幽门螺杆菌 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-06-15; 12(6): 1317-1320
在线出版日期: 2004-06-15. doi: 10.11569/wcjd.v12.i6.1317
携带幽门螺杆菌中性粒细胞激活蛋白活减毒鼠伤寒沙门菌口服重组DNA疫苗株的构建
孙波, 何苗, 杨骅, 金晶, 满晓华, 龚燕芳, 屠振兴, 杜奕奇, 李兆申
孙波, 杨骅, 金晶, 满晓华, 龚燕芳, 屠振兴, 杜奕奇, 李兆申, 中国人民解放军第二军医大学附属长海医院消化内科 上海市 200433
何苗, 中国人民解放军第二军医大学医技检验专升本26队 上海市 200433
孙波, 男, 1972-04-29生, 山东省平度市人, 汉族. 1999年第二军医大学毕业, 博士生, 主治医师, 讲师. 主要从事幽门螺杆菌疫苗防治研究.
基金项目: 国家自然科学基金资助项目, No. 30170427.
通讯作者: 杜奕奇, 200433, 上海市长海路174号, 中国人民解放军第二军医大学附属长海医院消化内科. duyiqi@hotmail.com
电话: 021-25070552 传真: 021-25070552
收稿日期: 2003-12-17
修回日期: 2004-01-09
接受日期: 2004-02-09
在线出版日期: 2004-06-15

目的: 构建携带人幽门螺杆菌(H. pylori)中性粒细胞激活蛋白(HP-NAP)基因(napA)的重组活减毒鼠伤寒沙门菌口服DNA疫苗.

方法: 应用基因工程技术扩增全长napA, 测序并同源性分析后, 将其亚克隆入真核表达载体pIRES, 鉴定正确后将重组质粒转化活减毒鼠伤寒沙门菌.

结果: 重组质粒经PCR及双酶切, 证实成功构建了携带HP-NAP基因的重组真核表达质粒pIRES-napA, 后者成功转化活减毒鼠伤寒沙门菌SL7207. 所克隆435bp napA与GenBank中SS1-napA核苷酸和蛋白质的同源性均为98%.

结论: 成功构建并鉴定了携带HP-NAP基因的重组活减毒鼠伤寒沙门菌口服DNA疫苗, 为多价抗H. pylori口服DNA疫苗的研制奠定了基础.

关键词: N/A
引文著录: 孙波, 何苗, 杨骅, 金晶, 满晓华, 龚燕芳, 屠振兴, 杜奕奇, 李兆申. 携带幽门螺杆菌中性粒细胞激活蛋白活减毒鼠伤寒沙门菌口服重组DNA疫苗株的构建. 世界华人消化杂志 2004; 12(6): 1317-1320
Construction of an oral recombinant DNA vaccine strain of live attenuated Salmonella typhimurium carrying H. pylori neutrophil activating protein
Bo Sun, Miao He, Hua Yang, Jing Jin, Xiao-Hua Man, Yan-Fang Gong, Zhen-Xing Tu, Yi-Qi Du, Zhao-Shen Li
Bo Sun, Hua Yang, Jing Jin, Xiao-Hua Man, Yan-Fang Gong, Zhen-Xing Tu, Yi-Qi Du, Zhao-Shen Li, Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Miao He, Division of, Medical Technology and Laboratory (Brigade 26), Second Military Medical University, Shanghai 200433, China
Supported by: the Natural Science Foundation of the Ministry of Health of China, No. 30170427.
Correspondence to: Yi-Qi Du, Department of Gastroenterology, Changhai Hospital, Second Military Medical University, 174 Changhai Road, Shanghai 200433, China. duyiqi@hotmail.com
Received: December 17, 2003
Revised: January 9, 2004
Accepted: February 9, 2004
Published online: June 15, 2004

AIM: To construct a live attenuated Salmonella typhimurium (S. typhimurium) strain carrying H. pylori-neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine.

METHODS: By genetic engineering method, a 435 bp napA gene (encoding HP-NAP) was subcloned into an eukaryotic expression vector pIRES. After sequencing and BLAST analysis, the identified recombinant plasmid was then transformed into a live attenuated S. typhimurium strain SL7207.

RESULTS: By using polymerase chain reaction (PCR) and restriction enzyme digestion, a recombinant eukaryotic expression plasmid pIRES-napA containing napA gene of H. pylori was constructed, and the recombinant plasmid was transformed successfully into the live attenuated S. typhimurium strain SL7207. Most of the H. pylori-napA sequences in recombinant plasmid pIRES-napA were homologized with that of SS1 strain reported by GenBank, and the homology of nucleotide and protein was 98%, respectively.

CONCLUSION: A recombinant live attenuated S. typhimurium strain carrying HP-NAP gene as DNA vaccine is successfully constructed. The results lay the foundation for developing multivalent oral DNA vaccine against H. pylori infection.

