摘要
目的：
幽门螺杆菌(Helicobacter pylori, H. pylori)为人类胃部疾病的重要致病菌之一,世界范围内感染率达到50%。Ⅰ型H. pylori菌株因携带cag致病岛(cag pathogenicity island, cag-PAI)而致其毒性较强,流行病学研究显示：Ⅰ型菌株与多种胃肠道疾病的发生关系更为密切。cag致病岛编码的Ⅳ型分泌系统能够合成并转运-细胞毒素相关基因A蛋白(Cytotoxin-associated gene A, CagA)进入到宿主细胞内导致细胞功能紊乱。目前已知致病岛可编码27种蛋白,但多数蛋白的功能尚未十分清楚。本文选择了致病岛中的hp0520作为研究对象,通过微生物学、分子生物学、免疫学和生物信息学等技术探讨hp0520基因的功能,为深入研究cag致病岛的功能和致病机制奠定基础。
方法：
1.根据Genbank收录的H. pylori26695全基因序列,利用Primer Premier6.0设计hp0520全长克隆基因引物,通过T-A克隆的方法,获得目的基因片段并测序；并对序列进行生物信息学分析研究。
2.构建原核表达载体pET-32a-hp0520,并转化入表达工程菌Rosetta (DE3);经IPTG诱导后,使用QIAGEN系统Ni2+-NTA柱纯化获得融合蛋白。经SDS-PAGE电泳、Western blot分析和质谱方法鉴定目的蛋白。
3.纯化后的HP0520融合蛋白免疫BALB/c小鼠,利用细胞融合技术将免疫小鼠的脾细胞与小鼠骨髓瘤细胞(SP2/0)融合获得杂交瘤细胞；通过数轮亚克隆和间接ELISA筛选能稳定分泌单克隆抗体(monoclonal antibody, mAb)的杂交瘤细胞；腹腔接种阳性杂交瘤细胞于BALB/c小鼠,制备腹水型mAb,并进行纯化；用ELISA法鉴定mAb的免疫球蛋白类型；间接ELISA法测定杂交瘤细胞培养上清、腹水的抗体效价；间接ELISA法和Western blot法鉴定mAb与抗原的反应特异性。
4.利用同源重组原理,构建hp0520基因缺陷自杀质粒pBlueKM40-△hp0520::Kmr,电转化进入H. pylori NCTC11637中,通过抗性筛选和PCR及Western-blot方法验证,获得hp0520基因缺失株。
5.收集并裂解H. pylori NCTC11637细菌菌体,使用超高速低温离心方法得到不同的亚细胞组分：膜组分(包括内膜和外膜)、胞质组分和周质组分,使用Western-blot方法,检测HP0520蛋白亚细胞定位；同时将h. pylori NCTC11637与胃上皮细胞GES-1共培养后,分离GES-1细胞组分成细胞膜蛋白和细胞浆部分,检测共培养后HP0520蛋白能否进入GES-1细胞中。
6.利用细菌双杂交体系,寻找cag致病岛中与HP0520蛋白具有相互作用的可疑蛋白。收集并裂解hp0520基因缺失株与野生株,用Western-blot方法检测cag致病岛编码蛋白CagX、CagM、CagL和CagA,研究hp0520基因对这些蛋白稳定性的影响。
7.将hp0520基因缺失株与野生株分别与胃上皮细胞GES-1共培养后,检测其对细胞形态,细胞因子分泌和CagA蛋白转运的影响。
结果：
1. H. pylori NCTC11637菌株hp0520基因全长为348bp,编码蛋白长度为115aa,其相对分子质量为12.42kDa,等电点为8.58。该蛋白属于稳定且亲水性的蛋白。在蛋白两端存在多个亲水区域,而蛋白的中段则是疏水的跨膜区域。预测该蛋白存在信号肽,位置在N端的前21个碱基,在21至22的位置处断裂。并且存在一个跨膜区域,位于蛋白中段,两端亲水,极可能游离于膜外。系统进化树分析表明H. pylori NCTC11637菌株的hp0520基因与hpEurope DU52:2及hpEurope101UK位于同一分支,同源性最高。
2.构建原核表达载体pET-32a-hp0520,通过IPTG诱导及QIAGEN系统纯化获得大量目的蛋白。
3.将纯化后的蛋白免疫小鼠获得了一株鼠抗HP0520融合蛋白的单克隆抗体HP20-1,效价为1：3.2×105。
4.成功构建hp0520基因敲除重组自杀质粒：pBlueKM40-△hp0520::kan。电转化自杀质粒进入H. pylori NCTC11637菌体内,通过卡那霉素抗性筛选及PCR、 Western-blot验证得到H. pylori hp0520基因缺失株：Ahp0520。
5.以单克隆抗体HP20-1为一抗作Western-blot分析,确定H. pylori在自然情况下会合成HP0520,且HP0520位于菌体膜部分,不会通过分泌进入到细胞内。
6.细菌双杂交结果筛选到HP0521、HP0525、HP0526及HP0538与HP0520蛋白具有相互作用。
7.比较H. pylori野生株和△hp0520基因缺陷株中四种已知Cag-TFSS重要的外膜结构蛋白及效应蛋白的表达情况。结果显示缺失株中CagX、CagM、CagL和CagA蛋白表达水平与野生株没有明显差异。
8.H. pylori与GES-1细胞共培养,检测细胞形态、CagA蛋白转运量及细胞因子分泌,结果显示hp0520基因缺失株可抑制CagA转运及细胞因子的分泌。
结论：
1.成功克隆了hp0520全长基因,使用生物信息学方法了解其编码蛋白的理化性质,并预测了其存在信号肽和跨膜区域。
