CN113348241A - 表达外源蛋白的微生物及其用途 - Google Patents
表达外源蛋白的微生物及其用途 Download PDFInfo
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- CN113348241A CN113348241A CN201980086881.4A CN201980086881A CN113348241A CN 113348241 A CN113348241 A CN 113348241A CN 201980086881 A CN201980086881 A CN 201980086881A CN 113348241 A CN113348241 A CN 113348241A
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- lactobacillus
- vip
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- promoter
- microorganism
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Abstract
提供一种表达血管活性肠肽(VIP)基因的微生物、包括该微生物的用于预防或治疗引起胃肠道损伤的疾病的组合物、试剂盒、以及预防或治疗引起胃肠道损伤的疾病的方法。
Description
技术领域
涉及一种表达VIP基因的微生物、包括该微生物的用于预防或治疗引起胃肠道损伤的疾病的组合物、试剂盒、以及预防或治疗引起胃肠道损伤的疾病的方法。
背景技术
血管活性肠肽(vasoactive intestinal peptide:VIP)是在肠内具有血管活性的肽激素。VIP是具有28个氨基酸残基的肽,所述28个氨基酸残基属于胰高血糖素/胰泌素超家族II类G蛋白-偶联受体的配体。
在人类中,人类基因组上的染色体6的q25区编码长度为170个氨基酸的胰泌素家族的成员。长度为170个氨基酸的该成员在翻译后被截短以形成血管活性肠肽(VIP)。VIP多肽的活性形式能够降低血压、增加血管壁的扩张(vasodilation)、松弛(relax)呼吸系统和胃肠道组织中的平滑肌、不仅减少Th1反应更通过促进Th2减少免疫反应、调节先天性和获得性免疫反应或促进内脏中电解质的分泌。此外,作为神经递质,发现VIP在中枢神经系统中和与淋巴细胞的通讯中活跃。VIP的生物活性通过三种已知的受体传递:VIP1R、VIP2R和PAC1R。已知这些受体会引起细胞中内钙的产生以及cAMP的浓缩。像VIP一样,它们对胰泌素的亲和力取决于亚型和配体的氨基酸序列。人类天然VIP在血液中具有约2分钟的半衰期,因此其具有很短的半衰期。
美国专利9,561,262公开了一种用于治疗患者高血压的方法,其包括向患者施用VIP和抗高血压剂。
US 2016-0045557 A1公开了作为用于改善肠神经系统的益生菌菌株,增加VIP水平的乳酸杆菌和双歧杆菌菌株,但是这些菌株是在包括上皮细胞(epithelial cell)和肠神经细胞(enteric neuronal cell)的共培养(co-culture)模型中筛选的菌株,并且不是遗传重组的菌株。
CN 10875360 A公开了一种多功能复合微生态学的制剂纳米-硒-重组表达VIP-乳酸乳球菌及其制备方法,为了表达抗菌性肽VIP,使用乳酸乳球菌以及使用乳链菌肽诱导型启动子和USP45信号序列的表达系统,并公开了其作为动物饲料添加剂的用途。
即使根据现有技术,也需要表达VIP基因的微生物及使用其在治疗炎症性肠病中。
发明内容
技术问题
一方面提供一种重组微生物,其包括增加编码VIP的基因的表达的基因修饰。
另一方面提供一种用于预防或治疗在人类中引起胃肠道(gastrointestinaltract)损伤的疾病的组合物,其包括所述微生物。
另一方面提供一种用于预防或治疗引起胃肠道(gastrointestinal tract)损伤的疾病的试剂盒,其包括所述微生物和TNF-α阻断剂。
另一方面提供一种预防或治疗引起胃肠道损伤的疾病的方法。
技术方案
在本说明书中,术语“VIP”、“VIP蛋白”或“VIP多肽”是指具有如本文所述的生物学活性中的一种或多种的生物学活性多肽。在本说明书中所述的VIP包括模仿天然VIP的功能的变异体。所述变异体包括具有比天然VIP更长的半衰期并且具有同等或大于天然VIP的生物学活性的变异体。
本发明的VIP包括但不限于人VIP、重组人VIP、鼠VIP和/或重组小鼠VIP。所述VIP可以具有与SEQ ID NO:1氨基酸序列的50%或更多、60%或更多、70%或更多、85%或更多、90%或更多、95%或更多、98%或更多、99%或更多或100%的同一性。所述VIP可以具有分别为SEQ ID NO:1、2、3、4、5、6、7、8和9的氨基酸序列。SEQ ID NO:2的VIP是人源的天然VIP。具有SEQ ID NO:1、3、4、5、6、7、8和9的氨基酸序列的VIP为人源的天然VIP变异体VIP1、VIP2、VIP3、VIP4、VIP5、VIP6、VIP7和VIP8。
在本文中使用的所述VIP多肽可以从诸如人类组织或其他来源的多种来源分离,或可以通过重组或合成方法制备。术语“VIP多肽”还包括VIP多肽的变异体。可以以形成包括与另一异源多肽或氨基酸序列融合的VIP的嵌合分子的方式修饰本发明的VIP。
一方面提供一种重组微生物,其包括增加编码VIP的基因的表达的基因修饰。
所述微生物可以包括增加VIP表达的基因修饰。所述基因修饰可以增加编码VIP的基因的拷贝数。增加所述拷贝数可以是由于将外源基因转导至微生物所致。所述转导可以由形质转换、形质转导、形质感染或电穿孔进行。所转导的外源基因可以合并于宿主细胞的基因组中,也可以不合并于宿主细胞的基因组中而存在。
所述微生物可以包括编码VIP的外源基因。所述基因可以可操作地连接于调节其表达的调节序列。所述调节序列可以是启动子、操纵子、终止子或编码信号肽的核苷酸序列。所述启动子可以是组成型启动子(constitutive promoter)。所述启动子在乳酸菌中的转录起始效率可以等于或大于常规启动子,例如,P11启动子。所述启动子也可以是诱导型启动子。所述诱导型启动子可以是例如乳链菌肽诱导型启动子(Nisin induciblepromoter)。所述启动子可以是源自副干酪乳杆菌(Lactobacillus paracasei)的PR4。所述PR4启动子可以具有SEQ ID NO:10的核苷酸序列。所述PR4启动子于2018年11月8日提出国际申请的国际公开WO 2019/132231A1中公开,所述国际申请的内容通过引用整体并入本文。当编码所述信号肽的核苷酸序列与外源蛋白基因框内连接时,将外源蛋白分泌到细胞外部的能力可能大于常规信号肽,例如USP45信号肽。所述信号肽可以是源自副干酪乳杆菌(Lactobacillus paracasei)的SP4。所述SP4可以具有SEQ ID NO:11的氨基酸序列。所述SP4于2018年11月8日提出国际申请的国际公开WO 2019/132231A1中公开,所述国际申请的内容通过引用整体并入本文。所述微生物可以分泌VIP到细胞外。
