CN115161395A - WaaL作为药物靶标在制备药物中的应用 - Google Patents
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Abstract
本发明属于医学工程技术领域,具体涉及WaaL作为药物靶标在制备药物中的应用。本发明还提供了一种敲除waaL基因的方法。本发明发现利用同源重组敲除幽门螺杆菌基因组中的waaL基因后,幽门螺杆菌的致瘤性明显降低,对临床幽门螺杆菌相关胃癌的防治具有重要的理论和实际意义。
Description
技术领域
本发明属于医学工程技术领域,具体涉及WaaL作为药物靶标在制备药物中的应用。
背景技术
幽门螺杆菌(Helicobacter pylori,H.pylori)是一种定植于胃黏膜的微需氧革兰染色阴性螺旋形细菌,感染全球约一半的人群。H.pylori感染能引起严重的慢性感染性疾病,破坏胃的结构和功能,是胃萎缩、消化性溃疡、胃腺癌和胃黏膜相关淋巴组织淋巴瘤的主要病因。H.pylori被世界卫生组织定义为I类致癌因子,全球约90%的非贲门胃癌可归因于H.pylori感染。共识指出:H.pylori相关性胃炎是一种感染性疾病,是导致胃癌最重要的因素,如不存在抗衡因素,无论有无症状,对所有患者都应进行根除治疗,从而预防胃癌的发生。
随着H.pylori对常用抗生素的耐药率不断上升,尽管根除方案不断改进,根除失败仍时有发生;并且即使根除成功,仍有部分人群发生再感染。因此,我们需要其他的替代策略以减少胃癌发病率。由于H.pylori感染导致胃癌的发生在很大程度上与细菌和宿主之间的相互作用有关,加强对相互作用的关键分子在H.pylori致胃癌发生中的作用研究,将可能为H.pylori相关胃癌的预防提供新思路。
脂多糖是H.pylori外膜最重要的组成成分,其分子结构由三部分组成:1)脂质A;2)核心多糖;3)O-抗原。H.pylori脂多糖独特的化学结构及生物合成中的代谢产物有助于H.pylori的慢性感染和致病性,研究脂多糖生物合成通路中的关键酶有利于阐明H.pylori感染的致病机制。在H.pylori脂多糖合成过程中,O-抗原起始酶WecA通过将GlcNAc转移到Und-PP载体以形成Und-PP-GlcNAc来启动O-抗原的组装。随后,连续的糖基转移酶添加相应的糖基以形成O-抗原。在胞浆组装完成后,O-抗原翻转酶Wzk将O-抗原经内膜转移到周浆间隙。同样在胞浆组装后,核心-脂质A由翻转酶MsbA翻转到周浆间隙,然后进行去磷酸化和去乙酰化修饰。由waaL基因编码的定位于细菌内膜上的O-抗原连接酶WaaL连接O-抗原与核心-脂质A,形成完整的脂多糖。
目前,未见H.pylori脂多糖合成过程对幽门螺杆菌导致胃癌发挥作用的报道。
发明内容
本发明通过比较基因组学发现,WaaL在H.pylori不同菌株中保守存在。敲除waaL基因后,H.pylori感染后的致炎及致胃上皮细胞DNA损伤的作用明显减轻、H.pylori感染所致的细胞凋亡增加从而减少了DNA受损细胞的堆积、H.pylori感染所致的细胞增殖作用减弱、并且H.pylori致蒙古沙鼠胃癌发生的作用也明显减少。
基于以上研究内容,本发明提供一种WaaL作为药物靶标在制备药物中的应用,具体的,
H.pylori脂多糖O-抗原连接酶WaaL作为药物靶标在制备药物中的用途,所述药物为以下任一或多个:
(1)治疗H.pylori相关胃炎的药物;
(2)治疗消化性溃疡的药物;
(3)预防或治疗H.pylori相关胃癌的药物。
本发明还提供抑制H.pylori脂多糖O-抗原连接酶WaaL表达的试剂在制备预防、治疗胃部疾病方面的应用。
其中,所述试剂包括敲除waaL基因的试剂、使waaL基因沉默的试剂或waaL基因的抑制剂。
所述抑制剂表现为降低WaaL表达水平或抑制WaaL生物活性。其中,所述抑制剂包括但不限于特异性或非特异性降低WaaL的表达水平或使WaaL生物活性失活的抑制剂。即只要药物利用降低WaaL的表达或使WaaL生物活性降低,均属于本发明的保护范围。
可选地,所述抑制剂选自化学药物、生物大分子、多肽、单链抗体、反义寡聚核苷酸、小发夹RNA、小干扰RNA、基因编辑体系。
其中,上述化学药物包括可降低WaaL表达水平或抑制WaaL生物活性的化合物或其药学上可接受的盐。
所述胃部疾病为由H.pylori菌株引起,H.pylori菌株指幽门螺杆菌。
所述胃部疾病包括慢性胃炎、消化性溃疡和/或胃癌等。
所述胃癌包括管状腺癌、乳头状腺癌、黏液腺癌、印戒细胞癌、混合癌。
所述敲除waaL基因的试剂包含SEQ ID NO.1所示的核苷酸序列,
SEQ ID NO.1所示序列为:
GCATGCCCTTATCATCAATAAGCCCTGAATTTAAATCCGCATCAATCGCCATTTGCTTTCCTGGCATAGCGTCTAGGGCAAATCGCGCCCTAACTTCAGTAACCCTAGTAGAGCCATTAGTAACCACTAATAAATTCACTAACACTAAAATACTAAAGATAATCGCCCCAATCACATAATTCCCGCTCACGCTAAATTCCCCAAACGCCGTGATAATATCGCTCACCGCGCTAGGCCCTTTATAGCCTTGCGTTAAAATCATTCTGGTGGTGGCGACATTTAAAGCCAAGCGGTATAAGGTTACAATGAGGAGCAAAGTAGGGAAAGCGCTAAAATCAGTAGGCTTGTCAATATAAAGCCCGATTAAAATAATCAACACCGATAGCGCGATAGAAATCGTGAGTAAAAAATCCAACACAAAAGGCGGTAACGGCACGATAATGATCGCTAAAATAGCGATCACAAAGACCACAAGGGCTAAGTCTTTGGATTGCAAAAAGCGTTTAAAGACAGGGAAAGTCTTTTTAAAAGCTAATTTGGAGCGTTCGTTTGCCATAATCTAAAATCATGCCTTAATCTGGTAGGTGGTTTGTCTCTTATTATAATATAAAGTTTAAAAGTATGGCACAAAAAAGCTTATTTTACGCTATAATGCGATCTTTATTAAATTACCAATTAGGAGGTCGTTATGGCTTTGAATCTGGAGAAAAAACAAGAAATCATTAAGGCGTTTGCTACTAAGGAAAACGATACGGGTTCTTGTGAGGTGCAAGTGGCGTTGTTGAATGAAAGGATCAAGCTTTTAACCGAGCATTTAAAGGCTAACCCCAAAGATCATTCCAGTCGTTTAGGGCTTTTAAAATTAGTCGCTCAAAGACGCAACTTGTTGAAATACATCAAACGCACCGCTCATGCGCGTTATGTGGTTTTGATTGAAAAGTTAGGCATTAAAGACAGATAATTTTTATCGGTTTTAGGGTGTGGCGAGTTTGTGTTGAAAGAGCGTTgTCGACTCTAGCTAGAGGATCGATCCGGTTTTTGTTAATCCGCCATATTGTGTTGAAACACCGCCCGGAACCCGATATAATCCGCCCTTCAACAGATCCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAGGATCcGCGCTTTTTAGCATCAAAGAGTTTGATGTTAGTCAAGCGGTAGTTTCTTGTGATGCGCTCTTAGGGGGATTTAAGGGGTAAGATAAAAACTTATGCTATAATGCGGTTTCATTCCATATTCAACAAGGAGAAATAAATTAATGAAAATTTTAGTGATTCAAGGGCCTAATTTAAACATGTTAGGACACAGAGACCCAAGACTTTATGGCATGGTAACCTTAGACCAAATCCATGAAATCATGCAAACTTTCGTGAAACAAGGCAATTTAGATGTGGAATTAGAGTTTTTTCAAACCAATTTTGAGGGCGAAATCATTGATAAAATTCAAGAGAGCGTGGGCAGCGATTATGAAGGGATCATCATTAACCCTGGAGCGTTTTCGCACACTTCTATTGCGATTGCAGATGCGATCATGCTAGCGGGCAAACCCGTTATTGAAGTGCATCTCACTAACATTCAAGCCAGAGAGGAATTCAGGAAAAATTCTTACACTGGAGCGGCTTGTGGAGGCGTGATCATGGGATTTGGCCCGCTTGGCTACAACATGGCTTTAATGGCGATGGTCAATATTTTAGCCGAGATGAAAGCGTTCCAAGAAGCCCAAAAAAACAACCCTAATAACCCCATTAACAATCAAAAATAAAAGGGGGCTACCCATGAAAGGATTAGAAAGAGAATCGCATTTCACGCTCAATGAAAACGCGATGTTTTTTGAGTGTGCTTATAGTTGCGATAACGCTTTATTTTTGCAATTAGACGATCGCTCGTTTTTTATCACTGATTCTCGCTACACTCAAGAAGCTAAAGAAAGCGTTCAGCCTAAAAATGGCGTTTTAGCGGAAGTGGTAGAATCTAGCGATTTAGTGCAAAGCACGATTGATTTGATCGCTAAAAGTTCGGTTAAAAAGCTCTTTTTTGACCCCAATCAAGTGAATTTACAAACCTACAAGCGTTTAAATTCAGCGCTTGGGGATAAGGTTACTTTAGAGGGTGTGCCTAGTTACCCCGGG
本发明还提供一种用于治疗H.pylori相关胃病的制剂,所述制剂含有敲除waaL基因的试剂、使waaL基因沉默的试剂或waaL基因的抑制剂,所述制剂为药物、药物组合物、保健品、食品或添加剂。
上述制剂中可包括药物赋形剂、稀释剂或载体,并可通过任何适合的方式用于需要治疗的患者。其中,适合方式包括但不限于口服、直肠、鼻部、局部(包括口腔和舌下)、皮下或胃肠外(包括皮下、肌肉、静脉、皮内、鞘内)施用。
本发明还提供了一种waaL基因敲除细菌模型的制备方法,它是利用同源重组系统对H.pylori菌株waaL基因进行敲除,具体方法如下:
a、PCR扩增G27菌株中waaL基因的上下游侧翼片段WaaL up和WaaL down(各约1000bp DNA);
b、将waaL基因上游片段WaaL up插入到pUC19载体的多克隆位点SphI和SalI之间,得到质粒pUC19-WaaL up;
c、通过T4 DNA连接酶连接cat抗氯霉素片段以及waaL基因下游片段WaaL down,将连接片段插入到pUC19-WaaL up的多克隆位点SalI和XmaI之间,得到waaL敲除质粒(包含SEQ ID NO.1所示序列,如图2);
d、将步骤c的敲除质粒经自然转化进入H.pylori菌体;
e、72小时收集细菌,鉴定并确认细菌中的waaL基因被敲除,获得waaL基因敲除菌株。
其中,所述细菌为H.pylori菌株,如H.pylori G27、7.13、NCTC11637。
本发明还提供了上述方法获得的waaL基因敲除细菌模型:G27ΔwaaL、7.13ΔwaaL、NCTC11637ΔwaaL。
本发明的有益效果:
本发明通过实验发现,敲除H.pylori的waaL基因后,H.pylori感染所致的胃黏膜上皮细胞炎症反应减轻、DNA损伤减少、细胞凋亡增加、细胞复制周期减慢、细胞“蜂鸟”样表型减少,H.pylori感染所致的蒙古沙鼠胃癌和癌前病变发生率减少。
本发明建立了敲除H.pylori菌株waaL基因的细菌模型,可以应用在H.pylori致病机制的研究中,对临床H.pylori相关胃癌的防治工作具有重要的理论和实际意义。
本发明提供的敲除waaL基因的试剂能有效减少H.pylori致胃癌发生的作用,从而预防H.pylori感染患者胃癌发生。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1显示敲除H.pylori G27、7.13和NCTC11637菌株中的waaL基因的鉴定图;其中,A为PCR扩增G27的waaL侧翼序列:Lane 1:上游1.0kb DNA片段,Lane 2:下游1.0kb DNA片段;B为单克隆PCR鉴定waaL上游片段克隆到pUC19的SphI和SalI位点:1、5、6号为正确的单克隆;C为SphI和SalI酶切鉴定B图中1、5、6号单克隆正确;D为SalI和XmaI酶切鉴定cat和waaL下游片段克隆到pUC19-WaaL up中:1、2号单克隆正确,得到waaL敲除质粒(包含SEQ IDNO.1所示序列);E为PCR鉴定H.pylori G27、7.13和NCTC11637菌株中的waaL基因的敲除情况;F为脂多糖银染鉴定waaL基因的敲除情况。
图2显示敲除waaL基因后H.pylori感染所致的IL-8水平变化。
图3显示敲除waaL基因后H.pylori感染所致的细胞DNA损伤变化;其中,A为G27、7.13和NCTC11637野生菌株及相应waaL基因敲除菌株感染后,AGS细胞发生DNA损伤的比例(一次代表性实验),未受感染的AGS细胞作为空白对照(CTRL);FL2+:DNA损伤细胞;wt:野生型;ΔwaaL:waaL基因敲除;NCTC:NCTC11637;B显示统计分析得到的DNA损伤细胞比例(n=3);***P<0.001。
图4显示敲除waaL基因后H.pylori感染所致的细胞凋亡率及抗凋亡蛋白表达变化;其中,A为G27、7.13和NCTC11637(NCTC)野生菌株及相应waaL敲除菌株感染AGS细胞6h后处于凋亡期细胞的比例(一次代表性实验);Q2和Q3:凋亡细胞;B为统计分析得到的凋亡细胞比例(n=3);***P<0.001;C为野生菌株及相应waaL敲除菌株感染后,凋亡标志蛋白Cleaved-caspase3及抗凋亡蛋白MCL1的表达情况。
