CN116732072A - Mnr-mbp蛋白质的发酵和纯化工艺 - Google Patents
Mnr-mbp蛋白质的发酵和纯化工艺 Download PDFInfo
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- CN116732072A CN116732072A CN202210195621.4A CN202210195621A CN116732072A CN 116732072 A CN116732072 A CN 116732072A CN 202210195621 A CN202210195621 A CN 202210195621A CN 116732072 A CN116732072 A CN 116732072A
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Abstract
本发明公开了表达MNR‑MBP蛋白质的菌株构建和融合蛋白发酵和纯化工艺。本发明提供的方法和菌株既能够减少重组融合蛋白的降解、又不会对重组融合蛋白的生物活性产生显著不良影响,对于提高使用重组融合蛋白的产品稳定性、降低单位剂量成本具有重要意义。
Description
技术领域
本发明涉及生物医药领域,具体而言,本发明涉及MNR-MBP蛋白质的发酵和纯化工艺。
背景技术
肿瘤细胞疫苗有可能激活免疫系统,让它们直面癌症细胞。肿瘤疫苗诱导肿瘤特异的免疫反应,激活免疫系统进攻带有特殊抗原的癌细胞。在众多免疫抗原中,粘蛋白MUC1被赋予希望。最初发现MUC1具有保护和润滑上皮的功能。后来陆续发现它在细胞信号传导以及从恶性细胞转化到肿瘤扩散的肿瘤发生的所有阶段均起着重要作用。MUC1是一种高度糖基化的跨膜蛋白,是一种I型跨膜蛋白,具有高度糖基化的胞外结构域,从细胞表面延伸200-500纳米。全长分为胞外区、跨膜区和胞内区。胞外区由脯氨酸,苏氨酸和富含丝氨酸的(PTS)域和SEA域组成。PTS域,也叫可变数目串联重复序列(VNTR)区,由高度多态的外显子编码,该外显子编码多个20-21个氨基酸序列重复序列组成,而MUC1的胞内区(CT)是高度保守的。MUC1的过表达通常与结肠癌,乳腺癌,卵巢癌,肺癌和胰腺癌有关。MUC1被证明存在于各种腺癌中。在一项原发性肝癌的病人治疗研究中,MUC1高表达的病人比例高达68%。同时手术后复发的比例也是最高,跟MUC1表达强度呈正相关。另外,MUC1在一些血液恶性肿瘤的也是过度表达。肿瘤组织和正常组织的MUC1不仅在表达量上有差异,肿瘤与正常组织的MUC1糖基化也存在差异。低糖基化的MUC1是在肿瘤细胞的整个表面过度表达,使肿瘤细胞粘附力的降低,为肿瘤转移提供了便利。已经形成的肿瘤中,大量MUC1可以抑制NK细胞对肿瘤细胞的杀伤作用,还可以抑制细胞毒淋巴细胞(CTL)的增殖,甚至可以诱导CTL凋亡。
疫苗接种可以为宿主提供长期的保护作用,且几乎没有副作用,是治疗癌症的重要方法之一。理论上MUC1应该是一个非常好的疫苗抗原,但在人体临床试验中的临床效果并不理想。疫苗产生的抗体大多不能与肿瘤细胞很好地结合,无法有效保护机体杀灭肿瘤。
发明内容
为解决上述问题,申请人研制的针对人体癌症细胞的注射免疫用MUC1重组蛋白MNR-MBP,其中MBP是蛋白质表达促溶成分,有助于蛋白质的纯化,也有研究表明MBP可以诱导机体免疫系统的T淋巴细胞活化。在此基础上,本发明进一步提供其发酵和纯化工艺,对于提高使用重组融合蛋白的产品稳定性、降低单位剂量成本具有重要意义。
本发明第一方面提供一种表达融合蛋白菌株的构建方法,所述方法包括利用融合蛋白基因连接载体、转化、诱导表达和/或所分离的菌体进行发酵的步骤。
根据本发明,所述融合蛋白包括麦芽糖结合蛋白MBP和粘蛋白MUC1-N。
优选地,所述融合蛋白由麦芽糖结合蛋白MBP和粘蛋白MUC1-N串联而成。
更进一步地,所述MUC1-N的氨基酸序列如SEQ ID NO.1所示,所述MBP的氨基酸序列如SEQ ID NO.3所示。优选地,所述MUC1-N的基因核苷酸序列如SEQ ID NO.2所示,所述MBP的基因核苷酸序列如SEQ ID NO.4所示。
更进一步地,所述融合蛋白的氨基酸序列如SEQ ID NO.5所示。根据本发明,所述载体优选pET载体。所述转化使用的感受态细胞,优选BL21(DE3)。所述诱导表达使用IPTG诱导。优选地,所述菌株包含核苷酸序列如SEQ ID NO.6所示的基因。
本发明第二方面提供一种表达融合蛋白的菌株,所述菌株是通过上述构建方法获得的。更优选,通过测定所述菌株的蛋白表达量进行进一步筛选。
本发明第三方面提供一种融合蛋白的发酵方法,所述方法包括利用融合蛋白发酵、再用所述发酵液和/或所分离的菌体进行发酵的步骤。
其中,所述融合蛋白包括麦芽糖结合蛋白MBP和粘蛋白MUC1-N。
优选地,所述融合蛋白由麦芽糖结合蛋白MBP和粘蛋白MUC1-N串联而成。
更优选地,所述MUC1-N的氨基酸序列如SEQ ID NO.1所示,所述MBP的氨基酸序列如SEQ ID NO.3所示。优选地,所述MUC1-N的基因核苷酸序列如SEQ ID NO.2所示,所述MBP的基因核苷酸序列如SEQ ID NO.4所示。
更优选地,所述融合蛋白的氨基酸序列如SEQ ID NO.5所示。
根据本发明,优选利用本发明构建的表达融合蛋白的菌株。优选地,所述菌株包含核苷酸序列如SEQ ID NO.6所示的质粒。所述发酵体系的配方优选5.0g/L酵母提取物、5.0g/L NaCl、3.5g/L KH2PO4、6.6g/L K2HPO4.3H2O、3.5g/L(NH4)2HPO4、1.0g/LMgSO4.7H2O、1mL消泡剂灭菌后加入除菌过滤的0.1g/L VB1、10.0g/L葡萄糖。更优选的,所述发酵体系的配方还含有10.0g/L胰蛋白胨和/或微量元素。
其中,所述微量元素为CuSO450μmol/L,ZnSO430μmol/L,MnSO450μmol/L,FeSO430μmol/L。
根据本发明,优选本发明所述发酵温度37℃。所述发酵培养诱导前培养0-2h的搅拌转速为400rpm,2-3h为550rpm,3-11h为700rpm。空气关联溶氧,起始为1-2vvm,氧气在2vvm后和溶氧关联。葡萄糖低于2g/L开始补料,3-5h补料速率为35mL/L/h,5-7h补料速率为65mL/L/h,7-11h为35mL/L/h。
其中,所述补液为:250g/L酵母提取物,250g/L葡萄糖。所述发酵培养OD600nm值达到50时开始加入IPTG终浓度为0.5mM,37℃,诱导培养4h。
本发明第四方面提供一种融合蛋白的制备方法,所述方法包括发酵含有能表达所述融合蛋白的表达载体的微生物,其中,所述方法还包括利用分离的发酵液中的融合蛋白、将所分离的菌体进行发酵的步骤。
根据本发明,所述方法还包括利用本发明所述的发酵方法对本发明表达融合蛋白的菌株进行发酵的步骤。
本发明第五方面提供一种分离和/或纯化融合蛋白的方法,其特征在于,所述方法包括:
(1)发酵本发明的表达融合蛋白的菌株;
(2)菌体破碎后澄清。
优选地,对步骤(2)菌体破碎后通过过滤澄清。优选用微米过滤澄清,更优选地,深层过滤后用0.2m2、0.2微米滤器过滤澄清。所述深层过滤为用3M、90SP深层膜包过滤,通量为90L/m2。
根据本发明,所述步骤(2)澄清后的澄清液用亲和层析柱进行纯化。优选地,所述亲和层析柱为Dextrin Beads 6FF。
根据本发明,所述步骤(2)澄清后的澄清液进行亲和层析步骤,例如,先用结合缓冲液平衡亲和层析柱Dextrin Beads 6FF,上样超声破碎,再用结合缓冲液洗涤,最后用洗脱缓冲液洗脱。其中,所述结合缓冲液为20mM Tris-HCl,0.2M NaCl,1mM EDTA,pH7.4,洗脱缓冲液为20mM Tris-HCl,0.2M NaCl,1mM EDTA,10mM麦芽糖,pH7.4。