所述微生物可以是细菌。所述细菌可以是革兰氏阳性细菌或革兰氏阴性细菌。所述革兰氏阳性细菌可以是乳酸菌。所述乳酸菌可以属于乳杆菌(Lactobacillus)、乳球菌(Lactococcus)、比菲德氏菌(bifidobacteria)、链球菌(Streptococcus)、明串珠菌(Leuconostoc)、魏斯氏菌(Weissella)、片球菌(Pediococcus)或肠球菌(Enterococcus)属。所述乳酸菌可以是副干酪乳杆菌(Lactobacillus paracasei)、短乳杆菌(Lactobacillus brevis)、植物乳杆菌(Lactobacillus plantarum)或乳酸乳球菌(Lactococcus lactis)。所述乳酸菌可以是植物乳杆菌LMT1-9(KCTC 13421BP)、副干酪乳杆菌LMT1-21(KCTC 13422BP)或短乳杆菌LMT1-46(KCTC 13423BP)。
另一方面提供一种用于预防或治疗引起胃肠道(gastrointestinal tract)损伤的疾病的组合物,其包括所述微生物。
所述疾病可以引起胃肠道炎症。所述疾病可以选自炎症性肠病(Inflammatorybowel disease:IBD)和结肠炎(colitis)中的一种或多种。所述炎症性肠病可以为溃疡性结肠炎(ulcerative colitis)或克罗恩病(Crohn's disease)。
所述组合物可以包括药学上或食物上可接受的载体,赋形剂或稀释剂。所述药学上或食物上可接受的载体是指任何标准的药学上或食物上的载体,例如磷酸缓冲生理盐水(phosphate buffered saline solution)、葡萄糖的5%水溶液和乳状液(例如油/水或水/油乳状液)。赋形剂的非限制性实例包括佐剂、结合剂、填充剂、稀释剂、崩解剂、乳化剂、湿润剂、润滑剂、滑泽剂、甜味剂、香料和着色剂。优选的药学上或食物上载体取决于活性剂的预期施用方式。典型的施用方式包括肠内(例如,口服)施用。所述组合物可以是单位施用剂型(unit dosage form)。所述组合物可以具有口服施用剂型。所述组合物可以是食物或药物组合物。所述组合物可以包括所述微生物的干燥物。所述组合物可以包括所述微生物的的培养基。
如本说明书所用,“药学上可接受的或食物上可接受的(pharmaceuticallyacceptable or acceptable for food)”是指在施用于个体时基本上不引起副作用(adverse reactions)。所述副作用可以是毒性、过敏或免疫反应。
另一方面提供用于预防或治疗引起胃肠道损伤的疾病的试剂盒,其包括所述重组微生物和用于治疗胃肠道损伤疾病的其他药物。所述重组微生物如上所述。所述重组微生物可以是上述组合物的形式。所述试剂盒可以包括说明书,其指导以使用重组微生物和用于治疗引起胃肠道损伤的疾病的其他药物来预防或治疗引起胃肠道损伤的疾病。
只要是用于治疗引起胃肠道损伤的疾病的药物,所述治疗引起胃肠道损伤的疾病的其他药物可以包括任何药物,例如治疗炎症性肠病的药物。治疗引起胃肠道损伤的疾病的药物可以包括以下药物:
(ⅰ)甾体抗炎剂
地塞米松(dexamethasone)、己雌酚(hexestrol)、甲巯基咪唑(methimazole)、倍他米松(betamethasone)、曲安奈德(triamcinolone)、丙炎松(triamcinoloneacetonide)、醋酸氟轻松(fluocinonide)、氟轻松(fluocinolone acetonide)、泼尼松龙(predonisolone)、甲泼尼龙(methylpredonisolone)、乙酸可的松(cortisone acetate)、氢化可的松(hydrocortisone)、氟米龙(fluorometholone)、倍氯米松(beclomethasonedipropionate)、雌甾三醇(estriol)、醋酸帕拉米松(paramethasone acetate)、醋酸氟氢可的松(fludrocortisone acetate)、丙酸氯倍他索(clobetasol propionate)、醋酸双氟拉松(diflorasone acetate)、丙酸地塞米松(dexamethasone propionate)、二氟泼尼酯(difluprednate)、二丙酸倍他米松(betamethasone dipropionate)、布地奈德(budesonide)、戊酸二氟可龙(diflucortolone valerate)、安西奈德(amcinonide)、哈西奈德(halcinonide)、糠酸莫米松(mometasone furoate)、丁酸丙酸氢可的松(hydrocortisone butyrate propionate)、新戊酸氟米松(flumetasone pivalate)、丁酸氯倍他松(clobetasone butyrate)、醋酸地塞米松(dexametasone acetate)等。
(ⅱ)5-氨基水杨酸
柳氮磺胺吡啶(sulfasalazine)、美沙拉嗪(mesalazine)、奥沙拉秦(olsalazine)、巴沙拉嗪(balsalazide)等。
(ⅲ)免疫调节剂(immunomodulator)或免疫抑制剂(immunosuppressant)
甲氨蝶呤(methotrexate)、环磷酰胺(cyclophosphamide)、MX-68、盐酸阿替莫德(atiprimod dihydrochloride)、BMS-188667、CKD-461、利美索龙(rimexolone)、环孢菌素(cyclosporine)、他克莫司(tacrolimus)、胍立莫司(gusperimus)、硫唑嘌呤(azathiopurine)、抗淋巴细胞血清(antilymphocyte serum)、冷冻干燥磺化免疫球蛋白(freeze-dried sulfonated normal immunoglobulin)、红细胞生成素(erythropoietin)、集落刺激因子(colony stimulating factor)、白细胞介素(interleukin)、干扰素(interferon)等。
(ⅳ)JAK抑制剂
托法替尼(tofacitinib)、芦可替尼(ruxolitinib)等。
(ⅴ)TNF抑制剂
重组TNF-α-受体IgG-Fc融合蛋白(依那西普(etanercept))、英夫利昔单抗(infliximab)、阿达木单抗(adalimumab)、赛妥珠单抗(certolizumab pegol)、戈利木单抗(golimumab)、PASSTNF-α、可溶性TNF-α受体、TNF-α结合蛋白、抗TNF-α抗体、CDP571等。
