CN113355313B - 一种聚合物微球及其制备与应用 - Google Patents
一种聚合物微球及其制备与应用 Download PDFInfo
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- CN113355313B CN113355313B CN202110457533.2A CN202110457533A CN113355313B CN 113355313 B CN113355313 B CN 113355313B CN 202110457533 A CN202110457533 A CN 202110457533A CN 113355313 B CN113355313 B CN 113355313B
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Abstract
本发明公开了一种聚合物微球及其制备与在制备抗体纯化填料中的应用,所述聚合物微球是以聚乙烯醇和海藻酸钠为原料,加入水在80‑100℃溶解后,降温至40℃后加入含ZZ融合蛋白的重组大肠杆菌菌液制成水相;再将液体石蜡和表面活性剂混合为油相,水相缓慢滴加到油相中,30‑70℃搅拌形成乳液后,再加入饱和硼酸氯化钙水溶液,制备得到所述的聚合物微球;所述ZZ融合蛋白是将ZZ蛋白通过连接肽与锚定蛋白(LppOmpA)融合而成;本发明方法制备的聚合物微球的直径为50‑250nm,超大孔径为1μm至2μm,原料廉价,成本低,操作简单,省略了常规配基的表达、纯化、接枝等制备过程,解决了配基昂贵等问题。
Description
(一)技术领域
本发明涉及一种聚合物微球及其制备与应用。
(二)背景技术
抗体类药物因其具有靶向性强、特异性高和毒副作用小等特点,在临床被广泛应用于疾病的诊断、治疗及免疫等方面,应用前景广阔。然而,抗体药物生产技术门槛较高,涉及到抗体筛选、抗体重组、高表达细胞株构建、大规模悬浮培养及下游纯化等核心技术,随着分子生物学技术的飞速发展,在抗体筛选、重组、人源化等上游技术方面已没有太多障碍,目前的困难主要集中在中游的大规模生产及下游纯化等方面。当前,约80%的下游工艺采用Protein A亲和层析进行快速捕获,由于Protein A对抗体具有良好的特异结合能力,而且操作简单,因此被广泛的利用。然而,Protein A因分子量较大,表达困难,而且表达后的Protein A还需要一系列的分离纯化,接枝修饰等过程,造成Protein A亲和填料的市场化价格比较昂贵,目前国内成规模生产Protein A亲和填料的公司很少,加之国内抗体培养表达的规模较小,色谱操作处理能力受限,因此生产成本居高不下,缺少市场竞争力。
因此,研究可替代载体及纯化技术具有重要的现实意义,近年来研究替代蛋白A的小分子功能基逐渐增多,其中最典型的疏水电荷诱导层析,利用疏水作用吸附抗体;调节pH值,使杂环带电实现洗脱,成为比较有潜力的替代蛋白A的纯化抗体模式。另外,Protein A亲和介质其配基主要来源于金黄色葡萄球菌蛋白A(StaphyIococcal Protein A,SPA),其作为一种“广泛二抗”,能与抗体分子的Fc片段具有良好的亲和特性,被广泛的应用于抗体纯化领域。SPA是位于革兰氏阳性金黄色葡萄球菌表面的细胞壁相关蛋白结构域,蛋白分子量为42kDa,由16种氨基酸(不含半胱氨酸及胱氨酸)组成,无二硫键及复杂的三级结构,因此天然结构十分稳定。由于SPA蛋白较大,表达过程中可能需要面临蛋白质二级结构发生折叠变形等问题。因此人们通过对SPA蛋白的基因结构进行分析,认为其与IgG结合区主要由E、D、A、B和C五个高度同源的IgG结合结构域组成,都能与IgG1,IgG2和IgG4的Fc段独立结合,且都显示具有较强的免疫球蛋白亲和活性,其中B结合结构域的结合力最强,同时为了提高B结构域对羟基胺介导的融合蛋白位点特异性化学裂解的耐受性,对B结构域进行基因改造(一个氨基酸残基发生突变)成Z结构域,可应用于固定化亲和配体捕捉IgG中,同时可通过对Z结构域首尾串联聚合,使其具有更强的捕捉抗体的能力,同时可将其作为一种可自我再生的固定化蛋白基质用于抗体纯化亲和色谱载体的大批量制备。
