CN116217680B - 碱稳定性高的免疫球蛋白结合蛋白及其应用 - Google Patents
碱稳定性高的免疫球蛋白结合蛋白及其应用 Download PDFInfo
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Abstract
本发明公开碱稳定性高的免疫球蛋白结合蛋白及其应用,本发明提供了由葡萄球菌蛋白A的Z、D、C结构域的部分肽段拼接得到的对碱稳定性提高的免疫球蛋白结合蛋白(zdc结构域);所述免疫球蛋白结合蛋白氨基酸序列如SEQ IDNO:1所示;进一步研究发现,对zdc结构域的第3、6、9、15、28、37位氨基酸进行了突变,可获得了对碱稳定性进一步提高的蛋白A突变体;本发明提供的免疫球蛋白结合蛋白制备的亲和层析填料能耐受0.5~1.0M NaOH在线清洗,具有良好的应用前景。
Description
技术领域
本发明属于免疫球蛋白分离纯化技术领域,涉及免疫球蛋白结合蛋白,具体涉及碱稳定性高的免疫球蛋白结合蛋白及其应用。
背景技术
免疫球蛋白(例如单克隆抗体或多克隆抗体)一类重要的生物活性蛋白,是当今研发和制造中最热门的生物制药产品。自1986年第一个单克隆抗体药物上市以来,全球已有超过100种单抗药物获批上市。国内已获批的进口单抗药物超过40个,国产单抗药物超过30个。经过30多年的研究和发展,单抗药物以其高度特异性和疗效确切的特点,在肿瘤、自身免疫、代谢、病毒感染等疾病领域显示出独特的优势和广阔的应用前景。
ProteinA亲和层析(或蛋白A亲和层析)是纯化免疫球蛋白或Fc融合蛋白的关键步骤之一,对蛋白纯度、纯化成本、纯化效率都有影响。但是Protein A亲和层析介质(或填料)在抗体的纯化过程中会出现配基脱落、结合载量降低等问题。另外,Protein A亲和层析在再生和在位清洗(CIP)时通常会使用高浓度碱(例如0.5-1.0M NaOH溶液)冲洗,从而除去附着在介质上的内毒素、蛋白沉淀物、核酸、病毒等残余污染物,在这个过程中高浓度碱会破坏Protein A亲和层析介质,大幅降低结合载量,缩短了介质使用寿命。为此,大量研究人员尝试使用不同结构域的蛋白A,对蛋白A进行突变等方面进行大量研究,期望找到对碱稳定性提高的蛋白A。
葡萄球菌蛋白A(Staphylococcal protein A,SPA)简称蛋白A,是金黄色葡萄球菌表面的一种蛋白质,通过共价键连接到胞壁肽聚糖。蛋白分子包含E、D、A、B、C五个结构域,能和人及多种哺乳动物IgG的Fc片段结合,因此被称为抗体结合区。其中B结构域对抗体的结合力最稳定,且本身结构也比较稳定,因此研究最为广泛。1987年,Nilsson等人将葡萄球菌蛋白A的B结构域中第1位丙氨酸替换为缬氨酸、第29位氨基酸替换为丙氨酸,得到了Z结构域(Protein Science(2004),13:549–554)。用含Z结构域的蛋白A制成的Protein A亲和层析介质,与B结构域相比,显示出了对碱稳定性提高的特点。研究人员有在Z结构域基础上继续做了大量研究,获得碱稳定性进一步改进的蛋白A突变体。
在本领域中,获得能够结合免疫球蛋白的新型对碱稳定蛋白的需求持续存在。本发明提供了新型的对碱稳定的免疫球蛋白结合蛋白,能够用于免疫球蛋白的亲和层析。具体地,与亲本蛋白相比,本发明的免疫球蛋白结合蛋白的显著优点是它们在高浓度碱下具有改善的稳定性。
发明内容
针对上述问题,本发明的目的是提供碱稳定性高的免疫球蛋白结合蛋白及其应用。
为实现上述目的,本发明采取的技术方案为:碱稳定性高的免疫球蛋白结合蛋白,所述的免疫球蛋白结合蛋白的氨基酸序列包含氨基酸序列如SEQ IDNO:1所示的序列或其突变序列,所述突变序列包含以下至少一个位点突变:第3、6、9、15、28、37位。
SEQ ID NO:1(zdc-domain)
VDNKFNKEQQSAFYEILHLPNLTEEQRNAFIQSLKDDPSV KELLAEAKKLNDAQAPK
本发明人通过分析葡萄球菌蛋白A的E、D、A、B、C、Z结构域的空间构象、保守序列以及氨基酸组成特点,选取了Z、D、C结构域中对碱稳定较高的肽段,并按特定顺序拼接起来,最终从获得了新型的对碱稳定性提高的蛋白A单体(氨基酸序列如SEQ ID NO:1所示),又称zdc融合结构域或zdc fusion domain(简称为zdc结构域)。为了进一步提高对碱的稳定性,本发明人还对zdc结构域的第3、6、9、15、28、37位氨基酸进行了突变,获得了对碱稳定性进一步提高的蛋白A突变体。