Key Words: N/A
0 引言

幽门螺杆菌(Helicobacter pylori, H. pylori)感染为慢性活动性胃炎和消化性溃疡的主要病因[1-8], 与肠型胃癌和胃黏膜相关淋巴样组织淋巴瘤的发生密切相关[9-16]. 以抗生素为基础的药物治疗为H. pylori感染的主要治疗方案, 但随着细菌耐药、药物不良反应及药效经济学等问题的突出, 迫切需要其他方法以防治H. pylori感染, 其一便是研制有效的黏膜疫苗.中性粒细胞激活蛋白(neutrophil activating protein, HP-NAP)为新近发现的H. pylori主要毒力因子之一[17]. 目前对HP-NAP的免疫原性及保护性研究尚处于起步阶段, 国内尚未见报道. 我们将HP-NAP基因亚克隆入真核表达载体pIRES, 并成功将重组质粒导入活减毒鼠伤寒沙门菌SL7207, 为进一步研究多价抗H. pylori口服DNA疫苗奠定了基础.

1 材料和方法
1.1 材料

H. pylori标准株CCUG 17874 (即NCTC 11638)由意大利IRIS 研究中心提供, 以弯曲菌选择培养基(Merck公司)37 ℃微需氧培养. 大肠杆菌DH 5a及活减毒鼠伤寒沙门菌LB 5000, SL 7207为本室保存. 胰蛋白胨及酵母提取物购自Oxoid公司. 克隆载体pBT为上海生工生物工程技术服务有限公司产品, 真核表达载体pIRES购自Clontech公司. EX Taq TM限制性内切酶XhoⅠ, MluⅠ购自TaKaRa公司. 小量细菌基因组DNA及质粒抽提、纯化试剂盒购自上海华舜生物工程有限公司. DNA Ligation Kit Ver.2及DNA Marker购自TaKaRa公司. 氨苄青霉素(Amp)、IPTG及X-gal为Amresco公司产品.

1.2 方法

1.2.1 重组真核表达质粒pIRES-napA的构建及鉴定: 根据GenBank中H. pylori-napA序列, 设计上游引物P1: 5'-GTC CTC GAG ATG AAA ACA TTT GAA ATT TTA AAA CAT TTG CAA GCG-3', 含XhoⅠ酶切位点; 下游引物P2: 5'-GTC ACG CGT TTA AGC CAA ATG GGC TTG CAA CAT CC-3', 含MluⅠ酶切位点, 由上海生工生物工程技术服务有限公司合成, 确保克隆基因开放读码框正确. CTAB法[18]小量抽提H. pylori基因组DNA为模板, 热循环仪行PCR: 94 ℃预变性5 min, 94 ℃变性60 s, 60 ℃复性60 s, 72 ℃延伸60 s, 30循环后再延伸10 min. 15 g/L琼脂糖凝胶电泳观察扩增结果. 将纯化PCR产物TA克隆入载体pBT(pBT-napA), 转化受体菌DH 5a感受态细胞(氯化钙法[18]), 蓝白斑筛选并扩增Amp抗性克隆, 碱裂解法[18]小量抽提质粒, 测序鉴定. pBT-napA经XhoⅠ及MluⅠ消化后切胶回收napA, 将其亚克隆入pIRES之相同双酶切片段(pIRES-napA), 挑取阳性克隆, 抽提质粒行PCR及双酶切鉴定.

1.2.2 测序及生物信息学分析: 重组质粒pBT-napA由上海生工生物工程技术服务有限公司进行序列测定. 测序结果与GenBank中H. pylori-napA核苷酸和蛋白质序列进行BLAST分析. Antheprot V5.0软件分析其生物学特性.

1.2.3 口服DNA疫苗株的构建及鉴定: 将重组质粒pIRES-napA转化LB 5000进行修饰, 挑取阳性菌落, 提取质粒进一步电击转化(25 mF, 2.5 kV, 200 W, 5 ms)[18] 终宿主菌SL 7207, 挑取阳性菌落于含100 mg/L Amp LB培养液扩增60代, 抽提质粒行PCR及双酶切鉴定.

2 结果
2.1 H. pylori-napA基因的扩增

PCR自H. pylori基因组DNA中扩增出一435 bp条带, 与预计相符(图1泳道2).

图1
图1 重组质粒pBT-napA及pIRES-napA鉴定图谱. 1: 100 bp DNA Ladder marker; 2: PCR amplified product of HP-NAP; 3: Recombinant plasmid pBT-napA; 4: pBT-napA/Xho I + Mlu I; 5: Plasmid pIRES; 6: Recombinant vector pIRES-napA; 7: pIRES-napA/Xho I + Mlu I; 8: PCR amplified product of pIRES-napA; 9: Plasmid SL 7207/pIRES-napA extracted from ending-host SL 7207; 10: SL 7207/pIRES-napA/Xho I + Mlu I; 11: PCR amplified product of SL 7207/pIRES-napA; 12: DNA marker DL 2000 + 15 000.
2.2 重组质粒的鉴定

重组质粒分别命名为pBT-napA及pIRES-napA(图2). 经双酶切和PCR鉴定, pBT-napA及pIRES-napA均含有435 bp的目的片段(图1), 表明重组质粒构建成功.