根据本发明,所述方法还包括亲和层析步骤后的一级精纯步骤,例如,使用Diamond MIXA、Bestarose Q HP、Capto MMC impres、POROS XS或Superdex S 75进行一级精纯。其中,Diamond MIXA的上样缓冲液为50mM Tris-HCl(pH7.0),洗脱缓冲液为50mMTris-HCl,0.5M NaCl,(pH7.0),0-100%梯度洗脱。Bestarose Q HP的上样缓冲液为50mMTris-HCl(pH 8.0),洗脱缓冲液为50mM Tris-HCl,0.5M NaCl(pH 8.0);Capto MMC impres的结合缓冲液为20mM PB(pH 4.6-6.0),洗脱缓冲液为20mM PB,1M NaCl(pH 4.6-6.0);POROS XS的结合缓冲液为20mM柠檬酸-柠檬酸钠(pH 4.6),洗脱缓冲液为20mM柠檬酸-柠檬酸钠,0.5M NaCl(pH 4.6);Superdex75的缓冲液为20mM PB,150mM NaCl(pH 7.5)。
根据本发明,所述方法还包括一级精纯之后的二级精纯步骤,例如使用Bestarosephenyl HP、POROS XS或Capto MMC impres二级精纯。优选地,使用Bestarose Q HP与POROSXS组合精纯。
根据本发明,所述步骤(2)包括如下内容:
(2.1)菌体破碎后通过过滤澄清;任选地,
(2.2)所述澄清后的澄清液的亲和层析步骤,例如用亲和层析柱进行纯化;具体地,所述澄清后的澄清液先用结合缓冲液平衡亲和层析柱Dextrin Beads 6FF,上样超声破碎,再用结合缓冲液洗涤,最后用洗脱缓冲液洗脱;任选地,
(2.3)一级精纯步骤;任选地,
(2.4)二级精纯步骤。
本发明第六方面提供一种融合蛋白制剂,其特征在于,所述制剂包括本发明制备的融合蛋白或本发明分离和/或纯化的融合蛋白。
根据本发明,所述制剂还包含佐剂,所述佐剂优选为氢氧化铝。优选,pH4.5-6.5。本发明的有益效果:
第一、本发明在优化重组MNR-MBP融合蛋白分子设计的基础上,进一步通过分子生物学的方法获得了高效表达该融合蛋白的菌株。
第二、本发明进一步利用该菌株对下游发酵和纯化工艺的改进来减少目的蛋白的降解、提高目的蛋白的稳定性。通过对表达该融合蛋白的菌株的发酵条件优化,确定优化发酵体系配方、发酵培养工艺。
第三、本发明在对MNR-MBP融合蛋白在结构、生物活性、理化形状等方面进行系统分析的基础上,结合上游的诱导发酵工艺、下游的分离纯化工艺,通过理化条件的协同,对于发酵液处理的操作条件、参数进行了多级优化选择,本发明提供的方法获得的重组融合蛋白既具有最好的聚集体及片段去除能力、又能保证较高的序列覆盖率和正确性,对于提高使用重组融合蛋白的产品稳定性以及降低单位剂量成本具有重要意义。
第四、本发明制备的融合蛋白形成的制剂,实验结果显示,pH4.5-6.5时,吸附率达到90%以上。
附图说明
图1.MNR-MBP表达工程菌株的PCR鉴定。
图2.SDS-PAGE检测菌株菌体目的蛋白质表达量(M:Marker,250、130、100、70、55、35、25、15、10KD;P:诱导前;1-20:1-20号克隆)。
图3.SDS-PAGE检测细菌菌体和培养液中目的蛋白质表达量(M:Marker,250、130、100、70、55、35、25、15、10KD;1:菌体沉淀;2:菌体上清)。
图4.SDS-PAGE检测细菌菌体和培养液中目的蛋白质表达量(M:Marker,250、130、100、70、55、35、25、15、10KD;1-5:5号菌P1、P5、P10、P15、P20蛋白质表达;6-10:6号菌P1、P5、P10、P15、P20蛋白质表达;11-15:9号菌P1、P5、P10、P15、P20蛋白质表达;16-20:17号菌P1、P5、P10、P15、P20蛋白质表达;21-25:19号菌P1、P5、P10、P15、P20蛋白质表达)。
图5.SDS-PAGE检测9号菌株在不同发酵培养基中的表达(M:Marker,250、130、100、70、55、35、25、15、10KD;1:生长7小时诱导前的F1;2:诱导4小时F1的菌泥;3-4:F1破碎上清;5:F1破碎沉淀;6:生长7小时诱导前的F2;7:诱导4小时F2的菌泥;8-9:F2破碎上清;10:F2破碎沉淀)。
图6.SDS-PAGE检测不同发酵程序发酵菌体纯化的蛋白质纯度(M:Marker,250、130、100、70、55、35、25、15、10KD;1:F1诱导前;2:F1诱导4h;3:F1诱导6h;4:F2诱导前;5:F2诱导4h;6:F2诱导6h;7:F3诱导前;8:F3诱导4h;9:F3诱导6h;10:F1菌体超声上清;11:F1菌体超声上清穿柱液;12:F1洗脱蛋白;13:F2菌体超声上清;14:F2菌体超声上清穿柱液;15:F2洗脱蛋白;16:F3菌体超声上清;17:F3菌体超声上清穿柱液;18:F3洗脱蛋白)。
图7.SDS-PAGE检测不同发酵菌株发酵目标蛋白质占比(M:Marker,250、130、100、70、55、35、25、15、10KD;1:9号诱导前;2:9号诱导4h后;3:19号诱导前;4:19号诱导4h后)。
图8.HPLC分析MNR-MBP。
图9.质谱分析MNR-MBP。
图10.MNR-MBP肽图。
具体实施方式
为了便于理解本发明,下面将对本发明进行更详细的描述。但是,应当理解,本发明可以许多不同的形式来实现,并不限于本文所描述的实施方式或实施例。相反地,提供这些实施方式或实施例的目的是使对本发明的公开内容的理解更加透彻全面。
下文中,仅仅是为说明,只在实施例中描述了其中一少部分,然而不应将其理解为对本发明的限制。除非特殊说明,否则本发明中所使用的试剂都是市售可购买的。
实施例1.MNR-MBP菌株基因鉴定
从-70℃超低温冰柜中取出一管(50μl)感受态菌,置于冰浴备用。加入5μl连接好的质粒(所述质粒是通过发明人的MNR-MBP融合基因与pET载体连接、转化,提取质粒,测序获得。其中MNR-MBP融合基因构建及组成参见CN202111301004.X)混合液,质粒序列如SEQID.NO.6所示:
atccggatatagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatctcagtggtggtggtggtggtgctcgagtgcggccgcaagcttgtcgacggagctcgaattcTTAGTGCGCCGGTGGCGCCGTACTGCCCGGCGCCGGGCGGGTATCCGGGGCACTCGTAACGCCATGCGCCGGTGGGGCCGTGCTGCCCGGCGCCGGACGGGTATCCGGCGCACTCGTCACACCGTGCGCCGGTGGGGCGGTGCTGCCCGGCGCCGGGCGGGTGTCCGGGGCGCTCGTCACACCGTGCGCCGGCGGCGCCGTACTGCCCGGCGCCGGACGCGTATCCGGCGCGCTCGTCACACCATGCGCCGGTGGGGCCGTACTGCCCGGCGCCGGGCGGGTATCCGGGGCGCTCGTAACACCATGCGCCGGCGGGGCGGTACTACCCGGCGCCGGACGCGTATCCGGCGCACTCGTAACACCGTGGGCCGGCGGCGCCGTGCTACCCGGCGCCGGGCGGGTGTCCGGGGCGCTCGTCACACCACTAATACGGCCTTCAATGCCCAGATTGTTGTTATTATTATTGTTATTGTTATTGCTACTGCTGTTGGTCTGCGCATCTTTCAGCGCTTCATCCACGGTCTGACGGCCGCTCGCGGCATTAATCACCGCGGTGCGCACCGCATACCAAAACGCGCTCATCTGCGGAATGTTCGGCATAATTTCGCCTTTCTGCGCGTTTTCCATCGTCGCCGCAATGCGCGGATCTTTCACCAGTTCCTCTTCATAGCTTTTCAGCGCCACCGCGCCCAGCGGTTTATCTTTGTTCACCGCTTCCAGGCCTTCATCGGTCAGCAGATAGTTTTCCAGAAATTCTTTCGCCAGTTCTTTGTTCGGGCTCGCCGCGTTAATGCCCGCGCTCAGCACGCCCACAAACGGTTTGCTCGGCTGACCTTTAAAGGTCGGCAGCACGGTCACGCCATAGTTCACTTTGCTCGTATCAATGTTGCTCCACGCCCACGGGCCGTTAATGGTCATCGCGGTTTCGCCTTTGTTAAACGCCGCTTCCGCAATGCTATAGTCGGTATCCGCGTTCATATGTTTGTTTTTAATCAGATCCACCAGAAAGGTCAGGCCCGCTTTCGCGCCCGCGTTATCCACGCCCACATCTTTAATATCATATTTGCCGTTTTCATATTTAAAGGCATAACCGCCATCCGCCGCAATCAGCGGCCAGGTAAAATACGGTTCTTGCAGGTTAAACATCAGCGCGCTTTTGCCTTTCGCTTTCAGTTCTTTATCCAGCGCCGGAATTTCTTCCCAGGTTTTCGGCGGGTTCGGCAGCAGATCTTTGTTATAAATCAGGCTCAGCGCTTCCACCGCAATCGGATACGCAATCAGTTTGCCGTTATAGCGCACCGCATCCCAGGTAAACGGATACAGTTTATCTTGAAACGCTTTATCCGGGGTAATTTCCGCCAGCAGGCCGCTTTGCGCATAGCCGCCAAAGCGATCATGCGCCCAAAAAATAATATCCGGGCCATCGCCGGTCGCCGCCACTTGCGGAAATTTTTCTTCCAGTTTATCCGGATGTTCCACGGTCACTTTAATGCCGGTATCTTTTTCAAATTTTTTGCCCACTTCCGCCAGGCCGTTATAGCCTTTATCGCCGTTAATCCAAATCACCAGTTTGCCTTCTTCAATTTTcatatggtatatctccttcttaaagttaaacaaaattatttctagaggggaattgttatccgctcacaattcccctatagtgagtcgtattaatttcgcgggatcgagatctcgatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgagatcccggacaccatcgaatggcgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtaagttagctcactcattaggcaccgggatctcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaagaattaattcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgcca
轻轻混匀后放置冰上30min。轻轻摇匀后插入42℃水浴中60秒进行热休克,然后迅速放回冰中,静置3min。在超净工作台中向上述各管中分别加入250μl SOC培养基(不含抗菌素)轻轻混匀,摇床,220rpm,37℃培养1小时。在超净工作台中取上述转化混合液50μl,滴到含50ug/mL kana的固体LB平板培养皿中,用酒精灯烧过的玻璃涂布棒涂布均匀。待LB平板上液体在超净台中干燥后,倒置放入37度温箱中过夜静置培养。
12小时后挑取转化后平板中20株单菌落,分别接种10ml LB培养基(含50ug/mlkana)过夜培养用于菌株扩增。37℃,200rpm培养16h,菌体OD值分别为3.42、3.43、3.65、3.74、3.78、3.68、3.74、3.66、3.78、3.75、3.77、3.64、3.77、3.72、3.90、3.69、3.93、3.36、3.92、3.93。菌株生长状况相似,其中5、9、15、17、19、20略高。同时对20株进行PCR鉴定,结果显示1-20号克隆均带有约2000bp的目的基因(图1)。
实施例2.MNR-MBP菌株摇瓶蛋白质表达鉴定
将20株候选克隆按1:10接种比例分别接种10ml的LB-kana(50ml离心管中)摇管,并经过终浓度为1mM的IPTG、37℃、200rpm诱导表达,诱导起始OD600nm为0.7,诱导时间为4.5小时。诱导结束后测定菌液OD600nm值,见下表。
表1.MNR-MBP表达工程菌株的菌体表达产量的筛选
同时用SDS-PAGE检测目的蛋白质表达量,见上表和图2。2、4、5、6、7、8、9、10、13、17、19克隆的蛋白质表达较高,定为高产细胞株进行后续研究。
经过SDS-PAGE分析,细菌上清液中无明显目的蛋白质表达,目的蛋白质主要表达于菌体中(图3)。后续纯化主要从菌体中纯化蛋白质,不考虑上清液。
挑选杂蛋白较少的5、6、9、17、19克隆的P1、P5、P10、P15、P20代菌种,按照1:1000比例分别接种到5ml LB培养基中过夜,第二天按照1:10比例接种到10ml LB培养基(含50ug/ml kana)中,37℃,180rpm培养到OD值到2。加入IPTG终浓度为0.5mM,30℃,180rpm诱导培养4h,取样做SDS-PAGE分析。P15表达较高的是9、19号,P20表达较高的是19号(图4)。而不加卡那霉素传代则不能表达目的蛋白。
实施例3.MNR-MBP菌株质粒保有率测定
将9、19号克隆P1、P10、P20复苏种子涂布于不含抗生素的LB培养基上,37℃培养过夜,分别挑取单菌落50个到含与不含卡那霉素的LB培养基,37℃培养过夜,计算工程菌传代时质粒保有率。质粒保有率=含卡那霉素LB平板上菌落数/不含卡那霉素LB平板上菌落数×100%。
表2.MNR-MBP菌株质粒保有率
结果显示19号克隆的质粒保有率略高(表2)。
实施例4.MNR-MBP菌株发酵条件优化
5L发酵体系配方优化,F1配方:5.0g/L酵母提取物、10.0g/L胰蛋白胨、5.0g/LNaCl、3.5g/L KH2PO4、6.6g/L K2HPO4.3H2O、3.5g/L(NH4)2HPO4、1.0g/L MgSO4.7H2O、1mL消泡剂灭菌后加入除菌过滤的0.1g/L VB1、10.0g/L葡萄糖;F2配方:5.0g/L酵母提取物、10.0g/L胰蛋白胨、5.0g/L NaCl、3.5g/L KH2PO4、6.6g/L K2HPO4.3H2O、3.5g/L(NH4)2HPO4、1.0g/LMgSO4.7H2O、1mL消泡剂灭菌后加入除菌过滤的0.1g/L VB1、10.0g/L葡萄糖,微量元素液(CuSO450μmol/L,ZnSO430μmol/L,MnSO450μmol/L,FeSO430μmol/L)。
