CN112481181A - 具有肿瘤抑制效应的产sting激动剂的工程益生菌 - Google Patents
具有肿瘤抑制效应的产sting激动剂的工程益生菌 Download PDFInfo
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Abstract
本发明公开了一种产STING激动剂的工程益生菌及其制备方法,其基因型为Nissle 1917(ΔdapA ΔthyA exo/cea:tetR‑Ptet‑dacA‑rrnB‑T1T2)。本发明的工程益生菌能够用于制备免疫应答相关疾病和STING活性相关疾病的治疗药物。
Description
技术领域
本发明属于基因工程领域和医药领域,具体地说,涉及一种具有肿瘤抑制效应的产STING激动剂的工程益生菌、其制备方法及其在制备免疫应答相关疾病和/或STING活性相关疾病治疗药物中的应用。
背景技术
干扰素基因刺激因子(stimulator of interferon gene,STING或小写sting)是一种胞内模式识别受体,激活cGAS-STING信号通路能诱导I型干扰素(interferon,IFN)等细胞因子产生,从而调节适应性免疫。STING也称作MITA、TMEM73、ERIS、NET23、MPYS等,是目前药物研发的一个非常热门的靶点。STING激动剂作为癌症、肥胖、病毒感染、肝损伤、糖脂代谢紊乱等诸多疾病的热门新兴治疗药物研究方向,备受业界关注,特别是在肿瘤的治疗上,STING激动剂作为免疫疗法成为了十分活跃的研究领域。
STING可通过cGAS-STING通路促进抗肿瘤作用。研究显示,对抗肿瘤来说,STING主要参与免疫过程,尤其是在T细胞介导的肿瘤免疫过程中起枢纽作用。研究人员已经在结肠癌、黑素瘤、缺乏端粒酶等多种癌症相关疾病中检测到cGAS-STING途径可有效抑制癌细胞转移。近期的研究发现,NK细胞依赖性肿瘤排斥反应在很大程度上也依赖于非肿瘤细胞STING的激活。相关机制研究均给了靶向STING进行抗肿瘤药物开发巨大的前景。目前,STING激动剂类抗肿瘤药物开发取得了较大的进展,多个药物已经进入临床研究。包括ImmuneSensor Therapeutics公司旗下的IMSA-101,葛兰素史克旗下的GSK3745417,百时美施贵宝公司旗下的BMS-986301,Spring Bank Pharmaceuticals公司旗下的SB-11285,默沙东公司旗下的MK-1454。在适应症方面,目前主要是针对实体瘤,但也已经开始向非实体瘤开发,例如MK-1454就针对淋巴瘤在进行开发。
cGAS(环鸟腺苷酸合成酶)是胞浆DNA识别受体,当cGAS识别胞质中积累的DNA时,cGAS与这些DNA结合,cGAS活性位点构象发生改变,催化ATP和GTP合成环二核苷酸2’,3’-cGAMP(cyclic guanosine monophos-phate-adenosine monophosphate,cGAMP)。cGAMP作为第二信使,与内质网(endoplasmic reticulum,ER)膜上的STING蛋白结合,STING迅速发生二聚化被激活,活化的STING从ER转移到高尔基体,在高尔基体中招募TANK结合激酶1(TANK-binding kinase 1,TBK1)和IκB激酶((IκB kinases,IKK)等激酶,这些激酶分别磷酸化IRF3和核因子-κB(nuclear factor kappa-B,NF-κB)抑制剂IκBα。磷酸化的IRF3会发生二聚化并转移到细胞核中,激活编码I型IFN的基因转录。IκBα磷酸化导致NF-κB转移到细胞核,并在其中激活编码促炎细胞因子白细胞介素-6(interleukin 6,IL-6)、肿瘤坏死因子(tumor necrosis factor,TNF)和I型IFN基因转录,发挥免疫调节作用,影响机体病毒防御、炎症和肿瘤治疗。