图5显示敲除waaL基因后H.pylori感染所致的细胞复制周期速率变化;其中,A显示G27野生型及waaL敲除菌株G27ΔwaaL感染后AGS细胞周期分布(一次代表性实验)及S+G2/M比例统计分析(n=3);B显示7.13、NCTC11637(NCTC)野生型及waaL敲除株感染后AGS细胞周期分布(一次代表性实验)及S+G2/M比例统计分析(n=3);***P<0.001,**P<0.01,*P<0.05。
图6显示敲除waaL基因后H.pylori引起的细胞“蜂鸟”样表型变化;其中,A为G27野生菌株和waaL基因敲除菌株感染AGS后,相差显微镜观察细胞“蜂鸟表型”情况;B为7.13、NCTC11637(NCTC)野生型及waaL敲除株感染AGS细胞后,经FITC偶联的鬼笔环肽染色细胞骨架,通过荧光显微镜观察到的细胞“蜂鸟表型”情况;标尺=50μm。
图7显示敲除waaL基因后H.pylori致癌能力变化;其中,A为银染鉴定H.pylori定植于蒙古沙鼠胃黏膜上皮细胞表面(红色箭头所示);标尺=100μm;B为H.pylori 7.13野生菌株及waaL基因敲除菌株感染8周和12周,蒙古沙鼠胃黏膜疾病发生情况;IEN:上皮内瘤变;C为H.pylori 7.13野生菌株及waaL基因敲除菌株感染12周,蒙古沙鼠胃黏膜H&E染色表现;标尺=100μm。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
1实验材料
1.1细胞系:本发明所用细胞系AGS为人胃腺癌细胞系,购于ATCC。
1.2菌株和质粒
本发明中使用的H.pylori菌株G27、NCTC11637由西澳大利亚大学Barry Marshall教授惠赠,7.13由范德堡大学Richard Peek教授惠赠。E.coli工程菌Top10作为本研究所用的分子克隆宿主菌。使用的质粒pUC19由慕尼黑大学Rainer Haas教授惠赠。
1.3寡核苷酸引物
利用CLC Main Workbench 7(CLC Bioinformatics Software Company,Denmark)软件设计寡核苷酸引物,并由天津擎科生物技术有限公司(中国)或metabion GmbH(Germany)合成。本发明使用的所有引物序列如表1。
表1寡核苷酸引物
1.4试剂
所有试剂的使用和储存都根据制造商的说明进行,其详细描述和供应商列于表2。
表2本发明所用的试剂
LB,Luria-Bertani Medium;BB,Brucella Broth;FCS,Fetal Calf Serum.
1.5抗体
本发明所用的一抗和二抗列于表3。
表3本发明所用的一抗和二抗
1.6实验耗材
细胞培养瓶、培养板(BD Falcon),1ml、2ml、5ml、10ml、25ml灭菌移液管(BDFalcon);细胞刮刀(BD Falcon);玻底培养皿(NEST);细菌培养皿(BD Falcon);PVDF膜(Bio-Rad);15ml、50ml锥形管(Corning);流式管(BD Falcon);组织包埋盒(MeVid);细胞冻存管(Corning);组织防脱载玻片(MeVid)。
1.7实验主要仪器和设备
激光共聚焦扫描显微镜(Olympus FV1000);流式细胞仪(Millipore Guava,FC500,FACS Cantoll);peqSTAR 96PCR仪(Isogen Life Science);核酸电泳仪(Bio-Rad);Molecular Imager Gel Doc XR System(Bio-Rad);垂直电泳槽(Bio-Rad);CO2恒温培养箱(Thermo Fisher Scientific);生物安全柜(Thermo Electron Corporation);-80℃冰箱(SANYO);CO2培养箱专用震荡器(INFORS HT);滚轴混匀器(SU Lab);紫外分光光度计(Beckman);高速低温台式离心机(Eppendorf);Thermomixer comfort(Eppenforf);DNASpeed Vac(Savant);病理切片机(Leica)。
1.8实验动物
SPF级、体重40-50g、5-7周龄雄性蒙古沙鼠购自浙江省实验动物中心。
本发明通过实施例作进一步说明敲除waaL基因的方法以及waaL基因敲除后H.pylori的致病作用变化:
首先,通过同源重组敲除H.pylori菌株中的waaL基因。敲除waaL基因后,结合酶联免疫吸附试验检测IL-8分泌情况、流式细胞术观察细胞DNA损伤、细胞凋亡和细胞周期变化、相差显微镜和免疫荧光显微镜观察细胞“蜂鸟”表型情况,了解WaaL在H.pylori致胃黏膜上皮细胞炎症反应、DNA损伤、细胞凋亡、细胞周期改变和细胞“蜂鸟”表型形成中的作用;最后利用H.pylori感染蒙古沙鼠实验,观察敲除waaL基因后H.pylori致胃癌前病变及胃癌发生能力的变化,明确WaaL在H.pylori致癌中的作用。
实施例1分析不同H.pylori菌株中waaL基因的存在情况
1.1数据准备
本发明对266例H.pylori菌株进行测序,并从公共数据库Enterobase获取927例H.pylori菌株的全基因组测序数据。
1.2H.pylori菌群类型分配并分析WaaL的存在及序列情况
使用Prokka(v1.13.3)对H.pylori菌株基因进行注释,分析每一例菌株waaL基因的CDS存在情况。使用STRUCTURE(v.2.3.4)对H.pylori的7个管家基因(atpA、efp、mutY、ppa、trpC、ureI、yphC)序列进行多位点序列分析(MLST),该分析采用贝叶斯方法来推断种群结构。在MAFFT软件中采用Needleman-Wunsch算法对不同H.pylori菌株的WaaL氨基酸序列进行多序列比对。
使用GraphPad Prism 5软件进行统计分析。定量显示的数据为至少两次独立实验得到的均数(Mean,M)以及均数的标准误(Standard Error Mean,SEM),使用Student’s t-检验进行差异的显著性检验,P<0.05时有统计学意义。分类数据以率表示,使用Fisher确切概率法进行差异的显著性检验,P<0.05时有统计学意义。
通过对上述1193例完成全基因组测序的H.pylori菌株的waaL基因进行比较基因组学分析,发现在这1193例菌株中,waaL基因都存在,说明waaL在不同H.pylori菌株的基因组中保守存在。
实施例2敲除H.pylori菌株中的waaL基因
2.1细菌DNA提取
E.coli质粒DNA提取:为了筛选阳性克隆,根据制造商说明用PureLinkTMQuickPlasmid Miniprep Kit从生长过夜的E.coli中提取质粒DNA。从琼脂平板中刮取细菌,溶于0.5ml PBS中,8000rpm室温离心5min,弃上清。重悬细菌沉淀于添加有RNase A的缓冲液1中。然后使用碱式SDS/NaOH方法裂解菌体,通过以13000rpm离心10min收集澄清的裂解液(上清液)。利用膜结合纯化系统进行质粒DNA的纯化,最后洗脱DNA于40μl超纯水中,-20℃保存。
