幽门螺杆菌vacA基因型和cagA基因及其表达产物与胃十二指肠疾病的关系

幽门螺杆菌(Helicobacter pylori,Hp)感染是慢性胃炎(chronic gastritis,CG)和消化性溃疡(peptic ulcer,PU)的病因之一。也是胃癌(gastric cancer,GC)的危险因素。Hp的空泡形成毒素基因(vacuolating toxin gene,vacA)型和细胞毒素相关基因(cytotoxin-associated gene,cagA)及其表达产物——空泡形成细     

印迹基因PEG10在胃癌中的表达及其与幽门螺杆菌感染的关系

目的探讨印迹基因PEG10在胃癌组织中的表达特点及其与幽门螺杆菌（Hp）感染的关系。方法采用逆转录聚合酶链反应（RT-PCR）检测40例胃癌、癌旁组织中印迹基因PEG10的mRNA水平;采用免疫组织化学染色技术及Warthin-Starry银染法检测42例胃癌、癌旁组织及6例正常胃组织中PEG10蛋白的表达及Hp感染情况。结果RT-PCR结果显示20例胃癌组织中9例（45.0%）PEG10mRNA表达,对应的癌旁组织仅有2例（10%）表达;免疫组织化学染色结果显示42例胃癌组织中22例（52.38%）PEG10蛋白表达阳性,对应的癌旁组织仅有5例（11.90%）PEG10蛋白表达阳性,正常胃组织无PEG10蛋白表达;Hp银染法结果显示42例胃癌组织中25例（59.52%）为Hp感染阳性,癌旁组织20例（47.62%）Hp感染阳性;22例PEG10阳性表达胃癌组织中20例Hp感染阳性。结论PT-PCR结果与免疫组织化学染色结果具有一致性,即PEG10mRNA及蛋白在胃癌组织和癌旁组织中的表达率比较差异有显著性（P〈0.05）;PEG10在胃癌组织中的表达与Hp感染呈正相关（P〈0.05）。     

幽门螺杆菌抗原基因ureB在乳酸菌中的克隆表达与免疫反应性研究

目的为了开发幽门螺杆菌（Helicobacter pylori,Hp）口服疫苗,将Hp尿素酶B（UreB）在食品级乳酸乳球菌NZ3900菌株中进行表达,并研究其免疫反应性。方法 PCR扩增Hp MEL-HP27菌株ureB基因（基因号：FJ436980）,将其克隆入大肠杆菌-乳酸乳球菌穿梭质粒pNZ8110中并转化乳酸乳球菌NZ3900;采用正交试验确定目的蛋白表达的适宜条件;应用western-blot鉴定其免疫反应性。结果成功扩增了Hp MEL-HP27菌株ureB基因,构建了ureB基因的乳酸菌NICE（Nisin-controlled expression）原核表达系统;UreB蛋白适宜的表达条件为：在ureB重组菌生长至对数生长前期（OD600≈0.3～0.4）加入终浓度为40ng/mL的nisin,诱导表达5h,可溶性UreB蛋白表达量最高,可达27.26μg/mL培养基。可溶性UreB蛋白的表达占上清蛋白的比例最高可达20.19%;Western-blot结果显示乳酸乳球菌表达的UreB抗原蛋白具有良好的免疫反应性。结论结果提示应用乳酸乳球菌构建幽门螺杆菌食品级疫苗可能具有较好前景。     

Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori (H pylori) neutrophil-activating protein (NAP) and E. coli maltose binding protein (MBP) and to evaluate its immunoreactivity and immunogenicity. METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis. Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment, Western blotting with human H pylori anti-sera. RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG. The molecular weight of rBMP-NAP was about 57 kD, accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture. The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself. CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.     

靶向siRNA抑制幽门螺杆菌vacA表达

目的:观察靶向siRNA能否抑制幽门螺杆菌(H pylori)vacA基因表达．方法:合成靶向H pyiori vacA基因5对特异 siRNA(实验组:vacA-S1,vacA-S2,vacA-S3, vacA-S4,vacA-S5)和1对非特异siRNA(对照组)．通过电穿孔法使siRNA转化至H pylori内,观察转化效率和转化1,6,12,24,48 h mRNA和蛋白表达的抑制率,并将PCR产物克隆和测序. 结果:电穿孔转化效率平均为89％．vacA-S2、 vacA-S4组在转化1 h vacA mRNA表达抑制率达最大值,1,6,12 h抑制率分别为65％和77％,43％和50％,17％和9％,24和48 h时 vacA mRNA表达无抑制效应,与vacA-S1、 vacA-S3、vacA-S5、vacA-S6组相比有显著差异 (P<0．05),因为vacA-S1、vacA-S3、vacA-S5、 vacA-S6组转化后各时间点mRNA表达无变化．vacA-S2和vacA-S4组在转化1,6,12,24 h时 vacA蛋白表达抑制率分别为26％和17％,47％和40％,70％和75％,33％和30％,与vacA-S1、 vacA-S3、vacA-S5、vacA-S6组相比有显著差异 (P<0．05);转化48 h则无抑制效应．PCR产物克隆和测序与相应报道序列比较,同源性为99％．结论:靶向siRNA可以通过电穿孔法转化并特异地抑制H pylori vacA基因表达.     