本发明利用DNA重组技术将抗体结合域ZZ蛋白通过具有展示大分子蛋白潜能的Lpp-OmpA展示系统展示在大肠杆菌表面,不经过解析、纯化,通过多孔载体包埋重组大肠杆菌简单培养,快速获得大量廉价的抗体亲和色谱填料并用于抗体纯化,对部分解决现有填料制备工艺复杂,成本高昂的局限性,降低生产成本具有重要意义。
(三)发明内容
本发明目的是提供一种用于IgG纯化的聚合物微球及其制备方法与应用,以聚乙烯醇和海藻酸钠为原料,加入重组大肠杆菌后与饱和硼酸氯化钙溶液进行交联得到的微球,通过重组大肠杆菌的简单培养就可以快速获得大量价廉的抗体亲和色谱填料,作为色谱介质应用于抗体分离纯化中,操作简化,价格低廉。
本发明采用的技术方案是:
本发明提供一种用于纯化IgG抗体的聚合物微球,所述聚合物微球是以聚乙烯醇和海藻酸钠为原料,加入水在80-100℃(优选95℃)溶解后,降温至40℃后加入含ZZ融合蛋白基因的重组大肠杆菌菌液制成水相;再将液体石蜡和表面活性剂混合为油相,水相缓慢滴加到油相中,30-70℃(优选50℃)搅拌形成乳液后,再加入饱和硼酸氯化钙水溶液,搅拌制备得到所述的聚合物微球;所述表面活性剂为span80;所述ZZ融合蛋白是将ZZ蛋白通过连接肽与锚定蛋白(LppOmpA)融合而成;所述ZZ蛋白氨基酸序列如SEQ ID NO.1所示,连接肽氨基酸序列为GIPG;所述锚定蛋白氨基酸序列如SEQ ID NO.3。
本发明所述ZZ蛋白编码基因核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.1:
RQHDEAVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAN。
SEQ ID NO.2:
CGTCAGCACGATGAAGCGGTTGATAACAAATTCAACAAAGAACAGCAGAACGCGTTCTACGAAATCCTGCACCTGCCGAACCTGAACGAAGAACAGCGTAACGCGTTCATTCAGAGCCTGAAAGATGATCCGTCTCAGAGCGCGAACCTGCTGGCGGAAGCGAAAAAACTGAACGATGCGCAGGCACCGAAAGTTGATAACAAATTCAACAAAGAACAGCAGAACGCGTTCTACGAAATCCTGCACCTGCCGAACCTGAACGAAGAACAGCGTAACGCGTTCATCCAGTCTCTGAAAGATGATCCGTCTCAGTCTGCGAACCTGCTGGCGGAAGCGAAAAAACTGAACGATGCGCAGGCGCCGAAAGTTGATGCGAAC。
所述ZZ融合蛋白的编码基因核苷酸序列为SEQ ID NO.4所示。
本发明含ZZ融合蛋白基因的重组大肠杆菌是将ZZ蛋白通过连接肽与锚定蛋白(LppOmpA)融合后的基因,用NcoI和HindIII限制性内切酶,转入载体pET28a(购自生工),构建pET28a-LppOmpA-ZZ质粒,转化E.coli BL21(DE3)制备得到。本发明所述重组大肠杆菌利用细胞表面展示技术把ZZ蛋白表达在大肠杆菌表面。所述细胞表面展示技术就是利用DNA重组技术将抗体结合域Z蛋白通过具有展示大分子蛋白潜能的Lpp-OmpA展示系统展示在大肠杆菌表面。所述的结构域Z蛋白是由B结构域进行基因改造而来的,而B结构域是IgG结合结构域结合力最强的一部分。ZZ蛋白是指把Z结构域首尾串联聚合,在亲和层析中ZZ蛋白有更温和的洗脱条件,这种人工修饰改造基因的方式,不仅使整个基因序列得到了缩短,能有效的保留SPA抗体亲和结构域与抗体的亲和活性,同时大大提高了蛋白分泌表达的效率。