(ⅵ)整合素抑制剂
那他珠单抗(natalizumab)、维多珠单抗(vedolizumab)、AJM300、TRK-170、E-6007等。
(ⅶ)白细胞介素-12/23抑制剂
尤特克单抗(ustekinumab)、布雷奴单抗(briakinumab)(抗白细胞介素-12/23抗体)等。
(ⅷ)非甾体抗炎剂(NSAID)
经典的NSAID
阿氯芬酸(alcofenac)、醋氯芬酸(aceclofenac)、舒林酸(sulindac)、托美汀(tolmetin)、依托度酸(etodolac)、非诺洛芬(fenoprofen)、噻洛芬酸(thiaprofenicacid)、甲氯芬那酸(meclofenamic acid)、美洛昔康(meloxicam)、替诺昔康(tenoxicam)、氯诺昔康(lornoxicam)、萘丁美酮(nabumeton)、乙酰氨基酚(acetaminophen)、非那西丁(phenacetin)、乙水杨胺(ethenzamide)、舒尔必宁(sulpyrine)、安替比林(antipyrine)、米格来宁(migrenin)、阿司匹林(aspirin)、甲芬那酸(mefenamic acid)、氟芬那酸(flufenamic acid)、双氯芬酸钠(diclofenac sodium)、酮保泰松(ketophenylbutazone)、洛索洛芬钠(loxoprofen sodium)、保泰松(phenylbutazone)、吲哚美辛(indomethacin)、布洛芬(ibuprofen)、酮洛芬(ketoprofen)、萘普生(naproxen)、奥沙普秦(oxaprozin)、氟比洛芬(flurbiprofen)、芬布芬(fenbufen)、普拉洛芬(pranoprofen)、夫洛非宁(floctafenine)、吡罗昔康(piroxicam)、(替诺昔康tenoxicam)、依匹唑(epirizole)、噻拉米特盐酸盐(tiaramide hydrochloride)、扎托布洛芬(zaltoprofen)、甲磺酸加贝酯(gabexate mesylate)、卡莫司他(camostat mesylate)、乌司他丁(ulinastatin)、秋水仙碱(colchicine)、丙磺舒(probenecid)、磺吡酮(sulfinpyrazone)、布可隆(bucolome)、苯溴马隆(benzbromarone)、别嘌醇(allopurinol)、金硫丁二钠(sodium aurothiomalate)、透明质酸钠(hyaluronate sodium)、水杨酸钠(sodium salicylate)、水杨酸(salicylicacid)、阿托品(atropine)、东莨菪碱(scopolamine)、左啡诺(levorphanol)、羟氢吗啡酮(oxymorphone)或其盐等。
(b)环氧合酶抑制剂(COX-1选择性抑制剂、COX-2选择性抑制剂等)
水杨酸衍生物(例如,塞来昔布(celecoxib)和阿司匹林(aspirin))、依托考昔(etoricoxib)、伐地考昔(valdecoxib)、双氯芬酸钠(diclofenac sodium)、吲哚美辛(indomethacin)、洛索洛芬(loxoprofen)等。
(c)释放一氧化氮的NSAID(nitric oxide-releasing NSAIDs)
另外,可以包括趋化因子抑制剂、聚类分化群(CD)抑制剂、白细胞介素、RIP激酶抑制剂、smad7抑制剂,MadCAM抑制剂等。
另一方面提供一种预防或治疗引起胃肠道损伤的疾病的方法,其包括向个体施用对预防或治疗引起胃肠道损伤的疾病有效的量的所述微生物。
所述方法还可包括向个体施用对预防或治疗引起胃肠道损伤的疾病有效的量的TNF-α阻断剂。所述向个体施用TNF-α阻断剂的步骤可以在所述向个体施用微生物的步骤的同时,之前或之后进行。所述TNF-α阻断剂可以以与施用所述微生物的途径相同或不同的途径施用。例如,所述TNF-α阻断剂可以经口服或肠胃外施用。所述肠胃外可以是诸如血管注射,腹腔或皮下注射的注射。例如,所述TNF-α阻断剂可以以腹腔内施用,并且所述微生物可以以口服施用。可以在口服施用所述微生物后,腹腔内施用所述TNF-α阻断剂。每次可以以5mg至100mg、5mg至50mg、5mg至40mg、10mg至50mg或20mg至50mg的量施用所述TNF-α阻断剂。
在所述方法中,所述疾病可以引起胃肠道炎症。所述疾病可以选自炎症性肠病(inflammatory intestinal disease)、自身免疫性疾病(autoimmune disease)、由辐射(radiation)引起的胃肠道损伤或移植物抗宿主病(graft versus host disease:GVHD)、炎性肠病(inflammatory bowel disease:IBD)以及包括慢性结肠炎的结肠炎(colitis)中的一种或多种。所述炎症性肠病可以为溃疡性结肠炎(ulcerative colitis)或克罗恩病(Crohn's disease)。
在本说明书中,所述“施用(administering或administration)”是指对生理系统(个体,或体内、体外或离体的细胞、组织和器官)给予所述微生物、包括其的组合物或治疗处理(therapeutic treatment)的行为。因此,在所述方法中,所述微生物可以是所述组合物的形式。对人体可接受的施用途径(route)可以是口或粘膜(例如肠粘膜,口粘膜或口腔)。所述施用可以与另外的治疗应用剂(therapeutic agent)组合施用。所述组合施用包括同时和连续中的任何顺序。
在本说明书中,术语“治疗(treatment)”是指预防性治疗(prophylactictreatment)或治疗性治疗(therapeutic treatment)。在特定实施例中,“治疗”是指出于治疗或预防目的向个体施用微生物或组合物。
“治疗性(therapeutic)”治疗是为了减轻或消除征兆(signs)或症状(symptoms)而对表现出病理征兆或症状的个体进行给予的治疗。征兆或症状可以是生化的、细胞的、组织学的、功能的或物理的、主观的或客观的。
“预防性(prophylactic)”治疗是为了降低发生病理学(pathology)的风险而对不显示疾病征兆或或仅显示疾病早期征兆的个体给予的治疗。在本说明书中所述的微生物或组合物可以作为预防性治疗而提供,以减少病理发生的可能性(likelihood)或在已经发生时最小化所述病理的严重程度。
在本说明书中,“治疗有效量(therapeutically effective amount)”是指足以实现所述目的的量。有效量可以通过试验确定。例如,可以根据所述微生物将VIP分泌到细胞外的程度来确定有效量。例如,对于体重为60公斤的人来说,可能是每天可以分泌0.01至300mg或0.5至100mg的VIP的所述微生物数量。例如,对于个体,每个个体在每次施用可以为1×107至1×1011cfu、1×107至1×1010cfu、1×108至1×1011cfu、1×108至1×1010cfu、2.5×108至7.5×109cfu、5.0×108至5.0×109cfu、7.5×108至2.5×109cfu或约1×109cfu。