图2
图2 重组质粒pBT-napA及pIRES-napA构建示意图.
2.3 目的片段序列测定及生物信息学分析

将测序结果与GenBank中H. pylori-napA核苷酸和蛋白质序列进行BLAST分析, 结果显示我们所克隆之H. pylori-napA与GenBank中H. pylori SS1株napA基因的同源性为98% (427/435), 其中C→T置换3个, A→G置换4个(图3). 与其他常见H. pylori菌株, 如5D, 5A, 2B, 2A, RHP901a, DB2, 1811a等同源性均为97% (426/435, 425/435). 克隆序列的碱基置换多数未改变氨基酸的编码, 与GenBank中SS1株的蛋白质同源性为98%(142/144), 与其他常见H. pylori菌株的同源性均为97%. Antheprot软件显示其蛋白质抗原性较SS1株无明显改变(图4).

图3
图3 幽门螺杆菌-napA核苷酸序列及其推导的氨基酸序列.
图4
图4 HP-NAP抗原性分析(Welling法). A: H. pylori SS1 HP-NAP; B: 目的序列.
2.4 口服DNA疫苗稳定性及鉴定

携带重组质粒pIRES-napA之SL 7207扩增60代后未见明显质粒丢失. 以抽提质粒为模板, P1, P2为引物, PCR扩增出约435 bp目的基因; XhoⅠ/MluⅠ双酶切亦可见线性化载体片段和目的基因(图1泳道10, 11).

3 讨论

胃黏膜感染H. pylori以中性粒细胞和单核细胞浸润为特征, 其损害程度与中性粒细胞浸润密切相关[19]. H. pylori水提取物150 ku蛋白有促进中性粒细胞黏附内皮细胞及诱导中性粒细胞产生反应性氧自由基的作用, 命名为中性粒细胞激活蛋白(HP-NAP)[20]. HP-NAP位于H. pylori菌体内, 通过自溶释放后, 结合于细菌表面, 于此起到黏附素的作用, 介导与粘蛋白的结合[21], 或者与多形核白细胞神经鞘磷脂结合[22].纯化的重组HP-NAP不含大肠杆菌脂多糖, 对人类中性粒细胞和单核细胞具有化学趋化作用[17], 并诱导内皮细胞移行所必需的b2整合素的表达[20], 提示HP-NAP具有使上述细胞积聚于H. pylori 感染部位的作用. HP-NAP为反应性氧自由基的强刺激剂, 其与特异受体结合, 级联激活细胞内事件, 胞质Ca2+浓度及蛋白质磷酸化增加, 进而通过PTX敏感途径(包括ERK和p38-MAPK), 导致中性粒细胞表面功能性NADPH氧化酶的富集[23]. 细胞因子TNF和IFN-γ可增强HP-NAP诱导的活性氧产生, 加重胃黏膜的损伤[24]. HP-NAP可促进纤维蛋白沉积和由H. pylori引起的胃黏膜炎症反应[25]. HP-NAP亦可穿过上皮单层, 序贯激活肥大细胞脱颗粒和释放前炎症细胞因子IL-6[26].此外通过促进中性粒细胞和单核细胞募集和激活而直接作用于二者.

HP-NAP高度保守, 60%的H. pylori感染患者体内存在HP-NAP特异抗体. Satin et al[17]. 以纯化重组HP-NAP口服免疫10只小鼠, 结果80%个体获得保护性免疫, 高于CagA(70%), 低于H. pylori超声裂解物(90%).提示HP-NAP为H. pylori主要的毒力因子, 可作为有效的保护性抗原用于H. pylori感染的疫苗防治.

DNA疫苗因可诱导全面的免疫应答、提供同种异株交叉保护作用、易于制备多价疫苗、兼有预防和治疗作用等特点在多种疾病的防治中显示出巨大的应用潜力.而与传统疫苗相比, 活减毒鼠伤寒沙门菌这一新型口服疫苗载体释放系统亦具有无需纯化抗原, 无需佐剂, 可避免抗原在胃内降解和变性等优点[27-29]. 我们将HP-NAP基因亚克隆入真核表达载体pIRES, 以其转化活减毒鼠伤寒沙门菌SL 7207, 经PCR和双酶切鉴定, 成功构建了HP-NAP活减毒鼠伤寒沙门菌口服DNA疫苗; 所克隆HP-NAP全长DNA序列经BLAST分析与GenBank中相关序列具有高度同源性, 生物信息学分析显示其具有良好抗原性.为多价基因工程疫苗的后继研究奠定了重要的实验基础.

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