H.pylori基因组DNA提取:提取生长1-2天的H.pylori基因组DNA。细菌量大约为1个接种环的细菌重悬于0.5ml PBS中,8000rpm室温离心5min,弃上清。然后用QIAampTissue Kit提取基因组DNA。最后洗脱DNA于40μl超纯水中,-20℃保存。
2.2 PCR扩增DNA片段
利用质粒DNA或基因组DNA作为模板进行扩增。本发明使用Takara公司提供的ExTaq DNA聚合酶及相应缓冲液进行PCR扩增。所有引物冻干粉溶于超纯水,制成浓度为100μM的储存液,置于-20℃保存,稀释为10μM的工作液,置于4℃保存。利用琼脂糖凝胶电泳对PCR产物进行鉴定。所有反应都使用peqSTAR 96PCR仪进行。使用Ex Taq DNA聚合酶的PCR反应体系和反应条件分别列于表4和表5。
表4使用Ex Taq DNA聚合酶的PCR反应体系
表5使用Ex Taq DNA聚合酶的PCR反应条件
2.3 DNA电泳和片段纯化
DNA电泳:利用琼脂糖凝胶电泳对PCR产物或酶切消化后的DNA片段进行鉴定。将5μl DNA样品加载于1%(w/v)琼脂糖凝胶,同时加载GeneRulerTM 1kb DNA ladder以评估DNA的片段大小。然后,在TAE电泳液中、70V电压条件下电泳60min(利用较低电压电泳更长时间可使不同大小的DNA条带更好地分离)。电泳完成后将凝胶置于1mg/L溴化乙锭中,15min后在260nm紫外光下分析DNA条带(Molecular Imager Gel Doc XR System,Bio-Rad)。
从PCR反应体系、酶切体系及琼脂糖凝胶中纯化DNA片段:本发明使用GEHealthcare公司的Illustra GFX PCRDNA and Gel Band Purification Kit纯化DNA片段,实验操作根据试剂说明书进行。特别地,对于琼脂糖凝胶中的DNA片段纯化,经紫外光分析片段大小正确后,将更多体积的DNA样品在琼脂糖凝胶中电泳更长时间。然后将胶置于0.1%亚加蓝中室温染色10min,蒸馏水洗3次、10min/次后在自然光下观察条带,用刀片切取目的DNA片段,置于500μl Capture buffer type 3中。在60℃温度下使胶完全溶解后,使用相同方法提取琼脂糖胶中的DNA。洗脱DNA于40μl超纯水中,-20℃保存。
2.4重组DNA克隆构建质粒
(1)限制性内切酶消化
本发明使用New EnglandBiolabs公司的限制性内切酶进行DNA片段的双酶切。若需要对构建的质粒进行鉴定,则使用10μl酶切体系在37℃孵育1.5h后进行DNA电泳鉴定;若需要将酶切后的DNA片段用于克隆,则使用50μl酶切体系在37℃孵育3h后进行DNA片段纯化。
(2)DNA连接
为了将DNA插入片段连接到载体上,本发明使用T4 DNA连接酶在10μl连接体系4℃孵育过夜进行DNA连接,然后将连接后的质粒转化到E.coli菌株Top10感受态细胞中。
(3)利用热激法将DNA转化到感受态E.coli Top10。
1)从-70℃取出感受态Top10,于冰上融化后与DNA连接体系(10μl)轻柔混合,将混合液置于冰上30min。
2)42℃水浴锅中热激90s,再次放于冰上2min。
3)菌液中加入1ml LB肉汤培养基,37℃、需氧、110rpm持续摇菌1h。
4)取100μl菌液接种于添加有合适抗生素的LB琼脂平板;其余900μl 5000rpm室温离心5min,100μl上清液重悬菌体,接种于添加同样抗生素的LB琼脂平板,37℃过夜培养。
2.5 H.pylori自然转化
将夜过培养的细菌重悬于0.5ml BB/10%FCS中,测量菌液浓度,取6x107(OD550=0.2)细菌重悬于1ml BB/10%FCS中,在37℃、10%CO2、110rpm摇动孵育2h。然后将E.coli中提取的500至1000ng质粒DNA(约5μl洗脱液)加入到菌液中,继续在37℃、10%CO2、110rpm摇动孵育4h。将DNA-细菌混合液在5000rpm离心5min,弃大部分上清液,重悬菌体于剩余的100μl BB/10%FCS。最后接种到相应的选择性GC琼脂平板,在37℃微需氧条件下孵育3-4天后,筛选转化阳性的单克隆菌株。
2.6 LPS银染鉴定waaL基因敲除
(1)将从琼脂平板中刮取的H.pylori(OD550=3.0)重悬于100μl LPS裂解液中。
(2)将样品置于100℃金属浴10min,置于冰上冷却。
(3)加入5μl 20mg/ml的蛋白酶K,55℃水浴锅中消化过夜。
(4)LPS在15%SDS-PAGE凝胶电泳后用200ml去离子蒸馏水洗胶2次,10min/次。
(5)将凝胶放置于含40%乙醇、5%冰乙酸、0.7%高碘酸的氧化液中,70rpm、室温氧化20min。
(6)蒸馏水洗胶3次,10min/次。
(7)加入新鲜配制的染色液,70rpm、室温染色10min。
(8)蒸馏水洗胶5次,10min/次。
(9)将凝胶转移至200ml含10mg柠檬酸、0.1ml 37%甲醛的显色液中,静置显色2-10min。
(10)倒掉显色液,加入含10%冰乙酸的中止液中止显色1min,然后拍摄图像。
2.7实验结果
分别用引物TXQ6、TXQ7以及TXQ8、TXQ9扩增G27基因组中waaL开放阅读框的上游1000bp DNA片段(WaaL up)及下游1000bp DNA片段(WaaL down)(图1A)。在pUC19的SphI和SalI位点插入上游片段,得到pUC19-WaaL up,用引物TXQ6和TXQ7进行克隆PCR鉴定(图1B)以及酶切鉴定(图1C)。
将cat盒子及下游片段通过SalI和XmaI位点克隆到pUC19-WaaL up,SalI和XmaI对质粒进行酶切鉴定,得到waaL敲除质粒pUC19-WaaL up-cat-WaaL down(图1D,包含SEQ IDNO.1所示敲除waal序列)。
将pUC19-WaaL up-cat-WaaL down转化进G27、7.13和NCTC11637,接种于氯霉素筛选平板,挑取单克隆接种于新鲜的氯霉素平板进行再次筛选,然后传代培养到非选择性平板,使用基因组DNA作为模板、引物TXQ6和TXQ9行PCR鉴定得到waaL基因敲除菌株G27ΔwaaL、7.13ΔwaaL、NCTC11637ΔwaaL(图1E)。
提取H.pylori菌株LPS,银染显示waaL基因敲除菌株的LPS缺失O-抗原,进一步说明waaL基因敲除成功(图1F)。
实施例3通过H.pylori感染AGS细胞实验验证WaaL的作用
从冻存管中挑取少许H.pylori G27、7.13和NCTC11637野生菌株(G27wt、7.13wt和NCTC11637wt)和相应waaL基因敲除菌株接种在添加有8%马血清、1%vitamin mix的GC琼脂平板,置于37℃微需氧条件下(85%N2、10%CO2、5%O2)培养48h后进行传代。细菌传代2-3次后进行体外感染AGS细胞实验,如下所述:
(1)感染前一天将细胞接种于6孔板中,使细胞汇合率在第二天实验时达到80-90%。