在所述方法中,所述个体可以是哺乳动物。所述哺乳动物可以是人、马、猪、牛、狗、猫、猴子、黑猩猩、绵羊或山羊。所述个体可以为人。所述个体可以为非人类哺乳动物。
有益效果
根据一方面的重组微生物可以产生和分泌VIP。
根据另一方面的组合物或试剂盒可用于预防或治疗引起胃肠道损伤的疾病。
根据另一方面,根据预防或治疗引起胃肠道损伤的疾病的方法,可以有效地预防或治疗引起胃肠道损伤的疾病。
附图简单说明
图1为示出pMT447载体的构成的图。
图2为示出对用pMT447载体形质转换的植物乳杆菌(L.plantarum)LMT1-9、副干酪乳杆菌(L.paracasei)LMT1-21和短乳杆菌(L.brevis)LMT1-46菌株的培养上清液进行蛋白印迹法的结果的图。
图3为以疾病活性指数(DAI)评分示出源自植物乳杆菌LMT1-9形质转换菌株的VIP在由硫酸葡聚糖(DSS)诱导的小鼠肠炎症模型中的治疗效果的图。
图4为以DAI评分示出源自副干酪乳杆菌LMT1-21形质转换菌株的VIP在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。
图5为以DAI评分示出源自短乳杆菌LMT1-46形质转换菌株的VIP在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。
图6为以DAI AUC评分示出源自形质转换菌株的VIP在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。
图7为在组织病理学上示出源自形质转换菌株的VIP在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。
图8为以DAI评分示出源自LAB形质转换菌株的VIP和依那西普(Enbrel,韩国辉瑞制药)的组合施用在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。
图9为以DAI AUC评分示出源自LAB形质转换菌株的VIP和依那西普的组合施用在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。
图10为示出源自LAB形质转换菌株的VIP和依那西普的组合施用在由DSS诱导的小鼠肠炎症模型中的治疗效果的的组织病理学结果的图。
最佳模式
在下文中,将参考实施例更详细地描述本公开。然而,这些实施例仅用于说明本公开,并且本公开的范围不限于这些实施例。
实施例1:表达VIP基因的乳酸菌细胞及其对IBD的治疗效果
在本实施例中,确认了将VIP基因导入乳酸菌细胞中,并且所述基因被表达,产物被分泌到细胞外。此外,将由所述VIP基因形质转换的乳酸菌细胞对个体以口服施用来确定其对IBD治疗是否有效,并确认其症状改善。VIP用作具有氨基酸序列SEQ ID NO:1的人VIP变异体。所述VIP变异体是其中在具有SEQ ID NO:2的氨基酸序列的人天然VIP中的4个氨基酸残基被取代的,并且在人血流中具有增加的半衰期。
1.制备包括VIP基因的载体
由SEQ ID NO:14的核苷酸序列组成的VIP基因通过合成来自Macrogen公司(韩国)的DNA来使用。通过分别在SalI和XhoI限制酶的存在下,使合成的VIP基因片段和乳酸菌(Lactic Acid Bacteria:LAB)-大肠杆菌穿梭载体pMT54-PR4-SP4(SEQ ID NO:13)接触并对其进行切割。使用凝胶纯化试剂盒(gel purification kit)(GeneAll公司)在反应溶液中纯化切割的基因片段和载体后,切割的基因片段和载体在碱性磷酸酶存在下培养并去磷酸化,从而获得去磷酸化的切割的VIP基因片段和切割的pMT54-PR4-SP4载体。
在试管中混合1ul的制备的载体DNA、3ul的VIP基因DNA、0.5ul的T4 DNA连接酶(Takara)、1ul缓冲溶液和5.5ul蒸馏水以使总体积为10ul,然后在16℃下培养12小时来连接VIP基因DNA和载体片段,从而获得包括VIP基因的载体。通过使用Sambrook等的方法(Sambrook et al.,Molecular Cloning:A Laboratory Manual,第二版,1989),将该载体形质转换到大肠杆菌Top10感受态细胞(TOP10 Competent Cells)中。
将形质转换的大肠杆菌涂抹在含有包括10ug/ml的氯霉素的Luria-Bertani(LB)-琼脂的平板上,并进行培养。结果,选择并培养形成的菌落,并从该培养物中分离载体。该载体被命名为pMT447。在pMT447载体中,VIP基因可操作地连接至PR4启动子和SP4的信号肽序列。因此,用pMT447载体形质转换的细胞高效表达VIP基因并将分泌到细胞外。
图1为示出pMT447载体的构成的图。在图1中,启动子是SEQ ID NO:10的PR4启动子序列,SP表示编码SEQ ID NO:11的SP4分泌信号序列的核苷酸序列(SEQ ID NO:12),VIP表示编码SEQ ID NO:1的人VIP变异体的基因(SEQ ID NO:14)。HA和6xHis表示编码人流感病毒血凝素(human influenza hemagglutinin)标签和6个组氨酸的核苷酸序列,其用于检测、分离和纯化蛋白。大肠杆菌ori、Rep和CM分别代表大肠杆菌的复制起始点、乳酸菌的复制起始点(replication origin)和氯霉素抗性标记基因。
2.将重组载体导入乳酸菌细胞
将从1.中制备的重组载体pPM447载体分别导入至植物乳杆菌LMT1-9(KCTC13421BP)、副干酪乳杆菌LMT1-21(KCTC 13422BP)和短乳杆菌LMT1-46(KCTC 13423BP),并且确认是否分泌了VIP蛋白。
将所述菌株的每一个在含有50mL的MRS培养基(Difco Co.,美国)的烧瓶中培养直至OD600变为0.5,然后在4℃和7,000rpm下离心10分钟,并使用25mL的冷电穿孔溶液(ice-cold electroporation solution(EPS))(含有1mM K2HPO4、1mM KH2PO4、pH 7.4、1mM MgCl2和0.5M蔗糖)洗涤细胞团两次。MRS也称为De Man、Rogosa和Sharpe琼脂培养基。
洗涤后,将细胞重悬于1ml冷EPS中,制备用于电穿孔的感受态细胞,并将其保管在-80℃的深冷柜(deep freezer)中。将40μl的感受态细胞和1μl的每种载体DNA(1μg/μl)置于比色皿中,并在冰上放置5分钟。在25μF,8kV/cm和400ohms的条件下施加电脉冲后,立即将其添加到1ml的MRS液体培养基中,并在37℃下培养1小时。然后,将培养的细胞涂抹在含有10μg/ml氯霉素的MRS培养基(也称为“MRS-CM”)上,并在37℃下培养48小时。