能激活cGAS-STING通路的药物被认为有可能诱导下游I型IFN等细胞因子产生,在抗肿瘤免疫治疗中,驱动肿瘤内抗原呈递细胞(APC)激活,进一步激活效应T细胞。天然的STING激动剂中,2’,3’-cGAMP来源真核细胞,而原核生物则产生c-di-AMP(简写CDA)、c-di-GMP和3’,3’-cGAMP。这些天然环二核苷酸的分子量大,净电荷和极性分布强烈限制了其膜通道和细胞吸收,且磷酸二酯键易于酶解。磷酸二酯基团的修饰后的候选药物ADU-S100目前正处于II期临床试验。最近有研究发现,小分子STING激动剂的缺陷是它们可能导致激活“脱靶”,例如它还同时激活了效应T细胞中的STING通路,从而导致细胞凋亡,最终阻碍免疫记忆的形成。将STING激动剂靶向肿瘤内APC的不同方法可以减少这些非特异性的全身性脱靶效应。细菌能被APC主动吞噬,并具有通过刺激模式识别受体(PRR)(例如TLR)来触发互补免疫途径的额外好处。由于细菌的c-di-AMP还参与细胞凋亡等生理活动,因此利用工程细菌产c-di-AMP需要精确调至合适的剂量。
环二腺苷酸(c-di-AMP,CDA)是细菌中广泛存在的第二信号分子。c-di-AMP在细菌中的代谢受二腺苷酸环化酶A(DacA)和磷酸二酯酶(PDE)的精密调控。c-di-AMP不仅调节细菌生长、细胞壁稳态、离子转运多种生理过程,而且能够被真核宿主胞内多种感应子/受体蛋白识别,从而调控抗感染免疫。细菌c-di-AMP参与调控宿主I型干扰素应答、NF-κB信号通路活性、自噬以及炎症小体应答等固有免疫应答。此外,c-di-AMP作为黏膜佐剂可诱导宿主适应性免疫。c-di-AMP被认为是一种新发现的病原体相关的分子模式(PAMP),已成为细菌疫苗和药物研究中的新靶点。
发明内容
益生菌是一大类药物,主要通过口服活菌制剂达到治疗疾病和康复保健效果。益生菌药物制剂的优点在于给药方便,口感一般较好,病人乐于接受,顺从性高,更重要的是能够持续地在肠道内增殖从而稳定地发挥治疗作用。
大肠杆菌属Nissle 1917(EcN)是一种非致病性大肠杆菌,是1917年从一次志贺菌痢大爆发时未出现腹泻的士兵的粪便中分离得到的。EcN是非乳酸益生菌中研究最多的益生菌,血清型为O6:K5:H1,具有独特的基因组、半粗糙O6-脂多糖(LPS)表型、K5型荚膜、3种不同的菌毛(F1A、F1C和卷曲菌毛)。EcN具有小菌素和铁摄取系统等特殊适应性因子,在与其他微生物竞争中起关键作用。EcN能长期稳定定植于肠道,与肠上皮细胞相互作用,被广泛用于预防传染性腹泻、炎性肠疾病如溃疡性结肠炎和克罗恩病,防止新生儿消化道内病原菌定殖等,和发挥免疫调节生物学功能。
为了寻求新的抗肿瘤药物,发明人利用基因工程技术来改造大肠杆菌Nissle1917(EcN),使其能够生产STING激动剂CDA,构建出了一株经动物实验证明具有肿瘤抑制效应的产STING激动剂的工程益生菌。具体而言,本发明包括如下技术方案:
一种产STING激动剂的工程益生菌,其是大肠杆菌Nissle 1917的衍生菌,基因型为Nissle 1917(ΔdapAΔthyA exo/cea::tetR-Ptet-dacA-rrnB-T1T2)。该基因型也可表述为EcNΔdapAΔthyA exo/cea::tetR-Ptet-dacA-rrnB-T1T2。为简要起见,本文中将该工程益生菌简写为EcN-STING。
上述STING激动剂是环二腺苷酸(c-di-AMP,CDA)。
本发明的第二个方面提供了一种构建上述工程益生菌的方法,其特征在于,包括以下步骤:
A.以大肠杆菌Nissle 1917为底盘菌,敲除dapA基因(二氢吡啶二羧酸合成酶基因)和thyA基因(胸腺嘧啶核苷合成酶(Thymidylate synthase,胸苷酸合成酶)基因),获得Nissle 1917ΔdapAΔthyA菌株;为简要起见,本文中将该菌株简写为EcNΔDT。
B.对于步骤A中获得的EcNΔDT菌株,在其基因组的exo/cea位点插入四环素诱导的启动子抑制蛋白tetR和启动子Ptet转录的dacA(二腺苷酸环化酶A)基因,得到菌株EcN-STING。
在一种优选的实施方式中,上述步骤A中dapA基因和thyA基因的敲除通过基因编辑技术实施,所述基因编辑采用CRISPR-Cas9系统、CRISPR-Cpf1系统、CRISPR-Cas相关的转座系统INTEGRATE系统或者CAST系统。
上述INTEGRATE系统是指Sam Sternberg研究组开发的基因编辑工具(Insertionof transposable elements by guide RNA-assisted targeting,引导RNA辅助靶向的转座元件插入);CAST系统是指张锋研究组开发的基因编辑工具(CRISPR-associatedtransposase,CRISPR相关转座酶)。
上述步骤A例如可以包括下述步骤:
A-1.构建质粒pTargetF-dapA;
