CN116497050A - 敲除sigW基因在提高地衣芽胞杆菌生产Protein A蛋白中的应用 - Google Patents
敲除sigW基因在提高地衣芽胞杆菌生产Protein A蛋白中的应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,公开了敲除sigW基因在提高地衣芽胞杆菌生产Protein A蛋白中的应用。本发明通过敲除地衣芽胞杆菌BL10中sigW基因构建了重组菌株BL10ΔsigW,在此基础上引入来源于金黄色葡萄球菌重组蛋白Protein A表达载体。相比对照菌株,缺失sigW基因可以特异性提高了重组Protein A的表达量。本发明的重组Protein A可广泛用于免疫球蛋白亲和层析技术中。本本发明首次使用芽胞杆菌表达重组Protein A,无细菌内毒素产生,敲除sigW极大地提高了Protein A的产量,显著降低生产成本,为Protein A的生产提供了新的思路和方法。
Description
技术领域
本发明属于基因工程技术领域,具体涉及敲除sigW基因在提高地衣芽胞杆菌生产Protein A蛋白中的应用。
背景技术
金黄色葡萄球菌蛋白A(Protein A)是金黄色葡萄球菌细胞壁上的一种膜蛋白,能够特异性与免疫球蛋白的Fc结构域结合。Protein A是目前研究最多的免疫球蛋白结合蛋白之一,免疫球蛋白结合蛋白是由细菌产生的特异结合宿主抗体的细菌免疫球蛋白结合蛋白。研究表明细菌免疫球蛋白结合蛋白的抗体结合区,是由多个序列高度同源的结构域以头尾相连组成,每个单结构域具有与分子完全相同的结合特性,形成免疫球蛋白结合功能的基本单位。
天然Protein A分子量42KD,由三个部分组成,自N端开始分别为:信号肽S、Ig结合结构域和C-末端X蛋白。Protein A含有五个高度同源的单结构域,自N端起分别为E、D、A、B和C,每个单结构域约含58个氨基酸残基,各结构域形成3股反向平行排列的α-螺旋,可以和哺乳动物IgG抗体重链中,第二及第三恒定区间的分界面结合,以螺旋1和螺旋2与Fc的疏水作用为主,并通过两个分子之间的四对氢键得到进一步稳定。
Protein A亲和层析法是利用Protein A蛋白可与IgG上的Fc段恒定区特异性地结合,早期的Protein A柱结合的都是天然Protein A。天然Protein A由5个IgG结合域和其它未知功能的非Fc结合域组成。这种柱子对IgG的亲和能力很强,可以吸附大量的IgG。但同时,天然Protein A的其他非结合域会和非目标蛋白结合,这样被洗脱下来的蛋白质纯度不够,会影响到后续的试验。
但是,现有技术中的Protein A还存在以下问题:现有技术中的Protein A重组蛋白大部分是用大肠杆菌原核表达系统制备的,内毒素含量比较高,需要去除内毒素;导致现有技术中的Protein A亲和纯化技术介质价格比较昂贵,提高了实验或生产的成本。地衣芽胞杆菌是公认的生物安全菌株,被作为益生菌使用,不产生内毒素,因其具有蛋白表达能力强、抗噬菌体和杂菌污染、溶剂耐受性强、生产效率快等优势,被广泛应用于生产酶制剂,因此,地衣芽胞杆菌是目前异源蛋白工业化生产的优良宿主。
Sigma W是芽胞杆菌胞外sigma因子的一种,调节芽胞细胞胞外的一些生理活动,如生物膜的形成等。然而,其表达水平是否影响异源蛋白表达是未知的。本发明通过在地衣芽胞杆菌BL10中敲除sigW,显著提高了重组Protein A的表达水平,说明敲除sigW是提高地衣芽胞杆菌发酵生产Protein A水平的有效策略。
发明内容
本发明就是为了解决上述技术问题,本发明提供了敲除sigW基因在提高地衣芽胞杆菌生产Protein A蛋白中的应用,所述的sigW基因的序列为SEQ ID NO.1所示。
本发明是另一个目的是提供了利用上述应用得到的Protein A蛋白的应用。
为了达到上述目的,本发明采取以下技术措施:
敲除sigW基因在提高地衣芽胞杆菌生产Protein A蛋白中的应用,包括利用本发明的常规方法,将地衣芽胞杆菌中的sigW基因敲除,获得地衣芽胞杆菌sigW基因缺失株,然后在该缺失株中转入Protein A表达载体后进行发酵表达即可。
以上所述的应用中,优选的,所述的Protein A为SEQ ID NO.2所示,编码其的基因为SEQ ID NO.3所示。
以上所述的应用中,优选的,所述的地衣芽胞杆菌为地衣芽胞杆菌BL10。
