幽门螺杆菌儿童分离株尿素酶基因B的克隆及序列分析

周珍文; 邓秋连; 夏慧敏; 耿岚岚; 梁伟河; 谢永强; 黄勇; 龚四堂

PDF(1308 KB)

中国当代儿科杂志 ›› 2009, Vol. 11 ›› Issue (12) : 877-880.

论著·临床研究

幽门螺杆菌儿童分离株尿素酶基因B的克隆及序列分析

周珍文，邓秋连，夏慧敏，耿岚岚，梁伟河，谢永强，黄勇，龚四堂

作者信息 +

Cloning and sequence analysis of UreB of Helicobacter pylori isolated from children

ZHOU Zhen-Wen, DENG Qiu-Lian, XIA Hui-Min, GENG Lan-Lan, LIANG Wei-He, XIE Yong-Qiang, HUANG Yong, GONG Si-Tang

Author information +

文章历史 +

摘要

目的：克隆幽门螺杆菌（Hp）临床儿童分离株尿素酶B（UreB）基因入Pgex-4T-1表达载体，并进行测序及基因比对分析，为以UreB作为Hp疫苗分子的口服疫苗研制奠定基础。方法：根据GenBank中Hp UreB序列，设计一对特异性引物PCR扩增Hp临床儿童分离株UreB全长基因，EcoR I及Not I酶切后与做相应酶切的pGEX-4T-1连接，转化大肠杆菌BL21，提取质粒进行双酶切鉴定及基因测序，并对测序结果进行比对分析。结果：以Hp儿童分离株GZCH1为模板，成功扩增了UreB基因，基因大小为1 710 bp,重组pGEX-4T-1-Ure双酶切鉴定可见目的片段，测序结果显示UreB在正确阅读框中，序列比对分析显示其与相关报道序列核苷酸和氨基酸一致性达98%。Hp儿童分离株GZCH1 UreB序列已登录GenBank（登录号：FJ455126）。结论：从Hp儿童分离株GZCH1中成功克隆了UreB基因，为UreB Hp口服疫苗研制奠定了基础。［中国当代儿科杂志，2009，11（11）：877-880］

Abstract

OBJECTIVE: To clone UreB gene of Helicobacter pylori (H. pylori) isolated from children to pGEX-4T-1 expression plasmid, and do sequence analysis. METHODS: A pair of specific primer was designed according to H. pylori UreB gene in the GenBank. Using H. pylori strains isolated from children as a template, a UreB gene was obtained by PCR. After EcoR I and Not I digestion, the PCR production was linked with pGEX-4T-1 which was digested with the same enzymes. The recombinant plasmid was transformed into E.coli BL21 and identified by double enzyme digestion and sequence analysis. The sequence results were compared with the gene sequence in the GenBank. RESULTS: A UreB gene was successfully amplified from children′s H. pylori strain GZCH1. It was 1 710 bp in size. The objective band was identified by double enzyme digestion. DNA sequence showed that UreB was in the correct open reading frame. The sequence comparison analysis showed that DNA and amino acid sequence identities of UreB gene with other strains were 98%. The sequence of UreB of H. pylori strain GZCH1 was submitted to GenBank (accession number：FJ455126). CONCLUSIONS: UreB of H. pylori strain GZCH1 is successfully cloned to pGEX-4T-1, which provides a basis for research of oral H. pylori vaccine.［Chin J Contemp Pediatr, 2009, 11 (11):877-880］

导出引用

周珍文, 邓秋连, 夏慧敏, 耿岚岚, 梁伟河, 谢永强, 黄勇, 龚四堂. 幽门螺杆菌儿童分离株尿素酶基因B的克隆及序列分析[J]. 中国当代儿科杂志. 2009, 11(12): 877-880

ZHOU Zhen-Wen, DENG Qiu-Lian, XIA Hui-Min, GENG Lan-Lan, LIANG Wei-He, XIE Yong-Qiang, HUANG Yong, GONG Si-Tang. Cloning and sequence analysis of UreB of Helicobacter pylori isolated from children[J]. Chinese Journal of Contemporary Pediatrics. 2009, 11(12): 877-880

中图分类号： R394-33

参考文献

［1］Warren J, Marshall B. Unidentified curved bacilli on gastric epithelium inactive chronic gastritis［J］. Lacent, 1983, 1(8336):1273-1275.
［2］Stephenie CS, Alfonso JB. Helicobacter pylori strikes again-gastric mucosa-associated lymphoid tissue(MALT) lymphoma［J］. Gastroenterol Nurs, 2007, 30(5):348-354.
［3］Danesh J. Helicobacter pylori infection and gastric cancer: systematic review of epidemiological studies［J］. Aliment Pharmacol Ther, 1999, 13(7):851-856.
［4］王娟,王润.中国幽门螺杆菌感染流行病学杂志Mata分析［J］.中华流行病学杂志, 2003, 24(6):443-446.
［5］耿岚岚,龚四堂,区文玑,潘瑞芳,陈宝心,丘小汕.两种非侵入性诊断方法在儿童幽门螺杆菌感染中的应用［J］.实用儿科临床杂志, 2003, 18(7):538-539.
［6］关克涛,徐文泉,刘云锋,卢铭江,颜幕霞.酶联免疫吸附测定检测儿童粪便幽门螺杆菌抗原［J］.广州医学院学报, 2006, 34(4):43-45.
［7］黄烈平,庄满利,顾承萍.36株青少年幽门螺杆菌耐药分析［J］.中国当代儿科杂志, 2009,11(3):210-212.
［8］袁小澎,邹全明,柏杨,杨珺,郭鹰,张卫军,等. 幽门螺杆菌B亚单位功能片段的纯化及活性研究［J］.南方医科大学学报, 2007, 27(7):959-962.
［9］赵煜,徐晓华,刘风霖,张书红,司徒爱明.儿童幽门螺杆菌感染与相关性疾病的临床研究［J］. 中国当代儿科杂志, 2008, 10(3):403-404.
［10］Myers GA, Ermak TH, Georgakopoulos K, Tibbitts T, Ingrassia J, Gray H, et al. Oral immunization with recombinant Helicobacter pylori urease confers long-lasting immunity against Helicobacter felis infection［J］.Vaccine, 1999, 17(12):1394-1403.
［11］Liu X, Hu J, Zhang X, Fan D. Oral immunization of mice with attenuated Salmonella typhimurium espressing Helicobacter pylori urease B subunit［J］. Chin Med J, 2002, 115(10):1513-1516.
［12］Michetti P, Kreiss C, Kotloff KL, Pota N, Bachmann D, Herranz M, et al. Oral immunization with urease and Escherichia coli heat-labile enterotoxin is safe and immunogenic in Heliobacter pylori-infected adults［J］. Gastroenterology, 1999, 116(4):804-812.
［13］周珍文,胡旭初,余新炳.枯草杆菌芽孢载体疫苗的研究及其在寄生虫病防治中的应用前景［J］.热带医学杂志, 2007, 7(9):935-937.
［14］Zhou ZW, Xia HM, Hu XC, Huang Y, Li YW, Li L, et al. Oral administration of a Bacillus subtilis Spore-based vaccine expressing Clonorchis sinensis tegumental protein 22.3 kDa confers protection against Clonorchis sinensis［J］.Vaccine, 2008, 26(15):1817-1825.
［15］Zhou ZW, Xia HM, HuXC, Huang Y, Ma CL, Chen XX, et al. Immunogenicity of recombinant Bacillus subtilis spores expressing Clonorchis sinesis tegumental protein［J］. Parasitol Res, 2008, 102(2):293-297.