进一步,所述含ZZ融合蛋白基因的重组大肠杆菌菌液按如下方法制备:将含ZZ融合蛋白基因的重组大肠杆菌均匀涂布在含有50μg/mL卡那霉素(Kan)抗性的LB平板上,在30℃下放置培养过夜。从LB平板上挑取单菌落,以体积浓度1%的接种量接种于含Kan(50μg/mL)抗性的LB液体培养基中,37℃,180rpm摇床培养过夜至OD600为0.62,获得重组菌E.coliBL21(DE3)/pET-28a-LppOmpA-ZZ菌液。
进一步,所述海藻酸钠与聚乙烯醇质量比为1:6,所述水体积用量以海藻酸钠质量计为90-100mL/g,所述重组大肠杆菌菌液中湿菌体加入量与海藻酸钠质量比为1-5:100,优选2.9:100。所述液体石蜡与表面活性剂体积比为10-30:1.5,优选20:1.5;所述油相与水相体积比为10-30:20,优选21.5:20,所述油相与饱和硼酸氯化钙水溶液体积比为20-60:21.5,优选40:21.5。
本发明方法制备的聚合物微球为多孔聚合物微球,所述多孔聚合微球是溶解后的聚乙烯醇和海藻酸钠与饱和硼酸氯化钙进行双交联,通过悬浮乳化法制备得到,其中双交联可制得超大孔径互穿网络结构的微球。具体所述聚合物微球按如下方法制备:
(1)将聚乙烯醇(优选PVA1799型),海藻酸钠(SA)粉末和双蒸水,在95℃条件下搅拌至溶液澄清透明,得到PVA/SA混合溶液;冷却至40℃后加入重组大肠杆菌菌液,在常温(20-25℃)下均匀搅拌30min,获得菌体混合液,即为水相;
(2)将液体石蜡和表面活性剂在300r/min转速机械搅拌30min后作为油相;向油相逐滴缓慢加入步骤(1)水相,在50℃、500r/min条件下水浴搅拌1h,形成白色乳液,边搅拌边降温,待温度降至40℃时快速加入饱和硼酸氯化钙水溶液(优选滴加速度40mL/5s),40℃、100r/min水浴加热搅拌反应2h,采用50-65目(孔径为250μm-270μm)的筛网过滤,滤液与异丙醇混合后离心(优选4000rpm高速离心2min),沉淀用蒸馏水清洗3-5次,获得包埋重组大肠杆菌的PVA/SA微球,即为所述的聚合物微球;所述异丙醇与水相体积比为1:1。
本发明还提供一种所述聚合物微球在制备抗体纯化填料中的应用,所述应用是将聚合物微球作为填料放入空层析柱,缓冲液经0.45μm膜过滤超声除尽气泡备用。用至少5个柱床体积的结合缓冲液(pH=7.2、0.02mol/L的Tris-HCl缓冲液)平衡柱。待基线跑平后,向柱中加样,用结合缓冲液冲洗,直到紫外吸收曲线稳定在基线,用洗脱缓冲液(0.1mol/L甘氨酸,0.01mol/L NaCl,溶剂是蒸馏水,pH=3.0)洗脱,获得目标蛋白。用结合缓冲液重生柱子。所述抗体包括IgG抗体。
与现有技术相比,本发明有益效果主要体现在:本发明方法制备的聚合物微球的直径为50-250nm,超大孔径为1μm至2μm,原料廉价,成本低,操作简单,省略了常规配基的表达、纯化、接枝等制备过程,解决了配基昂贵等问题。所述聚合物微球动态吸附IgG的速度非常快,60min内就达到了吸附平衡,在pH为7.2时,最大吸附容量为30.4mg/g。
(四)附图说明
图1是实施例1pET28a-LppOmpA-ZZ的质粒图谱(a)及酶切电泳图(b),其中M:marker;1:LppOmpA-ZZ。
图2是实施例1重组菌诱导表达后的蛋白SDS-PAGE电泳分析图;M:Marker;1:重组菌诱导表达后沉淀;2:重组菌诱导表达后破碎上清;3:重组菌诱导表达破碎后沉淀;4:空质粒上清;5:空质粒沉淀;6:空质粒破碎后上清;7:空质粒破碎后沉淀。
图3是实施例2制备的聚合物微球的SEM图,A:聚合物微球外部形貌;B:对照聚合物微球内部结构(未包埋大肠杆菌);C:聚合物微球内部结构(包埋大肠杆菌)。
图4是实施例3中聚合物微球结合性能荧光显微镜图;A:实验组,聚合物微球(包埋重组大肠杆菌),加上FITC-IgG抗体溶液;B:对照组,聚合物微球(包埋重组大肠杆菌),未加FITC-IgG抗体溶液;C:空白对照组,对照聚合物微球(包埋普通大肠杆菌),加入FITC-IgG抗体溶液。
