幽门螺杆菌HspA亚基的表达与纯化
刘秀丽, 张兆山, 陶好霞, 展德文, 刘纯杰, 北京生物工程研究所 北京市 100071
刘秀丽,女, 1971-10-10生, 河北省固安县人, 汉族, 2002年北京生物工程研究所博士研究生毕业, 助理研究员, 主要从事幽门螺杆菌疫苗的研究.
国家高技术研究发展计划资助项目(863计划), No. 2004AA215213
项目负责人: 刘纯杰, 100071, 北京市丰台东大街20号, 北京生物工程研究所. Liucj@ nic.bmi. ac.cn
电话: 010-66948834 传真: 010-63833521
收稿日期: 2004-12-17 接受日期: 2005-01-26
Expression and purification of recombinant heat shock protein A subunit of Helicobacter pylori
Xiu-Li Liu, Zhao-Shan Zhang, Hao-Xia Tao, De-Wen Zhan, Chun-Jie Liu
Xiu-Li Liu, Zhao-Shan Zhang, Hao-Xia Tao, De-Wen Zhan, Chun-Jie Liu, Beijing Institute of Biotechnology, Beijing 100071, China
Supported by the Hi-tech Research and Development Program of China, No.2004AA215213
Correspondence to: Chun-Jie Liu, Beijing Institute of Biotechnology, 20 Dongda Street, Beijing 100071, China. Liucj@ nic.bmi.ac.cn
Received: 2004-12-17 Accepted: 2005-01-26
Abstract
AIM: To express and purify recombinant heat shock protein A subunit (HspA) of Helicobacter pylori (H pyloi).
METHODS: Gene hspA was amplified by PCR, and inserted into the prokaryotic expression vector pET22b, pQE-60 and pGEX-4T-2 respectively. The plamids were transformed into the Escherichia coli JM109 and BL21 (DE3). Expression of hspA gene was induced by IPTG and was analyzed by SDS-PAGE. The recombinant proteins were purified through nickel-affinity chromatography. Antigenicity of the recombinant proteins was analyzed by western blot.
RESULTS: The hspA gene was expressed in two forms (HspA and HspA-His6) in pQE-60. The expressed protein accounted for above 40% of the total bacterial protein. The purity of HspA and HspA-His6 were 88.63% and 86.32% respectively after purification. Western blot proved that the recombinant proteins could be recognized by the anti-H pylori serum.
CONCLUSION: HspA of H pylori has been expressed and purified with high efficiency, which can be used for vaccine development and immunological investigation.
Key Words: Helicobacter pylori; Heat shock protein A; Recombined expression; Purification
Liu XL, Zhang ZS, Tao HX, Zhan DW, Liu CJ. Expression and purification of recombinant heat shock protein A subunit of Helicobacter pylori. Shijie Huaren Xiaohua Zazhi 2005;13(5):626-630
摘要
目的: 利用原核表达载体高效表达幽门螺杆菌热休克蛋白A(HspA)，并进行纯化.
方法:PCR扩增hspA基因，将其克隆至原核表达载体pET22b，pQE-60，pGEX-4T-2中，转化大肠杆菌，经IPTG诱导后，SDS-PAGE分析表达.利用镍离子亲和层析对表达产物进行纯化.免疫印迹检测蛋白的抗原性.
结果:PCR扩增得到hspA基因，在载体pQE-60中以HspA和HspA-His6两种形式得到表达，表达量高达菌体总蛋白的40%以上.经镍离子亲和层析后，纯化率分别为88.63%和 86.32%，但HspA-His6在纯化过程中发生降解.免疫印迹实验表明，HspA和HspA-His6都有很好的抗原性.
结论: 实现了HspA蛋白的高效表达和纯化，为幽门螺杆菌HspA亚单位疫苗的免疫实验奠定基础.
