CN113717262A - 一种截短版肺炎球菌表面蛋白a的发酵工艺及纯化方法 - Google Patents
一种截短版肺炎球菌表面蛋白a的发酵工艺及纯化方法 Download PDFInfo
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Abstract
本发明公开了一种适用于截短版肺炎球菌表面蛋白A，即PspA4 (αHD+PRD)的发酵工艺、纯化和保存的方法。所述纯化方法包括精纯化的步骤，即将所述含PspA4 (αHD+PRD)蛋白的粗产物经弱阴离子交换层析、疏水层析和阳离子交换层析后，获得精纯化的PspA4 (αHD+PRD)蛋白；所述PspA4 (αHD+PRD)蛋白的氨基酸序列如SEQ ID NO:2所示。通过本发明制造的重组PspA4 (αHD+PRD)精纯蛋白产量可达204.9mg/L发酵液，纯度达98％以上，内毒素含量低于检测限度。本发明公开的PspA4 (αHD+PRD)蛋白制造工艺具有产量高、纯度高、收率高、易放大、工艺稳定性强等特点，在重组PspA4 (αHD+PRD)蛋白的生产制造领域具有极为广阔的应用前景。
Description
技术领域
本发明涉及利用基因工程技术生产重组蛋白药物，具体涉及一种截短版肺炎球菌表面蛋白A(PspA4 (αHD+PRD))的高效可溶高表达和制造工艺，属于基因工程技术和医药技术领域。
背景技术
肺炎链球菌是一种常见的革兰氏阳性菌，含荚膜，其主要的致病原也来源于丰富的荚膜多糖和多种毒力蛋白。肺炎链球菌常诱发老人和儿童感染肺炎、脑膜炎、中耳炎及败血症等疾病，是威胁老人和儿童生命健康的重要病原菌之一。
目前已分离鉴定出的肺炎球菌型别有90多种，其中有20多种血清型对应的肺炎球菌具有较强的感染力。针对这些较强感染力的肺炎球菌，科学家们研制出了23价多糖疫苗(Ppv23)和多糖蛋白结合疫苗(PCV-7，PCV-13)。相较于多糖疫苗(Ppv23)，多糖蛋白结合疫苗能激发机体免疫记忆效应，对65周岁以上的老人和5周岁以下的儿童的保护率有显著提升。但随着流行菌株血清型的改变，出现了疫苗保护之外的血清型肺炎球菌感染，需开发价数更高的疫苗产品才能得以保护。但制备生产更多价数的多糖蛋白结合疫苗(如PCV-23等)工艺极为复杂，且成本较高，这在今后的疫苗研发战略中也不切实际。因此，研制不受肺炎球菌型别限制的新型肺炎球菌疫苗具有极大的应用前景。
肺炎球菌表面蛋白A(Pneumococcal Surface Protein A，PspA)是肺炎球菌毒力相关的一种重要表面抗原，几乎分布于所有临床分离的肺炎球菌中。PspA蛋白能够抑制机体的补体沉淀，从而避免机体吞噬调理作用。动物学实验表明，PspA产生的特异性抗体能帮助小鼠避免肺炎球菌侵袭。PspA在肺炎球菌中保守性较低，其主要由3个结构域组成：(1)280-380个氨基酸的N-末端区域，该区域由大量α螺旋卷曲结构组成(α-helix domin,αHD)；(2)紧接着是约90个氨基酸的脯氨酸富集区(Prolin-rich domin,PRD)；(3)最后是一个含有约200个氨基酸的胆碱结合区，且带着约17个疏水氨基酸尾巴的C-末端区域。PspA的多样性主要体现在αHD，该区域的氨基酸序列具有高度变异性，其中靠近PRD附近的100个氨基酸被定义为分枝定义区(CDR)，该分枝定义区将PspA蛋白分为3个家族(Family)和6个分枝(Clade)，分别为：家族1，包含分枝1和分枝2；家族2，包含分枝3，分枝4和分枝5；家族3，由分枝6组成。大量研究表明，同一分枝的PspA能产生高强度的免疫保护反应，同一家族不同分枝的PspA也能产生较高强度的免疫交叉保护，而不同家族的PspA则不能总是产生一定强度的免疫交叉保护反应。基于流行病学研究，含家族2中PspA的肺炎球菌广泛流行于西班牙、美国、加拿大、瑞典及法国等国及亚洲局部，而家族1中的PspA则广泛流行于英国、日本和澳大利亚等国。综上，地区差异性导致了PspA型别的差异性。
研究表明，PspA的N-末端的αHD和PRD均具有较好的免疫原性，如专利WO2018102774A3公开了一种包含αHD和PRD的PspA，结果表明以该种方式构建的PspA最大程度上提供了对不同血清型肺炎球菌的保护效力。因此，基于PspA的αHD和PRD两个区域进行新型肺炎疫苗的开发具有重要意义。
非专利文献1(卢井才.肺炎链球菌重组蛋白疫苗的研究[D].吉林大学,2015)成功对PspA4的32-450位氨基酸在大肠杆菌进行了重组表达(含组氨酸标签)，免疫原性实验发现，PsaA-PspA融合蛋白与PspA4蛋白在免疫血清中的特异性IgG抗体效价均能达106以上，且都能刺激小鼠脾细胞分泌IL-17A细胞因子。广谱性交叉免疫实验发现，不论是Western-blot技术还是流式细胞技术均显示PspA4蛋白与家族1和家族2中的所有菌体结合，表明了PspA4的交叉免疫原性极强。