(2)将过夜生长的H.pylori重悬于已复温的0.5ml PBS/10%FCS中,测量细菌OD550,按照每孔需要OD550=0.2的细菌量计算所需菌液,即感染复数(multiplicityofinfection,MOI)=100。
(3)弃细胞培养基,按每孔所需菌液加入到细胞中,然后加入PBS/10%FCS使总体积达2ml/孔;置于37℃、5%CO2培养。
3.1细胞上清IL-8测定
(1)H.pylori感染AGS细胞6h后收集上清,用PBS/10%FCS稀释10倍。
(2)倍比稀释准备以下浓度的标准品:100pg/ml,50pg/ml,25pg/ml,12.5pg/ml,6.25pg/ml,3.125pg/ml,1.56pg/ml,0.7pg/ml。
(3)将标准品和稀释后的上清液加入到板中,100μl/孔,每个样重复一次。
(4)将板置于4℃孵育过夜。
(5)PBS/0.05%Tween-20洗板四次,400μl/孔。
(6)加入100μl/孔的检测性抗体(Biotin Mouse Anti-human IL-8),室温孵育1h。
(7)PBS/0.05%Tween-20洗板四次,400μl/孔。
(8)加入100μl/孔的conjugate,室温孵育1h。
(9)PBS/0.05%Tween-20洗板四次,400μl/孔。
(10)加入按照A:B液为1:1比例准备的TMB底物试剂,100μl/孔,避光室温孵育约30min。
(11)当反应颜色明显变蓝时,加入50μl/孔的终止液终止反应。
(12)在450nm波长读取结果。
实验结果:检测炎症介质IL-8的分泌水平发现,与野生菌株感染后引起明显的IL-8分泌相比,相应waaL基因敲除菌株感染所致的IL-8分泌水平都明显下降(较野生菌株下降40%-50%,P<0.001)(图2),说明敲除waaL基因能够减轻H.pylori所致的炎症反应。
3.2流式细胞术检测细胞DNA损伤
(1)吸弃细胞培养基,PBS洗细胞2次,加入不含EDTA的胰酶消化细胞(AGS需要在37℃消化7min),用胰酶两倍体积的血清中止消化,2000rcf离心4min。
(2)冰上用Cytofix/Cytoperm固定通透细胞30min。
(3)Perm/Wash缓冲液洗细胞2次,然后按1:50稀释比例加入藻红蛋白标记的兔源多克隆抗pH2AFX(Ser139)抗体,室温避光孵育25min。
(4)设置流式细胞仪激发波长488nm、发射波长575nm,检测荧光强度。
(5)Flowjo软件分析阳性细胞比例。
H.pylori G27wt、7.13wt和NCTC11637wt感染AGS细胞12h后,检测到pH2AFX(DNA损伤标志因子)阳性细胞比例,分别为5.54±0.15%,6.76±0.13%和9.32±0.18%,显著高于未感染组的0.86±0.06%(P<0.001);而G27ΔwaaL、7.13ΔwaaL和NCTC11637ΔwaaL感染AGS细胞12h后,所致的pH2AFX阳性细胞比例分别为3.84±0.11%,2.89%±0.10和4.83±0.11%,较相应的野生菌株分别下降30-50%(P<0.001)(图3),说明敲除waaL基因能够减少H.pylori感染致细胞DNA损伤的作用。
3.3流式细胞术及Western blot检测细胞凋亡情况
流式细胞术检测细胞凋亡:
(1)吸弃6孔板中的细胞培养基,PBS洗细胞2次,加入不含EDTA胰酶消化细胞(AGS需在37℃消化7min),用胰酶两倍体积的血清中止消化。
(2)轻轻吹打细胞制成细胞悬液,2000rcf、4℃离心4min后弃上清,PBS洗细胞两次。
(3)100μl 1x Binding Buffer重悬细胞,制成单细胞悬液。
(4)在单细胞悬液中加入5μl FITC-AnnexinV及5μl PI混匀;室温下避光孵育15min。
(5)根据细胞量加入1xBinding Buffer,使细胞浓度调整到约为1x106/ml。
(6)1h内用流式细胞仪检测(双通道,分别设置激发波长485nm、发射波长519nm以及激发波长535nm、发射波长617nm)
(7)使用Flowjo 10分析细胞凋亡结果。
Western blot检测凋亡相关蛋白:
本发明使用10%的凝胶,其标准厚度为0.75mm。将裂解产物加样到凝胶孔中,同时加载4μl蛋白大小标记物,先在90v电压下电泳30min,然后将电压调至120v继续电泳,电泳时间根据所要分析的蛋白大小而定。电泳结束后将蛋白转移至PVDF膜并用于免疫印迹分析,或转移至尼龙膜后用于HiBiT检测。
SDS-PAGE后,利用电场垂直穿过凝胶从而将已分离开的带负电荷蛋白转移到PVDF膜上。当蛋白转移到PVDF膜表面时变为疏水性和极性,从而使其紧密结合到膜上。本研究使用半干转膜法,具体如下:首先将PVDF膜用甲醇活化后置于转膜液中待用。然后两层厚的和两层薄的blot纸用Western转膜液浸泡后铺在blot装置的负极,活化后的PVDF膜置于blot纸上面,再将凝胶置于膜上。最后,将另外两层薄的和两层厚的blot纸置于凝胶上,并将blotting装置的正极盖在整个系统上。每块凝胶用50mA电流转膜90min。
蛋白印迹结束后,取出载有蛋白的PVDF膜置于50ml管子,加入5ml TBST/5%(w/v)脱脂奶粉(blotting级别)室温封闭2h,以避免非特异性的抗体结合。然后根据特异性的抗体信息将一抗以1:1000的稀释比例加入到封闭液,室温过夜孵育。一抗孵育后用TBST洗4次,10min/次。然后室温孵育二抗(1:3000的稀释比例)1h。孵育二抗后再次用TBST洗4次,10min/次。最后,根据二抗所带的标签选用碱性磷酸酶曝光液或化学发光液显色。用碱性磷酸酶曝光液显色蛋白条带,直到条带显现后用水中止反应。
实验结果:
检测G27wt、7.13wt和NCTC11637wt感染AGS细胞6h后细胞凋亡比例,分别为12.53±0.69%,27.60±1.13%和14.25±0.34%,显著高于未感染AGS细胞中的凋亡细胞比例2.667±0.28%(P<0.001)。G27ΔwaaL、7.13ΔwaaL和NCTC11637ΔwaaL感染AGS 6h后,凋亡细胞比例分别为39.43±0.67%,61.10±0.58%和38.47±0.73%,较相应野生菌株感染组增加2倍、1倍和2倍(图4A和B)。
另外,提取各组细胞蛋白质后经Western blot检测后发现,G27wt、7.13wt和NCTC11637wt感染AGS细胞后凋亡标志蛋白Cleaved-caspase3以及抗凋亡蛋白MCL1较未感染组升高,而G27ΔwaaL、7.13ΔwaaL和NCTC11637ΔwaaL感染后Cleaved-caspase3水平较相应野生株感染组更高,MCL1水平较相应野生株感染组更低(图4C)。说明waaL基因敲除后H.pylori感染AGS细胞后细胞凋亡增加,H.pylori的抗凋亡作用减少。
3.4流式细胞术检测细胞周期
实验按照BD Biosciences公司细胞周期检测试剂操作说明进行:
(1)弃去6孔板中的细胞培养基,PBS洗细胞2次,加入不含EDTA的胰酶消化细胞(AGS需要在37℃消化7min),用胰酶两倍体积的血清中止消化。