关键词: 幽门螺杆菌; 热休克蛋白A; 重组表达; 纯化
刘秀丽, 张兆山, 陶好霞, 展德文, 刘纯杰. 幽门螺杆菌HspA亚基的表达与纯化. 世界华人消化杂志 2005;13(5):626-6301 (PDF) 克隆至pETH、pQEH上的Hsp A抗原的基因序列和氨基酸序列.
图2 (PDF) 重组质粒的酶切图. M1: DL2000; 1: pGEXH / XhoI+BamH I ; 2:pQEHS/Nco I+BamHI ; 3: pQEH/Nco I+BamHI; 4:pETH/Nco I+BamHI ; M2: DL15 000.
2.2 重组Hsp A蛋白的表达 重组的基因工程菌经IPTG诱导后,经SDS-PAGE检测发现:与对照JM109菌株相比pQEH和pQEHS均在14 000处表达处一条蛋白带.初步判断应为重组HspA蛋白.HspA蛋白占总蛋白的42.51%,HspA-His6蛋白占总蛋白的43.07%.与对照BL21相比pGEXH在43 ku左右表达处3条带,而pETH没有表达带(图3).
图3 (PDF) HspA重组蛋白的电泳分析图. M: Marker,(66 200,43 000,31 000,20 100,14 400); 1:JM109,2: HspA-His6(pQEHS),3:HspA(pQEH),4: HspA(pGEXH),5: HspA(pETH),6: BL21.
2.3重组Hsp A蛋白的纯化 质粒pETH未表达目的蛋白,而重组pGEXH未明原因的表达三条带.本实验以pQEH和pQEHS为纯化的研究对象.取大量培养的超声离心上清和沉淀样品、纯化时的上样穿流峰样品、平衡峰样品和洗脱峰样品进行SDS-PAGE电泳分析,结果发现pQEH表达的HspA蛋白均以可溶形式表达,而pQEHS表达的HspA-His6蛋白部分以可溶形式表达,部分以包涵体形式表达.HspA蛋白与镍柱结合效果好,在穿流峰中未检测到HspA蛋白.而HspA-His6蛋白与镍柱结合效果差,在穿流峰中检测到HspA-His6蛋白,且在洗脱峰中HspA-His6蛋白出现降解带.纯化HspA蛋白的纯度为88.63%,而HspA-His6蛋白的纯度为86.32%(包括降解带).从400 mL的培养菌中可得到HspA约12 mg,相同的菌量HspA-His6只能得到3 mg左右(图4).
图4 (PDF) HspA纯化蛋白的电泳分析结果. A: Expression and purification of HspA; B: Expressionand purification of HspA-His6. M:Marker(66 200,43 000,31 000,20 100,14 400); 1: Centrifuged Supernatant; 2:Centrifuged precipitate; 3: loading peak fraction; 4: Equilibrating peakfraction; 5: Eluting peak fraction.
2.4 HspA和HspA-His6蛋白的免疫印迹分析 以150 g/L的SDS-PAGE将HspA和HspA-His6蛋白进行分离,然后用抗H pylori血清进行免疫印迹,结果发现HspA纯化产物在14 KD左右有一阳性印迹,而HspA-His6纯化产物在14 ku左右有2条阳性印迹带,由此可见HspA-His6在纯化过程中有降解现象(图5).
图5 (PDF) 重组蛋白HspA-His6and HspA的免疫印记分析. M: Marker(97 400,66 200,43 000,31 000,20 100,14 400);1: HspA-His6; 2: HspA.
