pTe-F,20ng/ul) | 1 |
dNTP(10mM) | 4 |
EasyA聚合酶 | 0.5 |
10xEasyA缓冲液 | 5 |
正向引物(10uM) | 2 |
反向引物(10uM) | 2 |
双蒸水 | 35.5 |
总体积 | 50 |
扩增产物电泳结果如图1所示,图1说明PCR扩增片段长度为1488bp。
2、构建包含F片段的亚克隆质粒
所述构建包含F片段的亚克隆质粒是将F片段与pGEM-Teasy载体连接,进而构建包含F片段的亚克隆质粒pTe-F;
具体地,
所述RSV-F的真核表达质粒将所述pTe-F与表达载体pSEC分别酶切纯化后进行连接而构建;
PCR产物经胶回收纯化后,与pGEM-Teasy连接,构建含有RSV基因片段的亚克隆载体pTe-F,EcoRI酶切结果表明两条片段大小之和与插入片段大小相当,如图2所示。经测序鉴定,插入片段的基因序列与目的基因序列100%同源。
3、构建RSV-F的真核表达质粒
所述RSV-F的真核表达质粒将所述pTe-F与表达载体pSEC分别酶切纯化后进行连接而构建,酶切鉴定结果如图3所示。
具体地,
RSV-F的亚克隆质粒和真核表达质粒的空载体pSEC经Age I/Kpn I双酶切,电泳后进行胶回收纯化目的片段和载体臂,经连接转化,构建RSV-F的真核表达质粒(pSEC-F)。经测序鉴定,插入片段的基因序列与目的基因序列100%同源。使用Endo-free Plasmid MaxiKit大量制备真核表达质粒,具体方法参见Endo-free Plasmid Maxi Kit说明书(Endo-free Plasmid Maxi Kit购自QIAGEN)。
4、转染所述RSV-F的真核表达质粒于HEK293E细胞中,在培养基中培养
所述RSV-F的真核表达质粒通过Lipofectamine2000或PEI法转染于HEK293E细胞中,即采用的转染试剂为Lipofectamine2000或PEI;
具体地,
以细胞浓度为1×106cells/mL,细胞液、转染试剂Lipofectamine2000或PEI与RSV-F的真核表达质粒以1.5ml-2ml/1μg的转染条件下转染RSV-F的真核表达质粒于HEK293E细胞中;
具体地,
所述培养基为无血清培养基;
具体地,
以细胞密度达到2×106cells/ml时按0.5×106cells/ml接种细胞到含有2%谷氨酰胺的F17培养液,放置在37℃、5%CO2摇床中225rpm条件下培养细胞;
具体地,
在转染后第5天收集培养液上清;
具体地,
是以浓缩柱浓缩4倍,浓缩培养液上清,进而以4℃条件下500xg离心10min收集培养液上。
实验表明:使用Lipofectamine2000将表达质粒转染于HEK293E细胞,转染方法参见Lipofectamine2000说明书,接着,对转染细胞的密度和重组蛋白的收获时间进行优化:
将20μg pSEC-Fecto和40μl上转染试剂进行混合,转染不同密度的30ml HEK293E细胞,在转染后的不同时间进行收获重组蛋白,结果表明当细胞密度为1×106cells/ml时,第5天收获的重组蛋白,蛋白表达量较高。
收获细胞培养液上清中的目的重组蛋白:将细胞培养液收集于离心管中,4℃条件下500xg离心10min,收集细胞培养液上清;加至超滤浓缩柱浓缩4倍,将浓缩液放入4℃冰箱保存,待重组蛋白纯化使用。
进一步地,
对RSV-F重组蛋白的SDS-PAGE和Western blot分析,鉴定结果如图4所示。
具体地,
取100μl培养液的上清浓缩液,分别加入SDS-PAGE上样缓冲液后,煮沸用于SDS-PAGE和Western blot分析;
具体地,
样品分别点样在两块不同的胶上,其中一块SDS-PAGE电泳后直接考马斯亮蓝染色和脱色液脱色。另一块进行Western blot分析,检测时一抗使用1:1000倍稀释的羊抗RSV多抗;
二抗使用HRP标记羊抗鼠IgG,加入等体积的液体A和B配制的显色液,将显色液逐滴加至PVDF膜上进行显色,并置于凝胶成像仪中拍照。
采用SDS-PAGE和Western blot分析确认了RSV-F重组蛋白的成功表达,鉴定结果如图4所示。
实施例2、RSV-F重组蛋白的纯化方法
将实施例1中表达的RSV-F重组蛋白进行纯化,纯化步骤为:
1、阴离子交换纯化:使用Capto Q阴离子交换柱纯化,先使用15倍柱体积的结合缓冲液A(0.04M NaH2PO4,0.06M Na2HPO4,pH为7.0)平衡柱子;然后用缓冲液A稀释含有目的蛋白的培养液上清的浓缩液,调节样品的pH与缓冲液pH相同;用上样泵上样,设置上样体积(500ml)、流速(3ml/min)、柱前压(0.3MPa)、柱压差(0.2MPa)等参数,使样品在设定条件下流过柱子,收集流穿液做SDS-PAGE分析。层析图如图5所示,电泳图如图8所示。
2、阳离子交换纯化:采用Sepharose sp hp阳离子交换柱纯化,用15倍柱体积的缓冲液B(柠檬酸-柠檬酸钠,pH为6.0)平衡柱子,流速10ml/min;将收集的流穿液浓缩后,再用缓冲液B稀释,调节样品的pH;用上样泵上样,设置上样体积(200ml)、流速(2ml/min)、柱前压(0.3MPa)、柱压差(0.2MPa)等参数,使样品在设定条件下流过柱子;上样结束后,用缓冲液C(1M NaCl)进行线性梯度洗脱,离子浓度从1%-100%,用10倍柱体积洗脱;收集各洗脱峰并进行SDS-PAGE和Western Blot分析,获得阳性的洗脱成分。层析图如图6所示,电泳图如图8所示。