将由此获得的导入有pMT447载体的每个菌株在MRS液体培养基中在37℃静止培养16小时。将培养物接种到MRS液体培养基中,然后在相同温度下静置培养8小时,直至其浓度为3(v/v)%。将1ml的培养物以7,000rpm离心5分钟,并取上清液。将100μl的三氯乙酸添加至1ml的上清液中,并在4℃下静置1小时以浓缩培养成分。将反应物在4℃和13,000rpm离心10分钟后,用1ml冷丙酮洗涤团粒,在室温下干燥10分钟,并用100μl的三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液(pH 8.8)洗脱。
向洗脱液中加入4x上样缓冲液(Thermo)和10x还原剂(Thermo),然后在十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE凝胶)上进行电泳。使用Trans-blot半干细胞(semi-dry cell)(伯乐(bio-rad))将所述凝胶转移到硝酸纤维素膜上,并进行蛋白质印迹法。具体地,用含有1%脱脂乳(skim milk)的TBST缓冲液封闭所述膜1小时,在室温下与抗HA抗体(Santa cruz)反应2小时,然后用TBST洗涤3次5分钟,并通过增强化学发光法(enhancedchemiluminescence:ECL)检测。在pMT447载体中,VIP基因在其3'末端侧与血凝素(hemagglutinin:HA)基因可操作地连接,并因此以HA标记的状态表达。
图2为示出对用pMT447载体形质转换的植物乳杆菌(L.plantarum)LMT1-9、副干酪乳杆菌(L.paracasei)LMT1-21和短乳杆菌(L.brevis)LMT1-46菌株的培养上清液进行蛋白印迹法的结果的图。在图2中,行2、3和4分别表示用pMT447载体形质转换的植物乳杆菌LMT1-9、副干酪乳杆菌LMT1-21和短乳杆菌LMT1-46,行1表示标准蛋白阶梯。
如图2所示,所述形质转换的菌株有效地分泌了hVIP突变体。
3.分泌VIP的乳酸菌在由DSS诱导的模型中对IBD的效果
将在2.中所述的形质转换的植物乳杆菌LMT1-9、副干酪乳杆菌LMT1-21和短乳杆菌LMT1-46以口服施用于由硫酸葡聚糖(Dextran Sulfate Sodium Salt:DSS)诱导的小鼠结肠炎(colitis)模型后,通过确认小鼠的生存率和疾病活性指数(disease activityindex:DAI)来评估所述菌株的治疗肠道炎症的效果。
(1)重组微生物的单独施用
具体地,将所述形质转换的菌株在含有包括10ug/ml的氯霉素的10ml MRS培养基的烧瓶中在37℃下24小时进行第一次静止培养。第二天,将培养的菌株接种在300mL至600ml的MRS CM培养基中,以使OD600为0.01,并在与第一次培养相同的条件下进行第二次培养。约16至18小时后,当培养物的OD600达到2至3时,将培养物以7,000rpm离心10分钟以去除上清液,并将细胞悬浮于1xPBS中,以便可以每只每一次施用1×109cfu。将含有该细胞的PBS溶液以口服施用于个体(每只每一次1×109cfu),并且与饮水施用并行每天一次,共进行16天。
首先,将8至10周龄的雄性Balb/c小鼠(20至25g,每组10只小鼠)(ORIENTBIO公司)用作实验动物。将最初菌株施用日设定为第7天,并将DSS在无菌水中稀释至2(v/v)%,并在0至6天之内将其作为饮水提供给除PBS组以外的所有组。小鼠自愿摄取饮水,但通常每天摄取少于6mL。在第9天处死小鼠。
施用DSS后,每隔2天检查小鼠的生存率,并通过检查体重,毛发的倒竖,动物的活动程度和腹泻的存在来计算DAI评分。
DAI(Disease activity index:DAI)评分(scoring)是参考了Ameho,Gut 1997;41:487~493和Wallace,Gastroenterology 1989;96:29~36。结果示于图3至6中。
图3为以DAI评分示出源自植物乳杆菌LMT1-9形质转换菌株的VIP在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。在图3中,1-9VIP代表在植物乳杆菌LMT1-9中用pMT447形质转换的菌株。1-9载体代表在植物乳杆菌LMT1-9中用对照载体(没有VIP基因的母载体)形质转换的菌株。
图4为以DAI评分示出源自副干酪乳杆菌LMT1-21形质转换菌株的VIP在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。在图4中,1-21VIP代表在副干酪乳杆菌LMT1-21中用pMT447形质转换的菌株。1-21载体代表在副干酪乳杆菌LMT1-21中用对照载体(没有VIP基因的母载体)形质转换的菌株。
图5为以DAI评分示出源自短乳杆菌LMT1-46形质转换菌株的VIP在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。在图5中,1-46VIP代表在短乳杆菌LMT1-46中用pMT447形质转换的菌株。1-46载体代表在短乳杆菌LMT1-46中用对照载体(没有VIP基因的母载体)形质转换的菌株。
在图3至图5中,CyA是环孢菌素A(cyclosporinA)的缩写,并用作试验的阳性对照。PBS是仅用DSS处理而不处理菌的对照组。正常代表未处理任何菌和DSS的组。
图6为以DAI AUC评分示出源自形质转换菌株的VIP在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。上面提到了每种菌株和化学物质的描述,DAI AUC表示直到第8天DAI评分曲线下的面积(area under the curve)。
如图3至图6所示,表达VIP的形质转换的乳杆菌菌株在由DSS诱导的肠炎模型中具有治疗作用。
图7为在组织病理学上示出源自形质转换菌株的VIP在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。在图7中,正常,PBS,LMT1-9载体、MT1-9 VIP,LMT1-46载体,LMT1-46VIP,LMT1-21载体和LMT1-21 VIP如在图1至6中所述。
如图7所示,在由DSS诱导的肠炎小鼠模型中,通过显微镜对结肠进行了组织病理学检查,可以确认,与对照组相比,施用了表达VIP的乳杆菌菌株的小鼠具有治疗效果。
具体而言,如图7所示,在由DSS诱发的肠炎小鼠模型中,通过显微镜对结肠进行了组织病理学检查,在DSS组中,单核细胞浸润和粘膜损伤最严重,而在LMT 1-9载体,LMT 1-21载体和LMT 1-46载体施用组中未能看出对病症的明显的改善效果。另一方面,在LMT 1-9VIP,1-21VIP和LMT1-46 VIP施用组中,组织损伤和炎性浸润的损伤显示出减少的趋势。这可以看出表达VIP的乳杆菌菌株在由DSS诱导的炎症性肠病小鼠模型中具有缓和炎症和保护功效。
