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100023 北京市2345信箱	世界华人消化杂志 2002年3月15日;10(3):271-274
Email: wcjd@public.bta.net.cn	世界华人消化杂志 ISSN 1009-3079 CN 14-1260/R
http:// www.wjgnet.com	版权归世界胃肠病学杂志社

⊙幽门螺杆菌⊙

幽门螺杆菌细胞毒素相关抗原A的表达纯化及其临床研究

李姁君, 阎小君，刘智广, 苏成芝

李姁君, 阎小君，刘智广, 苏成芝, 中国人民解放军第四军医大学全军基因诊断与技术应用研究所陕西省西安市 710032
李姁君，女, 1973-11-25生，湖北省孝感人，汉族，1995年中国人民解放军第四军医大学医疗系本科毕业，1998年中国人民解放军第四军医大学心血管药理专业硕士毕业，助教.1999-2002攻读分子生物学与生物化学专业博士学位, 主要从事幽门螺杆菌及其相关疾病的研究.
项目负责人 李姁君,710032，陕西省西安市，中国人民解放军第四军医大学全军基因诊断与技术应用研究所.
llxxjjsec@sina.com
电话: 029-3374771 传真: 029-2518328
收稿日期 2002-01-16 接受日期 2002-02-07

Expression, purification and clinical research of Helicobacter pylori cytotoxin-associated gene A

Xu-Jun Li, Xiao-Jun Yan, Zhi-Guang Liu, Cheng-Zhi SuXu-Jun Li, Xiao-Jun Yan, Zhi-Guang Liu, Cheng-Zhi Su, Research institute of gene diagnosis and application, fourth military medical university, Xi'an 710032, Shaanxi province, China
Correspondence to: Xu-Jun Li, Research institute of gene diagnosis and application, fourth military medical university, Xi'an 710032, Shaanxi province, China.
llxxjjsec@sina.com
Received 2002-01-16 Accepted 2002-02-07

Abstract

AIM:The relationship between cagA⁺ Hp infection and gastric cancer in Asia was reported inconsistently. Using recombinant cytotoxin-associated protein A (CagA) of Helicobacter pylori expressed in E.coli, we have established dot immunogold filtration assay (DIGFA) and enzyme-linked immunosorbent assay (ELISA) to detect serum anti-CagA antibody and then performed a study on the relationship between cagA⁺ Hp infection and gastric cancer.

METHODS:After the recombinant plasmid pRSETcagA was proved, the engineered bacteria were induced to express CagA protein by IPTG, and CagA inclusions were denatured, washed through Ni-column, renatured and dialyzed. The purity and activity was revealed by Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA. Antigen was added on nitrocellulose membrane of 0.45μm pore size or coated on ELISA plates, then anti-CagA antibodies in sera from gastric cancer patients and healthy controls were determined by DIGFA or ELISA respectively.

RESULTS:The recombinant CagA antigen was about M_r36000, revealed 98% purity and good activity. Anti-CagA antibody was present in 19/64 (29.7%) gastric cancer and in 31/50 (62%) healthy controls. The gastric cancer subjects were more likely than the healthy controls to have a positive anti-CagA antibody assay (P<0.01).

CONCLUSION:The newly developed anti-CagA antibody assays were highly reproducible. Expression of anti-CagA antibody was present in a significantly higher percentage of gastric cancer subjects than in healthy controls. It is suggested that cagA⁺ Hp infection will increase the possibility of cagA⁺ Hp infection subjects to develop gastric cancer. CagA can be a candidate antigen as vaccine to protect and cure cagA⁺ Hp infection. The detection of anti-CagA antibody in sera will provide patient early warning of developing gastric cancer in future.

Li XJ, Yan XJ, Liu ZG, Su CZ. Expression, purification and clinical research of Helicobacter pylori cytotoxin-associated gene A.
Shijie Huaren Xiaohua Zazhi 2002;10(3):271-274

摘要

目的:cagA⁺Hp的感染与胃癌的相关性在亚洲有不同报道，本研究应用重组幽门螺杆菌毒素相关抗原A (CagA)建立检测血清中抗CagA抗体的方法, 以探讨本地区cagA⁺Hp的感染与胃癌的相关性.