(2)轻轻吹打细胞制成细胞悬液,2000rcf、4℃离心4min后弃上清,PBS洗细胞两次后调整细胞浓度为1×106/ml,取1ml单细胞悬液。
(3)2000rcf、4℃离心4min,50μl PBS重悬细胞,加入1ml预冷的75%乙醇固定细胞(边加边混匀,4℃,固定过夜)。
(4)3000rcf离心8min,PBS洗细胞2次后离心弃上清。
(5)加入0.5ml PI/RNase染色液重悬细胞,室温孵育15min(避光)。
(6)设置流式细胞仪激发波长535nm、发射波长617nm,检测荧光强度。
(7)通过Modfit LT3.1得到细胞周期分布状态,计算Go/G1%,S%及G2/M%。
G27wt、7.13wt和NCTC11637wt感染AGS 6h后,处于S+G2/M期的细胞比例分别为65.59±1.43%,66.88±2.59%和70.49±0.70%,显著高于未感染组的49.17±1.27%(P<0.001),提示H.pylori感染AGS细胞后,导致细胞复制周期加快;而G27ΔwaaL、7.13ΔwaaL、NCTC11637ΔwaaL感染AGS后,处于S+G2/M期的细胞比例分别为56.56±0.56%,58.45±1.36%和62.39±0.71%,较相应野生菌株感染组降低约8%(P<0.05)(图5A和B),提示waaL基因敲除后H.pylori促进细胞复制加快的作用减弱。
3.5显微观察AGS细胞形态
相差显微镜观察到,G27wt感染引起AGS细胞出现明显的“蜂鸟”表型,而G27ΔwaaL感染组“蜂鸟”表型(出现这一表型,表明细胞出现上皮-间质转化,导致肿瘤发生)减轻(图6A)。通过荧光显微镜观察到7.13ΔwaaL和NCTC11637ΔwaaL较相应野生菌株感染AGS细胞引起的“蜂鸟”表型也明显减轻(图6B)。这些说明waaL基因敲除后H.pylori致细胞上皮-间质转化的作用减弱。
实施例4通过H.pylori感染蒙古沙鼠动物实验验证WaaL的作用
4.1H.pylori感染蒙古沙鼠
(1)蒙古沙鼠分组与处理:
将65只沙鼠随机分成三组,具体如下:感染8周:空白对照组:共5只,予以BB/10%FCS灌胃;7.13wt感染组:共13只,予以7.13野生菌株悬浮液灌胃;7.13ΔwaaL感染组:共12只,予以waaL敲除菌株悬浮液灌胃。感染12周:空白对照组:共7只,予以BB/10%FCS灌胃;7.13wt感染组:共14只,予以7.13野生菌株悬浮液灌胃;7.13ΔwaaL感染组:共14只,予以waaL敲除菌株悬浮液灌胃。
(2)H.pylori感染蒙古沙鼠:灌胃前一天用25ml细胞培养瓶液体摇菌,调整起始菌液浓度0.1OD550,5ml/瓶,100rpm,37℃微需氧条件下培养16h,并在灌胃前将沙鼠禁食12h,禁水4h。细菌液体培养后用Beckman圆底管离心细菌(4000rpm,5min),然后用新鲜配制并已复温的BB/10%FCS重悬细菌,测量菌液浓度,调整浓度到2×109/ml;用18G灌胃针灌胃,0.5ml/只,对照组以相同方式灌胃等量的BB/10%FCS。灌胃后禁食及禁水4h。隔日再次以相同方式灌胃,共3次。
4.2蒙古沙鼠胃组织取材
(1)动物准备:取材前12h动物禁食。
(2)解剖操作:1)通过断颈法处死蒙古沙鼠;2)备皮;3)消毒;4)解剖。5)剖胃:用尖镊子夹住胃,从胃底沿胃大弯向胃窦部纵向剖开,摊开全胃,用灭菌生理盐水轻轻冲洗胃内残留物,肉眼观察胃黏膜大体情况;沿纵轴将胃分成左右两半,一半放于组织包埋盒浸入4%多聚甲醛,用于固定、脱水、石蜡包埋和组织切片,银染和苏木素-伊红(Hematoxylin&Eosin,H&E)染色;另一半再均分成两块,一块放于冻存管浸入液氮中冻存备用,一块剪碎后置于匀浆管中。
4.3胃组织石蜡切片制备
将组织从固定液取出在通风橱内用手术刀将目的部位组织修平整,将修切好的组织和对应的标签放于脱水盒内,进行脱水,包埋,切片。切片漂浮于摊片机40℃温水上将组织展平,用载玻片将组织捞起,并放进60℃烘箱内烤片。待水烤干及蜡烤化后取出,常温保存备用。
4.4胃黏膜组织学检测
胃黏膜组织银染:
本发明使用bielschowsky法镀银染色观察蒙古沙鼠胃黏膜H.pylori定植,以鉴定H.pylori感染蒙古沙鼠模型的建立。具体的镀银染色实验步骤如下:
(1)石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ20min-二甲苯Ⅱ20min-无水乙醇Ⅰ10min-无水乙醇Ⅱ10min-95%乙醇5min-90%乙醇5min-80%乙醇5min-70%乙醇5min-蒸馏水洗。
(2)硝酸银反应:切片浸入20%硝酸银加盖于暗处反应20min。
(3)蒸馏水洗(在此期间配制银氨溶液):染色第2步用过的20%硝酸银,逐滴加入氢氧化铵,边滴边搅拌,直到开始形成的白色沉淀又消失,银液变清即为银氨溶液。
(4)银氨液处理:切片浸入银氨液作用15min(在此期间配制稀氨水,银氨液用于下步配制显影工作液)。稀氨水配制:蒸馏水30ml加氢氧化铵3滴。
(5)稀氨水中浸泡:切片移入稀氨水中浸泡(在此期间配制显影工作液)。
(6)显影储备液:甲醛液20ml+蒸馏水100ml+硝酸1滴+柠檬酸0.5g。显影工作液:取第4步用过的银氨液30ml,加入显影储备液3滴,混合均匀,现配现用不能保存。
(7)显色:切片浸入显影工作液中显色,约3-7min,镜下控制显色,金黄色的背景,神经纤维呈黑色时即取出。切片浸入第5步用过的稀氨水中浸泡1min。流水冲洗1min。
(8)去除未反应的银离子:5%硫代硫酸钠处理2min(固定已反应的银盐和除去未反应的银离子)。流水冲洗5min。
(9)脱水封片:将切片依次放入95%乙醇I 5min-95%乙醇II 5min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-二甲苯Ⅰ5min-二甲苯Ⅱ5min中脱水透明,将切片从二甲苯拿出来稍晾干,中性树胶封片。
(10)显微镜镜检,图像采集分析。
胃黏膜组织H&E染色:
使用H&E染色法观察蒙古沙鼠胃黏膜组织病理改变情况,具体实验步骤如下:
(1)石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ20min-二甲苯Ⅱ20min-无水乙醇Ⅰ10min-无水乙醇Ⅱ10min-95%乙醇5min-90%乙醇5min-80%乙醇5min-70%乙醇5min-蒸馏水洗。
(2)苏木素染色细胞核:切片入Harris苏木素室温染3-8min,自来水洗,1%的盐酸乙醇分化数秒,自来水冲洗,0.6%氨水返蓝,流水冲洗。
(3)伊红染细胞质:切片入伊红染液中室温染色1-3min。
(4)脱水封片:将切片依次放入95%乙醇I 5min-95%乙醇II 5min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-二甲苯Ⅰ5min-二甲苯Ⅱ5min中脱水透明,将切片从二甲苯拿出来稍晾干,中性树胶封片。