非专利文献2(Hualong X,Jinfei Y,Qing S,et al.Expression andpurification of pneumococcal surface protein a of clade 4in Escherichia coliusing hydroxylapatite and ion-exchange column chromatography[J].ProteinExpression and Purification,2018,151:56-61.)成功实现了截短版PspA4的无标签可溶性表达，占全菌体表达量约17％，经羟基磷灰石、阴离子和阳离子三步纯化后，纯度达96％以上，内毒素0.125EU/ug左右，产量为22.8mg PspA4/L发酵液。
非专利文献1和非专利文献2均对截短版PspA4进行了可溶性表达，但前者存在融合标签去除和质粒抗性问题，后者存在产量及放大困难问题。在真正的重组蛋白生产制造中，高产量、高收率、高纯度、低热原等均是生产线上追求的每一个关键点。目前，尚未见有关于适用PspA蛋白生产制造的工艺方法报道。
发明内容
本发明通过对基因序列的密码子优化、载体筛选及发酵条件的摸索，成功构建了一套截短版重组的无标签、高效可溶性表达体系；通过对粗处理和纯化工艺的优化和验证，获得了稳定、高产、易放大的PspA4 (αHD+PRD)生产制造工艺；获得的截短版重组PspA4 (αHD+PRD)精蛋白可用于载体蛋白、疫苗组分或免疫学检测应用等方面。
为解决上述技术问题，本发明提供的技术方案之一为：一种PspA4 (αHD+PRD)蛋白的纯化方法，其包括以下步骤：
(I)精纯化：将所述含PspA4 (αHD+PRD)蛋白的粗产物经弱阴离子交换层析、疏水层析和阳离子交换层析后，获得精纯化的PspA4 (αHD+PRD)蛋白；所述PspA4 (αHD+PRD)蛋白的氨基酸序列如SEQ ID NO:2所示；
所述弱阴离子交换层析使用DEAE Sepharose FF，所述疏水层析使用PhenylBestarose HP，所述阳离子交换层析使用SP Sepharose FF。
优选地，所述PspA4 (αHD+PRD)蛋白来自菌株表达PspA4 (αHD+PRD)可溶蛋白后收集的菌体；所述纯化方法还包括(II)粗处理：使用精氨酸处理所述的菌体，获得粗产物；其中，所述精氨酸的终浓度为0.5～0.75M，所述菌体的终浓度为0.2～0.5g/mL，例如0.3g/mL。
在某一具体实施例中，步骤(II)中，所述精氨酸处理后还包括低渗处理，以获得所述粗产物。优选地，步骤(II)包括以下步骤：
(a)将含所述菌体的发酵液进行超滤浓缩，收集菌体浓缩液；
(b)在所述菌体浓缩液中加入精氨酸，室温搅拌例如3h，形成高渗处理液；
(c)将所述高渗处理液按照1:10体积比例与低渗液混合，室温静置例如静置过夜或静置12小时以上，获得混合液；所述低渗液为50mmol/L Tris-HCl、pH8.0、含1mmol/LEDTA；
(d)将所述混合液进行过滤，收集澄清流穿液；
(e)将所述澄清流穿液进行浓缩，收集浓缩截留液；
(f)将所述浓缩截留液使用置换液1进行置换，置换2-5次，获得浓缩置换液；所述置换液1为20mmol/L PB、pH6.5、含1mmol/L EDTA和0.2mol/L NaCl。
在某一具体实施例中，步骤(a)中所述菌体浓缩液中菌体浓度为0.3g菌体/mL；和/或，步骤(c)中所述高渗处理液中精氨酸的终浓度为0.5mol/L；和/或，步骤(d)中所述过滤使用孔径为0.22～0.45μm的膜包；和/或，步骤(a)中所述超滤浓缩、步骤(e)中浓缩使用型号10Kda～30Kda的膜包；和/或，步骤(f)中所述置换进行4次。
在某一具体实施例中，步骤(1)包括：
(1)将(f)中获得的所述浓缩置换液上样于经平衡后的阴离子交换层析柱，冲洗流穿后进行洗脱，收集洗脱液1，所述平衡、冲洗流穿和洗脱使用PB；
(2)将所述洗脱液1调节电导后上样于经平衡后的疏水层析柱，冲洗流穿后进行洗脱，收集洗脱液2，所述平衡、冲洗流穿和洗脱使用PB。
优选地，步骤(1)中所述平衡和冲洗流穿均使用20mmol/L PB、pH6.5、含1mmol/LEDTA和0.2mol/L NaCl的制剂1，所述洗脱使用20mmol/L PB、pH6.5、含1mmol/L EDTA和0.28mol/L NaCl洗脱缓冲液1；
步骤(2)中所述调节电导后的电导为180ms/cm，所述平衡和所述冲洗流穿均使用20mmol/L PB、pH7.5、含1mmol/L EDTA和1.4mol/L(NH4)2SO4的制剂2，所述洗脱使用20mmol/L PB、pH7.5、含1mmol/L EDTA和0.5mol/L(NH4)2SO4的洗脱缓冲液2。