3、凝胶过滤纯化:采用Superdex 200(10/300)凝胶过滤色谱柱纯化,用缓冲液D(Tris,NaCl,Tween 20,pH为7.4)平衡柱子1.5倍柱体积,流速为0.45ml/min;将阳离子交换获得含目的样品的组分浓缩后取500ml上样;用缓冲液D(TBS pH为7.4)洗脱1.5倍柱体积,流速为0.45ml/min,分别收集各洗脱峰并进行SDS-PAGE和Western Blot分析,获得阳性的洗脱成分。层析图如图7所示,电泳图如图8所示。
实施例3、分析RSV-F重组蛋白的纯度
1、用BCA法测定蛋白含量
用PBS稀释标准蛋白BSA(2mg/ml),制备标准品系列为0,25,125,250,500,750和1000μg/ml。取4ul各系列标准品和经适当稀释重组蛋白的纯化样品加到96孔板的标准品孔中,每孔各加入200ml的BCA工作液,室温放置5min,用酶标仪测量595nm的吸光度。用标准品浓度和吸光度绘制标准曲线(y=1.2787x-0.516),其中为蛋白y为蛋白浓度,x为吸光度,计算出样品浓度。
2、用灰度扫描仪分析蛋白的纯度
纯化表达的目的蛋白,三步纯化后收获的目的蛋白做SDS-PAGE,用灰度扫描仪分析表达蛋白的纯度,三批表达蛋白的纯度分别92.2%、96.7%、98.7%。
实施例4、分析RSV-F重组蛋白的免疫原性
为了证实RSV-F重组蛋白的免疫原性,选取16-18g周龄为6的BALB/C雌鼠36只,分为6组,每组6只,分别在第0和28天通过肌肉注射接种TBS,1ugF,1ugF(铝佐剂),5ugF,5ugF(铝佐剂),FI-RSV(1.78mg),分别在接种后的第14、28、42和49天在小鼠尾部采集静脉血。根据标准程序,通过ELISA对的血清样品中抗原特异性IgG抗体效价。96孔板包埋纯化的灭活RSVLong,4℃温育过夜。血清样品在封闭缓冲液中梯度稀释,初始血清稀释浓度为1:10,此后以2倍的系列稀释成不同的浓度。还有空白对照(血清稀释液),阴性对照(1:100稀释的未免动物血清)和阳性血清(1:6000稀释的鼠抗RSV血清),将上述抗血清稀释液每孔100μl加入酶标板,37℃温育1小时。用偶联辣根过氧化物酶(HRP)的抗小鼠IgG(1:2000)二抗。3,3A,5,5A-四甲基联苯胺(TMB,BD Opt EIATM,BD Biosciences,ON)用作HRP的底物。每孔加入50μl 1M H2SO4终止反应。用微量培养板读板仪(Molecular Devices,USA)检测450nm处的吸光值,吸光值结果如图9所示。结果表明RSV-F具有高免疫原性,免疫应答呈剂量依赖效应,且铝佐剂具有增强免疫应答效应。
尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
序列表
<110> 兰州生物制品研究所有限责任公司
<120> 一种重组蛋白及其表达方法、纯化方法及用途
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1563
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggggatcc ttcccagccc tgggatgcct gcgctgctct ccctcgtgag ccttctctcc 60
gtgctgctga tgggttgcgt agctgaaacc ggtcagaata tcaccgagga gttctaccag 120
tccacatgtt ctgccgtgag caagggctac ctgagcgccc tgaggacagg atggtatacc 180
tctgtgatca caatcgagct gagcaacatc aaggagaaca agtgcaatgg caccgacgcc 240
aaggtgaagc tgatcaacca ggagctggat aagtacaaga atgccgtgac agagctgcag 300
ctgctgatgc agtccaccac agccgccaac aatcgggccc ggagagagct gccacggttc 360
atgaactata ccctgaacaa taccaagaag acaaatgtga ccctgagcaa gaagaggaag 420
aggcgcttcc tgggctttct gctgggagtg ggatccgcca tcgcctctgg catcgccgtg 480
tccaaggtgc tgcacctgga gggcgaggtg aacaagatca agagcgccct gctgtccacc 540
aacaaggccg tggtgtctct gagcaatggc gtgtctgtgc tgacaagcaa ggtgctggac 600
ctgaagaatt atatcgataa gcagctgctg cccatcgtga acaagcagtc ctgtaggatc 660
tctaatatcg agaccgtgat cgagttccag cagaagaaca ataggctgct ggagatcaca 720
cgcgagtttt ctgtgaacgc cggcgtgacc acacctgtga gcacctacat gctgacaaat 780
agcgagctgc tgtccctgat caacgacatg ccaatcacca atgatcagaa gaagctgatg 840