方法:构建表达幽门螺杆菌CagA抗原的表达载体，工程菌经IPTG诱导，表达的CagA包含体经Ni柱纯化, 变性、复性、透析并经活性鉴定后，抗原被点在硝酸纤维素膜上或用以包被ELISA板，建立检测正常人及胃癌患者血清抗CagA抗体的DIGFA (斑点金免疫渗滤试验, 简称滴金法) 和ELISA法.

结果:重组克隆pRSETcagA的工程菌可表达M_r36000的目的蛋白，表达形式为包含体；CagA包含体经纯化后SDS-PAGE显示纯度达98％以上, 活性经ELISA证实; 正常人64例及胃癌患者血清50例中, cagA⁺Hp的感染率分别为29.7％和62%，胃癌患者cagA⁺Hp的感染率明显高于正常人(P<0.01).

结论:我们建立的检测血清抗CagA抗体的DIGFA和ELISA均具有良好的可重复性; 胃癌患者cagA⁺ Hp的感染比例明显高于正常人, CagA阳性幽门螺杆菌的感染与胃癌有关, CagA可作为制备防治cagA⁺ Hp感染的疫苗的后备抗原. CagA可作为幽门螺杆菌疫苗制备的候选抗原，检测患者血清幽门螺杆菌CagA抗体，有可能为胃癌的发生提供预警作用.

李姁君, 阎小君, 刘智广, 苏成芝. 幽门螺杆菌细胞毒素相关抗原A的表达纯化及其临床研究.世界华人消化杂志 2002;10(3):271-274

0 引言

幽门螺杆菌与多种胃病关系密切^［^1-10^］，在过去10a中，大约有90％以上的十二指肠溃疡和75%以上的胃溃疡是与Hp相关的, 虽然目前这个比例在西方国家中已下降到60～65％；但在许多其他国家包括中国, Hp感染相关的消化性溃疡比例仍然很高；此外, Hp还被世界卫生组织确认为1类致癌因子, cagA⁺ Hp致病力更强^［^11-15^］.但在一些亚洲国家包括我国，cagA⁺ Hp蹬感染十分普遍，加之cagA的变异，因此，其cagA⁺ Hp的感染与胃癌的关系受到质疑^［¹⁶^］.我们通过重组技术表达CagA抗原，并进行纯化，建立检测方法，检测正常人及胃癌患者血清, 从而探讨cagA⁺ Hp的感染与胃癌的相关性.

1 材料和方法

1.1 材料 正常及胃癌患者血清的获取：正常62例及胃癌患者血50例取自中国人民解放军第四军医大学附属西京医院. Hp抗原：Hp43504菌株，Dako公司产品.

1.2 方法 CagA重组表达载体的构建：采用pRSETA His6表达载体，为组氨酸融合表达载体，将已构建的pUCcagA载体酶切后，所切下的cagA基因亚克隆入pRSETAHis6表达载体，转化大肠杆菌BL21，酶切鉴定重组克隆.cagA引物按照标准菌株Hp26695 的cagA基因序列设计^［¹⁷^］. IPTG诱导目的蛋白表达; 采用分级分离对诱导全菌的外周质，胞质，及包含体进行分析，判断目的蛋白表达形式.

1.2.1 包含体变性和折叠复性 变性液为50mmol·L^-1Tris, 8mol·L^-1尿素, 10mmol·L^-1 2-巯基乙醇（pH8.0），5mL·g^-1包含体, 室温过夜充分融解包含体; 包含体变性液上Ni柱，用含0.5mol·L^-1咪唑, 50mmol·L^-1Tris, 10mmol·L^-1NaCl（pH8.0）的洗脱液洗脱; 20mmol·L^-1咪唑, 50mmol·L^-1Tris, 0.5mol·L^-1NaCl（pH8.0）复性液复性，最后对0.01mol·L^-1 PBS（pH7.4）4℃透析过夜.