在某一具体实施例中，步骤(1)中所述平衡和所述冲洗流穿使用3倍柱体积的制剂1；和/或，所述洗脱使用3倍柱体积的洗脱缓冲液1；
步骤(2)中所述平衡和所述冲洗流穿使用3倍柱体积的制剂2，和/或，所述洗脱使用3倍柱体积的洗脱缓冲液2。
在某一具体实施例中，还包括以下步骤：
(3)用置换液2置换所述的洗脱液2，收集置换液3，平衡后将所述的置换液3上样阳离子层析柱，冲洗流穿后进行洗脱，收集洗脱液3，所述平衡、冲洗流穿和洗脱均使用NaAC缓冲液；所述置换液2为50mmol/L NaAC、pH4.8、含1mmol/L EDTA。
优选地，步骤(3)中所述置换、平衡和冲洗流穿均使用50mmol/L NaAC、pH4.8、含1mmol/L EDTA的制剂3，所述洗脱使用50mmol/L NaAC、pH4.8、含1mmol/L EDTA和0.097mol/L NaCl的洗脱缓冲液3。
更优选地，步骤(3)中所述置换、平衡和冲洗流穿使用3倍柱体积的制剂3；和/或，所述洗脱使用3倍柱体积的洗脱缓冲液3。
为解决上述技术问题，本发明提供的技术方案之二为：一种PspA4 (αHD+PRD)蛋白的发酵工艺，其包括以下步骤：
(i)培养表达氨基酸序列如SEQ ID NO:2所示的PspA4 (αHD+PRD)蛋白的宿主细胞，所述宿主细胞为大肠杆菌；
(ii)接种前加入硫酸卡那霉素和无机盐溶液，所述无机盐为K2HPO4和/或MgSO4。优选地，所述硫酸卡那霉素的终浓度为30μg/mL；
(iii)发酵工艺参数为：发酵温度32～37℃，摇床转速100～600rpm。优选地，所述发酵温度为35℃；
(iv)当A600nm值达到9～11时，添加诱导剂；所述诱导剂为IPTG，诱导温度例如为28～36℃，诱导时长为4h～12h。优选地，所述诱导温度为32℃，所述IPTG的终浓度为0.05～0.5mmol/L。
优选地，所述宿主细胞为BL21(DE3)，所述宿主细胞包括表达载体，所述表达载体为pET27b或pET30a。
更优选地，所述重组表达载体包含如SEQ ID NO:1所示分离的核酸，所述表达载体为pET27b。
进一步更优选地，所述大肠杆菌为BL21(DE3)。
为解决上述技术问题，本发明提供的技术方案之三为：一种PspA4 (αHD+PRD)蛋白的保存方法，所述PspA4 (αHD+PRD)蛋白的氨基酸序列如SEQ ID NO:2所示，且用pH8.0的硼酸盐缓冲液对如技术方案之一所述的纯化方法制得的洗脱液3进行置换后保存。
为解决上述技术问题，本发明提供的技术方案之四为：一种重组表达载体，所述重组表达载体包含如SEQ ID NO:1所示分离的核酸。优选地，所述表达载体为pET27b或pET30a。
为解决上述技术问题，本发明提供的技术方案之五为：一种表达菌株，所述表达菌株包含如技术方案之四所述的重组表达载体。优选地，所述表达菌株的宿主菌为大肠杆菌。更优选地，所述大肠杆菌为BL21(DE3)。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明通过密码子优化及发酵条件摸索使得PspA4 (αHD+PRD)蛋白大量可溶性表达，30L发酵罐规模下，可溶性PspA4 (αHD+PRD)产量可达36.8mg PD/g菌体或1.42g PD/L发酵液；三步纯化后，精蛋白产量可达204.9mg/L发酵液，SDS-PAGE电泳和毛细管电泳纯度均达98％以上，内毒素含量低于检测限度；这意味本发明在PspA4 (αHD+PRD)的放大生产过程中具有极其广阔的应用前景。
附图说明
图1是密码子优化前后重组PspA4 (αHD+PRD)蛋白核苷酸序列对比图。
图2是pET27b-PspA4 (αHD+PRD)载体构建示意图。
图3是重组PspA4 (αHD+PRD)表达株摇瓶诱导表达验证SDS-PAGE电泳图谱。
图4是重组PspA4 (αHD+PRD)蛋白纯化过程柱层析层析图谱。
图5是重组PspA4 (αHD+PRD)精蛋白精确分子量测定图谱。
图6是重组PspA4 (αHD+PRD)精蛋白N端测序结果图。
图7是重组PspA4 (αHD+PRD)精蛋白SDS-PAGE电泳纯度分析图谱。
具体实施方式
下面通过以下具体实施方式对本发明做进一步阐述，但是本发明的内容完全不局限于此。
实施例1.重组PspA4 (αHD+PRD)表达株构建
根据GenBank NO.U89711.1公布的肺炎链球菌PspA蛋白的氨基酸序列，对其αHD和PRD区对应的氨基酸的密码子(32-450位氨基酸)进行优化，将稀有密码子替换为大肠杆菌常用密码子，并在两端分别加入NcoI和XhoI酶切位点，进行全片段化学合成；
将合成好的核苷酸序列连接至T载体，并转化至DH5α感受态细胞，将测序正确的菌液用试剂盒提取质粒，再将质粒转化至BL21(DE3)感受态细胞，再次提取质粒，测序正确后保种，-80℃储存(载体构建示意图见图2)。