图5是实施例3中聚合物微球静态吸附图。
图6是实施例3中聚合物微球动态吸附图。
图7是实施例4中聚合物微球分离抗体的色谱图(A)及电泳图(B),B中M:marker;1:洗脱峰。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1重组菌株的构建及诱导表达
1、重组菌株的构建
ZZ结构域氨基酸序列为(SEQ ID NO.1):
RQHDEAVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAN。
ZZ结构域与LppOmpA蛋白连接肽氨基酸序列为:GIPG。
锚定蛋白(LppOmpA)氨基酸序列为(SEQ ID NO.3):
MKATKLVLGAVILGSTLLAGCSSNAKIDQGINPYVGFEMGYD7WLGRMPYKGSVENGAYKAQGVQLTAKLGYPITDDLDIYTRLGGMVWRADTKSNVYGKNHDTGVSPVFAGGVEYAITPEIATRLEYQWTNNIGDAHTIGTRPDN。
(1)pET28a-LppOmpA-ZZ质粒
根据ZZ结构域基因编码序列(AAB00807.1,核苷酸序列如SEQ ID NO.2),通过连接肽(GIPG)与锚定蛋白(LppOmpA)融合,并由生工生物工程(上海)股份有限公司进行密码子优化及全基因合成(核苷酸序列如SEQ ID NO.4所示),采用NcoI和HindIII限制性内切酶,转入载体pET28a(购自生工),构建pET28a-LppOmpA-ZZ质粒,如图1中a。
SEQ ID NO.4:
CCATGGGCAAAGCGACCAAACTGGTTCTGGGCGCGGTTATCCTGGGTAGCACCCTGCTGGCGGGTTGCTCTAGCAACGCGAAAATCGATCAGGGTATTAACCCGTACGTTGGTTTCGAAATGGGTTACGATTGGCTGGGCCGTATGCCGTATAAAGGCTCTGTTGAAAACGGTGCATACAAAGCGCAGGGCGTTCAGCTGACCGCTAAACTGGGCTACCCGATCACCGATGATCTGGATATCTACACCCGTCTGGGTGGTATGGTTTGGCGTGCGGATACCAAATCTAACGTTTACGGTAAAAACCACGATACCGGTGTGTCTCCGGTTTTCGCGGGCGGTGTTGAATACGCGATCACCCCGGAAATCGCGACCCGTCTGGAATACCAGTGGACCAACAACATTGGTGATGCGCACACCATTGGTACCCGTCCGGATAACGGTATTCCGGGTCGTCAGCACGATGAAGCGGTTGATAACAAATTCAACAAAGAACAGCAGAACGCGTTCTACGAAATCCTGCACCTGCCGAACCTGAACGAAGAACAGCGTAACGCGTTCATTCAGAGCCTGAAAGATGATCCGTCTCAGAGCGCGAACCTGCTGGCGGAAGCGAAAAAACTGAACGATGCGCAGGCACCGAAAGTTGATAACAAATTCAACAAAGAACAGCAGAACGCGTTCTACGAAATCCTGCACCTGCCGAACCTGAACGAAGAACAGCGTAACGCGTTCATCCAGTCTCTGAAAGATGATCCGTCTCAGTCTGCGAACCTGCTGGCGGAAGCGAAAAAACTGAACGATGCGCAGGCGCCGAAAGTTGATGCGAACTAAGCTT。
(2)重组菌株