按照1:1000比例接种37℃、200rpm培养14小时制备种子,再按照1:10比例接种到2L发酵培养基37℃培养,搅拌转数为400-700rpm,诱导前溶氧为30%,关联搅拌。pH为7.0,关联磷酸和氨水。OD600nm值达到30时开始诱导。加入IPTG终浓度为0.5mM,28℃,诱导培养4h,葡萄糖低于1.0g/L添加补料培养基(200g/L酵母提取物、200g/L葡萄糖),速度为1-2%。诱导结束后取样,并经过超声破碎后做SDS-PAGE分析,结果表明:诱导产生的蛋白质位于破碎上清中,两种配方表达的蛋白质含量接近。2L发酵液的菌泥产量F1为152g,F2为164g,菌泥单位产量分别为69、75g/L。F2配方较优,但考虑到微量元素添加对菌泥增加量有限,而灭菌较为复杂,在后续实验中不建议使用。
进一步优化5L发酵体系发酵温度,F1发酵程序:5.0g/L酵母提取物、5.0g/L NaCl、3.5g/L KH2PO4、6.6g/L K2HPO4.3H2O、3.5g/L(NH4)2HPO4、1.0g/L MgSO4.7H2O、1mL消泡剂灭菌后加入除菌过滤的0.1g/L VB1、10.0g/L葡萄糖;OD600nm值达到50时开始诱导。加入IPTG终浓度为0.5mM,30℃,诱导培养5h,葡萄糖低于1.0g/L添加补料培养基;F2发酵程序:5.0g/L酵母提取物、10.0g/L胰蛋白胨、5.0g/L NaCl、3.5g/L KH2PO4、6.6g/L K2HPO4.3H2O、3.5g/L(NH4)2HPO4、1.0g/L MgSO4.7H2O、1mL消泡剂灭菌后加入除菌过滤的0.1g/L VB1、10.0g/L葡萄糖;OD600nm值达到50时开始诱导。加入IPTG终浓度为0.5mM,37℃,诱导培养5h,葡萄糖低于1.0g/L添加补料培养基;F3发酵程序:5.0g/L酵母提取物、10.0g/L胰蛋白胨、5.0g/L NaCl、3.5g/L KH2PO4、6.6g/LK2HPO4.3H2O、3.5g/L(NH4)2HPO4、1.0g/L MgSO4.7H2O、1mL消泡剂灭菌后加入除菌过滤的0.1g/L VB1、10.0g/L葡萄糖;OD600nm值达到50时开始诱导。加入IPTG终浓度为0.5mM,30℃,诱导培养5h,葡萄糖低于1.0g/L添加补料培养基。发酵结束后,收集F1、F2、F3菌泥,按照1:10加入破碎缓冲液进行超声破碎,离心后收集上清上亲和柱(1g菌泥,超声上清10ml)。
表3.MNR-MBP表达工程菌纯化出的蛋白质产量
结果显示F2发酵程序蛋白质表达在诱导后4-6小时稳定,捕获的蛋白质产量最高(表3),即37℃培养温度较30℃可明显提高蛋白表达量;基础培养基中添加蛋白胨利于蛋白表达,但是蛋白质的纯度稍低,但考虑到微量元素添加对菌泥增加量有限,而灭菌较为复杂,在后续实验中不建议使用(图5)。提高诱导表达OD(50vs 30)可增加菌泥生产总量进而提高蛋白表达量。确定发酵培养工艺诱导前培养0-2h的搅拌转速为400rpm,2-3h为550rpm,3-11h为700rpm。空气关联溶氧,起始为1-2vvm,氧气在2vvm后和溶氧关联。葡萄糖低于2g/L开始补料,3-5h补料速率为35mL/L/h,5-7h补料速率为65mL/L/h,7-11h为35mL/L/h。
实施例5.MNR-MBP高产菌株筛选
挑选9、19克隆的P1代菌种进行5L发酵对比实验,发酵配方为:5.0g/L酵母提取物、10.0g/L胰蛋白胨、5.0g/L NaCl、3.5g/L KH2PO4、6.6g/L K2HPO4.3H2O、3.5g/L(NH4)2HPO4、1.0g/L MgSO4.7H2O、1mL消泡剂灭菌后加入除菌过滤的0.1g/L VB1、10.0g/L葡萄糖;补液为:250g/L酵母提取物,250g/L葡萄糖。发酵培养OD600nm值达到50时开始加入IPTG终浓度为0.5mM,37℃,诱导培养4h。进行SDS-PAGE和灰度计算目标蛋白质占比。结果显示19号菌株产率和目标蛋白质占比都较高(表4,图6),确定为生产菌株。
表4.MNR-MBP表达菌株发酵筛选
实施例6.MNR-MBP的初纯、一级中纯
菌体破碎后采用方案1:离心+300K超滤(10000rpm离心20min,再用科佰特0.1平米膜超滤膜包);方案2:离心+深层过滤(10000rpm离心20min,再用3M、90SP深层膜包过滤,通量为90L/m2);方案3:深层过滤+0.2微米过滤(深层过滤后用0.2m2、0.2微米滤器过滤)。方案1、2、3最后澄清的样本浊度小于10、160和15NTU(Nephelometric turbility unit),最后采用方案3进行破碎菌体的澄清。
澄清液先用结合缓冲液(20mM Tris-HCl,0.2M NaCl,1mM EDTA,pH7.4)平衡天地人和公司亲和层析柱Dextrin Beads 6FF体积157ml,上样超声破碎来源于102g菌体的上清157ml,再用结合缓冲液洗涤,最后用洗脱缓冲液(20mM Tris-HCl,0.2M NaCl,1mM EDTA,10mM麦芽糖,pH7.4)洗脱目的蛋白质,得到133mg蛋白质。再使用Diamond MIX A、BestaroseQ HP、Capto MMC impres、POROS XS、Superdex S 75进行一级精纯。Diamond MIXA的上样缓冲液为50mM Tris-HCl(pH7.0),洗脱缓冲液为50mM Tris-HCl,0.5M NaCl,(pH7.0),0-100%梯度洗脱。Bestarose Q HP的上样缓冲液为50mM Tris-HCl(pH 8.0),洗脱缓冲液为50mM Tris-HCl,0.5M NaCl(pH 8.0);Capto MMC impres的结合缓冲液为20mM PB(pH 4.6/5.2/6.0),洗脱缓冲液为20mM PB,1M NaCl(pH4.6/5.2/6.0);POROS XS的结合缓冲液为20mM柠檬酸-柠檬酸钠(pH 4.6),洗脱缓冲液为20mM柠檬酸-柠檬酸钠,0.5M NaCl(pH4.6);Superdex75的缓冲液为20mM PB,150mM NaCl(pH 7.5)。结果显示Diamond MIX A、Bestarose Q HP、POROS XS、Capto MMC impres、Superdex S 75都具有一定的纯化效果(表5)。
表5.MNR-MBP蛋白质的一级精纯工艺效果比较
实施例7.MNR-MBP的二级精纯
Diamond MIX A+Bestarose phenyl HP精纯组合,SEC纯度难以达到95%以上;Bestarose Q HP+Bestarose phenyl HP精纯组合,Bestarose phenyl HP提纯效果较差;Bestarose Q HP+Capto MMC impres精纯组合有一定的聚集体及片段去除能力;BestaroseQ HP+POROS XS精纯组合有最好的聚集体及片段去除能力(表6)
表6.MNR-MBP蛋白质的二级精纯工艺效果比较
实施例8.MNR-MBP蛋白质确证
Bestarose Q HP+POROS XS二级精纯的的蛋白质经过HPLC分析,在11.47分钟洗脱出紫外峰,峰面积占比为96%(图7),经过质谱分析确定其分子量为55792(图8),和理论预测值60KD接近。
肽图分析发现97%的序列可以被覆盖,总离子流色谱图(Total IonsChromatograph)显示正确,69.61处显示的是漏切肽段(图9)。
实施例9.MNR-MBP的制剂应用
氢氧化铝佐剂对MNR-MBP吸附率的测定时先将蛋白质和氢氧化铝佐剂混合,10分钟后13800×g离心5min,取上清测定蛋白质浓度,计算吸附率。结果显示吸附率较好,pH4.5-6.5时,吸附率达到90%以上(表7)。
表7.MNR-MBP蛋白质的佐剂吸附性能