A-2.设计敲除dapA基因的供体DNA片段、引物dapA-F(5’-gacttttgaacagagtaagccatcaaatctccctaaactgggccatcctctgtgcaaac-3’,SEQ ID NO:1)和dapA-R(5’-tgcttttaatgccataccaaacgtaccattgagacacttgtttgcacagaggatggccc-3’,SEQ ID NO:2),两条引物使用PCR退火配对获得片段ΔdapA;
A-3.将pCas质粒转入Nissle 1917菌株,然后转入构建的pTargetF-dapA质粒和片段ΔdapA,通过PCR验证和测序确定已将dapA基因敲除,获得Nissle 1917ΔdapA菌株;
A-4.构建质粒pTargetF-thyA;
A-5.设计敲除thyA基因的供体DNA片段、引物thyA-F(5’-tatcgtcgcagcccacagcaacacgtttcctgaggaacctggaccggtggcgacacgca-3’,SEQ ID NO:3)和thyA-R(5’-aattgcagatgagtttgatccatatggttgctgtagagatgcgtgtcgccaccggtcca-3’,SEQ ID NO:4),两条引物使用PCR退火配对,获得片段ΔthyA;
A-6.将pCas质粒转入步骤A-3中获得的Nissle 1917ΔdapA菌株,然后再转入pTargetF-thyA质粒和片段ΔthyA,通过PCR验证和测序确定已将thyA基因敲除,获得Nissle1917ΔdapAΔthyA菌株(简写为EcNΔDT)。
上述步骤B可以包括下述步骤:
B-1.构建质粒pTargetF-exo/cea;
B-2.将步骤B-1中得到的pTargetF-exo/cea质粒用EcoRI/HindIII酶切得到片段A;PCR扩增获得exo/cea上游同源臂、tetR-Ptet片段、dacA片段、rrnB终止子片段和exo/cea下游同源臂,将上述5个片段和所述片段A用DNA assembly方法连接,获得质粒pTarget-exo/cea-tetR-Ptet-dacA-rrnB-T1T2;
B-3.将pCas质粒转入步骤A中获得的Nissle 1917ΔdapAΔthyA菌株,然后转入步骤B-2中得到的pTarget-exo/cea-tetR-Ptet-dacA-rrnB-T1T2质粒,通过PCR验证和测序确定在exo/cea位点整合入tetR-Ptet-dacA-rrnB-T1T2片段,获得目的菌株Nissle 1917ΔdapAΔthyA exo/cea::tetR-Ptet-dacA-rrnB-T1T2(简写为EcN-STING)。
在一种实施方式中,上述质粒pTargetF-dapA的构建可以包括下述步骤:引物N20-dapA-F(5’-TAGTAGATCGCAGCCAGTACGGGA-3’,SEQ ID NO:5)与N20-dapA-R(5’-AAACTCCCGTACTGGCTGCGATCT-3’,SEQ ID NO:6)使用PCR退火配对,pEcgRNA质粒(SEQ IDNO:14)用BsaI酶切,然后和前述退火配对所得的双链用T4DNA连接酶连接,获得质粒pTargetF-dapA。
上述pEcgRNA的构建包括如下步骤:
(1)以pTargetF为模板,正向引物ccdB-pTargetF-F(5’-CTGTTATCTGGCTTTTAGTAAGCCGGATCCCTGGGTCTCGGTTTTAGAGCTAGAAATAG-3’)和反向引物ccdB-pTargeF-R(GAAGAACATTTGGAAGGCTGTCGGTCGACCTGGGTCTCAACTAGTATTATACCTAG GAC-3’)进行PCR扩增,获得片段1;
(2)以基因合成的ccdB基因为模板,正向引物pTargetF-ccdB-F(5’-AGCTCAGTCCTAGGTATAATACTAGTTGAGACCCAGGTCGACCGACAGCCTTCCAAAT G-3’)和反向引物pTargeF-ccdB-R(5’-TAACTTGCTATTTCTAGCTCTAAAACCGAGACCCAGGGATCCGGCTTACTAAAAGCCA G-3’)PCR扩增ccdB基因,获得片段2。
片段1和片段2用DNA assembly连接(DNA assembly Kit购自全式金),转化E.coliDB3.1,获得pEcgRNA质粒(图1)。