(2)重组微生物与TNF-α阻断剂的组合施用效果
图8为以DAI评分示出源自LAB形质转换菌株的VIP和依那西普(Enbrel,韩国辉瑞制药)的组合施用在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。在图8中,正常是未进行任何处理的组。PBS是仅处理DSS的组。依那西普是处理DSS和依那西普的组。1-46VIP是向由DSS诱导的小鼠提供在短乳杆菌LMT1-46中用pMT447形质转换的菌株的组。1-46VIP+依那西普是在由DSS诱导的小鼠中一起处理在短乳杆菌LMT1-46中用pMT447形质转换的菌株和依那西普的组。
图8是通过以下过程而获得的结果。为了评估源自形质转换的菌株的VIP蛋白的治疗效能,将形质转换的菌株以口服施用于由DSS诱导的小鼠肠炎模型,然后确认生存率或DAI评分。首先将形质转换的菌株在10ml的含10ug/ml氯霉素的MRS培养基中进行第一次培养一天,然后将培养的菌株接种在300至600ml的MRS CM培养基中,以使OD600为0.01。在16至18小时后,当OD600达到2至3时,根据施用的小鼠总数和进食次数回收菌株。通过以7,000rpm离心10分钟以除去培养上清液,并将回收的菌株悬浮于1xPBS中,使得每只每一次可以施用1×109cfu。每天进行一次菌株施用,进行共16天。
将最初菌株施用日设定为第7天以开始治疗效能试验,并将DSS在无菌水中稀释至2(v/v)%,并在0至6天之内将其作为饮水提供给除PBS组以外的所有组。在第9天,处死小鼠。DSS处理后,每隔2天检查生存率,并通过检查体重,毛发的倒竖,动物的活动程度和腹泻的存在来计算DAI分数。另外,为评估与依那西普(肿瘤坏死因子阻断剂(TNF blocker;依那西普)组合施用的效能,将依那西普以10mg/kg在0、2、4和6天以腹膜内(IP)施用。
图9为以DAI AUC评分示出源自LAB形质转换菌株的VIP和依那西普的组合施用在由DSS诱导的小鼠肠炎症模型中的治疗效果的图。上面描述了每种菌株和化学物质,DAIAUC代表了直到第8天DAI评分曲线下的面积。
如图8和9所示,当将表达VIP的菌株和依那西普的组合施用时,与仅处理菌株或依那西普的组相比,DAI评分降低。
在本试验中使用依那西普是用于治疗炎性肠病的示例性药物,并且可以将其他用于治疗炎性肠病的药物与源自LAB形质转换菌株的VIP组合使用。
(3)组织病理学(histopathology)试验方法
在试验的第9天处死试验动物后,对距离2cm的远端结肠的两部分分别取出0.5cm,固定在10%福尔马林溶液中后,以石蜡包埋。用切片机(microtome)以5μm的间隔切割包埋的组织以制备载玻片,然后进行苏木精-伊红(H&E)染色。对于染色的组织,通过参考Erben等,Int J Clin Exp Pathol 2014;7(8):4557-4576的评估方法来评估组织的严重性,并且对于每个样本评估任何3或4个或更多部位。
简要描述评估方法,分别评估肠上皮细胞损伤和炎性细胞侵润程度,将各部分的得分相加,结果显示为0至8分。
首先,根据以下标准评估炎症细胞的浸润程度:
0=正常(normal),
1=轻度-围绕隐窝基础渗透(mild-infiltrate around crypt basis),
2=中等度-浸润到粘膜肌板(moderate-infiltrate reaching to L.muscularismucosae),
3=明显-大范围浸润到粘膜肌板和所述粘膜增厚(marked-extensiveinfiltration reaching the muscularis mucosae and thickening of the mucosa),
4=严重-浸润到浸润到下层(severe-infiltration of the L.submucosa)。
此外,根据以下标准评估对上皮细胞的损伤程度:
0=正常,1=焦点脱落-杯状细胞损失(focal erosions-loss of gobletcells),
2=脱落-大面积杯状细胞的损失,
3=脱落/溃疡-隐窝(焦点)的损失(erosion/ulceration-loss of crypts(focal))
4=扩展的溃疡-肉芽组织、肉芽组织隐窝的损失(extended ulceration-granulation tissue,loss of crypts in large areas)。
图10为示出源自LAB形质转换菌株的VIP和依那西普的组合施用在由DSS诱导的小鼠肠炎症模型中的治疗效果的的组织病理学结果的图。在图10中,正常、PBS、依那西普、LMT1-46 VIP和LMT1-46 VIP+依那西普如图9中所述。
如图10所示,当将表达VIP的菌株和依那西普的组合施用时,与仅处理菌株或依那西普的组相比,治疗效能得到改善。
具体而言,如图10所示,在由DSS诱发的肠炎小鼠模型中,通过显微镜对结肠进行了组织病理学检查,在DSS组中,单核细胞浸润和粘膜损伤最严重,而在单独施用依那西普的组中未能看出对病症的明显的改善效果。另一方面,与用作阳性对照组的CyA施用组一样,在LMT1-46 VIP单独施用和LMT 1-46 VIP&依那西普组合施用组中,表现出减少组织学损伤和炎性浸润病变的趋势。这可以看出表达VIP的乳杆菌菌株在由DSS诱导的炎症性肠病小鼠模型中具有缓和炎症和保护功效。
序列表
<110> 玫帝托克斯股份有限公司
<120> 表达异性蛋白的微生物及其用途
<130> PX060551OV
<150> KR1020180173063
<151> 2018-12-28
<160> 14
<170> PatentIn version 3.5
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His Ser Asp Ala Val Phe Thr Ala Asn Tyr Thr Arg Leu Arg Arg Gln
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tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 660
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 720
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 780
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 840
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 900