将含有重组pET28a-LppOmpA-ZZ质粒的E.coli DH5α,100μL涂布于含Kan(50μg/mL)抗性的LB平板上,在30℃下放置培养过夜。从LB平板上挑取单菌落,接种于100mL含Kan(50μg/mL)抗性的LB液体培养基中,30℃,180rpm摇床培养过夜,按体积比1:100的比例将菌液接种于新的100mL的LB液体培养基中,在30℃下摇瓶培养过夜。按照SanPrep柱式质粒DNA小量抽提试剂盒(购自生工生物工程(上海)股份有限公司,试剂盒编号B518191-0050)的操作方法取3-4mL的菌液进行质粒提取。
将于-80℃冰箱保存的100μL大肠杆菌E.coliBL21(DE3)感受态细胞取出,置于冰上解冻。将10μL质粒加入到E.coli BL21(DE3)融化的感受态细胞中,轻轻混匀,冰浴30min。42℃精确热激90s后,立刻冰浴3min。加入900μL无抗性的LB液体培养基,37℃振荡培养1h。将孵育的菌液均匀涂布在含有Kan(50μg/mL)抗性的LB平板上,在30℃下放置培养过夜,进行细胞转化。
从LB平板上挑取单菌落,接种于100mL含Kan(50μg/mL)抗性的LB液体培养基中,37℃,180rpm摇床培养过夜至OD600为0.62,获得重组菌E.coliBL21(DE3)/pET-28a-LppOmpA-ZZ菌液,进行重组菌的培养。同样条件构建对照菌株E.coli BL21(DE3)/pET-28a菌液。
(3)琼脂糖凝胶电泳检测
步骤(2)的E.coli BL21(DE3)/pET-28a-LppOmpA-ZZ菌液,遵循SanPrep柱式质粒DNA小量抽提试剂盒(购自生工生物工程(上海)股份有限公司,试剂盒编号B518191-0050)的操作方法,取3-4毫升质粒通过琼脂糖凝胶电泳检测,结果见图1中b(M:marker;1:LppOmpA-ZZ)。在琼脂糖凝胶800bp到900bp之间有与目的条带分子量一致的条带,因此表明重组质粒pET-28a-LppOmpA-ZZ表达载体构建成功。
2、蛋白的诱导表达及检测
将E.coli BL21(DE3)/pET-28a-LppOmpA-ZZ菌株接种到1mL含Kan(50μg/mL)抗性的LB液体培养基中,37℃过夜培养后,培养液以体积浓度2%的比例接种到10mL含Kan抗性(50μg/mL)的LB液体培养基中,在30℃,180rpm下震荡培养2-3h至OD600值为0.6时,加入终浓度为1mmol/L的IPTG,继续放入摇床诱导培养4h。取1mL菌液8000rpm离心5min,弃去上清,菌体用PBS缓冲液洗涤2次后加40μL的PBS缓冲液悬浮,记为1号样品。
取1ml的1号样品,在功率200W、超声5s、间隔6s条件下超声破碎30min,8000rpm离心5min后,获得上清和沉淀;取40μL上清,记为2号样品,沉淀加40μL PBS缓冲液悬浮,记为3号样品。
以相同的方法对E.coli BL21(DE3)/pET-28a菌体进行处理,分别将菌体离心后的上清取40μL,记为4号样品,菌体悬浮液记为5号样品,菌体悬浮液超声破碎后的上清液记为6号样品,超声破碎后的沉淀悬浮液记为7号样品。
1-6号样品分别加10μL 5×SDS上样缓冲液,震荡混匀菌体,沸水浴条件下煮沸5min后冷却,在4℃、10000rpm条件下离心2min,取上清作SDS-PAGE分析。SDS-PAGE胶电泳分析检测蛋白质如图2(M:Marker;1:重组菌诱导表达后沉淀;2:重组菌诱导表达后破碎上清;3:重组菌诱导表达破碎后沉淀;4:空质粒上清;5:空质粒沉淀;6:空质粒破碎后上清;7:空质粒破碎后沉淀)。IPTG诱导培养的菌体在1、2、3泳道31kDa左右范围内有一条明显蛋白条带,与预估的目标蛋白分子量30.37kDa相符,而在4、5、6、7泳道在此区域未发现明显条带,可以初步判断重组表达菌株可以成功诱导表达重组蛋白LppOmpA-ZZ。
实施例2、聚合物微球的制备及表征