3 讨论H pylori中含有一个编码13 ku的热休克蛋白A(HspA),他与大肠杆菌GroES高度同源动物实验研究表明用HspA(佐剂为霍乱毒素CT)免疫小鼠,可使小鼠对猫胃螺杆菌攻击的保护率为80%,说明HspA是有效的保护性抗原[21].另外由于HspA蛋白序列在不同H pylori菌株间高度保守,因此将其单独使用或与其他抗原联用,均可激发机体产生保护免疫,是H pylori疫苗的理想组分[22].我们将hspA基因克隆至不同的表达载体中,目的是实现该基因的高度表达并利于纯化,在pET22b中,克隆的基因经测序后序列正确,但未能实现表达.将该基因克隆至pGEX-4T-2中,使其与GST融合,然后利用GST亲和层析对其纯化.虽然克隆的基因序列完整无误,但融合蛋白在表达或在检测处理过程中出现降解,使得纯化出现困难.可喜的是 HspA在pQE-60中以HspA和 HspA-His6的形式得到高效表达,因此在纯化过程中我们以纯化HspA和HspA-His6为主.
Hsp A 位于H pylori的表面,其C端含有4个组氨酸和4个半胱氨酸,能够特异性结合镍离子,起到镍离子库的作用[23-29].尿素酶是H pylori中重要的定植因子,他通过分解尿素形成氨以中和胃酸,为H pylori的定值和生存创造条件.尿素酶的活性与镍离子的存在有密切关系.因此Ure和Hsp A之间通过镍离子建立起一定的联系,在决定H pylori定植效率方面起协同作用[30].我们将hspA基因克隆至pQE-60中,因pQE-60中含有6个组氨酸,以利于用镍亲和柱纯化,因此我们将hspA基因以带有和不带有his6尾巴两种形式表达.通过纯化实验发现,HspA无论在表达形式(可溶表达或包涵体)还是在与亲和柱结合上比HspA-His6的效果都要好.这可能是his6尾巴的加入不但没有增强与镍亲和柱的结合,反而干扰了HspA可溶性表达和HspA原有的结合镍离子的作用区域,实属画蛇添足.实验研究表明HspA对镍离子有很强的网络能力,这为H pylori含有HspA的融合疫苗通过镍离子亲和层析纯化打下基础.实验中所获得的高纯度的HspA蛋白为进一步进行动物免疫实验提供了大量抗原.
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编辑 潘伯荣( 刘秀丽,张兆山, 陶好霞, 展德文, 刘纯杰)
刘秀丽,女, 1971-10-10生, 河北省固安县人, 汉族, 2002年北京生物工程研究所博士研究生毕业, 助理研究员, 主要从事幽门螺杆菌疫苗的研究.
国家高技术研究发展计划资助项目(863计划), No. 2004AA215213
项目负责人: 刘纯杰, 100071, 北京市丰台东大街20号, 北京生物工程研究所. Liucj@ nic.bmi. ac.cn
电话: 010-66948834 传真: 010-63833521
收稿日期: 2004-12-17 接受日期: 2005-01-26
Expression and purification of recombinant heat shock protein A subunit of Helicobacter pylori
Xiu-Li Liu, Zhao-Shan Zhang, Hao-Xia Tao, De-Wen Zhan, Chun-Jie Liu
Xiu-Li Liu, Zhao-Shan Zhang, Hao-Xia Tao, De-Wen Zhan, Chun-Jie Liu, Beijing Institute of Biotechnology, Beijing 100071, China
Supported by the Hi-tech Research and Development Program of China, No.2004AA215213
Correspondence to: Chun-Jie Liu, Beijing Institute of Biotechnology, 20 Dongda Street, Beijing 100071, China. Liucj@ nic.bmi.ac.cn
Received: 2004-12-17 Accepted: 2005-01-26
Abstract
AIM: To express and purify recombinant heat shock protein A subunit (HspA) of Helicobacter pylori (H pyloi).
METHODS: Gene hspA was amplified by PCR, and inserted into the prokaryotic expression vector pET22b, pQE-60 and pGEX-4T-2 respectively. The plamids were transformed into the Escherichia coli JM109 and BL21 (DE3). Expression of hspA gene was induced by IPTG and was analyzed by SDS-PAGE. The recombinant proteins were purified through nickel-affinity chromatography. Antigenicity of the recombinant proteins was analyzed by western blot.