SEQ ID NO:2(E-domain)
AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPK
SEQ ID NO:3(D-domain)
ADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK
SEQ ID NO:4(A-domain)
ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK
SEQ ID NO:5(B-domain)
ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK
SEQ ID NO:6(C-domain)
ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK
SEQ ID NO:7(Z-domain)
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK
所述免疫球蛋白结合蛋白氨基酸序列的N-和/或C-末端,还可以包含额外的氨基酸残基,例如,N-末端的前导序列,在N-或C-末端含有半胱氨酸。
作为本发明的优选实施方式,所述突变序列包含以下突变中的至少一种:
a)如SEQ ID NO:1所示氨基酸序列的第3、6、28位天冬酰胺突变为除天冬氨酸、谷氨酸、谷氨酰胺、脯氨酸、半胱氨酸、苯丙氨酸、色氨酸、组氨酸、酪氨酸以外的氨基酸,优选的氨基酸是丙氨酸、苏氨酸、丝氨酸;
b)如SEQ ID NO:1所示氨基酸序列的第9位谷氨酰胺突变为除天冬氨酸、天冬酰胺、谷氨酸、脯氨酸、半胱氨酸、苯丙氨酸、色氨酸、组氨酸、酪氨酸以外的氨基酸,优选的氨基酸是丙氨酸、苏氨酸、丝氨酸;
c)如SEQ ID NO:1所示氨基酸序列的第15位谷氨酸突变为除天冬氨酸、天冬酰胺、谷氨酰胺、脯氨酸、半胱氨酸、苯丙氨酸、色氨酸、组氨酸、酪氨酸以外的氨基酸,优选的氨基酸是苏氨酸、赖氨酸、精氨酸;
d)如SEQ ID NO:1所示氨基酸序列的第37位天冬氨酸突变为除谷氨酸、天冬酰胺、谷氨酰胺、脯氨酸、半胱氨酸、苯丙氨酸、色氨酸、组氨酸、酪氨酸以外的氨基酸,优选的氨基酸是丙氨酸、苏氨酸。
作为本发明的优选实施方式,所述突变的氨基酸序列为如SEQ ID NO:8~26所示的序列。
在zdc结构域的第3、6、9、15、28、37位氨基酸的一个或多个进行了突变,获得了碱稳定性较好的蛋白A突变体,这些突变体的氨基酸序列分别如SEQ IDNO:8~26所示:
SEQ ID NO:8zdc(N3A,N6T,Q9A,E15T,N28A)
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
SEQ ID NO:9zdc(N3A,N6T,Q9A,E15T,N28A,D37A)
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDAPSVSKELLAEAKKLNDAQAPK
SEQ ID NO:10zdc(N3A,N6T,Q9A,E15T,N28A,D37T)
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDTPSVSKELLAEAKKLNDAQAPK
SEQ ID NO:11zdc(N3A,N6T,Q9A,E15T,N28T)
VDAKFTKEAQSAFYTILHLPNLTEEQRTAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
SEQ ID NO:12zdc(N3A,N6T,Q9A,E15T,N28T,D37A)
VDAKFTKEAQSAFYTILHLPNLTEEQRTAFIQSLKDAPSVSKELLAEAKKLNDAQAPK
SEQ ID NO:13zdc(N3A,N6T,Q9A,E15T,N28T,D37T)