(1)在25ml茄形瓶中加入0.6g聚乙烯醇颗粒(PVA1799型),0.1g海藻酸钠(SA)粉末和9.3mL双蒸水,在95℃条件下搅拌至溶液澄清透明,得到PVA/SA混合溶液(其中PVA质量浓度6.0wt%,SA质量浓度1.0wt%);冷却至40℃后加入实施例1制备的重组菌E.coli BL21(DE3)/pET-28a-LppOmpA-ZZ菌液0.5ml(湿菌体含量5.8mg/mL),在常温(20-25℃)下均匀搅拌30min,获得菌体混合液10.5mL,作为水相。
(2)在100mL圆底烧瓶中加入20ml液体石蜡,1.5ml Span 80作为表面活性剂,300r/min转速机械搅拌30min后作为油相,逐滴缓慢加入步骤(1)方法制备的20.0mL水相,在50℃、500r/min条件下水浴搅拌1h,形成白色乳液,边搅拌边降温,待温度降至40℃时5s内快速加入40ml饱和硼酸氯化钙水溶液(使得CaCl2质量终浓度为5wt%),40℃、100r/min水浴加热搅拌反应2h。用60目(孔径为250μm)的筛网过滤去除大颗粒沉淀,取滤液(即筛分出来的微球),然后把滤液倒入50ml离心管中,加入20mL异丙醇,4000rpm高速离心2min,去除异丙醇后,沉淀用蒸馏水清洗3-5次,获得包埋重组大肠杆菌E.coli BL21(DE3)/pET-28a-LppOmpA-ZZ的PVA/SA微球5g,简称聚合物微球。
同样条件下,去除步骤(1)菌液制备对照聚合物微球,通过SEM扫描电镜观察微球内部结构和孔隙,结果如图3(A:聚合物微球外部形貌;B:对照聚合物微球内部结构(未包埋大肠杆菌);C:聚合物微球内部结构(包埋大肠杆菌))所示。图3中(A)为微球表面形状,微球粒径大概在100μm至200μm之间,可以看到微球表面有微小的通道可以让蛋白通过。图3中(B)为放大20000倍后的聚乙烯醇/海藻酸钠微球剖面结构,从图中可以看到微球内部存在大孔,孔径大概在1μm左右,孔径通道相互贯通,形成三维网状结构。说明该方法制备出的PVA/SA复合微球是多孔结构且蛋白能通过微球孔道。图3中(C)可以看到该方法制备的微球中存在大肠杆菌,说明微球能成功包埋大肠杆菌,且大肠杆菌形态完好。
(3)使用Mastersizer激光粒度仪2000进行粒径分布测定,使用前先用双蒸水清洗三遍,直到检测背景降到设定值。在1L烧杯中加入800mL无水乙醇,打开循环泵使无水乙醇在管路循环,加入10g步骤(2)制备的聚合物微球至遮光度为10%。点击测量样品按钮,测量结束后排出管路中无水乙醇,用无水乙醇清洗三遍,再用双蒸水清洗三遍管路直到背景恢复正常,保存并导出数据。80%体积分布在47.366μm至252.438μm之间,表面积平均直径为83.587μm,体积平均直径142.888μm。筛分后微球粒径体积分布在50μm至250μm。GEHealthcare SP Sepharose Fast Flow微球大小在45μm到165μm,大小基本符合预期结果。
实施例3、聚合物微球的性能及应用
1、聚合物微球的结合性能:
将实施例2制备的聚合物微球,用pH=7.2、0.02mol/L的Tris-HCl缓冲液清洗三次,取0.1g放入1.5mL离心管中,加入0.5mL用pH=7.2、0.02mol/L Tris-HCl缓冲液配制的浓度为0.2mg/mL的FITC标记的兔抗小鼠lgG溶液(H+L,购自生工生物工程(上海)股份有限公司),在25℃培养箱中孵育3h,然后用pH=7.2、0.02mol/L的Tris-HCl缓冲液迅速清洗2-3遍后,作为实验组进行荧光显微镜镜检。
同样条件下,以不添加FITC-IgG抗体溶液为对照组。以实施例2对照聚合物微球替换聚合物微球为空白对照组。
荧光显微镜镜检结果(λ=488nm)如图4所示,A:实验组,聚合物微球(包埋重组大肠杆菌),加上FITC-IgG抗体溶液;B:对照组,聚合物微球(包埋重组大肠杆菌),未加FITC-IgG抗体溶液;C:空白对照组,对照聚合物微球(包埋普通大肠杆菌),加入FITC-IgG抗体溶液。加入FITC-IgG的聚合物微球在荧光显微镜上可以看到明显的绿色荧光,说明兔抗小鼠IgG(H+L)能通过微球孔道,表达在大肠杆菌表面的蛋白能吸附IgG。实验组和空白对照组均加入了FITC-IgG抗体溶液,对照聚合物微球未看到明显的绿色荧光,说明普通大肠杆菌和PVA/SA微球对IgG基本没有吸附特性,排除了微球本身对IgG的非特异性吸附。进一步说明表达在大肠杆菌表面的蛋白ZZ能吸附lgG。