tctaacaatg tgcagatcgt gcgccagcag tcctattcta tcatgagcat catcaaggag 900
gaggtgctgg cctacgtggt gcagctgcca ctgtatggcg tgatcgacac cccctgctgg 960
aagctgcaca catcccctct gtgcaccaca aacaccaagg agggctctaa tatctgcctg 1020
acccggacag acagaggctg gtactgtgat aacgccggca gcgtgtcctt ctttccccag 1080
gccgagacct gcaaggtgca gagcaaccgg gtgttctgtg acaccatgaa ttctctgaca 1140
ctgccaagcg aggtgaacct gtgcaatgtg gacatcttta atcccaagta tgattgtaag 1200
atcatgacat ctaagaccga cgtgagcagc agcgtgatca ccagcctggg cgccatcgtg 1260
tcctgctacg gcaagacaaa gtgtaccgcc tccaacaaga atagaggcat catcaagaca 1320
ttctccaacg gctgcgacta cgtgagcaac aagggcgtgg ataccgtgtc cgtgggcaac 1380
acactgtact atgtgaacaa gcaggagggc aagtctctgt acgtgaaggg cgagcccatc 1440
atcaacttct atgaccccct ggtgttccct agcgacgagt ttgatgcctc tatcagccag 1500
gtgaacgaga agatcaatca gagcctggcc tttatcagga agtccgatga gctgctgtaa 1560
tga 1563
<210> 1
<211> 519
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Gly Ile Leu Pro Ser Pro Gly Met Pro Ala Leu Leu Ser Leu Val
1 5 10 15
Ser Leu Leu Ser Val Leu Leu Met Gly Cys Val Ala Glu Thr Gly Gln
20 25 30
Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys
35 40 45
Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr
50 55 60
Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala
65 70 75 80
Lys Val Lys Leu Ile Asn Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val
85 90 95
Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Thr Ala Ala Asn Asn Arg
100 105 110
Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn Thr
115 120 125
Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu
130 135 140
Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Ile Ala Val
145 150 155 160
Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser Ala
165 170 175
Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser
180 185 190
Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln
195 200 205
Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Arg Ile Ser Asn Ile Glu
210 215 220
Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr
225 230 235 240
Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr Tyr
245 250 255
Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile
260 265 270
Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val Arg
275 280 285
Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu Ala
290 295 300
Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp
305 310 315 320
Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser
325 330 335
Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala
340 345 350
Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser
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Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser Glu
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Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys
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Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser Leu
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Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn
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Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val
435 440 445
Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr
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Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile
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Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala
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Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile
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Arg Lys Ser Asp Glu Leu Leu
515
Claims (9)
1.一种重组蛋白的纯化方法,其特征在于,
所述重组蛋白为RSV-F蛋白;所述RSV-F蛋白的氨基酸序列为序列表SEQ ID No.2中的第32-519位的氨基酸序列;
所述方法包括:
(1)RSV-F编码片段的扩增;
(2)根据扩增后的RSV-F编码片段构建包含F蛋白的亚克隆质粒;
(3)根据所述亚克隆质粒构建RSV-F的真核表达质粒;
(4)转染所述RSV-F的真核表达质粒于HEK293E细胞中,在培养基中培养;
(5)收集培养液的上清RSV-F;
(6)将所述收集的上清RSV-F经Capto Q阴离子交换柱纯化;
(7)将步骤(6)纯化后的RSV-F进行Sepharose sp hp阳离子交换柱纯化;
(8)将步骤(7)纯化后的RSV-F进行Surperdex 200凝胶过滤色谱柱纯化。
2.根据权利要求1所述的重组蛋白的纯化方法,其特征在于,
所述RSV-F编码片段的扩增是以优化的RSV-F核苷酸序列为模板,PCR扩增RSV-F片段。
3.根据权利要求1所述的重组蛋白的纯化方法,其特征在于,
所述RSV-F核苷酸序列为序列表SEQ ID No.1第nt94-nt1563。
4.根据权利要求1所述的重组蛋白的纯化方法,其特征在于,
所述构建包含F蛋白的亚克隆质粒是将F蛋白与pGEM-Teasy载体连接,进而构建包含F蛋白的亚克隆质粒pTe-F。
5.根据权利要求1所述的重组蛋白的纯化方法,其特征在于,
所述RSV-F的真核表达质粒将所述pTe-F与表达载体pSEC分别酶切纯化后进行连接而构建。
6.根据权利要求1所述的重组蛋白的纯化方法,其特征在于,
所述RSV-F的真核表达质粒通过Lipofectamine2000或PEI法转染于HEK293E细胞中;
所述培养基为无血清培养基。
7.根据权利要求1所述的重组蛋白的纯化方法,其特征在于,
所述阴离子交换柱纯化使用A液平衡层析柱和层析系统,将A液稀释的含目标蛋白的浓缩上清进行上样,收集流穿液;
所述A液为NaH2PO4,Na2HPO4,pH 7.0。
8.根据权利要求1所述的重组蛋白的纯化方法,其特征在于,
所述阳离子交换柱纯化使用B液平衡层析柱和层析系统,使用C液进行线性梯度洗脱;
所述B液为柠檬酸-柠檬酸钠缓冲液,pH 6.0;C液为NaCl。
9.根据权利要求1所述的重组蛋白的纯化方法,其特征在于,
所述凝胶过滤色谱柱纯化使用D液平衡层析柱和层析系统,再用D液洗脱,去除痕量的杂蛋白;
所述D液为Tris缓冲液,NaCl,Tween 20,pH 7.4。
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