以上所述的应用中,优选的,应用过程为发酵反应,发酵培养基为:20-50g/L葡萄糖、5-15g/L骨蛋白胨、5-15g/L大豆蛋白胨、0-20g/L玉米浆,5-15g/L酵母粉,5-10g/L氯化钠,2-5g/L K2 HPO4,4-8g/L(NH4)2SO4,其余为水,pH 6.8~7.4。
以上所述应用过程生产的Protein A蛋白在制备亲和填料中的应用。
与现有技术相比,本发明具有以下优点:
本发明采用基因工程方法通过敲除地衣芽胞杆菌BL10中的sigW基因,在此菌株的基础上构建高效表达重组Protein A的重组芽胞杆菌株。相比对照菌株,Protein A的产量至少提高30%。本发明的重组Protein A可广泛用于免疫球蛋白亲和层析技术中。本发明还公开了一种由重组Protein A和琼脂糖凝胶相偶联而成的高产无内毒素抗体Protein A亲和填料。本本发明首次使用芽胞杆菌表达重组Protein A,无细菌内毒素产生,并通过敲除sigW基因极大地提高了Protein A的产量,显著降低生产成本,能够产生巨大的经济效益,为重组Protein A的生产提供了新的思路和方法。
附图说明
图1是本发明pHY-Protein A重组质粒的构建。
图2是本发明分泌的重组Protein A蛋白的纯化图。
图3是利用本发明制备的重组ProteinA亲和树脂纯化人IgG蛋白的纯化图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。本发明所述技术方案,如未特别说明,均为本领域的常规方案;所述试剂或材料,如未特别说明,均来源于商业渠道。
实施例1:
sigW基因缺失株地衣芽胞杆菌BL10△sigW的获得:
1、根据地衣芽胞杆菌WX-02(CN104630124A)基因组DNA序列中sigW基因序列,设计sigW基因的上游同源臂引物(sigW-AF、sigW-AR)和下游同源臂引物(sigW-BF、sigW-BR);并以地衣芽胞杆菌WX-02的基因组DNA为模板,分别以sigW基因的上游同源臂引物、下游同源臂引物进行PCR扩增得到sigW基因的上游同源臂片段和sigW基因的下游同源臂片段;
2、通过重叠延伸PCR将sigW基因的上游同源臂和下游同源臂连接到一起,得到目的基因片段;
3、以T2质粒为模板,以引物T2-T5-F和T2-T5-R进行PCR扩增得到T2骨架;
4、采用ExnaseⅡT5外切酶对步骤2中的目的基因片段和步骤3中的骨架进行反应;通过氯化钙转化法将该反应产物转入大肠杆菌DH5α,在37℃的条件下经含有四环素抗性的培养基进行筛选,筛选得到转化子,对转化子进行菌落PCR验证,所用引物为:T2-YF,T2-YR;将菌落PCR正确的阳性转化子,命名为:敲除载体T2-ΔsigW;
5、将敲除载体T2-ΔsigW转入地衣芽胞杆菌BL10(CN104630123A)感受态细胞中,在37℃的条件下经含有卡那青霉素抗性的培养基进行筛选,筛选得到转化子,对转化子挑质粒进行菌落PCR验证(所用引物为:T2-F和T2-R),获得阳性转化子(即转入了敲除载体T2-ΔsigW的地衣芽胞杆菌BL10);
6、将步骤5得到的阳性转化子在45℃条件下、含有卡那青霉素抗性的培养基上转接培养3次,每次培养12h,并以T2-F和ΔsigW-KYR为引物(或以T2-R与ΔsigW-KYF为引物)进行菌落PCR检测,获得单交换菌株;
7、将步骤6得到的单交换菌株在37℃、不含有卡那青霉素的培养基中经过数次转接培养,挑转化子进行菌落PCR验证(引物为ΔsigW-KYF和ΔsigW-KYR)。随后针对阳性转化子进行DNA测序进一步验证,得到双交换成功的sigW敲除菌株(即地衣芽胞杆菌BL10ΔsigW)。T2-YF:ATGTGATAACTCGGCGTA
T2-YR:GCAAGCAGCAGATTACGC
T2-T5-F:GGATCCCCCGGGCTGCAGGAATTC
T2-T5-R:GAGCTCGTAGAAAAGATCAAAGGA
sigW-AF:CTGCAGCCCGGGGGATCCCGCGACAAAGGGATCAAA
sigW-AR:CTCATTTCATCACCCCACATTTATCTAACCTCTGCCTT
sigW-BF:AAGGCAGAGGTTAGATAAATGTGGGGTGATGAAATGAG
sigW-BR:GATCTTTTCTACGAGCTCACTTGTCCTGCTGAAGCC
ΔsigW-KYF:ACATCGTCATCATCGGTG
ΔsigW-KYR:TTCACACCAAAAACCGAT
实施例2:
Protein A表达载体的构建:
1.以重组的Protein A氨基酸序列(SEQ ID NO.2)为模板,经过地衣芽胞杆菌密码子优化得到核酸序列SEQ ID NO.3所示,经过南京金斯瑞生物科技有限公司合成获得DNA序列。按照SEQ ID NO.3序列设计引物spa-F/spa-R扩增spa基因,得到spa基因序列1089bp;2.以载体pPykzA-P43-SAT为模板(Rao et al.,JBiotechnol,2020,312:1-10),利用引物P-F/P-R扩增PykzA-P43和信号肽SPywbN序列;
3.通过重叠延伸PCR将PykzA-P43(SEQ ID NO.4所示)与信号肽SPywbN序列(SEQID NO.5所示)和spa基因连接到一起,得到目的基因片段;
4.以pHY-300为模板,以pHY-T5-F、pHY-T5-R进行PCR扩增得到pHY-300骨架;
5.采用ExnaseⅡT5外切酶对步骤3中的融合片段和步骤4中的骨架进行酶切酶连得到连接产物;通过氯化钙转化法将该连接产物转入大肠杆菌DH5α,在37℃的条件下经含有四环素抗性的培养基进行筛选,筛选得到转化子,对转化子挑质粒进行菌落PCR验证,所用引物为:pHY-F,pHY-R;将菌落PCR正确的阳性转化子,进行DNA测序,测序正确后命名为:表达载体pHY-spa;
spa-F:CTTGTTCAGACTGCGGCTATGGCTGATAATAAATTTAAT
spa-R:TCCGTCCTCTCTGCTCTTTCAGTGGTGGTGGTGGTG
P-F:TTTTTTATAACAGGAATTGAAATATTGATGTGACACTTGA
P-R:TTAAACTTATTGTCCGCCATAGCCGCAGTCTGAACAAG
pHY-F:GTTTATTATCCATACCCTTAC
pHY-R:CAGATTTCGTGATGCTTGTC
pHY-T5-F:AATTCCTGTTATAAAAAAAGGATCA
pHY-T5-R:AAGAGCAGAGAGGACGGATTT
实施例3:
重组Protein A表达菌株构建及应用
将重组Protein A表达质粒pHY-spa电转入地衣芽胞杆菌BL10ΔsigW中,以四环素抗性作为筛选标记,并经过菌落PCR筛选,以pHY-F和pHY-R作为验证引物,PCR验证得到阳性转化子,获得重组表达菌株地衣芽胞杆菌BL10ΔsigW/pHY-spa;本实施例中,将重组Protein A表达质粒pHY-spa电转入地衣芽胞杆菌BL10中,以上述相同的方法筛选出基因工程菌重组Protein A表达菌株地衣芽胞杆菌BL10/pHY-spa作为对照。
本实施例通过向10种不同的发酵培养基中,分别接种上述2个重组基因工程菌,以考察不同的培养基对重组Protein A的影响,以及验证缺失了sigW基因的地衣芽胞杆菌确实能提高重组Protein A的表达量。所用的10中发酵培养基配方如表1所示。
重组Protein A表达发酵:将待发酵的重组地衣芽胞杆菌于含有四环素抗性的LB固体培养基上划线活化,37℃过夜培养。单菌落转接至添加有相应抗性的LB液体培养基(50mL),37℃摇床养10-14h,得到种子液。将种子液以2%(v/v)的接种量转接至50mL添加相应抗性的发酵培养基(表1所示),37℃培养36h,即得发酵液。反应结束后,12,000rpm离心10min,上清液置于4℃保存。
表1培养基组成
培养基类型 | 葡萄糖(g/L) | 骨蛋白胨(g/L) | 玉米浆(g/L) |
培养基1 | 20 | 5 | 10 |
培养基2 | 20 | 10 | 10 |
培养基3 | 20 | 15 | 10 |
培养基4 | 30 | 10 | 0 |
培养基5 | 30 | 10 | 10 |
培养基6 | 30 | 10 | 20 |
培养基7 | 40 | 10 | 10 |
培养基8 | 40 | 15 | 5 |
培养基9 | 40 | 5 | 10 |
培养基10 | 50 | 10 | 10 |
10种培养基中其他成分均为:10g/L大豆蛋白胨,10g/L酵母粉,10g/L氯化钠,3g/LK2HPO4,5g/L(NH4)2SO4,pH 7.2。
重组蛋白A蛋白浓度测定方法:BCA蛋白检测试剂盒:每个样取稀释相应倍数后的蛋白样20μl,试剂盒内A溶液和B溶液按50:1的比例混合,再每个样加混合后的A和B溶液200μL,放入37℃恒温培养箱静置30min,用酶标仪测OD=562nm的吸光值。
表2实验组和对照组在不同培养基条件下Protein A的产量