优化前后核苷酸序列对比如图1所示。
实施例2.重组PspA4 (αHD+PRD)表达株表达鉴定
将甘油菌用接种环三线法接种至固体LB(含Kana)培养基中，37℃倒置过夜培养；
挑取平板中长势较好的单克隆菌落至LB(含Kana)液体培养基中(15mL)，32℃，140rpm过夜培养；
将过夜培养的菌液按1:100接种比例接种至400mL新鲜液体LB(含Kana)培养基中，37℃，220rpm培养至A600nm＝0.6-0.8，约3h；加入IPTG，使得IPTG终浓度为0.1mmol/L，32℃，220rpm开始诱导，诱导前及诱导后每2小时取样一次，测定A600nm；
对诱导前样品和诱导后样品进行还原SDS-PAGE电泳分析；样品按照相应的A600nm值用0.9％NaCl复溶至5A600nm/管；
如图3所示，经凝胶成像分析软件得诱导2h、4h和6h的表达量分别为35.17％、32.55％和32.92％，表明PspA4 (αHD+PRD)的表达量在诱导2h时即达到最高。(M为Marker；泳道1为诱导0h样品；泳道2为诱导2h样品；泳道3为诱导4h样品；泳道4为诱导6h样品。)
实施例3.30L发酵罐规模下的重组PspA4 (αHD+PRD)蛋白发酵
取一支冻存的甘油菌，取0.5mL接种到装有1L培养基的三角瓶中进行培养；每瓶加入1mL 30mg/mL硫酸卡那霉素。摇床设定温度32℃，转速为140rpm，摇育过夜，A600nm达到1～3之间；
10L基础培养基(终体积20L)，待温度达到设定温度35±0.5℃，在火焰保护无菌状态下，开始接种。接种前加入20mL 30mg/mL硫酸卡那霉素(终浓度30μg/mL)，以及无机盐溶液(K2HPO4、MgSO4)，种子液接种比例为1:2.5。发酵工艺参数为：pH7.0；100～600rpm；300～400L/H；罐压0.05MPa；溶氧≥30％；
过程中加入消泡剂消除泡沫，当A600nm达到1以上，开始进行分批补料，每小时补料一次，分阶段进行，在对数期完成补料；每小时测A600nm，染色镜检；
A600nm值达到10左右时，开始降温至32℃，然后开始降温诱导；
加入已过滤除菌的诱导剂IPTG，至终浓度为0.1mmol/L，诱导4小时发酵结束；
诱导结束，停止发酵，放料收获发酵液。
实施例4.精氨酸提取法粗处理提取可溶性重组PspA4 (αHD+PRD)蛋白
重组PspA4 (αHD+PRD)蛋白为可溶性的细胞蛋白，利用不同浓度精氨酸造成的渗透压进行处理后释放并提取蛋白。
收获实施例3所得的发酵液20L，使用0.45μm膜包将发酵液浓缩至一定体积后收集，再加入一定体积纯水洗膜后收集，重复2次，收集菌体浓缩液，菌体浓缩液的菌体浓度约0.3g/mL；
在上述菌体浓缩液中边搅拌边加入精氨酸粉末，使其终浓度为0.5mol/L，在室温条件下，搅拌作用3h；
使用50mmol/L Tris-HCl(pH8.0)和1mmol/L EDTA缓冲液10倍体积稀释高渗处理液，在室温条件下，搅拌作用不少于12h；
使用0.45μm膜包对低渗处理液进行膜过滤处理，收集澄清流穿液；
使用10kDa膜包对澄清流穿液进行浓缩处理，当浓缩至一定体积后，使用20mmol/LPB(pH6.5)含1mmol/L EDTA和0.2mol/L NaCl缓冲液对浓缩截留液进行4次缓冲液置换处理，当最后一次缓冲液置换结束后，对样品进行收集，收集后加入一定体积的缓冲液对膜包进行润洗，重复4次，收集浓缩置换液。
实施例5.三步法纯化工艺纯化重组PspA4 (αHD+PRD)蛋白
第一步纯化：使用20mmol/L PB(pH6.5)含1mmol/L EDTA和0.2mol/L NaCl对DEAESepharose FF(BXK50/30)层析柱平衡3CV，粗处理获得的粗样上样DEAE Sepharose FF层析柱，使用20mmol/L PB(pH6.5)含1mmol/L EDTA和0.2mol/L NaCl冲洗流穿3CV，使用20mmol/L PB(pH6.5)含1mmol/L EDTA和0.28mol/L NaCl洗脱3CV，收集洗脱液1；该步骤柱层析上样量为6.6mg PspA4 (αHD+PRD)/mL gel；
第二步纯化：使用20mmol/L PB(pH7.5)含1mmol/L EDTA和1.4mol/L(NH4)2SO4平衡Phenyl Bestarose HP(BXK26/40)3CV，将洗脱液1用2mol/L(NH4)2SO4调节电导至180ms/cm后上样，并用20mmol/L PB(pH7.5)含1mmol/L EDTA和1.4mol/L(NH4)2SO4冲洗流穿3CV，使用20mmol/L PB(pH7.5)含1mmol/L EDTA和0.5mol/L(NH4)2SO4洗脱3CV，收集洗脱液2；该步骤柱层析上样量为14.9mg PspA4 (αHD+PRD)/mL gel；