2、聚合物微球静态吸附:
标准曲线:用pH=7.2、0.02mol/L的Tris-HCl缓冲液将兔抗小鼠lgG(H+L)分别配制成0.12mg/ml、0.5mg/L、1mg/L、1.5mg/L和2mg/L抗体溶液。在280nm分别测溶液的吸光值,以抗体溶液为横坐标,对应的吸光值为纵坐标绘制标准曲线,得到的方程式为y=0.4288x+1.8329
将实施例2制备的聚合物微球3g用30mL去离子水清洗并抽干后,放入30mL、0.05mol/L pH=7.2的Tris-HCl缓冲液中,用25℃摇床180rpm平衡15min后,真空抽干10min,获得抽干聚合物微球2g。依次称取0.01g抽干聚合物微球分别置于10支1.5mL离心管中,然后向每支离心管中加入1mL不同浓度的pH=7.2、0.02mol/LTris-HCl缓冲液配制的兔抗小鼠lgG(H+L)(购自生工生物工程(上海)股份有限公司)抗体溶液,浓度分别为0.25mg/L、0.5mg/L、0.75mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2mg/L、2.5mg/L和3mg/L。混合均匀后,离心管置于摇床中在25℃和170r/min下震荡反应8h。反应结束后,离心管在4000r/min下离心10min,收集上清液并用酶标仪在280nm下测定吸光值,根据标准曲线获得蛋白浓度,即为吸附平衡时上清液中抗体浓度。然后,根据物料平衡公式计算静态吸附量:
其中:Q表示聚合物微球上抗体吸附量(mg/g),C0表示初始抗体浓度(mg/mL),C表示吸附平衡时上清液中抗体浓度(mg/mL),V表示抗体溶液体积(mL),m表示抽干的聚合物微球质量(g)。
利用Langmuir方程进行拟合,得到等温吸附曲线,如下图5所示(其中横坐标表示初始IgG的浓度,纵坐标表示聚合物微球的吸附量。聚合物微球在pH为7.2、0.02mol/L的Tris-HCl缓冲液中,最大吸附容量为30.4mg/g。说明在pH为7.2的条件下,聚合物微球对IgG能有效的进行吸附。
3、聚合物微球吸附动力学:
以兔抗小鼠lgG(H+L)(购自生工生物工程(上海)股份有限公司)作为模型吸附蛋白。将实施例2制备的聚合物微球用蒸馏水洗涤,真空抽干后,取2g于20mL、pH=7.2、0.02mol/L的Tris-Hcl缓冲液中,25℃摇床180rpm平衡15min,真空抽干后,称取0.5g置于10ml离心管中,加入6mg/mL的pH=7.2、0.02mol/L Tris-HCl缓冲液配制的兔抗小鼠lgG(H+L)(购自生工生物工程(上海)股份有限公司)抗体溶液5mL,于水浴摇床中,25℃,180rpm震荡反应,每隔10min取200μL上清液于280nm处测定吸光度后,并迅速倒入原反应液中。根据步骤2的标准曲线,计算出不同时间下的IgG蛋白浓度变化,绘制动力学曲线。聚合物微球的吸附动力学曲线如下图6所示,可以看出,聚合物微球吸附兔抗小鼠lgG(H+L)的速度非常快,60min内就达到了吸附平衡。
实施例4、聚合物微球在抗体分离中的应用