VDAKFTKEAQSAFYTILHLPNLTEEQRTAFIQSLKDTPSVSKELLAEAKKLNDAQAPKSEQ ID NO:14 zdc(N3A,N6T,Q9A,E15T,N28S)
VDAKFTKEAQSAFYTILHLPNLTEEQRSAFIQSLKDDPSVSKELLAEAKKLNDAQAPKSEQ ID NO:15 zdc(N3A,N6T,Q9A,E15T,N28S,D37T)
VDAKFTKEAQSAFYTILHLPNLTEEQRSAFIQSLKDTPSVSKELLAEAKKLNDAQAPKSEQ ID NO:16 zdc(N3A,N6T,Q9A,E15K,N28A)
VDAKFTKEAQSAFYKILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPKSEQ ID NO:17 zdc(N3A,N6T,Q9A,E15K,N28A,D37A)
VDAKFTKEAQSAFYKILHLPNLTEEQRAAFIQSLKDAPSVSKELLAEAKKLNDAQAPKSEQ ID NO:18 zdc(N3A,N6T,Q9A,E15K,N28T,D37T)
VDAKFTKEAQSAFYKILHLPNLTEEQRTAFIQSLKDTPSVSKELLAEAKKLNDAQAPKSEQ ID NO:19 zdc(N3A,N6T,Q9A,E15K,N28S,D37A)
VDAKFTKEAQSAFYKILHLPNLTEEQRSAFIQSLKDAPSVSKELLAEAKKLNDAQAPKSEQ ID NO:20 zdc(N3A,N6T,Q9T,E15T,N28A)
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPKSEQ ID NO:21 zdc(N3A,N6T,Q9T,E15T,N28T)
VDAKFTKETQSAFYTILHLPNLTEEQRTAFIQSLKDDPSVSKELLAEAKKLNDAQAPKSEQ ID NO:22 zdc(N3A,N6T,Q9T,E15T,N28T,D37A)
VDAKFTKETQSAFYTILHLPNLTEEQRTAFIQSLKDAPSVSKELLAEAKKLNDAQAPKSEQ ID NO:23 zdc(N3A,N6T,Q9T,E15T,N28S,D37T)
VDAKFTKETQSAFYTILHLPNLTEEQRSAFIQSLKDTPSVSKELLAEAKKLNDAQAPK SEQ ID NO:24zdc(N3A,N6A,Q9A,E15T,N28A)
VDAKFAKEAQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
SEQ ID NO:25zdc(N3A,N6A,Q9A,E15T,N28T,D37A)
VDAKFAKEAQSAFYTILHLPNLTEEQRTAFIQSLKDAPSVSKELLAEAKKLNDAQAPK
SEQ ID NO:26zdc(N3A,N6A,Q9A,E15K,N28T,D37T)
VDAKFAKEAQSAFYKILHLPNLTEEQRTAFIQSLKDTPSVSKELLAEAKKLNDAQAPK
作为本发明的优选实施方式,所述免疫球蛋白结合蛋白为由包含氨基酸序列如SEQ ID NO:1所示的序列或其在第3、6、9、15、28、37位至少一个位点发生突变得到的突变序列构成的同源多聚体。
更优选地,所述免疫球蛋白结合蛋白为同源四聚体或六聚体。
作为本发明的优选实施方式,所述免疫球蛋白结合蛋白的氨基酸序列如SEQ IDNO:28~36所示。
所述SEQ ID NO:28~36为如SEQ ID NO:1所示氨基酸序列及其突变序列形成的四聚体或六聚体的氨基酸序列,具体如下:
SEQ ID NO:28zdc四聚体;
VDNKFNKEQQSAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDNKFNKEQQSAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDNKFNKEQQSAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDNKFNKEQQSAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKELLAEAKKLNDAQAPKCSEQ ID NO:29zdc(N3A,N6T,Q9A,E15T,N28A)四聚体;