1.2.2 复性蛋白检测 活性鉴定用ELISA检测, 以复性蛋白为包被抗原，0.03～15μg/孔，血清按1∶100稀释，博士德酶标羊抗人IgG1∶10000稀释，TMB显色；生物晶美Hp抗原为阳性对照，PBS为空白对照.

1.2.3 血清抗体检测 ELISA检测以复性蛋白为包被抗原，0.8μg/孔，血清按1∶100稀释，博士德酶标羊抗人IgG1∶10000稀释，TMB显色，检测血清抗CagA抗体，以A450nm>0.5为阳性,设立阴性和空白对照；滴金法检测将复性蛋白点样于硝酸纤维素膜（0.45μm）上(0.1μg), 干燥后50g·L^-1 BSA封闭，再用PBS洗涤3次，干燥后4℃保存；检测时滴加10～15μL待检血清，待渗入后，用PBS洗涤3次; 然后滴加制备好的免疫胶体金(15nm)，待渗入后，用PBS洗涤3次, 观察结果，膜中央出现明显红色斑点为阳性，否则为阴性.

统计学处理感染率用百分数表示，作χ²检验，P<0.05差异有显著意义.

2 结果

2.1 cagA基因重组表达载体的构建 重组质粒可切出975bp的外源片断(图1).

图1 重组质粒pRSETcagA的酶切鉴定. M:PCR分子量参照物; 2:质粒 pRSETA; 1,3:重组质粒pRSETcagA

2.2 表达形式鉴定 表达CagA His6 M_r36000，主要以包含体形式存在(图2).

图2 CagAHis6表达形式鉴定. M:蛋白质分子量参照物; 1:外周质; 2:胞质; 3,4:包含体

2.3 包含体纯化 经纯化的CagAHis6见纯度可达98％(图3).

图3 包含体纯化. M:蛋白质分子量参照物； 1:诱导空菌； 2:诱导工程菌； 3:纯化CagA；4:变性CagA包含体

2.4 复性蛋白的活性鉴定 经纯化的CagAHis6倍比稀释后包被ELISA板，用抗CagA抗体阳性血清（生物晶美Hp抗原筛出）测定其活性，可见纯化的CagAHis6具有良好的活性(图4).

图4 纯化CagA活性测定

2.5 DIGFA及ELISA检测血清抗CagA抗体 正常人64例及胃癌患者血清50例中, cagA⁺ Hp的感染率分别为29.7％和62%，胃癌患者cagA⁺ Hp的感染率明显高于正常人(P<0.01, 表1).

表1 胃癌和正常人cagA⁺ Hp感染率的比较

项目	胃癌(n=50)	正常人(n=64)
男/女	27/23	34/28
平均年龄(岁)	46	37
anti-CagA⁺
总体ELISA n(％)/DIGFA n(％)	31(62.0)^b/30(60.0)^b	19(29.7)^b/17(26.6)^b
男ELISA n(％)/DIGFA n(％)	17(63.0)/16(59.3)	11(32.4)/11(32.4)
女ELISA n(％)/DIGFA n(％)	13(60.9)/13(60.9)	8(28.6)/7(25.0)