第三步纯化：使用50mmol/L NaAC(pH4.8)含1mmol/L EDTA对洗脱液2置换3次，同时将SP Sepharose FF(BXK26/40)层析柱进行平衡，将置换完成的洗脱液2上样SP层析柱，用50mmol/L NaAC(pH4.8)含1mmol/L EDTA冲洗3CV，再用50mmol/L NaAC(pH4.8)含1mmol/LEDTA和0.097mol/L NaCl缓冲液洗脱3CV，收集洗脱液3(层析图谱见图4，A为DEAE柱层析，B为HIC柱层析，C为SP柱层析)；该步骤柱层析上样量为12mg PspA4 (αHD+PRD)/mL gel。
将洗脱液3用硼酸盐缓冲液(pH8.0)进行浓缩置换，便于保存。
实施例6.重组PspA4 (αHD+PRD)精蛋白部分质量检定
6.1重组PspA4 (αHD+PRD)精确分子量测定
精蛋白样品采用超高效液相色谱系统U3000(Thermofisher公司)进行分离，流动相A为0.1％FA水溶液，流动相B为0.1％FA的乙腈溶液。色谱柱为C4(Thermo MAbPacTMRP，2.1mmX50mm，4um)。流速为：0.3mL/min，柱温70℃。液相梯度如下：
用高分辨质谱仪Q Exactive Plus(Thermofisher公司)对其进行精确分子量测定，分析时长：15min；检测方式：正离子；扫描范围：900-3000m/z；分辨率：17500。原始数据由Biopharmafinder 1.0软件处理；实验类型:Manual RespectTM；质量容差:20ppm；干扰抑制:95％置信区间。
如图5所示，重组PspA4 (αHD+PRD)精蛋白精确分子量为47.005KDa，与理论分子量46.875KDa基本一致，由于重组蛋白均含首位Met(M)，故测出的PspA4 (αHD+PRD)精确分子量47.005KDa与理论分子量完全一致，该结果表明载体中自带的PelB信号肽在释放的过程中被信号肽酶完全切除。
6.2重组PspA4 (αHD+PRD)N端测序
将重组PspA4 (αHD+PRD)精蛋白送检第三方检测公司进行N末端的10个氨基酸测序，确认目标蛋白N末端的PelB是否完全切除干净。如图6所示，N端前10位氨基酸分别为MEEAPVANQS，除M外，与SEQ ID NO:2中的前9个氨基酸完全一致，再次表明PelB信号肽被完全切除。
6.3 SDS-PAGE法纯度测定
将10ug PspA4 (αHD+PRD)精蛋白分别进行还原和非还原SDS-PAGE电泳(分离胶浓度为12％)，80v电泳30分钟，120v电泳约1h，直至溴酚蓝条带到达分离胶底部时停止电泳；将凝胶剥离，加入考马斯亮蓝染色液染色2h，弃染色液，再加入脱色液脱色，期间每2h更换脱色液；待脱色至背景为透明时，停止脱色；使用凝胶成像仪进行SDS-PAGE图谱分析；
如图7所示，还原电泳条件下(泳道2、泳道3、泳道4)，PspA4 (αHD+PRD)精蛋白纯度为99.18％；非还原电泳条件下(泳道6、泳道7、泳道8)，PspA4 (αHD+PRD)精蛋白纯度为98.93％。
6.4动态浊度法内毒素含量测定
配制4组细菌内毒素工作标准品溶液，标准品溶液内毒素含量分别为10EU/mL、2.5EU/mL、0.625EU/mL和0.156EU/mL，每一浓度制备3份平行，同时设立阴性对照。将样品用细菌内毒素检测用水稀释至细菌内毒素浓度在0.156-10EU/mL之间，制备2份平行供试品溶液，供试品稀释倍数应不超过最大有效稀释倍数。设立阳性加标溶液，利用鲎试剂对样品进行检测，绘制标准曲线。标准曲线相关系数绝对值应≥0.980，加标回收率应在50％-200％之间，阴性对照内毒素含量应小于检测限，样品检测数据才有效；
经内毒素含量检测发现，洗脱液1中内毒素含量为628.43EU/ug PspA4 (αHD+PRD)，洗脱液2中内毒素含量为1.33EU/ug PspA4 (αHD+PRD)，洗脱液3中内毒素含量小于检测限，表明三步纯化能有效去除内毒素。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围。
SEQUENCE LISTING
<110> 江苏坤力生物制药有限责任公司
<120> 一种截短版肺炎球菌表面蛋白A的发酵工艺及纯化方法
<130> P21014460C
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1260
<212> DNA
<213> Artificial Sequence
<220>
<223> PspA4(αHD+PRD)蛋白的核苷酸序列
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gaagaagcac cggttgccaa tcagagtaaa gcagagaaag attatgatgc agccgttaag 60