2.确定了HP0520蛋白的亚细胞定位,位于菌体膜部位,且不能分泌进入细胞。
3.证明了HP0520蛋白可能与]HP0525、HP0526及HP0538构成TFSS的内膜蛋白具有相互作用,hp0520缺失后对CagX、CagM、CagL位于TFSS外膜蛋白和CagA效应蛋白的稳定性没有影响,说明HP0520蛋白位于细菌的细胞内膜上。
4.证明了hp0520基因缺失后使H. pylori刺激细胞分泌细胞因子的能力明显下降与空白对照组一致,CagA蛋白转运能力也明显下降但并没有完全消失,说明hp0520基因参与TFSS诱导细胞因子分泌及CagA转运过程,结合亚细胞定位结果证明HP0520蛋白在Ⅳ型分泌系统中是位于内膜上的结构蛋白。
The Helicobacter pylori (H. pylori) is one of the human gastric pathogens which infects half of the world's population. Type I H. pylori strains harboring the cag pathogenicity island (cag PAI) are highly relevant with several gastric diseases. The cag PAI encoded Type IV Secretion System (TFSS) is able to synthesize and deliver its major effector protein CagA into host cells for cell damage. So far, studies have shown that cag PAI encodes27proteins and the function of most genes is still unclear. We concentrated on hp0520, the No.l gene of cag PAI. Microbiology, molecular biology, immunology and bioinformatics methods were used for exploring the function of hp0520. All of these results will shed light on the function and pathogenic mechanism of cag PAI.
Methods:
1. A primer pair of hp0520gene was designed by Primer Premier6.0according to the complete gene sequence of H. pylori26695from Genebank. The target genome was obtained through T-A cloning method and studied using bioinformatics methods.
2. The expression vector pET-32a-hp0520was constructed and transformed into E. coli Rosetta (DE3). After induced by IPTG, the target protein was expressed and purified by Ni2+-NTA via QIAGEN system. The purified protein was identified via Western blot analysis and mass spectrum methods.
3. The purified HP0520protein was used to immunize BALB/c mice. Then the spleen cells were fused with myeloma SP2/0cells via cell fusion methods. The positive hybridoma cell lines, which were able to secret the target monoclonal antibody (mAb) were screened by means of several rounds of subclone and indirect ELISA methods. The positive hybridoma cell lines were intraperitoneal inoculated BALB/c mice to produce ascetic-type mAb. Immunoglobin isotype of the mAb was identified by ELISA methods. Indirect ELISA methods were used to test the titer of the mAb in cell culture supertanant and ascites. Indirect ELISA methods and Western blot analysis were used to indentify the specific reaction between the mAb and its antigen.
