李抄,陈定宇,张晓怡,赵艳,王琴容,周建奖,谢渊
LI Chao,CHEN Dingyu,ZHANG Xiaoyi,ZHAO Yan,WANG Qinrong,ZHOU Jianjiang,XIE Yuan

• 1:贵州医科大学地方病与少数民族性疾病教育部重点实验室贵州省医学分子生物学重点实验室贵州医科大学分子生物学重点实验室

摘要(Abstract)：

目的:构建幽门螺杆菌(Hp)毒力蛋白cagA基因敲除突变株并进行鉴定。方法:采用基因打靶技术,从Hp1004基因组扩增cagA基因的上、下游同源重组臂序列,从pACYC184质粒上扩增氯霉素(Cm)序列,通过融合聚合酶链式反应(PCR)技术获得cagA基因敲除打靶片段,克隆入载体pUCmT,得到打靶载体pUCmT-ΔcagA::Cm;再通过电转化法将pUCmT-ΔcagA::Cm质粒直接转入Hp1004,氯霉素抗性平板筛选cagA基因敲除株并命名为Hp/ΔcagA::Cm;采用PCR进行鉴定,并用Hp/ΔcagA::Cm和Hp/cagA感染原代胃癌细胞,检测胞内cagA的表达和对细胞形态的影响。结果:融合PCR构建打靶片段长度为1 794 bp,打靶载体转化Hp并筛选后,用cagA外侧引物PCR扩增Hp/ΔcagA::Cm和野生型Hp/cagA,产物长度分别为2 150 bp和5 014 bp;Hp/ΔcagA::Cm和Hp/cagA分别感染细胞后,细胞死亡数量多于感染Hp/ΔcagA::Cm突变株,差异有统计学意义(P<001);野生型Hp/cagA菌株及其感染细胞内cagA蛋白表达水平较Hp/ΔcagA::Cm突变型菌株及其感染细胞明显降低,差异有统计学意义(P<001)。结论:成功构建Hp/ΔcagA::Cm突变株,并在感染细胞中得到验证,表明cagA蛋白对细胞有一定的毒性作用。
Objective: To construct a knockout mutant strain of helicobacter pylori(Hp) virulenceproteincagAby gene targeting and identify it.Methods: Gene targeting technology was used toamplify thecagAgene's upstream and downstream homologous recombination arm sequences from theHp1004 genome, and the chloramphenicol( Cm) sequence was amplified from the pACYC184 plasmid. ThecagAgene knockout target fragment was obtained by fusion PCR technology and cloning.The vector pUCmT was inserted to obtain the target vector pUCmT-ΔcagA::Cm; and then the pUCmT-ΔcagA::Cmplasmid was directly transferred intoHp1004 by electroporation. The chloramphenicol-resistant plate was screened for thecagAgene knockout strain and namedHp/ΔcagA::Cm; PCR wasused to identify it,Hp/ΔcagA::Cmand infection ofHp/cagAoriginal generation of gastric cancercells were used to detect the expression of intracellularcagAand effect on the cell morphology.Results: Fusion PCR was used to construct the target fragment with a length of 1 794 bp. After thetarget vector was transformed intoHpand screened,Hp/ΔcagA::Cm and wild-typeHp/cagA were amplified by thecagAlateral primer PCR, the product lengths were 2 150 bp and 5 014 bprespectively. AfterHp/ΔcagA:: Cm andHp/cagAinfected cells respectively, cell deaths were morethan Hp infection/ΔcagA:: Cm mutant strains, and the difference was statistically significant(compared with WZKHp/cagA, YHDHp/cagAgroupP< 0. 01; compared with HZMHp/cagAgroupP< 0. 05); The expression level ofcagAprotein in wild-typeHp/cagAstrain and its infected cells wassignificantly lower than that ofHp/ΔcagA::Cm mutant strain and its infected cells, and the differencewas statistically significant.(P< 0. 01 compared with eachHp/ΔcagA:: Cm infection group).Conclusion: TheHp/ΔcagA::Cm mutant is successfully constructed and verified in infected cells,indicating that the cagA protein has a certain toxic effect on the cells.

关键词(KeyWords)： 螺杆菌,幽门;基因打靶;感染;细胞毒性相关蛋白cagA;聚合酶链式反应;电转化
Helicobacter pylori(Hp);gene targeting;infection;cagA;polymerase chain reaction(PCR);electrical transformation

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目（31660031,31960028）;; 贵州省科技计划项目黔科合平台人才项目[(2017)5652];; 贵阳市科技计划项目筑科合同项目[(2017)5-16];; 贵州省科技基金项目[黔科合LH字(2016)7347]

作者(Author): 李抄,陈定宇,张晓怡,赵艳,王琴容,周建奖,谢渊
LI Chao,CHEN Dingyu,ZHANG Xiaoyi,ZHAO Yan,WANG Qinrong,ZHOU Jianjiang,XIE Yuan