tttaaatcaa tctaaagtat atatgagtaa acttggtgaa ttcgcaaggt atagcccaat 960
atactttact tcgtaaagtt tgagctctgc gaggcaaaaa cgtcagatat tgaatctaaa 1020
caagagtata ataaacacag ttacacaaat aaaaacaaaa ggagtagatc ttaaaaatgg 1080
aagcaaatcc aaagtggcaa gaaacaggaa gacaacaaaa agtaaaacaa cttgactatt 1140
cagaaacatt agcagtaaga atagacaatg tggaattata caactcagaa aagaaattat 1200
attatctagt aattcaaacc cttcttggga atccccaaaa aaatgaatgg cgtatccagg 1260
gacaaagtca aatattaaaa gaatcaaaac aaaaaataat tgtgtcataa aattattgca 1320
cattaaaagt cctaaaaatt attttgcatt aaagatttca aaactcattt aatgtaaaat 1380
aattttttct ttgataatcc catttagcaa caatctcgcg taccacttgg tcaacctttt 1440
gatcatcact atccgtatta atcaaatcac cattctcaac gtcttctaat tgcaactgct 1500
tgcgaatttc tttcagcaaa ccgccatagc taatttgccg ggaaccagcc aaagctcgtt 1560
ccaaatcatc aattacttgt aggtcttgtt cttgattatt agtcaaaata tctttagatt 1620
ttacctgata tttagccgtt tcttgagcac tagccaataa cgaattttta tggcgattca 1680
tattcggttt aactgcctca acattcacca ttggtacata agtcaatttc attgcccgtt 1740
gccaaaaacc agtccattct acttgtgaaa tatagttatc agtcccctta aaataatggt 1800
tcttcacaaa aagcaaaaca tgcatatgat ggtggtacat tggctgaccc gcttcatgat 1860
taacagtcac ttcagtcgaa cgtacatagc ccaataaatt tttggcaact tttgtatact 1920
gaaaaatttt tgcaacagct cgtcccattt gacgtaactc actcttcaat tgatcaccag 1980
ttgtattctc aaccgtcaac gttaaaaata agaaccggcc cgtctttcgt tgtttaacag 2040
cttccgtcaa aatctgtgtt aactgattgg attgcttcat tgcccgccgc caattacata 2100
acggacacaa acgagaatgg caaaaccaag tctgcgccaa tttcttgtga ccatttttat 2160
cttccacaaa acgcaaaact tcgccacatt ctttaactcg atgagctttc ttataatgca 2220
atatttgtaa atagtcacca tactgcaagt tctccaactt gcgctcccgc cacggccgaa 2280
ctttccctga ctttgaccga tcaaccaaga ctttttcatt agccaaaata aaaactcccc 2340
tcaccaacca cgtgagaaga gttaatctgt tatgtacttg cttcacttaa atcagtcaga 2400
aggcttgacg gcaaagggtt caagctttaa actatgtcta gtaaatccaa atacgatttt 2460
tacaagatta actcacgcgt tcgaggtcgg caaactttcg aagctcacgt gggttttttt 2520
tatatttatt ttataccaca ataatacgcc taaacccagt tgtgtcaagg gtttacctca 2580
ctttttgaaa atgacgttgt ttctaatagt atcaagataa gaagaaaccg tcgaaaaaac 2640
gacggtttca aaccccaaaa agcagagaat tcggtacctg gagctgtaat ataaaaacct 2700
tcttcaacta acggggcagg ttagtgacat tagaaaaccg actgtaaaaa gtacagtcgg 2760
cattatctca tattataaaa gccagtcatt aggcctatct gacaattcct gaatagagtt 2820
cataaacaat cctgcatgat aaccatcaca aacagaatga tgtacctgta aagatagcgg 2880
taaatatatt gaattacctt tattaatgaa ttttcctgct gtaataatgg gtagaaggta 2940
attactatta ttattgatat ttaagttaaa cccagtaaat gaagtccatg gaataataga 3000
aagagaaaaa gcattttcag gtataggtgt tttgggaaac aatttccccg aaccattata 3060
tttctctaca tcagaaaggt ataaatcata aaactctttg aagtcattct ttacaggagt 3120
ccaaatacca gagaatgttt tagatacacc atcaaaaatt gtataaagtg gctctaactt 3180
atcccaataa cctaactctc cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt 3240
tatcaccctt gtcactaaga aaataaatgc agggtaaaat ttatatcctt cttgttttat 3300
gtttcggtat aaaacactaa tatcaatttc tgtggttata ctaaaagtcg tttgttggtt 3360
caaataatga ttaaatatct cttttctctt ccaattgtct aaatcaattt tattaaagtt 3420