RESULTS: The hspA gene was expressed in two forms (HspA and HspA-His6) in pQE-60. The expressed protein accounted for above 40% of the total bacterial protein. The purity of HspA and HspA-His6 were 88.63% and 86.32% respectively after purification. Western blot proved that the recombinant proteins could be recognized by the anti-H pylori serum.
CONCLUSION: HspA of H pylori has been expressed and purified with high efficiency, which can be used for vaccine development and immunological investigation.
Key Words: Helicobacter pylori; Heat shock protein A; Recombined expression; Purification
Liu XL, Zhang ZS, Tao HX, Zhan DW, Liu CJ. Expression and purification of recombinant heat shock protein A subunit of Helicobacter pylori. Shijie Huaren Xiaohua Zazhi 2005;13(5):626-630
摘要
目的: 利用原核表达载体高效表达幽门螺杆菌热休克蛋白A(HspA)，并进行纯化.
方法:PCR扩增hspA基因，将其克隆至原核表达载体pET22b，pQE-60，pGEX-4T-2中，转化大肠杆菌，经IPTG诱导后，SDS-PAGE分析表达.利用镍离子亲和层析对表达产物进行纯化.免疫印迹检测蛋白的抗原性.
结果:PCR扩增得到hspA基因，在载体pQE-60中以HspA和HspA-His6两种形式得到表达，表达量高达菌体总蛋白的40%以上.经镍离子亲和层析后，纯化率分别为88.63%和 86.32%，但HspA-His6在纯化过程中发生降解.免疫印迹实验表明，HspA和HspA-His6都有很好的抗原性.
结论: 实现了HspA蛋白的高效表达和纯化，为幽门螺杆菌HspA亚单位疫苗的免疫实验奠定基础.
关键词: 幽门螺杆菌; 热休克蛋白A; 重组表达; 纯化
刘秀丽, 张兆山, 陶好霞, 展德文, 刘纯杰. 幽门螺杆菌HspA亚基的表达与纯化. 世界华人消化杂志 2005;13(5):626-6301 (PDF) 克隆至pETH、pQEH上的Hsp A抗原的基因序列和氨基酸序列.
图2 (PDF) 重组质粒的酶切图. M1: DL2000; 1: pGEXH / XhoI+BamH I ; 2:pQEHS/Nco I+BamHI ; 3: pQEH/Nco I+BamHI; 4:pETH/Nco I+BamHI ; M2: DL15 000.
2.2 重组Hsp A蛋白的表达 重组的基因工程菌经IPTG诱导后,经SDS-PAGE检测发现:与对照JM109菌株相比pQEH和pQEHS均在14 000处表达处一条蛋白带.初步判断应为重组HspA蛋白.HspA蛋白占总蛋白的42.51%,HspA-His6蛋白占总蛋白的43.07%.与对照BL21相比pGEXH在43 ku左右表达处3条带,而pETH没有表达带(图3).
图3 (PDF) HspA重组蛋白的电泳分析图. M: Marker,(66 200,43 000,31 000,20 100,14 400); 1:JM109,2: HspA-His6(pQEHS),3:HspA(pQEH),4: HspA(pGEXH),5: HspA(pETH),6: BL21.
2.3重组Hsp A蛋白的纯化 质粒pETH未表达目的蛋白,而重组pGEXH未明原因的表达三条带.本实验以pQEH和pQEHS为纯化的研究对象.取大量培养的超声离心上清和沉淀样品、纯化时的上样穿流峰样品、平衡峰样品和洗脱峰样品进行SDS-PAGE电泳分析,结果发现pQEH表达的HspA蛋白均以可溶形式表达,而pQEHS表达的HspA-His6蛋白部分以可溶形式表达,部分以包涵体形式表达.HspA蛋白与镍柱结合效果好,在穿流峰中未检测到HspA蛋白.而HspA-His6蛋白与镍柱结合效果差,在穿流峰中检测到HspA-His6蛋白,且在洗脱峰中HspA-His6蛋白出现降解带.纯化HspA蛋白的纯度为88.63%,而HspA-His6蛋白的纯度为86.32%(包括降解带).从400 mL的培养菌中可得到HspA约12 mg,相同的菌量HspA-His6只能得到3 mg左右(图4).
