肝螺杆菌CdtB基因缺失株的构建与验证

doi:10.16431/j.cnki.1671-7236.2020.01.001

摘要/Abstract

摘要： 试验旨在敲除肝螺杆菌（Helicobacter hepaticus，H.hepaticus）的CdtB基因，采用pcDNA质粒构建自杀质粒，用于敲除肝螺杆菌ATCC 51449菌株CdtB基因。根据同源重组原理，通过电击转化方法将质粒转入肝螺杆菌构建肝螺杆菌CdtB基因缺失株（ΔCdtB）。采用PCR及测序技术对ΔCdtB缺失株进行鉴定。采用生化试验、生长曲线测定检测ΔCdtB缺失株与野生型肝螺杆菌区别。结果显示，获得以质粒pcDNA构建的同源重组敲除质粒pcDNA-ΔCdtB，电击转化肝螺杆菌后经过传代筛选可获得氯霉素阳性转化子。缺失株替换片段的PCR产物为964 bp，大于野生型肝螺杆菌对应产物884 bp，测序结果表明，氯霉素抗性基因已替换了CdtB基因。比较发现，ΔCdtB缺失株与野生型菌株的尿素酶试验结果均呈阳性，并且在生长速率方面与普通血琼脂平板上无显著差异，但最终缺失株在含氯霉素的血琼脂平板上生长更多。除此之外，将获得的缺失株持续传代并进行PCR鉴定未发现回复突变，有较好的遗传稳定性。综上所述，本研究构建的基因敲除质粒pcDNA-ΔCdtB实现了对肝螺杆菌中目标基因的敲除，为肝螺杆菌基因功能研究和致病因子的发掘提供了有效的基因操作工具。

关键词: 肝螺杆菌; 基因敲除; 质粒; 同源重组

Abstract: The experiment was aimed to knock out the CdtB gene of Helicobacter hepaticus (H.hepaticus).A suicide plasmid for knocking out the CdtB gene of H.hepaticus ATCC 51449 strain was constructed using the pcDNA plasmid.According to the principle of homologous recombination,the plasmid was transferred into H.hepaticus by electroporation to construct a H.hepaticus CdtB gene mutant strain (ΔCdtB).The ΔCdtB mutant strain was identified by PCR and sequencing.Using biochemical test and calculating the growth curve to detect the difference between the ΔCdtB mutant strain and the wild type H.hepaticus.The results showed that the homologous recombinant knockout plasmid pcDNA-ΔCdtb was constructed by plasmid pcDNA,and the chloramphenicol positive transformant was obtained by subculture and screening after the transformation.The PCR product of the mutant strain was 964 bp,which was larger than that of the corresponding product 884 bp of wild type H.hepaticus. It was found that the urease test results of ΔCdtB mutant strain and wild type strain were all positive,and there was no significant difference in growth rate between them on the common blood agar plate,but the ΔCdtB mutant strain grew more on the blood agar plate containing chloramphenicol.In addition,the deletion strain was passed on and identified by PCR,and no reverse mutation was found.To sum up,the gene knockout plasmid pcDNA-ΔCdtb constructed in this study could knock out the target gene of H.hepatica,and provide an effective gene manipulation tool for gene function research and pathogenic factor discovery of H.hepatica.

Key words: Helicobacter hepaticus; gene knockout; plasmid; homologous recombination

中图分类号:

R378

朱宸, 曹舒扬, 吴志浩, 殷俊, 朱立麒, 张泉. 肝螺杆菌CdtB基因缺失株的构建与验证[J]. 中国畜牧兽医, 2020, 47(1): 1-7.

ZHU Chen, CAO Shuyang, WU Zhihao, YIN Jun, ZHU Liqi, ZHANG Quan. Construction and Identification of Helicobacter hepaticus CdtB Gene Mutant Strain[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 1-7.

