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⊙研究原著⊙

幽门螺杆菌vac A基因毒性相关片段的克隆及序列分析

江  红  阎小君  苏成芝  韩锋产  冯永强  侯  瑜

中国人民解放军第四军医大学基因诊断技术研究所  陕西省西安市  710032
江红，女，1967-11-06生，辽宁省北票市人，汉族. 1989年陕西师范大学生物系本科毕业，1992年西南师范大学生物化学专业硕士毕业，现攻读第四军医大学分子生物学与生物化学专业博士学位，主要从事基因克隆与表达的研究. 发表论文5篇.
项目负责人  江红,710032,陕西省西安市,中国人民解放军第四军医大学基因诊断技术研究所.
Institute of Genetic Diagnosis, Fourth Military Medical University, Xi＇an 710038, Shaanxi Province, China
Correspondence to Hong Jiang, Institute of Genetic Diagnosis, Fourth Military Medical University, No 17 Changle Xilu, Xi＇an 710038, Shaanxi Province, China
Tel. 0086-29-3374771,  Fax. 0086-29-3216587
Email. jhmqf@263.net
收稿日期  2000-04-03  接收日期  2000-04-13

Cloning and sequence analysis of a fragment related to vacuolating cytotoxin of vacA of Helicobacter pylori

Hong Jiang, Xiao-Jun Yan, Cheng-Zhi Su, Feng-Chan Han, Yong-Qiang Feng and Yu Hou




Abstract

AIM  We clone and sequence a fragment related to vacuolating cytotoxin of vacA from Helicobacter pylori (Hp） strain 88022 for the clinical analysis of Hp vacA.

METHODS  A 903bp fragment was amplified by the polymerase chain reaction. The template DNA was purified from Hp strain 88022. We designed the primers located in 316bp-335bp and 1198bp-1218bp respectively, from the relatively reserved region of vacA gene. DNA fragment containing the sequence codes for amino acids 34-334, was digested by BamHⅠ and PstⅠ and cloned into pGEM-3Zf(-) plasmid. Its sequence was determined with an autosequencing instrument from two directions and compared with those having the corresponding region of vacA from other Hp strains.

RESULTS  The DNA sequence we got in this study is the same as that of Hp strain CCUG17874 (GeneBank gi 619248) and NCTC11638 (GeneBank gi 495469).

CONCLUSION  We got a recombined clone of Hp vacA related to vacuolating  cytotoxin which has laid the foundation for further studies.

Subject headings  Helicobacter pylori (Hp）; vacA gene; molecular clone; sequence analysis

Jiang H, Yan XJ, Su CZ, Han FC, Feng YQ, Hou Y. Cloning and sequence analysis of a fragment related to vacuolating cytotoxin of vacA of Helicobacter pylori.  Shijie Huaren Xiaohua Zazhi, 2000;8(7):728-732 


摘要

目的  vacA 基因编码的蛋白是幽门螺杆菌的一个重要毒力因子，通过空泡化作用损伤上皮细胞. vacA基因与Hp 感染者的临床发病有着密切的关系，vacA的基因型决定毒素蛋白体外表达水平的高低. 我们从幽门螺杆菌88022菌株中扩增出vacA基因毒性相关片段，进行序列测定和序列比较分析，为Hp 的临床检测奠定基础.

方法  以该菌株总DNA为模板，紧随vacA基因序列s区保守区域的引物（位于316bp～335bp和1198bp～1218bp）扩增vacA基因的一个903bp片段，编码的氨基酸为34～334位，用BamHⅠ和PstⅠ双酶切后克隆入pGEM-3Zf（-）质粒载体，以双脱氧法双向测定目的片段的序列，拼接出该片段的全序列，并与已知的Hp vacA基因序列作比较.

结果  所得序列与Hp 国际标准株CCUG17874 (GeneBank gi 619248)和NCTC11638 (GeneBank gi 495469)的vacA基因序列完全一致，氨基酸序列分析和所报道的结果一致.

结论  构建了Hp vacA基因毒性相关片段的重组克隆，为以后的进一步研究奠定了基础.