图4 (PDF) HspA纯化蛋白的电泳分析结果. A: Expression and purification of HspA; B: Expressionand purification of HspA-His6. M:Marker(66 200,43 000,31 000,20 100,14 400); 1: Centrifuged Supernatant; 2:Centrifuged precipitate; 3: loading peak fraction; 4: Equilibrating peakfraction; 5: Eluting peak fraction.
2.4 HspA和HspA-His6蛋白的免疫印迹分析 以150 g/L的SDS-PAGE将HspA和HspA-His6蛋白进行分离,然后用抗H pylori血清进行免疫印迹,结果发现HspA纯化产物在14 KD左右有一阳性印迹,而HspA-His6纯化产物在14 ku左右有2条阳性印迹带,由此可见HspA-His6在纯化过程中有降解现象(图5).
图5 (PDF) 重组蛋白HspA-His6and HspA的免疫印记分析. M: Marker(97 400,66 200,43 000,31 000,20 100,14 400);1: HspA-His6; 2: HspA.
3 讨论H pylori中含有一个编码13 ku的热休克蛋白A(HspA),他与大肠杆菌GroES高度同源动物实验研究表明用HspA(佐剂为霍乱毒素CT)免疫小鼠,可使小鼠对猫胃螺杆菌攻击的保护率为80%,说明HspA是有效的保护性抗原[21].另外由于HspA蛋白序列在不同H pylori菌株间高度保守,因此将其单独使用或与其他抗原联用,均可激发机体产生保护免疫,是H pylori疫苗的理想组分[22].我们将hspA基因克隆至不同的表达载体中,目的是实现该基因的高度表达并利于纯化,在pET22b中,克隆的基因经测序后序列正确,但未能实现表达.将该基因克隆至pGEX-4T-2中,使其与GST融合,然后利用GST亲和层析对其纯化.虽然克隆的基因序列完整无误,但融合蛋白在表达或在检测处理过程中出现降解,使得纯化出现困难.可喜的是 HspA在pQE-60中以HspA和 HspA-His6的形式得到高效表达,因此在纯化过程中我们以纯化HspA和HspA-His6为主.
Hsp A 位于H pylori的表面,其C端含有4个组氨酸和4个半胱氨酸,能够特异性结合镍离子,起到镍离子库的作用[23-29].尿素酶是H pylori中重要的定植因子,他通过分解尿素形成氨以中和胃酸,为H pylori的定值和生存创造条件.尿素酶的活性与镍离子的存在有密切关系.因此Ure和Hsp A之间通过镍离子建立起一定的联系,在决定H pylori定植效率方面起协同作用[30].我们将hspA基因克隆至pQE-60中,因pQE-60中含有6个组氨酸,以利于用镍亲和柱纯化,因此我们将hspA基因以带有和不带有his6尾巴两种形式表达.通过纯化实验发现,HspA无论在表达形式(可溶表达或包涵体)还是在与亲和柱结合上比HspA-His6的效果都要好.这可能是his6尾巴的加入不但没有增强与镍亲和柱的结合,反而干扰了HspA可溶性表达和HspA原有的结合镍离子的作用区域,实属画蛇添足.实验研究表明HspA对镍离子有很强的网络能力,这为H pylori含有HspA的融合疫苗通过镍离子亲和层析纯化打下基础.实验中所获得的高纯度的HspA蛋白为进一步进行动物免疫实验提供了大量抗原.
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编辑 潘伯荣( 刘秀丽,张兆山, 陶好霞, 展德文, 刘纯杰)