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPKCSEQ ID NO:30zdc(N3A,N6T,Q9A,E15T,N28A,D37A)四聚体;
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDAPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDAPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDAPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDAPSVSKELLAEAKKLNDAQAPKCSEQ ID NO:31zdc(N3A,N6T,Q9A,E15T,N28A,D37T)四聚体;
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDTPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDTPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDTPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRAAFIQSLKDTPSVSKELLAEAKKLNDAQAPKCSEQ ID NO:32zdc(N3A,N6T,Q9A,E15T,N28T)四聚体;
VDAKFTKEAQSAFYTILHLPNLTEEQRTAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRTAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRTAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRTAFIQSLKDDPSVSKELLAEAKKLNDAQAPKCSEQ ID NO:33zdc(N3A,N6T,Q9A,E15T,N28S)四聚体;
VDAKFTKEAQSAFYTILHLPNLTEEQRSAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRSAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRSAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKEAQSAFYTILHLPNLTEEQRSAFIQSLKDDPSVSKELLAEAKKLNDAQAPKCSEQ ID NO:34zdc(N3A,N6T,Q9T,E15T,N28A)四聚体;
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPKCSEQ ID NO:35zdc(N3A,N6A,Q9A,E15K,N28T,D37T)四聚体;
VDAKFAKEAQSAFYKILHLPNLTEEQRTAFIQSLKDTPSVSKELLAEAKKLNDAQAPK
VDAKFAKEAQSAFYKILHLPNLTEEQRTAFIQSLKDTPSVSKELLAEAKKLNDAQAPK
VDAKFAKEAQSAFYKILHLPNLTEEQRTAFIQSLKDTPSVSKELLAEAKKLNDAQAPK
VDAKFAKEAQSAFYKILHLPNLTEEQRTAFIQSLKDTPSVSKELLAEAKKLNDAQAPKCSEQ ID NO:36zdc(N3A,N6T,Q9T,E15T,N28A)六聚体;
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPK
VDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSVSKELLAEAKKLNDAQAPKVDAKFTKETQSAFYTILHLPNLTEEQRAAFIQSLKDDPSV SKELLAEAKKLNDAQAPKC
通过在多聚体序列的C端添加半胱氨酸“C”,可使其能够和空白微球偶联。上述多聚体碱稳定性比Z四聚体(氨基酸序列如SEQ ID NO:27所示)明显提高,其中各突变体的碱稳定性约为Z序列四聚体的2倍左右。
SEQ ID NO:27Z四聚体(Z-domain四聚体)
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKC
相应地,本发明还要求保护所述的免疫球蛋白结合蛋白的核苷酸序列。
根据本发明提供的氨基酸序列,本领域技术人员不难得到相应的核苷酸序列,并可根据不同生物的密码子特性对其核苷酸序列进行优化。
相应地,本发明还要求保护包含所述核苷酸序列的表达载体。
相应地,本发明还要求保护包含表达载体的细胞。
通过将所述核苷酸序列插入到合适的表达载体,并进一步转染细胞可得到用于表达所述免疫球蛋白结合蛋白的重组载体及细胞,用于所述免疫球蛋白结合蛋白的生产。
进一步地,本发明还要求保护所述免疫球蛋白结合蛋白,所述的核苷酸序列,所述的表达载体,所述的细胞的用途。
所述的核苷酸序列,所述的表达载体,所述的细胞可用于制备所述免疫球蛋白结合蛋白,所述免疫球蛋白结合蛋白可用于免疫球蛋白的亲和层析,特别是使用高浓度碱溶液的免疫球蛋白的亲和层析过程中。
本发明提供了一种由葡萄球菌蛋白A的Z、D、C结构域的部分肽段拼接得到的对碱稳定性提高的免疫球蛋白结合蛋白(zdc结构域);所述免疫球蛋白结合蛋白氨基酸序列如SEQ ID NO:1所示;进一步研究发现,对zdc结构域的第3、6、9、15、28、37位氨基酸进行了突变,可获得了对碱稳定性进一步提高的蛋白A突变体;本发明提供的免疫球蛋白结合蛋白制备的亲和层析填料能耐受0.5~1.0M NaOH在线清洗,具有良好的应用前景。
附图说明
图1为免疫球蛋白结合蛋白zdc四聚体的发酵液SDS-PAGE电泳结果。
图2为用免疫球蛋白结合蛋白亲本和突变体制备的免疫球蛋白结合蛋白亲和层析介质的碱稳定性(1.0M NaOH)的结果。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合附图和具体实施例对本发明作进一步说明。
实施例1免疫球蛋白结合蛋白的表达和纯化
本实施例主要以Z、D、C结构域拼接得到的zdc结构域(SEQ ID NO:1)的四聚体为例进行描述,其他免疫球蛋白结合蛋白,包括zdc结构域的六聚体以及其突变体蛋白(SEQ IDNO:29-SEQ ID NO:36)的四聚体和六聚体或其他聚体等,均按类似的方法进行表达和纯化。
1)工程菌株构建
根据免疫球蛋白结合蛋白zdc四聚体的氨基酸序列(SEQ ID NO:28)反向翻译获得核苷酸序列,根据密码子简并性和大肠杆菌密码子偏好性得到优化的核苷酸序列,连接至大肠杆菌表达质粒pET-30a(+),然后用氯化钙法将质粒导入大肠杆菌中,用含有50μg/mL卡那霉素的LB平板筛选转化子。挑取生长较好的单菌落,接种至含有50μg/mL卡那霉素的LB液体培养基,37℃,220rpm摇床培养,当菌液浓度生长至0.8~1.5之间时,制成含有15%甘油的菌液,-80℃保存,即得用于表达免疫球蛋白结合蛋白zdc四聚体工程菌株。
2)发酵
取工程菌株接种LB液体培养基,37℃,220rpm摇床培养5h,接种含有3L培养基的7.5L发酵罐,通入无菌空气,通过调节转速和通气量维持溶氧(DO)在20%,培养16h加入IPTG进行诱导表达,诱导16h放罐,离心收集菌体。期间取诱导0、12、16h的发酵液,用纯化水稀释10倍,超声破碎10min,离心分别收集破碎上清和破碎沉淀,沉淀用8M尿素溶解,然后进行SDS-PAGE电泳检测(见图1)。
图1从左往右依次为:
第1泳道:诱导0h破碎上清;第2泳道:诱导0h破碎沉淀;第3泳道:蛋白质标准品(从上到下大小为98KD、66.2KD、45KD、31KD、20KD、14.4KD);第4泳道:诱导12h破碎上清;第5泳道:诱导12h破碎沉淀;第6泳道:诱导16h破碎上清;第7泳道:诱导16h破碎沉淀。
由图1可见,免疫球蛋白结合蛋白zdc四聚体正确表达(箭头指示处),主要在上清液中,表达量约为12g/L发酵液。