(5)显微镜镜检,图像采集分析。
组织病理评判:
所有切片由一位对实验分组不知情的有经验病理医师进行读片。H&E染色切片参照临床诊断标准进行病理结果判定,评估是否存在胃黏膜萎缩、肠上皮化生、IEN和胃癌,其中IEN分为低级别IEN和高级别IEN。以胃组织区域内检查到的最严重病变作为最终的组织学诊断结果。镀银染色切片在高倍镜下观察,在胃黏膜黏液层、表面上皮、小凹上皮或腺管上皮表面观察到典型的小短杆状、黑色螺旋样菌体为H.pylori阳性,否则为阴性。
结果:经镀银染色鉴定,7.13wt感染组及7.13ΔwaaL感染组胃黏膜黏液层及上皮表面可见H.pylori(图7A)。敲除waaL减少7.13所致的蒙古沙鼠胃癌前病变及胃癌发生:灌胃后第8和12周,7.13ΔwaaL感染组中蒙古沙鼠胃黏膜上皮内瘤变和胃癌总发生率分别为33.3%(4/12)和35.7%(5/14),分别低于7.13wt感染组的69.2%(9/13)和78.6%(11/14)(图7B)。7.13wt感染12周的蒙古沙鼠发生黏膜内癌,细胞呈现出明显异形性,不规则的腺体拥挤排列并有浸润的趋势;而7.13ΔwaaL感染12周蒙古沙鼠示例仅显示出腺管密集,腺体细胞核深染,但无复杂结构(图7C)。从而说明,WaaL在H.pylori致蒙古沙鼠胃癌发生中起重要作用。
综上,通过比较基因组学发现,WaaL在H.pylori不同菌株中保守存在。敲除waaL基因后,H.pylori感染的致炎及致胃上皮细胞DNA损伤的作用明显减轻、H.pylori感染所致的细胞凋亡增加从而减少了DNA受损细胞的堆积、H.pylori感染所致的细胞增殖作用减弱、并且H.pylori致蒙古沙鼠胃癌发生的作用也明显减少。靶向敲除waaL基因的试剂或抑制waaL基因表达的试剂可用于制备相应药物以降低H.pylori的致瘤性,可用于预防H.pylori相关胃癌的发生,临床应用前景好。
序列表
<110> 四川大学华西医院
<120> WaaL作为药物靶标在制备药物中的应用
<141> 2022-04-27
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2811
<212> DNA
<213> Artificial Sequence
<400> 1
gcatgccctt atcatcaata agccctgaat ttaaatccgc atcaatcgcc atttgctttc 60
ctggcatagc gtctagggca aatcgcgccc taacttcagt aaccctagta gagccattag 120
taaccactaa taaattcact aacactaaaa tactaaagat aatcgcccca atcacataat 180
tcccgctcac gctaaattcc ccaaacgccg tgataatatc gctcaccgcg ctaggccctt 240
tatagccttg cgttaaaatc attctggtgg tggcgacatt taaagccaag cggtataagg 300
ttacaatgag gagcaaagta gggaaagcgc taaaatcagt aggcttgtca atataaagcc 360
cgattaaaat aatcaacacc gatagcgcga tagaaatcgt gagtaaaaaa tccaacacaa 420
aaggcggtaa cggcacgata atgatcgcta aaatagcgat cacaaagacc acaagggcta 480
agtctttgga ttgcaaaaag cgtttaaaga cagggaaagt ctttttaaaa gctaatttgg 540
agcgttcgtt tgccataatc taaaatcatg ccttaatctg gtaggtggtt tgtctcttat 600
tataatataa agtttaaaag tatggcacaa aaaagcttat tttacgctat aatgcgatct 660
ttattaaatt accaattagg aggtcgttat ggctttgaat ctggagaaaa aacaagaaat 720
cattaaggcg tttgctacta aggaaaacga tacgggttct tgtgaggtgc aagtggcgtt 780
gttgaatgaa aggatcaagc ttttaaccga gcatttaaag gctaacccca aagatcattc 840
cagtcgttta gggcttttaa aattagtcgc tcaaagacgc aacttgttga aatacatcaa 900
acgcaccgct catgcgcgtt atgtggtttt gattgaaaag ttaggcatta aagacagata 960
atttttatcg gttttagggt gtggcgagtt tgtgttgaaa gagcgttgtc gactctagct 1020
agaggatcga tccggttttt gttaatccgc catattgtgt tgaaacaccg cccggaaccc 1080
gatataatcc gcccttcaac agatccgaga ttttcaggag ctaaggaagc taaaatggag 1140
aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 1200
gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 1260
gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 1320
cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 1380
gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 1440
tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 1500
gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 1560
tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 1620
atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 1680
gtgctgatgc cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc 1740
agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaggatcc 1800
gcgcttttta gcatcaaaga gtttgatgtt agtcaagcgg tagtttcttg tgatgcgctc 1860