从表2可看出,在相同的发酵条件下,采用本发明的地衣芽胞杆菌BL10ΔsigW/pHY-spa的发酵液中重组Protein A表达量较对照菌均有大幅提升,均提高了30%以上。说明本发明中的基因工程改造方法在提高地衣芽胞杆菌异源蛋白生产方面具有重要的应用价值。
实施例4:分泌的重组Protein A蛋白的纯化与特性
重组Protein A蛋白经地衣芽胞杆菌工程菌发酵培养直接分泌到上清液。经离心将含有分泌出的重组Protein A蛋白上清液后,直接上汇研科技有限公司的IDA亲和层析介质,用柱层析法将目标蛋白质收集,纯化方法:20mM PB,500mM NaCl,pH 7.4平衡液平衡纯化柱并上样,20mM PB,500mM NaCl,20mM咪唑,pH 7.4将杂蛋白洗脱,20mM PB,500mM NaCl,250mM咪唑,pH 7.4,将目的蛋白洗脱。将目的蛋白洗脱,得到纯度达98%以上的重组Protein A。图2是本发明分泌的重组Protein A蛋白的纯化图,泳道M:蛋白质标准分子量。
实施例5:Protein A亲和填料制备和抗体纯化应用
Protein A亲和填料的制备方法,包括以下步骤:称取适量的活化的琼脂糖微球。将重组Protein A置换到NaHCO3溶液中,重组Protein A的浓度为10mg/ml,备用。将重组Protein A和上述处理后的琼脂糖微球混合(混合比例约10mg重组Protein A/1g琼脂糖微球),震荡过夜。静置后离心,用含氯化钠的柠檬酸缓冲液(pH4.0)和含氯化钠的PBS(pH8.0),交替洗涤,重复3次,再用大量的纯化水冲洗。
上述制备Protein A亲和填料用于抗体纯化,本发明制备的Protein A亲和填料纯化的抗体含量达到。图3是本发明Protein A亲和填料纯化抗体SDS-PAGE蛋白电泳图,泳道M:蛋白质标准分子量;泳道1:本发明中Protein A亲和填料纯化结果。表明提供了一种由重组Protein A和琼脂糖凝胶相偶联而成的高产无内毒素抗体Protein A亲和填料,可应用与抗体纯化。
对比例1:
按照实施例2,以pHY300为表达载体,以PykzA-P43为启动子和SPywbN为信号肽,以地衣芽胞杆菌碱性蛋白酶编码基因aprE为目标基因(SEQ ID NO.6所示),构建碱性蛋白酶表达载体pHY-aprE,按照实施例3的方法,分别电转化至地衣芽胞杆菌BL10和BL10ΔsigW中,获得BL10/pHY-aprE和BL10ΔsigW/pHYaprE重组菌株。并进行发酵培养,发酵培养基为:20g/L葡萄糖、5g/L骨蛋白胨,10g/L大豆蛋白胨,10g/L玉米浆,10g/L酵母粉,10g/L氯化钠,3g/L K2 HPO4,5g/L(NH4)2SO4,其余为水,pH 7.2,37℃培养36h,即得发酵液。反应结束后,12,000rpm离心10min,上清检测碱性蛋白酶的表达水平。结果显示,BL10/pHY-aprE和BL10ΔsigW/pHY-aprE中发酵产物碱性蛋白酶AprE酶活相差不大(3689±245vs 3595±312U/mL)。
aprE-F:CTTGTTCAGACTGCGGCTATGATGAGGAAAAAGAGTaprE-R:TCCGTCCTCTCTGCTCTTTTATTGAGCGGCAGCTTCG。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (5)
1.敲除sigW基因在提高地衣芽胞杆菌生产Protein A蛋白中的应用,应用过程包括将地衣芽胞杆菌中的sigW基因敲除,获得地衣芽胞杆菌sigW基因缺失株,然后在该缺失株中转入Protein A表达载体后进行发酵表达即可。
2.根据权利要求1所述的应用,所述的Protein A为SEQ ID NO.2所示,编码其的基因为SEQ ID NO.3所示。
3.根据权利要求2所述的应用,所述的地衣芽胞杆菌为地衣芽胞杆菌BL10。
4.根据权利要求3所述的应用,应用过程中的发酵,其发酵培养基为:20-50g/L葡萄糖、5-15g/L骨蛋白胨、5-15g/L大豆蛋白胨、0-20g/L玉米浆,5-15g/L酵母粉,5-10g/L氯化钠,2-5g/L K2 HPO4,4-8g/L(NH4)2SO4,其余为水,pH 6.8~7.4。
5.权利要求1所述的应用,所述的应用过程生产的Protein A蛋白在制备亲和填料中的应用。
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