catttgatat gcctcctaaa tttttatcta aagtgaattt aggaggctta cttgtctgct 3480
ttcttcatta gaatcaatcc ttttttaaaa gtcaatatta ctgtaacata aatatatatt 3540
ttaaaaatat cccactttat ccaattttcg tttgttgaac taatgggtgc tttagttgaa 3600
gaataaagac cacattaaaa aatgtggtct aagcttctgc aggatatccg atcgtccaca 3660
atcaaggtgc ttggcttttt cgatcgcgag gtcaccatgt acatcagtcg tgagagcatt 3720
gtgttgacag tgatcggcat cgtgttcggc tatctgctcg gcaatttgct gacagcctac 3780
attttgtatc aagccgaaac tgaggccgtg gtttttccac tcacgatcag cattgtcggc 3840
tacctcacgg ccacgttact catgttggcc ttcaccggcg tcgtcacctg gctcacgcat 3900
cgtcgactcc aacgggtgga catggtcgaa gccctgaaat caaacgaata acctacaatt 3960
ttgtcaggca gcgtcgtcac ggcgctgctt ttttcataca aaattcatca aaaattggga 4020
ttaaaaacgt tcatgatcgc aattttgaag cgcaaatgaa gattgagacc aactcctaac 4080
agtcctgtaa cgctgacgta acattgacac agtaaagtag cctttagtta atcaaattaa 4140
gggtgaggtc aaaaatgaaa ttcaataaag tcatgatcac gttggttgct gcagttacct 4200
tagcaggttc tgctagcgcc gtaacaccag ttttcgctga tacaagcgtc gacctcgag 4259
<210> 14
<211> 84
<212> DNA
<213> 人工序列
<220>
<223> VIP变体1
<400> 14
cattcagatg ctgttttcac cgataactat acccgtttgc gtcgtcaatt ggctgttcgt 60
cgttatttga actcaatctt gaac 84
Claims (22)
1.一种重组微生物,其包括启动子和编码与所述启动子可操作地连接的血管活性肠肽(VIP)的外源基因,其中,所述重组微生物属于乳杆菌(Lactobacillus)属。
2.根据权利要求1所述的微生物,其为副干酪乳杆菌(Lactobacillus paracasei)、短乳杆菌(Lactobacillus brevis)或植物乳杆菌(Lactobacillus plantarum)。
3.一种重组微生物,其包括组成型启动子(constitutive promoter)和编码与所述启动子可操作地连接的血管活性肠肽(VIP)的外源基因,其中,所述重组微生物为乳酸菌。
4.根据权利要求3所述的重组微生物,其中,所述组成型启动子为SEQ ID NO:10的启动子。
5.根据权利要求3所述的微生物,其中,所述乳酸菌为乳杆菌(Lactobacillus)属或乳球菌(Lactococcus)属。
6.根据权利要求5所述的微生物,其中,所述乳酸菌为副干酪乳杆菌(Lactobacillusparacasei)、短乳杆菌(Lactobacillus brevis)、植物乳杆菌(Lactobacillus plantarum)或乳酸乳球菌(Lactococcus lactis)。
7.根据权利要求1至6中任一项所述的微生物,其进一步包括在所述启动子和所述外源基因之间可操作地连接的信号序列。
8.根据权利要求7所述的微生物,其中,所述信号序列编码具有氨基酸序列SEQ ID NO:11的信号肽。
9.一种用于预防或治疗在人类中引起胃肠道(gastrointestinal tract)损伤的疾病的组合物,其包括重组微生物,其包括启动子和编码与所述启动子可操作地连接的血管活性肠肽(VIP)的外源基因,其中,所述重组微生物为乳酸菌。
10.根据权利要求9所述的组合物,其中,所述疾病引起胃肠道炎症。
11.根据权利要求10所述的组合物,其中,所述疾病选自炎症性肠病(IBD)和结肠炎(colitis)中的一种或多种。
12.根据权利要求11所述的组合物,其中,所述炎症性肠病为溃疡性结肠炎(ulcerative colitis)或克罗恩病(Crohn's disease)。
13.根据权利要求9所述的组合物,其为口服施用剂型。
14.根据权利要求9所述的组合物,其中,所述乳酸菌为乳杆菌(Lactobacillus)属或乳球菌(Lactococcus)属。
15.根据权利要求14所述的组合物,其中,所述乳酸菌为副干酪乳杆菌(Lactobacillusparacasei)、短乳杆菌(Lactobacillus brevis)、植物乳杆菌(Lactobacillus plantarum)或乳酸乳球菌(Lactococcus lactis)。
16.根据权利要求9至15中任一项所述的组合物,其中,所述启动子为组成型启动子。
17.根据权利要求16所述的组合物,所述组成型启动子由SEQ ID NO:10的核苷酸序列组成。
18.根据权利要求9至15中任一项所述的组合物,其进一步包括在所述启动子和外源基因之间可操作地连接的信号序列。
19.根据权利要求18所述的组合物,其中,所述信号序列编码具有氨基酸序列SEQ IDNO:11的信号肽。
20.根据权利要求9至15中任一项所述的组合物,其进一步包括治疗引起胃肠道损伤的疾病的其他药物。
21.根据权利要求20所述的组合物,其中,所述治疗药物选自甾体抗炎剂、5-氨基水杨酸、免疫调节剂、JAK抑制剂、TNF抑制剂、整合素抑制剂、白细胞介素抑制剂、趋化因子抑制剂、聚类分化群(CD)抑制剂、白细胞介素、RIP激酶抑制剂、smad7抑制剂、MadCAM抑制剂和非甾体抗炎剂(NSAID)。
22.根据权利要求21所述的组合物,其中,所述非甾体抗炎剂为经典的NSAID、环氧合酶抑制剂或释放一氧化氮的NSAID。
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PCT/KR2019/018596 WO2020139014A2 (ko) | 2018-12-28 | 2019-12-27 | 외래 단백질을 발현하는 미생물, 및 그의 용도 |
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