ttagggggat ttaaggggta agataaaaac ttatgctata atgcggtttc attccatatt 1920
caacaaggag aaataaatta atgaaaattt tagtgattca agggcctaat ttaaacatgt 1980
taggacacag agacccaaga ctttatggca tggtaacctt agaccaaatc catgaaatca 2040
tgcaaacttt cgtgaaacaa ggcaatttag atgtggaatt agagtttttt caaaccaatt 2100
ttgagggcga aatcattgat aaaattcaag agagcgtggg cagcgattat gaagggatca 2160
tcattaaccc tggagcgttt tcgcacactt ctattgcgat tgcagatgcg atcatgctag 2220
cgggcaaacc cgttattgaa gtgcatctca ctaacattca agccagagag gaattcagga 2280
aaaattctta cactggagcg gcttgtggag gcgtgatcat gggatttggc ccgcttggct 2340
acaacatggc tttaatggcg atggtcaata ttttagccga gatgaaagcg ttccaagaag 2400
cccaaaaaaa caaccctaat aaccccatta acaatcaaaa ataaaagggg gctacccatg 2460
aaaggattag aaagagaatc gcatttcacg ctcaatgaaa acgcgatgtt ttttgagtgt 2520
gcttatagtt gcgataacgc tttatttttg caattagacg atcgctcgtt ttttatcact 2580
gattctcgct acactcaaga agctaaagaa agcgttcagc ctaaaaatgg cgttttagcg 2640
gaagtggtag aatctagcga tttagtgcaa agcacgattg atttgatcgc taaaagttcg 2700
gttaaaaagc tcttttttga ccccaatcaa gtgaatttac aaacctacaa gcgtttaaat 2760
tcagcgcttg gggataaggt tactttagag ggtgtgccta gttaccccgg g 2811
Claims (8)
1.H.pylori脂多糖O-抗原连接酶WaaL作为药物靶标在制备药物中的用途,所述药物为以下任一个或多个:
(1)治疗H.pylori相关胃炎的药物;
(2)治疗消化性溃疡的药物;
(3)预防或治疗H.pylori相关胃癌的药物。
2.抑制H.pylori脂多糖O-抗原连接酶WaaL表达的试剂在制备预防、治疗胃部疾病方面的应用。
3.根据权利要求2所述的应用,其特征在于,所述试剂包括敲除waaL基因的试剂、使waaL基因沉默的试剂或waaL基因的抑制剂。
4.根据权利要求2所述的应用,其特征在于,所述胃部疾病为由H.pylori菌株引起的胃部疾病。
5.根据权利要求2或4所述的应用,其特征在于,所述胃部疾病包括慢性胃炎、消化性溃疡和/或胃癌。
6.根据权利要求5所述的应用,其特征在于,所述胃癌包括管状腺癌、乳头状腺癌、黏液腺癌、印戒细胞癌、混合癌。
7.根据权利要求3所述的应用,其特征在于,所述敲除waaL基因的试剂包含SEQ IDNO.1所示的核苷酸序列。
8.一种用于治疗H.pylori相关性胃炎的制剂,其特征在于,所述制剂含有敲除waaL基因的试剂、使waaL基因沉默的试剂或waaL基因的抑制剂,所述制剂为药物、药物组合物、保健品、食品或添加剂。
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Citations (2)
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US20120093869A1 (en) * | 2009-05-14 | 2012-04-19 | David Klumpp | Live-attenuated compositions for bacterial infections |
CN105671067A (zh) * | 2015-12-01 | 2016-06-15 | 滨州医学院 | 一种幽门螺杆菌基因敲除载体质粒的构建方法 |
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Patent Citations (2)
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US20120093869A1 (en) * | 2009-05-14 | 2012-04-19 | David Klumpp | Live-attenuated compositions for bacterial infections |
CN105671067A (zh) * | 2015-12-01 | 2016-06-15 | 滨州医学院 | 一种幽门螺杆菌基因敲除载体质粒的构建方法 |
Non-Patent Citations (3)
Title |
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ISABELLE HUG等: "Helicobacter pylori lipopolysaccharide is synthesized via a novel pathway with an evolutionary connection to protein N-glycosylation", 《PLOS PATHOG》, vol. 6, no. 3, 19 March 2010 (2010-03-19), pages 1000819 * |
KAI-WEN TENG等: "Helicobacter pylori employs a general protein glycosylation system for the modification of outer membrane adhesins", 《GUT MICROBES》, vol. 14, no. 1, 7 October 2022 (2022-10-07), pages 2130650 * |
丁相元等: "通过生物信息学分析发现胃癌的靶点基因和生物学特性", 《中华普通外科学文献》, vol. 16, no. 1, 1 February 2022 (2022-02-01), pages 20 - 26 * |
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