主题词  幽门螺杆菌；vacA基因；聚合酶链反应；克隆；序列分析

江红,阎小君,苏成芝,韩锋产,冯永强,侯瑜. 幽门螺杆菌vac A基因毒性相关片段的克隆及序列分析.世界华人消化杂志，2000；8(7)：728-732


0  引言

幽门螺杆菌（Helicobacter pylori，Hp）为革兰阴性螺旋杆状细菌，生存于人胃部（以幽门附近的胃窦最多），是慢性胃炎［1-3］和消化性溃疡［4-9］ 的病因之一，同时又是胃癌［10-16］和粘膜相关淋巴样组织性淋巴瘤的危险因素［17-19］. 1994年国际癌症研究机构 (IARC) 将Hp 列为人类Ⅰ类致癌原［20］，这是被认为与致癌有关的第一个细菌. 它是世界性分布的病菌，在发达国家有20%年龄小于30岁的人口被感染，50%小于60岁的人口被感染，在发展中国家则更高，有80%大于20岁的人口被感染. 细胞空泡毒素A（vacuolating cytotoxin A, vacA）基因编码可损伤上皮细胞的空泡毒素A. vacA存在于所有的Hp 菌株，但仅在50%～60%的菌株中表达［21,22］. vacA基因由s区（编码信号肽）和m区（编码中间区域）两部分组成，s区有sla，slb，slc和s2四种类型：m区有ml，m2a和m2b三种类型，因而vacA基因有多种基因型组合，不同的Hp 菌株有不同的vacA基因型组合. 有学者研究表明我国 vacA基因型多为slm2型［23-26］，不同Hp 菌株vacA基因的大小略有不同，多为3.8kb左右. 我们以Hp 菌株88022总DNA为模板扩增了vacA基因的一个片段，克隆入pGEM-3Zf(-)质粒载体，测定其核苷酸序列，并作同源性比较，为其进一步的表达和毒性鉴定打下基础.

1  材料和方法

1.1  材料  Hp 菌株88022为西京医院消化内科乔文博士赠送；BamHⅠ，PstⅠ，T4 DNA Ligase，DNA marker(DL2000)，dNTP混合物，异丙基硫代-β-D-半乳糖苷（IPTG），5-溴-4-氯-3-吲哚-β-D-半乳糖苷（X-gal）均购自日本TaKaRa公司；大肠杆菌JM109菌株及质粒载体pGEM-3Zf(-)系第四军医大学赵锦荣博士赠送；Taq酶和质粒提取试剂盒Promega Wizard Plus Miniprep DNA Purification System购自Promega公司；DNA回收试剂盒Nucleospin Extraction Kit购自Clontech公司；根据不同Hp 菌株序列的比较设计引物，上游引物5＇-CGC GGA TCC ATG GCC TTT TTC ACA ACC GTG AT-3＇，含有BamHⅠ酶切位点和起始码ATG，下游引物5＇-CGC CTG CAG TTA TTT ATC CTT ATA GCC ACC TTC-3＇，含有PstⅠ酶切位点和终止码TTA，由上海生物工程公司合成；PCR仪为美国Progene公司产品.

1.2  方法  制备模板：在Hp 平板上挑取生长良好的菌落，按颜子颖et al方法［27］，提取Hp 总DNA，溶于100μL无菌水中，稀释5倍作为模板. 目的片段的扩增：PCR反应的总体积为25μL，其中含模板2μL，每种引物各10pmol，2.5mmol/L dNTP混合物2μL，10×PCR Reaction buffer 2.5μL，25mmol/L MgCl2 2.5μL，耐热DNA聚合酶0.75U，加H2O补足25μL. PCR扩增反应条件为94℃预变性2min；94℃变性1min，50℃退火1min，72℃延伸3min，共循环40次. 反应结束后在10g/L琼脂糖凝胶上检查扩增结果，电泳缓冲液为0.5×TAE，溴化乙锭染色，并在紫外灯下切下目的条带. PCR产物克隆：用Clontech 公司DNA回收试剂盒回收PCR产物，溶于40μL NE溶液(5mMTris-HCl pH 8.5)中. 取0.5mL的EP管2支，其中一支加入含有20μL回收目的片段DNA，另一支加入含有1μg pGEM-3Zf(-)的溶液，然后各加入10×K buffer 2.5μL，BamHⅠ 1μL(12U)，PstⅠ 1μL(15U)，30℃温育2h之后37℃温育2h. 反应结束后进行10g/L琼脂糖凝胶电泳，同前切下目的条带. 将含双酶切目的片段的琼脂糖凝胶与含双酶切pGEM-3Zf(-)的凝胶按3∶1的比例放在同一1.5mL的EP管中，用Clontech公司DNA回收试剂盒回收，最后溶于30μL NE溶液中，向回收产物中加入4μL T4 DNA Ligase buffer, 1μL T4 DNA Ligase，于12℃保温16h，连接产物存于-20℃备用. 将连接产物5μL转化JM109感受态细胞，同时以水作空白对照，pGEM-3Zf(-)作阴性对照，在含有Amp(100mg/mL)的LB平板上加入20μL 0.1mol/L IPTG和16μL 5% X-Gal作蓝白筛选，挑取白色克隆，培养后用Promega公司的质粒提取试剂盒提取质粒，同前双酶切鉴定重组质粒. 序列测定：选择经双酶切鉴定阳性的含重组vacA-pGEM-3Zf(-)的菌株，由上海博亚生物技术有限公司用PE ABI 377测定序列.

2  结果

2.1  模板制备与扩增  从Hp 菌株88022中提取总DNA，用上、下游引物按照上述条件进行PCR反应，取反应产物10μL，经10g/L琼脂糖凝胶电泳，显示扩增出一条903bp的片段(图1).

2.2  PCR产物的克隆  将目的片段的PCR产物用Clontech试剂盒纯化，双酶切后与同样双酶切的pGEM-3Zf(-)连接，转化E.coli JM109，通过氨苄青霉素筛选和蓝白筛选挑选白色阳性克隆，用Promega Wizard Plus Minipreps DNA Purification System提取质粒，经双酶切鉴定含有插入的目的片段（图2）.