aaatcagaag ccgcaaagaa agattatgaa accgcaaaga agaaagccga agatgcacag 120
aagaaatatg atgaagatca gaagaagact gaagcaaagg cagagaaaga acgcaaagca 180
agtgagaaga ttgccgaagc aaccaaagaa gtgcagcagg catatctggc ctatctgcag 240
gccagtaatg aaagtcagcg caaagaagca gataagaaga ttaaagaagc cacccagcgt 300
aaagatgaag ccgaagccgc atttgcaacc attcgcacca ccattgtggt tccggaaccg 360
agcgaactgg cagaaaccaa gaagaaagca gaagaagcca ccaaagaagc cgaagttgcc 420
aagaagaaat cagaggaagc agccaaagaa gtggaagtgg agaagaataa gattctggaa 480
caggatgcag agaatgagaa gaagattgat gtgctgcaga ataaagttgc agatctggag 540
aaaggtattg caccgtatca gaatgaagtt gccgaactga ataaagaaat tgcacgcctg 600
cagagtgatc tgaaagatgc agaagagaat aatgtggaag attatattaa ggagggtctg 660
gaacaggcca ttaccaataa gaaagccgag ctggcaacca cccagcagaa tattgataag 720
actcagaaag atctggaaga tgccgaactg gaactggaga aagtgctggc caccctggat 780
ccggaaggta agactcagga tgaactggat aaagaagcag cagaagccga actgaacgag 840
aaagtggaag cactgcagaa tcaggttgca gaactggaag aagaactgag caaactggaa 900
gataatctga aagatgccga aaccaataat gtggaggatt atattaaaga gggtctggag 960
gaagccattg ccaccaagaa agccgaactg gagaagaccc agaaagaact ggatgccgca 1020
ctgaatgaac tgggcccgga tggtgatgaa gaagaaacac ctgcaccggc accgcagccg 1080
gagaaaccgg ctgaagaacc ggagaatccg gcacctgcac ctaaaccgga gaaatcagca 1140
gatcagcagg cagaagaaga ttatgcccgc cgcagtgaag aagaatataa tcgtctgacc 1200
cagcagcagc cgccgaaagc cgagaaaccg gcaccggcac ctcagccgga acagccttaa 1260
<210> 2
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<223> PspA4(αHD+PRD)蛋白的氨基酸序列
<400> 2
Glu Glu Ala Pro Val Ala Asn Gln Ser Lys Ala Glu Lys Asp Tyr Asp
1 5 10 15
Ala Ala Val Lys Lys Ser Glu Ala Ala Lys Lys Asp Tyr Glu Thr Ala
20 25 30
Lys Lys Lys Ala Glu Asp Ala Gln Lys Lys Tyr Asp Glu Asp Gln Lys
35 40 45
Lys Thr Glu Ala Lys Ala Glu Lys Glu Arg Lys Ala Ser Glu Lys Ile
50 55 60
Ala Glu Ala Thr Lys Glu Val Gln Gln Ala Tyr Leu Ala Tyr Leu Gln
65 70 75 80
Ala Ser Asn Glu Ser Gln Arg Lys Glu Ala Asp Lys Lys Ile Lys Glu
85 90 95
Ala Thr Gln Arg Lys Asp Glu Ala Glu Ala Ala Phe Ala Thr Ile Arg
100 105 110
