CN117343942B - 一种pagA重组蛋白及其制备方法 - Google Patents
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Abstract
本发明公开了一种pagA重组蛋白及其制备方法,属于生物技术领域。本发明提供一种用于制备pagA重组蛋白的核酸序列,其包括pagA全长序列和His标签序列。本发明还提供了一种pagA重组蛋白的制备方法,包括如下步骤:(1)用引物P1和P2对pagA进行PCR扩增;(2)将PCR产物与PET28a载体经NcoI和XhoI酶切后通过重组酶连接、转化、测序鉴定重组子;(3)将重组子转入大肠杆菌BL21(DE3)表达菌株中进行诱导培养;(4)纯化、复性得到pagA重组蛋白。本发明pagA重组蛋白的制备流程简单、产量较高、易于重复,适合大规模生产,且产物纯净。
Description
技术领域
本发明涉及生物技术领域,尤其是涉及一种pagA重组蛋白及其制备方法。
背景技术
炭疽毒素是炭疽杆菌的关键性致病因子,由保护性抗原(Protective antigen,PA)、水肿因子(Edema factor,EF)、致死因子(Lethal factor,LF)三种毒素因子组成,PA与宿主细胞表面的炭疽毒素受体结合经一系列的相互作用后,LF/EF被PA输送进宿主细胞细胞质中,从而发挥各自的毒素效应。
LFn(致死因子氨基末端结构域)由LF氨基端的前254aa组成,不含有LF的毒性区域,与PA具有很高的亲和结合力。基于炭疽毒素的分子结构和作用机制发展的PA/LFn转运系统,具有转运外源蛋白到胞质中的作用。当PA与LFn同时存在时,PA会和细胞表面受体结合,并与LFn形成多聚体,从而将LFn转运入细胞。PA蛋白与宿主细胞表面的受体结合,形成PA-受体复合物,PA-受体复合物会通过内吞作用进入宿主细胞内部,形成一个内吞体,内吞体会与细胞内液泡融合,形成一个通道,通过该通道释放PA蛋白,释放出的PA蛋白能够与细胞膜上的受体结合,引发一个剪切活化过程,将LFn从PA蛋白中释放出来,发挥毒性。
PA能够介导LFn-protein融合蛋白易位到细胞质中,激起机体的特异性免疫应答,在机体的抗病毒及抗肿瘤治疗中具有广阔的应用前景。目前PA蛋白基本由实验室自行制备,而课题组前期通过查询炭疽杆菌pagA的基因序列(序列如SEQ ID NO.4所示)直接制备PA蛋白未成功获得PA蛋白,即使对表达方法进行优化也未成功获得PA蛋白。
发明内容
本发明针对现有技术存在的上述不足,采用重组蛋白工程技术制备一种pagA重组蛋白。本发明提供了一种pagA重组蛋白的制备方法。具体是将优化后的pagA基因序列插入到表达载体中,然后转化到宿主细胞中进行表达,通过优化表达系统和调控表达条件,获得纯净的、高产量的pagA蛋白。
本发明的第一个目的是提供一种编码pagA蛋白的基因,所述基因的核苷酸序列如SEQ ID NO.2所示。
本发明的第二个目的是提供携带所述基因的重组质粒。
在一种实施方式中,所述重组质粒以pET系列载体为表达载体。
本发明的第三个目的是提供携带所述基因或所述重组质粒的重组细胞。
在一种实施方式中,所述重组细胞是以大肠杆菌、枯草芽孢杆菌、地衣芽孢杆菌或毕赤酵母为宿主。
本发明还提供了一种重组大肠杆菌,所述重组大肠杆菌表达了所述基因。
在本发明的一种实施方式中,所述重组大肠杆菌为,以pET-28a(+)为载体,在大肠杆菌BL21(DE3)中表达SEQ ID NO.2所示的pagA基因。
本发明还提供了一种制备pagA蛋白的方法,所述方法为将所述重组细胞或所述重组大肠杆菌接种于含有碳源的反应体系中进行诱导表达,收集发酵液,纯化获得pagA蛋白。
在一种实施方式中,所述诱导表达的条件为在18~20℃以下诱导表达3~5h。
在一种实施方式中,所述诱导表达的诱导剂为20~200 μM的IPTG。
本发明还提供了所述基因,或所述重组质粒,或所述重组细胞,或所述重组大肠杆菌在生产制备pagA蛋白中的应用。
本发明的有益效果:
本发明以氨基酸序列为SEQ ID NO.1所示的pagA蛋白为基础,优化得到了炭疽杆菌pagA的核苷酸序列(SEQ ID NO.2),设计合成带有8×His纯化标签的pagA基因(SEQ IDNO.3),无需开发特异性亲和配体即可完成蛋白的亲和层析蛋白纯化,成功实现了pagA蛋白的表达和纯化。采用本发明提供的重组蛋白工程技术制备的pagA蛋白,纯度较好,浓度为0.9 mg/mL (20mM PB缓冲液,pH 8.0),本发明克服了pagA蛋白表达和纯化困难的问题。
附图说明
图1 酶切鉴定;
图2 构建质粒;
图3 PA蛋白在BL21(DE3)中的表达验证;
图4 重组蛋白在不同诱导温度下上清和沉淀中的诱导表达;
图5 重组蛋白在不同浓度诱导剂下上清和沉淀中的诱导表达;
图6 重组蛋白的纯化与浓缩。
具体实施方式
实施例1 pagA基因的扩增
(1)重组菌的构建
根据PA蛋白的氨基酸序列(SEQ ID NO.1),化学合成优化后的pagA基因的核酸序列(SEQ ID NO.2)。
根据pagA基因设计引物,扩增得到带有8×His纯化标签的pagA基因(SEQ IDNO.3),在目的基因5’端引入NcoI酶切位点,在目的基因的3’端引入XhoI酶切位点,两条引物为:
上游引物P1(5’-3’):TAATACGACTCACTATAGGG;
下游引物P2(5’-3’):TGCTAGTTATTGCTCAGCGG;
引物稀释成0.1A/μL,然后进行PCR步骤。
PCR反应体系25μL:上/下游引物 2μL,10×Buffer(含有4mM MgCl2) 2.5μL,dNTP5μL,ddH2O 11μL,模板 2μL,Taq DNA聚合酶 0.5μL。
PCR反应条件:94℃ 5min,35个循环(94℃ 30sec,59℃ 30sec,72℃ 60sec),72℃10min。
反应结束后,取PCR产物进行1%琼脂糖凝胶电泳,紫外检测2235bp位置左右的一条亮带即为带有8×His纯化标签的pagA基因片段。
凝胶回收带有8×His纯化标签的pagA基因产物,同时用NcoI和XhoI处理pET28a载体的线性化载体,取8×His纯化标签的pagA基因产物6 μL与线性化载体4 μL,再加10 μL重组酶,50~55℃水浴40 min,取反应产物10 μL,转化至T1感受态细胞中。用Kan+抗性的LB平板筛选挑出单克隆,再用上下游引物筛选出含有2235 bp左右条带的阳性克隆。
随后利用分子克隆的碱裂解法提取质粒,利用酶切方法鉴定阳性质粒,酶切鉴定结果显示产生1167bp、6281bp的片段(图1)。最终将经过酶切鉴定的含有目的基因的重组质粒送样到公司测序,测序结果符合要求的重组质粒命名为pET28a-pagA(图2)。
将构建好的原核表达载体pET28a-pagA通过热激法转入大肠杆菌BL21(DE3)感受态细胞中,Kan+抗性筛选后,将所得抗性克隆进行重组蛋白表达验证。
(2)重组蛋白表达及验证
挑取4个抗性克隆于37℃、200 r/min条件下培养约14 h,按2.5%的体积比将所培养的菌液分别接种于4 mL带有Kan+抗性的LB液体培养基中,于37℃、200 r/min条件下振荡培养约1.5 h,加入终浓度为100 μM的IPTG。再经19℃、200 r/min条件下诱导培养约4 h后,离心,分别收集沉淀和上清,对沉淀进行超声破解,进行SDS-PAGE电泳,并进行考马斯亮蓝染色检测全菌表达情况及分子量。
SDS-PAGE检测结果显示,挑选的4个克隆样品孔道中均出现与预期蛋白大小一致的条带,见图3,可知重组质粒能够成功表达。
(3)重组蛋白诱导表达条件的优化
重组蛋白表达的步骤同步骤(2),仅是将表达蛋白的阳性克隆分别在19℃、37℃下,在20、50、100、200 μM的IPTG下诱导表达。诱导结束后,经12000 r/min离心3 min,分别取上清和沉淀进行SDS-PAGE电泳,并进行考马斯亮蓝染色检测表达情况,全菌作为阳性对照。
结果如图4显示,在19℃条件下,蛋白的表达量更高,但无论是在37℃还是19℃,该蛋白主要都是以包涵体的形式表达。
将重组菌分别在浓度20、50、100、200 μM的IPTG下,诱导4小时,见图5。结果显示,在100 μM的IPTG诱导下,蛋白的表达量更高。
实施例2 重组蛋白的纯化
根据实施例1中优化的诱导表达条件,进行重组蛋白的制备。从实施例1可知,pagA蛋白主要是以包涵体的形式表达表达,因此,将诱导所得的菌液分装于离心管,4℃低温条件下4000 r/min 离心15 min收集菌体,每10 mL菌液加入1 mL 20 mM的PBS缓冲液后冰浴30 min,在冰浴条件下超声破碎细胞。随后,在4℃低温条件下12000 r/min离心菌液20min,保留上清液,并将上清液在冰浴条件下转移至4℃低温层析柜中。将10 mL蛋白上清经孔径为0.45 μm的滤膜过滤后,缓慢添加至镍柱中,重复上样4次。用20 mM的PBS和0、10、20、30、40、200 mM的咪唑进行梯度洗脱,收集洗脱液,经处理后用SDS-PAGE电泳检测,初步确定最佳洗脱条件。
结果显示,最佳洗脱条件为20 mM的咪唑,随后进行大量洗脱,根据SDS-PAGE电泳检测结果,收集纯度符合要求的洗脱液用透析法进行复性。透析结束后,将蛋白放置于提前预冷的变色硅胶中,在4℃条件下将蛋白浓缩至1~2 mL,结果见图6,得到了纯度较好的重组蛋白,其浓度为0.9 mg/mL (20mM PB缓冲液,pH 8.0)。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (6)
1.一种重组大肠杆菌,其特征在于,所述重组大肠杆菌表达了核苷酸序列如SEQ IDNO.3所示的基因。
2.根据权利要求1所述的重组大肠杆菌,其特征在于,所述重组大肠杆菌以pET-28a(+)为载体。
3.一种制备pagA蛋白的方法,其特征在于,所述方法为将权利要求1或2所述重组大肠杆菌接种于含有碳源的反应体系中进行诱导表达,收集培养液,纯化获得pagA蛋白。
4.根据权利要求3所述的方法,其特征在于,所述诱导表达的条件为在18~20℃以下诱导表达3~5h。
5.根据权利要求3或4所述的方法,其特征在于,所述诱导表达的诱导剂为20~200 μM的IPTG。
6.核苷酸序列如SEQ ID NO.3所示的基因,或权利要求1或2所述重组大肠杆菌在生产制备pagA蛋白中的应用。
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