3)纯化
将1kg菌体用10L纯化水重悬,用700bar高压均质2次,离心收集上清液;调pH到2.0~2.5之间,离心收集上清液;用离子交换层析纯化,收集得到含有免疫球蛋白结合蛋白zdc四聚体的收集液;用5KD超滤膜包超滤除盐浓缩;最后用冷冻干机冻干得到免疫球蛋白结合蛋白zdc四聚体冻干粉。
实施例2免疫球蛋白结合蛋白亲和层析介质的制备
1)微球活化
取10g琼脂糖微球用布氏漏斗真空抽滤干净,然后使用400mL纯化水反复冲洗抽滤,尽量抽干;依次用20% DMSO(二甲基亚砜)、40% DMSO、60% DMSO缓慢冲洗微球并抽滤,尽量抽干;将微球转移至锥形瓶中,加入14.4mL DMSO、ECH(环氧氯丙烷)、2.4mL 9MNaOH,密封反应3h。将微球混合物倒入布氏漏斗,使用100mL 20%乙醇抽滤,然后用100mL纯化水冲洗,抽干至不滴水。
2)偶联
取10g活化后的微球置于干燥的锥形瓶中;向锥形瓶中加入15mL按实施例1方法制备的免疫球蛋白结合蛋白溶液(取0.3g免疫球蛋白结合蛋白,用15mL磷酸钠缓冲液溶解)和40mL磷酸钠缓冲液,充分混匀;用50%磷酸和6M氢氧化钠调节pH至8.5~8.6之间,密封,置于摇床中,37℃,振荡反应15~24h。
3)封闭
将偶联后的微球倒入布氏漏斗,抽滤除去反应液,用200mL去离子水冲洗。将微球转移至锥形瓶中,加入与微球等体积的0.2M碳酸钠溶液,再加入终浓度6%的巯基甘油,调pH至9.0~10.0之间,25℃反应4~8h。将反应后微球转移至布氏漏斗,抽滤除去反应液,用去离子水冲洗中性,再用200mL 0.5M氢氧化钠洗涤,抽干至不滴水,20%乙醇保存。
实施例3免疫球蛋白结合蛋白亲和层析介质的测试
1)免疫球蛋白(IgG)动态载量的测试
取5mL实施例2制备的免疫球蛋白结合蛋白亲和层析介质装柱,用平衡液(20mMPBS,150mM NaCl,pH7.2)平衡至基线平稳,上样人抗体IgG溶液(5mg/mL人IgG,20mM PBS,pH7.2),流速150cm/h,用紫外检测器监测流出液中的人IgG浓度突破10%时的人IgG吸附量,计算出动态结合容量(10%DBC),用洗脱液(100mM甘氨酸,pH3.0)洗脱结合的人IgG。
2)1M NaOH在位清洗(CIP)
将免疫球蛋白结合蛋白亲和层析柱循环暴露在1.0M NaOH和平衡液(20mM PBS,150mM NaCl,pH7.2)环境下,每个循环步骤如下:用1.0M NaOH溶液以1mL/min的速率冲洗层析柱,持续20min,将层析柱静置4h,暴露在室温(20~25℃)环境下;用平衡液以1mL/min的速率冲洗层析柱,持续60min。按此方式,共进行6个循环,即暴露在1.0M NaOH环境下的累积时间为24h。
将经过6个1M NaOH在位清洗(CIP)的免疫球蛋白结合蛋白亲和层析柱,按1)方法测试剩余的动态结合载量。结果见表1。
表1各免疫球蛋白结合蛋白的测试结果
由测试结果可得,本发明的免疫球蛋白结合蛋白相比原Z结构域组成的四聚体耐碱能力明显提高,zdc四聚体耐碱性能约为Z四聚体的130%,各突变体的耐碱性能约为Z四聚体的169%~209%。
最后应当说明的是,以上实施例仅用以说明本发明的技术方案,而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细地说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (5)
1.碱稳定性高的免疫球蛋白结合蛋白,其特征在于,所述的免疫球蛋白结合蛋白的氨基酸序列为如SEQ ID NO:8~26之一所示的序列的同源四聚体或同源六聚体。
2.碱稳定性高的免疫球蛋白结合蛋白,其特征在于,所述免疫球蛋白结合蛋白的氨基酸序列如SEQ ID NO:28~36之一所示。
3.包含如权利要求1或2所述的免疫球蛋白结合蛋白的编码核苷酸序列的表达载体。
4.包含如权利要求 3所述的表达载体的细胞。
5.如权利要求1或2所述的免疫球蛋白结合蛋白,或如权利要求3所述的表达载体,或如权利要求4所述的细胞的在制备免疫球蛋白结合蛋白亲和层析介质中的用途。
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