Thr Thr Ile Val Val Pro Glu Pro Ser Glu Leu Ala Glu Thr Lys Lys
115 120 125
Lys Ala Glu Glu Ala Thr Lys Glu Ala Glu Val Ala Lys Lys Lys Ser
130 135 140
Glu Glu Ala Ala Lys Glu Val Glu Val Glu Lys Asn Lys Ile Leu Glu
145 150 155 160
Gln Asp Ala Glu Asn Glu Lys Lys Ile Asp Val Leu Gln Asn Lys Val
165 170 175
Ala Asp Leu Glu Lys Gly Ile Ala Pro Tyr Gln Asn Glu Val Ala Glu
180 185 190
Leu Asn Lys Glu Ile Ala Arg Leu Gln Ser Asp Leu Lys Asp Ala Glu
195 200 205
Glu Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Gln Ala Ile
210 215 220
Thr Asn Lys Lys Ala Glu Leu Ala Thr Thr Gln Gln Asn Ile Asp Lys
225 230 235 240
Thr Gln Lys Asp Leu Glu Asp Ala Glu Leu Glu Leu Glu Lys Val Leu
245 250 255
Ala Thr Leu Asp Pro Glu Gly Lys Thr Gln Asp Glu Leu Asp Lys Glu
260 265 270
Ala Ala Glu Ala Glu Leu Asn Glu Lys Val Glu Ala Leu Gln Asn Gln
275 280 285
Val Ala Glu Leu Glu Glu Glu Leu Ser Lys Leu Glu Asp Asn Leu Lys
290 295 300
Asp Ala Glu Thr Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu
305 310 315 320
Glu Ala Ile Ala Thr Lys Lys Ala Glu Leu Glu Lys Thr Gln Lys Glu
325 330 335
Leu Asp Ala Ala Leu Asn Glu Leu Gly Pro Asp Gly Asp Glu Glu Glu
340 345 350
Thr Pro Ala Pro Ala Pro Gln Pro Glu Lys Pro Ala Glu Glu Pro Glu
355 360 365
Asn Pro Ala Pro Ala Pro Lys Pro Glu Lys Ser Ala Asp Gln Gln Ala
370 375 380
Glu Glu Asp Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn Arg Leu Thr
385 390 395 400
Gln Gln Gln Pro Pro Lys Ala Glu Lys Pro Ala Pro Ala Pro Gln Pro
405 410 415
Glu Gln Pro
Claims (10)
1.一种PspA4 (αHD+PRD)蛋白的纯化方法，其特征在于，其包括以下步骤：
(I)精纯化：将所述含PspA4 (αHD+PRD)蛋白的粗产物经弱阴离子交换层析、疏水层析和阳离子交换层析后，获得精纯化的PspA4 (αHD+PRD)蛋白；所述PspA4 (αHD+PRD)蛋白的氨基酸序列如SEQID NO:2所示；
所述弱阴离子交换层析使用DEAE Sepharose FF，所述疏水层析使用PhenylBestarose HP，所述阳离子交换层析使用SP Sepharose FF；
优选地，所述PspA4 (αHD+PRD)蛋白来自菌株表达PspA4 (αHD+PRD)可溶蛋白后收集的菌体；所述纯化方法还包括(II)粗处理：使用精氨酸处理所述的菌体，获得粗产物；其中，所述精氨酸的终浓度为0.5～0.75M，所述菌体的终浓度为0.2～0.5g/mL，例如0.3g/mL。
2.如权利要求1所述的纯化方法，其特征在于，步骤(II)中，所述精氨酸处理后还包括低渗处理，以获得所述粗产物；优选地，步骤(II)包括以下步骤：
(a)将含所述菌体的发酵液进行超滤浓缩，收集菌体浓缩液；
(b)在所述菌体浓缩液中加入精氨酸，室温搅拌例如3h，形成高渗处理液；
(c)将所述高渗处理液按照1:10体积比例与低渗液混合，室温静置例如静置过夜或静置12小时以上，获得混合液；所述低渗液为50mmol/L Tris-HCl、pH8.0、含1mmol/L EDTA；