DOI: 10.19367/j.cnki.1000-2707.2020.02.001

参考文献(References)：

• [1]JIN Y P,FORMAN D,WASKITO L A,et al.Epidemiology ofHelicobacter pyloriand cagA-positive infections and global variations in Gastric Cancer[J].Toxins,2018,10(4):163．
• [2]CHEN W,ZHENG R,BAADE P D,et al.Cancer statistics in china[J].Ca-a Cancer Journal for Clinicians,2016,66(2):115-132．
• [3]WHITE J R,WINTER J A,ROBINSON K.Differential inflammatory response toHelicobacter pyloriinfection:etiology and clinical outcomes[J].Journal of Inflammation Research,2015,8:137-147．
• [4]AMIEVA M,PEEK R M,Pathobiology ofHelicobacter pylori-Induced Gastric Cancer[J].Gastroenterology,2016,150(1):64-78．
• [5]PARK B,LIM J W,KIM H.Lycopene treatment inhibits activation of Jak1/Stat3 and Wnt/β-catenin signaling and attenuates hyperproliferation in gastric epithelial cells[J].Nutrition Research,2019,70(1):70-81．
• [6]NAUMANN M,SOKOLOVA O,TEGTMEYER N,et al.Helicobacter pylori:A paradigm pathogen for subverting host cell signal transmission[J].Trends In Microbiology,2017,25(4):316-328．
• [7]JANG S,SU H,BLUM F C,et al.Dynamic expansion and Contraction ofcagAcopy number inHelicobacter pylori impact development of Gastric Disease.[J].MBIO,2017,8(1):e01779-16．
• [8]HUANG L U,WANG Z Y,PAN D D,Penicillin-binding protein 1A mutation-positiveHelicobacter pyloripromotes epithelial-mesenchymal transition in gastric cancer via the suppression of microRNA-134[J].International Journal of Oncology,2019,54:916-928．
• [9]SILJA W,STEFFEN B.A novel basolateral type IV secretion model for the cagA oncoprotein ofHelicobacter pylori.[J].Microbial Cell,2018,5(1):60-62．
• [10]李景芬，袁野，于浩，等．猪MSTN基因敲除载体的构建[J]．江苏农业科学，2008(1):45-47．
• [11]陈相好，刘芳，王彩霞，等．高效严谨型大肠杆菌Targetron基因打靶系统的构建[J]．生物技术通报，2019,35(6):213-220．
• [12]CHEY W D,LEONTIADIS GRIGORIOS I,HOWDEN CW,et al.Correction:ACG Clinical Guideline:Treatment ofHelicobacter pyloriInfection.[J].The American Journal of Gastroenterology,2018,113(7):1102．
• [13]WANG Y Z,XIAO H J,WU H T,et al.G protein subunitαq regulates gastric cancer growth via the p53/p21and MEK/ERK pathways[J].Oncology Reports,2017,37(4):1998-2006．
• [14]CAI H K,CHEN X,ZHANG J B,et al.18β-glycyrrhetinic acid inhibits migration and invasion of human gastric cancer cells via the ROS/PKC-α/ERK pathway[J].Journal Of Natural Medicines,2018,72(1):252-259．
• [15]龙妮娅，熊林，周建奖，等．幽门螺杆菌东、西方株cagA的序列差异及其对胃癌细胞生长与凋亡的影响[J]．微生物学通报，2018,45(4):848-855．
• [16]FARZAM V,SAMIRA T,ABOLFAZL F,et al.New insights ofHelicobacter pylorihost-pathogen interactions:The triangle of virulence factors,epigenetic modifications and non-coding RNAs[J].World Journal of Clinical Cases,2018,6(5):64-73．
• [17]AMIEVA M R,VOGELMANN R,COVACCI A,et al.Disruption of the epithelial apical-junctional complex by Helicobacter pyloricagA[J].Science,2003,300(5624):1430-1434．
• [18]谢尚训，谢明琦，陈治池，等．利用基因打靶技术构建转基因小鼠及其在生物学中的应用[J]．生物医学工程与临床，2016(5):540-544．
• [19]贺飞燕，闫建俊，冯瑞云，等．基因组编辑技术的原理及应用[J]．应用与环境生物学报，2016,20(2):350-356．
• [20]KAWANO Y,HONDA A.Gene targeting in rabbits:single-step generation of knock-out rabbits by microinjection of CRISPR/Cas9 plasmids.[J].Methods in Molecular Biology,2017,1630:109-120．
• [21]吴璐，王磊，任远，等．基因组编辑技术研究进展[J]．生物技术通报，2014(11):84-90．
• [22]张丽娜，刘国安，杨红．基因打靶技术的研究进展[J]．生物技术通报，（9):55-59．
• [23]黄志刚，段广才，范清堂，等．cagA基因缺失的中国幽门螺杆菌突变菌株的构建及鉴定[J]．世界华人消化杂志，2006,14(33):3190-3194．
• [24]洪伟，陈学术，吴昌学，等．幽门螺旋杆菌cagA基因失活突变株的构建[J]．贵阳医学院学报，2015,40(12):1328-1324.(2019-12-06,2020-02-03)

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