胃癌组织中幽门螺杆菌cagA和vacA的表达及与其感染的相关性

目的:研究HpyloricagA和HpylorivacA在胃癌、胃黏膜不典型增生和胃炎组织中的表达及与Hpylori感染的相关性.方法:采用Warthin-Starry嗜银染色法检测胃癌组织39例,胃黏膜不典型增生组织24例和慢性胃炎组织33例中Hpylori感染情况;PCR法检测上述标本中HpyloricagA和HpylorivacA的表达.结果:胃癌组织中Hpylori,HpyloricagA 株和HpylorivacA 株感染率显著高于慢性胃炎组织(χ2=7.00,P<0.05;χ2=15.20,P<0.05;χ2=12.43,P<0.05);胃黏膜不典型增生组织中Hpylori,HpyloricagA 株和HpylorivacA 株感染率显著高于慢性胃炎组织(χ2=6.25,P<0.05;χ2=11.04,P<0.05;χ2=11.61,P<0.05);低分化胃癌组织中Hpylori,HpyloricagA 和HpylorivacA 株感染率显著高于高中分化胃癌组织(χ2=8.19,P<0.05;χ2=13.14,P<0.05;χ2=6.62,P<0.05).慢性胃炎、不典型增生和胃癌组织中Hpylori与HpyloricagA和HpylorivacA表达均呈正相关(慢性胃炎:r=0.56,P<0.01;r=0.64,P<0.01;不典型增生组织:r=0.64,P<0.01;r=0.92,P<0.01;胃癌:r=0.90,P<0.01;r=0.95,P<0.01).结论:Hpylori感染是慢性胃炎向胃黏膜不典型增生及胃癌发展的重要启动因子,Hpylori感染可能通过诱导cagA表达促使胃黏膜上皮细胞增殖加快,诱导vacA表达促使胃黏膜上皮细胞损伤;他们的协同作用可能在胃癌发生、发展过程中发挥了重要作用.     

Recombinant Helicobacter pylori catalase

AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori(H. pylon) and assay the activity of H. pylori catalase.METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E.coli strain which expressed catalase recombinant protein. The activity of H.pylori catalase was assayed by the Beers&amp;Sizers.RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank‘s research. The catalase recombinant protein amounted to 24.4% of the total bacterial protein after induced with IPTGf or 3 hours at 37℃ and the activity of H. pyloricatalase was high in the BL21 (DE3) E.coli strain.CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.     

Distribution of vacA and cagA genotypes of Helicobacter pylori in Kuwait

There is considerable geographical variation in the distribution of allelic types of Helicobacter pylori. This first study from Kuwait determined the prevalence of cagA and vacA genotypes among 117 unselected patients attending a gastroenterology center. We found that whereas vacA s1 and s2 types were equally likely to be present in biopsies obtained from patients of Middle-Eastern origin, African Arabs were predominantly infected with s2 type and South-Asians the s1 type. South Asians most frequently carried the cagA positive genotype with Bangladeshis showing the highest prevalence rate of 87%.     

Geographic distribution of vacA allelic types of Helicobacter pylori

BACKGROUND & AIMS: Distinct allelic types of Helicobacter pylori vacA have been defined. The geographic distribution of vacA alleles and cagA was assessed in this study. METHODS: A total of 735 cultures from patients in 24 countries were analyzed by polymerase chain reaction and reverse hybridization on a line probe assay (LiPA). RESULTS: In 124 (16.9%) of the 735 cultures, multiple vacA genotypes were detected, permitting analysis of 611 strains. In Europe, a distribution gradient of s1 subtypes was observed. In northern and eastern Europe, 89% were subtype s1a. s1a and s1b were equally present in France and Italy, whereas in Spain and Portugal 89% of strains were subtype s1b. s1a and s1b were approximately equally prevalent in North America. In Central and South America, virtually all s1 strains were subtype s1b. Subtype s1c was observed in 77% of the s1 isolates from East Asia. m1 and m2a have equal presence, except on the Iberian peninsula and in Central and South America, where m1 (86.2%) is more prevalent than m2 (13.8%). Subtype m2b was found exclusively among East Asian s1c strains. In all parts of the world, vacA s1/cagA-positive genotypes were associated with peptic ulcer disease (P < 0.001). CONCLUSIONS: These data indicate a geographic distribution of H. pylori genotypes and aid in understanding the relationship of H. pylori with disease.     

Helicobacter pylori vacA and cagA genotypes in Mexican adults and children

Studies examining associations between Helicobacter pylori virulence markers and disease have concentrated on adults in developed countries. This study assessed adults and children in Mexico. Ninety patients were recruited, 56 adults (37 with active peptic ulceration and 19 with no ulcers) and 34 children (all with recurrent abdominal pain and no ulcers). H. pylori was cultured from gastric biopsy specimens, and vacA alleles and cagA were typed by use of polymerase chain reaction from multiple colony sweeps. Multiple vacA types were common in single-biopsy isolates and were more frequent in adults with ulcers (95%) than in adults without ulcers (37%; P<.001) or in children (52%; P<.01). vacA s1b and cagA+ strains were more frequent in adults than in children. vacA s1 and cagA+ strains had similar frequencies in adults with and without ulcers. In conclusion, infection with multiple H. pylori strains, defined by different vacA genotypes, is common in Mexico. Such mixed infection is associated with ulcer disease. Strain populations infecting Mexican adults and children differ.