(d)将所述混合液进行过滤，收集澄清流穿液；
(e)将所述澄清流穿液进行浓缩，收集浓缩截留液；
(f)将所述浓缩截留液使用置换液1进行置换，置换2-5次，获得浓缩置换液；所述置换液1为20mmol/L PB、pH6.5、含1mmol/L EDTA和0.2mol/L NaCl。
3.如权利要求2所述的纯化方法，其特征在于，步骤(a)中所述菌体浓缩液中菌体浓度为0.3g菌体/mL；和/或，步骤(c)中所述高渗处理液中精氨酸的终浓度为0.5mol/L；和/或，步骤(d)中所述过滤使用孔径为0.22～0.45μm的膜包；和/或，步骤(a)中所述超滤浓缩、步骤(e)中浓缩使用型号10Kda～30Kda的膜包；和/或，步骤(f)中所述置换进行4次。
4.如权利要求2或3所述的纯化方法，其特征在于，步骤(1)包括：
(1)将(f)中获得的所述浓缩置换液上样于经平衡后的阴离子交换层析柱，冲洗流穿后进行洗脱，收集洗脱液1，所述平衡、冲洗流穿和洗脱使用PB；
(2)将所述洗脱液1调节电导后上样于经平衡后的疏水层析柱，冲洗流穿后进行洗脱，收集洗脱液2，所述平衡、冲洗流穿和洗脱使用PB；
优选地，步骤(1)中所述平衡和冲洗流穿均使用20mmol/L PB、pH6.5、含1mmol/L EDTA和0.2mol/L NaCl的制剂1，所述洗脱使用20mmol/L PB、pH6.5、含1mmol/L EDTA和0.28mol/L NaCl洗脱缓冲液1；
步骤(2)中所述调节电导后的电导为180ms/cm，所述平衡和所述冲洗流穿均使用20mmol/L PB、pH7.5、含1mmol/L EDTA和1.4mol/L(NH4)2SO4的制剂2，所述洗脱使用20mmol/L PB、pH7.5、含1mmol/L EDTA和0.5mol/L(NH4)2SO4的洗脱缓冲液2。
5.如权利要求4所述的纯化方法，其特征在于，步骤(1)中所述平衡和所述冲洗流穿使用3倍柱体积的制剂1；和/或，所述洗脱使用3倍柱体积的洗脱缓冲液1；
步骤(2)中所述平衡和所述冲洗流穿使用3倍柱体积的制剂2，和/或，所述洗脱使用3倍柱体积的洗脱缓冲液2。
6.如权利要求4所述的纯化方法，其特征在于，还包括以下步骤：
(3)用置换液2置换所述的洗脱液2，收集置换液3，平衡后将所述的置换液3上样阳离子层析柱，冲洗流穿后进行洗脱，收集洗脱液3，所述平衡、冲洗流穿和洗脱均使用NaAC缓冲液；所述置换液2为50mmol/L NaAC、pH4.8、含1mmol/L EDTA；
优选地，步骤(3)中所述置换、平衡和冲洗流穿均使用50mmol/L NaAC、pH4.8、含1mmol/L EDTA的制剂3，所述洗脱使用50mmol/L NaAC、pH4.8、含1mmol/L EDTA和0.097mol/L NaCl的洗脱缓冲液3；
更优选地，步骤(3)中所述置换、平衡和冲洗流穿使用3倍柱体积的制剂3；和/或，所述洗脱使用3倍柱体积的洗脱缓冲液3。
7.一种PspA4 (αHD+PRD)蛋白的发酵工艺，其特征在于，其包括以下步骤：
(i)培养表达氨基酸序列如SEQ ID NO:2所示的PspA4 (αHD+PRD)蛋白的宿主细胞，所述宿主细胞为大肠杆菌；
(ii)接种前加入硫酸卡那霉素和无机盐溶液，所述无机盐为K2HPO4和/或MgSO4；优选地，所述硫酸卡那霉素的终浓度为30μg/mL；
(iii)发酵工艺参数为：发酵温度32～37℃，摇床转速100～600rpm；优选地，所述发酵温度为35℃；
(iv)当A600nm值达到9～11时，添加诱导剂；所述诱导剂为IPTG，诱导温度例如为28～36℃，诱导时长为4h～12h；优选地，所述诱导温度为32℃，所述IPTG的终浓度为0.05～0.5mmol/L；
优选地，所述宿主细胞为BL21(DE3)，所述宿主细胞包括表达载体，所述表达载体为pET27b或pET30a；
更优选地，所述重组表达载体包含如SEQ ID NO:1所示分离的核酸，所述表达载体为pET27b；
进一步更优选地，所述大肠杆菌为BL21(DE3)。
8.一种PspA4 (αHD+PRD)蛋白的保存方法，其特征在于，所述PspA4 (αHD+PRD)蛋白的氨基酸序列如SEQ ID NO:2所示，且用pH8.0的硼酸盐缓冲液对如权利要求6所述的纯化方法制得的洗脱液3进行置换后保存。
9.一种重组表达载体，其特征在于，所述重组表达载体包含如SEQ ID NO:1所示分离的核酸；优选地，所述表达载体为pET27b或pET30a。
10.一种表达菌株，其特征在于，所述表达菌株包含如权利要求9所述的重组表达载体；优选地，所述表达菌株的宿主菌为大肠杆菌；更优选地，所述大肠杆菌为BL21(DE3)。
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