参考文献

[1] FOX J G,LI X,YAN L,et al.Chronic proliferative hepatitis in A/JCr mice associated with persistent Helicobacter hepaticus infection:A model of helicobacter-induced carcinogenesis[J].Infection and Immunity,1996,64(5):1548-1558.
[2] XU M,POKROVSKII M,DING Y,et al.c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont[J].Nature,2018,554(7692):373-377.
[3] FALSAFI T,MAHBOUBI M.Helicobacter hepaticus,a new pathogenic species of the Helicobacter genus:Similarities and differences with H.pylori[J].Iranian Journal of Microbiology,2013,5(3):185-194.
[4] NILSSON I,LINDGREN S,ERIKSSON S,et al.Serum antibodies to Helicobacter hepaticus and Helicobacter pylori in patients with chronic liver disease[J].Gut,2000,46(3):410-414.
[5] HAMADA T,YOKOTA K,AYADA K,et al.Detection of Helicobacter hepaticus in human bile samples of patients with biliary disease[J].Helicobacter,2009,14(6):545-551.
[6] CHOW J,MAZMANIAN S K.A pathobiont of the microbiota balances host colonization and intestinal inflammation[J].Cell Host Microbe,2010,7(4):265-276.
[7] CAHILL R J,FOLTZ C J,FOX J G,et al.Inflammatory bowel disease:An immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus[J].Infection and Immunity,1997,65(8):3126-3131.
[8] LARA-TEJERO M,GALAN J E.CdtA,CdtB,and CdtC form a tripartite complex that is required for cytolethal distending toxin activity[J].Infection and Immunity,2001,69(7):4358-4365.
[9] GUERRA L,CORTES-BRATTI X,GUIDI R,et al.The biology of the cytolethal distending toxins[J].Toxins,2011,3(3):172-190.
[10] SMITH J L,BAYLES D O.The contribution of cytolethal distending toxin to bacterial pathogenesis[J].Critical Reviews in Microbiology,2006,32(4):227-248.
[11] POTT J,KABAT A M,MALOY K J.Intestinal epithelial cell autophagy is required to protect against TNF-induced apoptosis during chronic colitis in mice[J].Cell Host & Microbe,2018,23(2):191-202.
[12] PERE-VEDRENNE C,CARDINAUD B,VARON C,et al.The cytolethal distending toxin subunit CdtB of Helicobacter induces a Th17-related and antimicrobial signature in intestinal and hepatic cells in vitro[J].Journal of Infectious Diseases,2016,213(12):1979-1989.
[13] PRATT J S,SACHEN K L,WOOD H D,et al.Modulation of host immune responses by the cytolethal distending toxin of Helicobacter hepaticus[J].Infection and Immunity,2006,74(8):4496-4504.
[14] ISHII Y,SHIBATA W,SUGIMORI M,et al.Activation of signal transduction and activator of transcription 3 signaling contributes to Helicobacter-associated gastric epithelial proliferation and inflammation[J].Gastroenterology Research & Practice,2018,2018:9050715.
[15] YOUNG V B,KNOX K A,SCHAUER D B.Cytolethal distending toxin sequence and activity in the enterohepatic pathogen Helicobacter hepaticus[J].Infection and Immunity,2000,68(1):184-191.
[16] JINADASA R N,BLOOM S E,WEISS R S,et al.Cytolethal distending toxin:A conserved bacterial genotoxin that blocks cell cycle progression,leading to apoptosis of a broad range of mammalian cell lineages[J].Microbiology,2011,157(Pt 7):1851-1875.
[17] MORI H,SUZUKI H,MATSUZAKI J,et al.Acquisition of double mutation in gyrA caused high resistance to sitafloxacin in Helicobacter pylori after unsuccessful eradication with sitafloxacin-containing regimens[J].United European Gastroenterology Journal,2018,6(3):391-397.
[18] MOU K T,PLUMMER P J.The impact of the LuxS mutation on phenotypic expression of factors critical for Campylobacter jejuni colonization[J].Veterinary Microbiology,2016,192:43-51.
[19] 刘明月,程古月,王旭,等.空肠弯曲杆菌鞭毛的研究进展[J].中国畜牧兽医,2018,45(9):2591-2599. LIU M Y,CHENG G Y,WANG X,et al.Research progress on the flagella of Campylobacter jejuni[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2591-2599.(in Chinese)
[20] GE Z,FENG Y,GE L,et al.Helicobacter hepaticus cytolethal distending toxin promotes intestinal carcinogenesis in 129Rag2-deficient mice[J].Cellular Microbiology,2017,19(7):e12728.
[21] GE Z,ROGERS A B,FENG Y,et al.Bacterial cytolethal distending toxin promotes the development of dysplasia in a model of microbially induced hepatocarcinogenesis[J].Cellular Microbiology,2007,9(8):2070-2080.
[22] YOUNG V B,KNOX K A,PRATT J S,et al.In vitro and in vivo characterization of Helicobacter hepaticus cytolethal distending toxin mutants[J].Infection and Immunity,2004,72(5):2521-2527.
[23] GE Z,FENG Y,WHARY M T,et al.Cytolethal distending toxin is essential for Helicobacter hepaticus colonization in outbred Swiss Webster mice[J].Infection and Immunity,2005,73(6):3559-3567.