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
SEQUENCE LISTING
<110> 元本(珠海横琴)生物科技有限公司
<120> MNR-MBP蛋白质的发酵和纯化工艺
<130> CPCN22110019
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aacaaacaca tgaacgctga caccgattat tctattgcgg aggcggcttt taacaagggc 660
gagaccgcaa tgaccatcaa cggtccgtgg gcttggtcta acatcgacac ctccaaagta 720
aattacggtg ttaccgtcct gccgaccttc aaaggtcaac cgagcaaacc gttcgtgggc 780
gtgctgtccg caggtatcaa cgctgcctcc ccaaacaaag agctggccaa agagttcctg 840
gaaaactatc tgctgaccga cgaaggcctg gaagctgtta ataaagacaa accgctgggt 900
gctgttgcac tgaaatccta tgaagaagaa ctggtcaaag atccgcgtat tgccgccact 960
atggagaacg cgcagaaagg tgaaatcatg ccgaacatcc cgcaaatgtc cgctttttgg 1020
tacgcggtgc gtaccgctgt aattaacgcg gcgtccggtc gtcagactgt cgatgaagcg 1080
ctgaaagatg ctcagactaa ctctagctct aacaataaca ataataacaa caacaacaat 1140
ctgggtattg aaggtcgcat ctct 1164
<210> 5
<211> 528
<212> PRT
<213> 人工合成
<400> 5
Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly
1 5 10 15
Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
20 25 30
Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro
35 40 45
Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His
50 55 60
Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr
65 70 75 80
Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
85 90 95
Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala
100 105 110
Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr
115 120 125
Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys
130 135 140
Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
145 150 155 160
Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr
165 170 175
Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
180 185 190
Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr
195 200 205
Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met
210 215 220
Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
225 230 235 240
Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys
245 250 255
Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn
260 265 270
Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
275 280 285
Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu
290 295 300
Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala Thr
305 310 315 320
Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met
325 330 335
Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser
340 345 350
Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn Ser
355 360 365
Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile Glu
370 375 380
Gly Arg Ile Ser Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
385 390 395 400
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
405 410 415
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
420 425 430
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
435 440 445
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
450 455 460
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
465 470 475 480
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
485 490 495
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
500 505 510
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
515 520 525
<210> 6
<211> 6859
<212> DNA
<213> 人工合成
<400> 6
atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60
ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120
tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180
cgacggagct cgaattctta gtgcgccggt ggcgccgtac tgcccggcgc cgggcgggta 240
tccggggcac tcgtaacgcc atgcgccggt ggggccgtgc tgcccggcgc cggacgggta 300
tccggcgcac tcgtcacacc gtgcgccggt ggggcggtgc tgcccggcgc cgggcgggtg 360
tccggggcgc tcgtcacacc gtgcgccggc ggcgccgtac tgcccggcgc cggacgcgta 420
tccggcgcgc tcgtcacacc atgcgccggt ggggccgtac tgcccggcgc cgggcgggta 480
tccggggcgc tcgtaacacc atgcgccggc ggggcggtac tacccggcgc cggacgcgta 540
tccggcgcac tcgtaacacc gtgggccggc ggcgccgtgc tacccggcgc cgggcgggtg 600
tccggggcgc tcgtcacacc actaatacgg ccttcaatgc ccagattgtt gttattatta 660
ttgttattgt tattgctact gctgttggtc tgcgcatctt tcagcgcttc atccacggtc 720
tgacggccgc tcgcggcatt aatcaccgcg gtgcgcaccg cataccaaaa cgcgctcatc 780
tgcggaatgt tcggcataat ttcgcctttc tgcgcgtttt ccatcgtcgc cgcaatgcgc 840
ggatctttca ccagttcctc ttcatagctt ttcagcgcca ccgcgcccag cggtttatct 900
ttgttcaccg cttccaggcc ttcatcggtc agcagatagt tttccagaaa ttctttcgcc 960
agttctttgt tcgggctcgc cgcgttaatg cccgcgctca gcacgcccac aaacggtttg 1020
ctcggctgac ctttaaaggt cggcagcacg gtcacgccat agttcacttt gctcgtatca 1080
atgttgctcc acgcccacgg gccgttaatg gtcatcgcgg tttcgccttt gttaaacgcc 1140
gcttccgcaa tgctatagtc ggtatccgcg ttcatatgtt tgtttttaat cagatccacc 1200
agaaaggtca ggcccgcttt cgcgcccgcg ttatccacgc ccacatcttt aatatcatat 1260
ttgccgtttt catatttaaa ggcataaccg ccatccgccg caatcagcgg ccaggtaaaa 1320
tacggttctt gcaggttaaa catcagcgcg cttttgcctt tcgctttcag ttctttatcc 1380
agcgccggaa tttcttccca ggttttcggc gggttcggca gcagatcttt gttataaatc 1440
aggctcagcg cttccaccgc aatcggatac gcaatcagtt tgccgttata gcgcaccgca 1500
tcccaggtaa acggatacag tttatcttga aacgctttat ccggggtaat ttccgccagc 1560
aggccgcttt gcgcatagcc gccaaagcga tcatgcgccc aaaaaataat atccgggcca 1620
tcgccggtcg ccgccacttg cggaaatttt tcttccagtt tatccggatg ttccacggtc 1680
actttaatgc cggtatcttt ttcaaatttt ttgcccactt ccgccaggcc gttatagcct 1740
ttatcgccgt taatccaaat caccagtttg ccttcttcaa ttttcatatg gtatatctcc 1800
ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct 1860
atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg 1920
tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg 1980
atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg 2040
tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg 2100
cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc 2160
ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg 2220
gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta 2280
tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag 2340
gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat 2400
tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt 2460
gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc 2520
gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc 2580
tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat 2640
ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta 2700
tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt 2760
acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg 2820
ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact 2880
cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt 2940
caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac 3000
gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg 3060
gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta 3120
accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 3180
ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga 3240
aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 3300
atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 3360
tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa 3420
cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt 3480
cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga 3540
ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt 3600
gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca 3660
ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag 3720
gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga 3780
gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt 3840
cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat 3900
gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt 3960
aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc 4020
cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat 4080
cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta 4140
cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag 4200
aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga 4260
catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt 4320
cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct 4380
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg 4440
tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat 4500
gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa ataccgcaca 4560
gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc 4620
tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 4680
tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 4740
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 4800
agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 4860
accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 4920
ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 4980
gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 5040
ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 5100
gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 5160
taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 5220
tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 5280
gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 5340
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 5400
agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac 5460
ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg 5520
ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat 5580
gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg 5640
tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta 5700
aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat 5760
gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa 5820
tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat 5880
tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg 5940
caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc 6000
tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca 6060
gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata 6120
ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta 6180
tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt 6240
attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa 6300
gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 6360
tccgcgcaca tttccccgaa aagtgccacc tgaaattgta aacgttaata ttttgttaaa 6420
attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa 6480
aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa 6540
caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca 6600
gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg 6660
taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc 6720
ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc 6780
aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca 6840
gggcgcgtcc cattcgcca 6859
Claims (10)
1.一种表达融合蛋白菌株的构建方法,其特征在于,所述方法包括利用融合蛋白基因连接载体、转化、诱导表达和/或所分离的菌体进行发酵的步骤。所述融合蛋白包括麦芽糖结合蛋白MBP和粘蛋白MUC1-N。
优选地,所述融合蛋白由麦芽糖结合蛋白MBP和粘蛋白MUC1-N串联而成。
更优选地,所述MUC1-N的氨基酸序列如SEQ ID NO.1所示,所述MBP的氨基酸序列如SEQID NO.3所示。优选地,所述MUC1-N的基因核苷酸序列如SEQ ID NO.2所示,所述MBP的基因核苷酸序列如SEQ ID NO.4所示。
更优选地,所述融合蛋白的氨基酸序列如SEQ ID NO.5所示。
2.根据权利要求1的构建方法,其特征在于,所述载体优选pET载体。所述转化使用的感受态细胞优选BL21(DE3)。所述诱导表达使用IPTG诱导。优选地,所述菌株包含核苷酸序列如SEQ ID NO.6所示的基因。
3.一种表达融合蛋白的菌株,其特征在于,所述菌株是通过权利要求1或2的构建方法获得的。更优选,通过测定所述菌株的蛋白表达量进行进一步筛选。
4.一种融合蛋白的发酵方法,其特征在于,所述方法包括利用融合蛋白、发酵、再用所述发酵液和/或所分离的菌体进行发酵的步骤。其中,所述融合蛋白包括麦芽糖结合蛋白MBP和粘蛋白MUC1-N。
优选地,所述融合蛋白由麦芽糖结合蛋白MBP和粘蛋白MUC1-N串联而成。
更优选地,所述MUC1-N的氨基酸序列如SEQ ID NO.1所示,所述MBP的氨基酸序列如SEQID NO.3所示。优选地,所述MUC1-N的基因核苷酸序列如SEQ ID NO.2所示,所述MBP的基因核苷酸序列如SEQ ID NO.4所示。
更优选地,所述融合蛋白的氨基酸序列如SEQ ID NO.5所示。
5.根据权利要求4的发酵方法,其特征在于,利用权利要求1或2的构建方法构建的表达融合蛋白的菌株。优选地,所述菌株包含核苷酸序列如SEQ ID NO.6所示的质粒。其中,所述发酵体系的配方优选5.0g/L酵母提取物、5.0g/L NaCl、3.5g/L KH2PO4、6.6g/LK2HPO4.3H2O、3.5g/L(NH4)2HPO4、1.0g/L MgSO4.7H2O、1mL消泡剂灭菌后加入除菌过滤的0.1g/L VB1、10.0g/L葡萄糖。更优选的,所述发酵体系的配方还含有10.0g/L胰蛋白胨和/或微量元素。
其中,所述微量元素为CuSO450μmol/L,ZnSO430μmol/L,MnSO450μmol/L,FeSO430μmol/L。
优选地,所述发酵温度37℃。所述发酵培养诱导前培养0-2h的搅拌转速为400rpm,2-3h为550rpm,3-11h为700rpm。空气关联溶氧,起始为1-2vvm,氧气在2vvm后和溶氧关联。葡萄糖低于2g/L开始补料,3-5h补料速率为35mL/L/h,5-7h补料速率为65mL/L/h,7-11h为35mL/L/h。
其中,所述补液为:250g/L酵母提取物,250g/L葡萄糖。所述发酵培养OD600nm值达到50时开始加入IPTG终浓度为0.5mM,37℃,诱导培养4h。
6.一种融合蛋白的制备方法,其特征在于,所述方法包括发酵含有能表达所述融合蛋白的表达载体的微生物,其中,所述方法还包括利用分离的发酵液中的融合蛋白、将所分离的菌体进行发酵的步骤。
优选地,所述方法还包括利用权利要求4或5所述的发酵方法对权利要求3所述的表达融合蛋白的菌株进行发酵的步骤。
7.一种分离和/或纯化融合蛋白的方法,其特征在于,所述方法包括:
(1)发酵权利要求3所述的表达融合蛋白的菌株;
(2)菌体破碎后澄清。
优选地,步骤(2)菌体破碎后,通过过滤澄清,优选地,用微米过滤澄清,更优选地,深层过滤后用0.2m2、0.2微米滤器过滤澄清。所述深层过滤为用3M、90SP深层膜包过滤,通量为90L/m2。
8.根据权利要求7所述方法,其特征在于,所述步骤(2)获得的澄清液用亲和层析柱进行纯化。优选地,所述亲和层析柱为Dextrin Beads 6FF。优选地,所述澄清液先用结合缓冲液平衡亲和层析柱Dextrin Beads 6FF,上样超声破碎,再用结合缓冲液洗涤,最后用洗脱缓冲液。其中,所述结合缓冲液为20mM Tris-HCl,0.2M NaCl,1mM EDTA,pH7.4,洗脱缓冲液为20mM Tris-HCl,0.2M NaCl,1mM EDTA,10mM麦芽糖,pH7.4。
优选地,所述方法还包括亲和层析步骤后的一级精纯步骤。优选使用Diamond MIX A、Bestarose Q HP、Capto MMC impres、POROS XS、Superdex S 75进行一级精纯。
其中,Diamond MIX A的上样缓冲液为50mM Tris-HCl(pH7.0),洗脱缓冲液为50mMTris-HCl,0.5M NaCl,(pH7.0),0-100%梯度洗脱。Bestarose Q HP的上样缓冲液为50mMTris-HCl(pH 8.0),洗脱缓冲液为50mM Tris-HCl,0.5M NaCl(pH 8.0);Capto MMC impres的结合缓冲液为20mM PB(pH 4.6-6.0),洗脱缓冲液为20mM PB,1M NaCl(pH 4.6-6.0);POROS XS的结合缓冲液为20mM柠檬酸-柠檬酸钠(pH 4.6),洗脱缓冲液为20mM柠檬酸-柠檬酸钠,0.5M NaCl(pH 4.6);Superdex75的缓冲液为20mM PB,150mM NaCl(pH 7.5)。
9.根据权利要求7或8所述方法,其特征在于,所述方法还包括一级精纯之后的二级精纯步骤。优选地,使用Bestarose phenyl HP、POROS XS或Capto MMC impres二级精纯。更优选地,使用Bestarose Q HP与POROS XS组合精纯。
10.一种融合蛋白制剂,其特征在于,所述制剂包括权利要求6制备的融合蛋白或权利要求7-9中任一方法分离和/或纯化的融合蛋白。
优选地,所述制剂还包含佐剂。其中,所述佐剂优选为氢氧化铝。优选地,pH4.5-6.5。
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