4. The suicide vector pBlueKM40-△hp0520::Kmr was constructed on the basis of an allelic exchange mutagenesis strategy and introduced into H. pylori NCTC11637by electroporation. Positive clones were selected based on antibiotic selection and identified by PCR and western blot analysis and an hp0520-deficient mutant was obtained.
5. The H. pylori NCTC11637bacteria cells were collected and separated into three individual fractions including periplasm, cytoplasm and membrane (including inner and outer membrane). Then the subcellular fractionation of the HP0520protein was determined by western blot analysis. Meanwhile, The H. pylori NCTC11637strains were co-cultured with human cell GES-1to determine whether the HP0520protein was secreted into the GES-1cell.
6. To explore the interaction of HP0520and other cag PAI proteins, the bacterial two-hybrid system was used. Then the hp0520gene mutant and wild type NCTC11637were collected and prepared for whole-cell lysates. Western blot analysis was used to detect the expression of CagX, CagM, CagL and CagA after hp0520gene mutation.
7. The hp0520gene mutant and wild type NCTC11637were co-cultured with GES-1cells respectively. Cell morphology, cytokines secretion and the ability of CagA translocation were detected.
Results:
1. H. pylori NCTC11637hp0520gene is348bp long and its encoded protein is115amino acids long. The relative molecular mass of HP0520is12.42kDa and the isoelectric point is8.58. There are multiple hydrophilic regions on the both sides of the protein, whereas hydrophobic transmembrane regions exist on the middle area. It is predicted that HP0520owns one signal peptide which is constituted with first21bases with a cleavage between the twenty-first and twenty-second base on the C-terminal.
2. The expression vector pET-32a-hp0520was constructed and the target protein was obtained by IPTG induction and purified via QIAGEN system.
3. The purified protein was used to immunize mice to obtain the HP20-1cell line, which was able to secret anti-HP0520monoclonal antibody. The titer of the antibody is1:3.2叁105.
4. The suicide vector pBlueKM40-A hp0520::kan was constructed and introduced into H. pylori NCTC11637.The H. pylori Ahp0520mutant was screened via kanamycin resistance, PCR and Western blotting.
5. The monoclonal antibody, HP20-1, was used for Western blot analysis. The results confirmed that H. pylori was able to synthesize HP0520under natural conditions. Moreover, HP0520was localized in the bacterial membrane and not secreted into host cells.
6. The bacterial two-hybrid system has screened that HP0520interacted with HP0521, HP0525, HP0526and HP0538, among which HP0525, HP0526and HP0538were located in the bacterial inner membrane.
7. The expression of4critical outer-membrane structural and effector proteins in the Cag-T4SS were compared between the hp0520gene mutant and wild type NCTC11637. The result suggested that deletion of hp0520had no significant influence on the expression of CagX, CagM, CagL and CagA.
0. The hp0520gene mutant and wild type NCTC11637were co-cultured with GES-1cells. Then Cell morphology, CagA translocation and cytokines secretion were detected. The data showed that deletion of hp0520gene decreased CagA translocation into host cells and cytokines secretion.
Conclusion:
1. The full-length hp0520gene was successfully cloned. Bioinformatics methods were used to understand the physico-chemical properties of the hp0520encoded protein and predict signal peptides and transmembrane regions.
2. The hp0520gene encoded protein was localized in the bacterial membrane and not able to be secreted into the host cell.
3. HP0520protein interacted with HP0525, HP0526and HP0538, which were chaperone-like proteins in the TFSS localized in the inner membrane. Moreover, deficiency of HP0520did not influence the stablization of CagX, CagM, CagL (inner membrane proteins) and CagA (effector protein). This result suggested that HP0520was located in the membrane of H. pylori.
4. Deletion of hp0520abolished the host cell ability of cytokines secretion and partial function of CagA translocation, which means the function of T4SS was partly weakened. These results suggested that HP0520was a structure protein in the TFSS localized in the inner membrane.
引文
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