将实施例2方法制备的聚合物微球3g,装入5ml层析柱空柱(内直径15.7mm,高度30mm),采用pH=7.2,0.02mol/L的Tris-Hcl缓冲液平衡柱子,待基线平稳后,向柱中加入用pH=7.2、0.02mol/L的Tris-Hcl缓冲液配制的牛血清白蛋白(10.0mg/ml)和兔抗小鼠IgG(H+L)(5.0mg/ml)的预混合液,上样量为500μL,用pH=7.2、0.02mol/L的Tris-Hcl缓冲液淋洗,直至基线平稳,随后用洗脱缓冲液(0.1mol/L甘氨酸,0.01mol/LNaCl,溶剂是蒸馏水,pH=3.0)以0.5ml/min流速洗脱7ml,紫外分光光度计测280nm处OD值,当洗脱峰出现时用10ml离心管进行接取样品,取样后迅速用pH=9.0的Tris-Hcl缓冲液中和样品溶液,用pH=7.2、0.02mol/L的Tris-Hcl缓冲液重生柱子。接取的样品随后进行SDS-PAGE电泳验证,用凝胶成像系统进行软件数据分析。抗体的回收率是86.7%,纯度97.6%。聚合物微球分离抗体的色谱图及电泳图(图7,M:marker;1:洗脱峰的电泳图)。图7可看到出现了穿透峰和洗脱峰。洗脱出的液体在还原性上样缓冲液处理后,在SDS-PAGE凝胶电泳25KD和55KD分别能看到清晰的条带,与兔抗的分子量大小一致。说明制备的聚合物微球可以吸附抗体并洗脱抗体。
序列表
<110> 浙江工业大学
<120> 一种聚合物微球及其制备与应用
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Arg Gln His Asp Glu Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln
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cgtcagcacg atgaagcggt tgataacaaa ttcaacaaag aacagcagaa cgcgttctac 60
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Claims (5)
1.一种用于纯化IgG抗体的聚合物微球的制备方法,其特征在于所述聚合物微球是以聚乙烯醇和海藻酸钠为原料,加入水在80-100℃溶解后,降温至40℃后加入含ZZ融合蛋白基因的重组大肠杆菌菌液制成水相;再将液体石蜡和表面活性剂混合为油相,水相缓慢滴加到油相中,30-70℃搅拌形成乳液后,再加入饱和硼酸氯化钙水溶液,搅拌制备得到所述的聚合物微球;所述表面活性剂为span80;所述ZZ融合蛋白是将ZZ蛋白通过连接肽与锚定蛋白LppOmpA融合而成;
所述ZZ蛋白氨基酸序列如SEQ ID NO.1所示,连接肽氨基酸序列为GIPG;所述锚定蛋白氨基酸序列如SEQ ID NO.3;
ZZ融合蛋白编码基因的核苷酸序列为SEQ ID NO.4所示;
所述含ZZ融合蛋白基因的重组大肠杆菌是将ZZ蛋白通过连接肽与锚定蛋白LppOmpA融合后的基因,用NcoI和HindIII限制性内切酶,转入载体pET28a,构建pET28a- LppOmpA-ZZ质粒,转化E.coli BL21(DE3)制备得到;
所述含ZZ融合蛋白基因的重组大肠杆菌菌液按如下方法制备:将含ZZ融合蛋白基因的重组大肠杆菌均匀涂布在含有50 μg/mL 卡那霉素的LB平板上,在30 ℃下放置培养过夜;从LB平板上挑取单菌落,以体积浓度1%的接种量接种于含50 μg/mL卡那霉素的LB液体培养基中,37 ℃,180 rpm摇床培养过夜至OD600为0.62,获得所述的菌液;
所述海藻酸钠与聚乙烯醇质量比为1:6,所述水体积用量以海藻酸钠质量计为90-100mL/g;所述重组大肠杆菌菌液以湿菌体重量计,则湿菌体加入量与海藻酸钠质量比为1-5:100;所述液体石蜡与表面活性剂体积比为10-30:1.5;所述油相与水相体积比为10-30:20,所述油相与饱和硼酸氯化钙水溶液体积比为20-60:21.5。
2.如权利要求1所述的方法,其特征在于饱和硼酸氯化钙水溶液滴加速度40mL/5s。
3.一种根据权利要求1所述方法制备的用于纯化IgG抗体的聚合物微球。
4.一种权利要求3所述聚合物微球在制备抗体纯化填料中的应用。
5.如权利要求4所述的应用,其特征在于所述抗体包括IgG抗体。
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