^bP<0.01, vs normal controls

3 讨论

大量研究显示，Hp菌株随着环境及感染人群不同表现出高度的基因多态性，但相反的是，其大多表型则是相对保守的，表型有差异的是CagA和空泡毒素VacA，并由此将Hp菌株大体分为两型，I型表达CagA和空泡毒素VacA, 即通常所谓的毒力株, II型则不表达CagA和空泡毒素^［¹⁸^］.最新研究发现，cagA是Hp致病岛的一部分，其编码蛋白可作为cagA致病岛的血清学标志^［^19，20^］，cagA基因由于其3'端含有数量不同的中间重复序列，其编码产物的大小不同，约为M_r（120～140）×10³, 但不影响其抗原性^［²¹^］.Hp致病岛基因编码IV型蛋白分泌系统，他在Hp黏附于胃上皮细胞后将C端酪氨酸磷酸化的CagA蛋白输入上皮细胞，触发一系列细胞内信号传递，包括核因子-κB的激活等，诱发炎症和免疫反应^［^22-24^］；和picB基因协同诱导产生IL-1β、IL-6以及TNF-α和IL-8, 致单核细胞和多核巨细胞浸润，蛋白酶和胶原酶释放最终引起胃黏膜损伤^［^25-27^］；临床研究也发现，CagA同Hp的更除率、胃黏膜萎缩病变、肠上皮化生有显著的相关性，一项随访研究表明，Hp的根除者胃窦和胃体炎症减退，胃体萎缩改善，降低服用非甾体类抗炎药致溃疡的危险性降低；通过cagA⁺ Hp的根除，某些MALT淋巴瘤可以彻底消退^［²⁸^］.
世界范围内，60～70％的Hp菌株中存在cagA，编码相应的CagA疏水性蛋白，cagA⁺ Hp的感染与慢性胃炎、消化性溃疡、胃癌及MALT 淋巴瘤密切相关^［²⁹^］；但在日本，韩国等亚洲国家, 90%以上的Hp含有cagA基因，并表达CagA蛋白，在流行病学上无法统计cagA⁺与疾病的相关性，因此，有部分研究认为，在韩国、日本、中国人群中，cagA⁺ Hp的感染率虽然很高，但并不能作为罹患胃炎、消化性溃疡、胃癌的危险性因子，他们并没有相关性^［^30-32^］.由于cagA⁺ Hp的感染可通过CagA血清抗体检测发现，我们选择cagA基因的保守区进行扩增，并选择pRSETA His6表达载体，高水平的表达His6-CagA，表达的CagA以包含体形式存在，不具有生物活性，可用含8M脲的变性液溶解，利用组氨酸和镍的亲和作用通过镍柱纯化，并进行重折叠，所得的重组CagA和阳性对照抗原比较具有良好的活性和抗原性，利用他来建立检测血清抗体的滴金法和ELISA，敏感性分别为95.5%和99%，特异性为98.5%和92％，可作为筛查cagA⁺ Hp的感染的简便有效的方法.
本研究对临床患者和正常人的血清抗体检测显示，胃癌患者感染cagA⁺ Hp的比例远高于正常人，其血清抗体滴度也高于正常人血清抗体滴度，因此，cagA⁺ Hp是潜在的致癌因子，CagA血清抗体可作为胃癌高位人群的标志，检测血清抗体可用以判断菌株类型，作为预测疾病未来转归的依据；一般而言，约有71%的非贲门癌源于cagA⁺ Hp的感染；一项对中国胃癌高发区林县181例胃癌病例的对照研究还发现，Hp的感染也和胃贲门癌的发生有关^［³³^］；只检测血清Hp抗体而不检测CagA血清抗体会漏掉一些近期或既往cagA⁺ Hp的感染，这也可能是一些研究的结论为胃癌与cagA⁺ Hp的感染无关的原因之一^［³⁴^］. Hp与胃黏膜细胞增生加速，cag致病岛与凋亡增加有密切联系; 而且, CagA可进入胃上皮细胞；cagA⁺ Hp的感染可上调许多生长因子的表达，如gastrin, 转化生长因子TNFα，生长因子HGF和COX-2，下调Bax，上调Bcl-2系统；和无Hp或cagA^- Hp的感染相比，只有cagA⁺ Hp的感染患者胃上皮细胞凋亡增加等^［^35－37^］，这些因素均直接或间接与胃癌的发生相关联.增生与凋亡在胃癌的产生过程中十分关键，研究证实Hp毒力因子包括LPS, Urease, VacA, NO均可诱导胃黏膜细胞凋亡，还有未知的其他因子参与其中；宿主因素似乎比Hp细菌本身对Hp感染个体的未来疾病转归更加重要.因此可以认为，胃上皮细胞增生与凋亡是一个包含多种途径的复杂的信号传递网络，其详细机制还有待发现^［^38－40^］.

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