图1  vacA基因PCR产物电泳结果.1. DNA marker(DL2000)；2. vacA基因PCR产物

图2  vacA-pGEM-3Zf(-)酶切鉴定结果.1. DNA marker (DL2000)；2. vacA-pGEM-3Zf双酶切产物(3.2kb+903bp)

2.3  测定结果  含重组质粒的阳性克隆在自动测序仪PE ABI 377上正、反双向测定，并拼接出完整序列，结果如下(奇数行为DNA序列，偶数行为氨基酸序列)：

2.4  同源性分析  将所得Hp 菌株88022 vacA基因的毒性相关片段与GeneBank中序列进行比较，结果显示，它与报道的Hp 国际标准菌株CCUG17874和NCTC11638的vacA基因序列完全一致.


3  讨论

幽门螺杆菌感染的普遍性高，且容易作为一个重要的致病因子引发消化系统的疾病，感染Hp 的个体是否发病，一方面与宿主基因易感性和环境因素有关［28］，另一方面与所感染Hp 菌株的型别（即存在不同毒力的Hp 菌株）［29-36］有关，在感染了Hp 的人群中大约只有15%的人发病［37］因而对Hp的检测特别是菌株型别的检测就尤为重要. VacA基因编码的蛋白质，是Hp 的一种重要致病因子［38］，也是Hp 毒力分型的标志［39-41］，与Hp 感染者的临床发病有着密切的关系.
    Leunk et al［42］于1988年首先报道了Hp 能产生使真核细胞空泡变性的毒素，并命名为vacA. 这种蛋白质对热稳定［43］，对胰蛋白酶敏感，可被酸性蛋白酶和碱性蛋白酶中和，能被硫酸胺沉淀. 目前对vacA的生物学功能研究得比较清楚，体外实验表明它能致许多哺乳动物细胞的广泛性空泡样变性［44-46］，虽然这种作用是非致死性的，即空泡化的细胞仍然是活的，但由于细胞的空泡化，细胞在数天后失去正常形态，发生皱缩，最后仍然死亡. 在感染Hp 的慢性活动性胃炎者的胃粘膜上皮也观察到类似的结果；小鼠口服vacA后，胃部组织出现明显的损害现象［47］. 于长清et al［48］对48例消化病患者分离得到42株Hp 菌，发现约有62%的菌株产生空泡毒素，并且不同疾病感染产毒株的比例也有差别，其中胃癌阳性率最高（80%），消化性溃疡患者阳性率为57.7%，而慢性胃炎感染率仅为1/8，这提示Hp 的vacA与较严重胃粘膜病变的发生密切相关［49-51］. 在体外，V型ATPase抑制剂可阻断或逆转细胞的空泡样变，而Na+-K+ATPase抑制剂可使其增强，提示细胞空泡毒素的作用机制是使靶细胞内阳离子的转运机制发生异常［52,53］，但vacA的确切致病机制仍不十分清楚［54］.
       VacA蛋白在分泌过程中，信号肽协助前体蛋白穿过细菌内膜后留在内膜，C末端插入细菌外膜协助蛋白的通过，C末端本身则留在外膜［55-57］，因此，vacA基因表达产物的空泡毒素活性主要与vacA基因信号肽和C末端之间的区域（即基因序列的中间部分）有关. Telford et al［47］曾以CCUG17874（s1m1型）为模板表达了五个vacA片段，其中C端的两个片段无活性，信号肽和C末端之间三个片段有活性；同样Pagliaccia  et al［58］曾以Hp 菌株95～54（s1m2型）为模板表达了一个对RK-13细胞具空泡毒素活性的2556bp长的片段，也是位于这个区间. 研究表明，Hp vacA的基因型对体外vacA蛋白的毒性高低具有决定性作用. 在体外，s1m1型Hp 菌株产生的毒性水平最高，s1m2型产生低至中等程度的毒性，s2m2型无毒性，而基因型为s2m1的菌株尚未发现［59］.Hp 在体外不产生活性vacA，可能是由于某些Hp 菌株在体外缺乏对vacA的表达，而在体内则可能表达；还可能是某些Hp 菌株完全缺乏具有活性功能的vacA［60-63］. 我们通过GeneBank比较了不同Hp 菌株CCUG17874（s1m1）,NCTC11637(s1m1)，NCTC11638(s1m1)，ATCC60190(s1m1)，95-54(s1m1)和 Tx30a (s2m2) 的vacA基因序列，发现s1m1，s1m2，s2m2型Hp 菌株的vacA基因在s区具有较高的同源性；其是C端区域，而在S区和C端之间的区域变异较大，我们在vacA基因序列的中间部分找出相对保守的区域，以CCUG17874为依据设计引物，上、下游引物的位置分别为316bp～335bp和1198bp～1218bp，选择克隆了紧随s区的316bp～1218bp间的一个片段，以期能获得具有空泡毒性的片段，为临床对该基因的检测奠定基础.另外，我们在设计引物时，通过对CCUG17874和NCTC11638菌株vacA基因序列的比较，发现这两个菌株vacA基因的编码区序列完全一致，提示它们可能是同一个菌株.

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