在一种实施方式中,上述质粒pTargetF-exo/cea的构建可以包括下述步骤:引物N20-exo/cea-F(5’-TAGTTATTGATATATTTACGTC-3’,SEQ ID NO:7)与N20-exo/cea-R(5’-AAACGACGTAAATATATCAATA-3’,SEQ ID NO:8)使用PCR退火配对;将pEcgRNA质粒用BsaI酶切,然后和前述退火配对所得的双链用T4DNA连接酶连接,获得质粒pTargetF-exo/cea。
本发明的第三个方面提供了上述工程益生菌在制备免疫应答相关疾病和/或STING活性相关疾病治疗药物中的应用、尤其是在制备抗肿瘤药物中的应用。该药物中,所述工程益生菌可激活人和动物比如鼠的先天免疫细胞活性,包括干扰素通路和NF-kB通路。
优选地,所述免疫应答相关疾病和/或STING活性相关疾病是癌症或肿瘤。
上述癌症选自下组:结肠癌、乳腺癌、肺癌、黑素瘤、肝癌、胃癌、宫颈癌、卵巢癌、纤维肉瘤和鳞状细胞癌、脑癌、脑癌、脊椎癌、头颈部癌、白血病和血癌、皮肤癌、生殖系统癌、胃肠系统癌、肝和胆管癌、肾癌和膀胱癌、骨癌、肺癌、恶性间皮瘤、肉瘤、淋巴瘤、腺癌、甲状腺癌、心脏肿瘤、生殖细胞肿瘤、恶性神经内分泌肿瘤、中线束癌和不明原发性癌症。优选上述癌症选自下组:结肠癌、淋巴瘤、黑色素瘤。
在一种实施方式中,上述药物可通过口服或瘤内注射给药。相应地,药物剂型为适用于瘤内注射或者口服、同时保持益生菌活性的剂型。
本发明构建的工程益生菌能够有效地产生适量的c-di-AMP,动物实验证明,该工程菌可抑制小鼠体内因移植A20、B16F10、MC38等癌细胞而产生的多种肿瘤,具有广谱的抗肿瘤活性,开发应用前景广阔。
附图说明
图1是质粒pEcgRNA的结构示意图。其核苷酸序列为SEQ ID NO:14。
图2是本发明构建的重组质粒pTarget-exo/cea-tetR-Ptet-dacA-rrnB-T1T2的结构示意图。
图3是本发明的工程菌对于小鼠淋巴瘤细胞A20皮下移植瘤抑制的A20肿瘤生长曲线图。其中,control是对照(未接种A20的小鼠)。
图4是本发明的工程菌对于小鼠淋巴瘤细胞A20皮下移植瘤的免疫记忆效应的肿瘤生长曲线图。其中,Naive mice是naive小鼠,Rechallenged mice是再移植小鼠。
图5是本发明的工程菌对于小鼠黑色素瘤B16F10皮下移植瘤抑制的B16F10肿瘤生长曲线。其中,control是对照(未接种B16F10的小鼠)。
图6是本发明的工程菌对于小鼠结肠癌MC38皮下移植瘤抑制的MC38肿瘤生长曲线图。其中,是Vehicle是溶媒对照组对照(未接种MC38的小鼠)。
具体实施方式
本发明的工程益生菌是通过对大肠杆菌Nissle 1917进行基因工程改造而产生的基因工程衍生菌,通过对原始大肠杆菌Nissle 1917的基因组进行改造,使得衍生菌株能够产生STING激动剂c-di-AMP。
为简要起见,本文中有时将“产STING激动剂的工程益生菌”简称为“工程益生菌”、“重组益生菌”、“(基因)工程菌”、“益生菌”或者EcN-STING,它们表示相同的意义,可以互换使用。
作为STING激动剂之一的c-di-AMP是本发明的工程菌在表达DacA(Diadenylatecyclase A,二腺苷酸环化酶A)时的代谢产物,通过构建Ptet-可变翻译起始区-dacA结构的基因表达盒,能够提高c-di-AMP的产量,达到精确调节c-di-AMP剂量的目的。
在本文中,为了描述简便,有时会将某种蛋白比如DacA(二腺苷酸环化酶A)与其编码基因(DNA)dacA名称混用,本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。
为了考察工程菌对肿瘤的抑制效果,在小鼠体内移植了小鼠淋巴瘤细胞A20、黑素瘤细胞B16F10、结肠癌细胞MC38等癌细胞,使小鼠体内产生肿瘤。通过向肿瘤内注射工程菌,验证了工程菌的抗肿瘤效果。
以下结合具体实施例对本发明做进一步详细说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
实施例
材料和方法
本文中的全基因合成、引物合成及测序皆由生工生物工程(上海)股份有限公司完成。
本文中的分子生物学实验包括质粒构建、酶切、感受态细胞制备、转化等主要参照《分子克隆实验指南》(第三版),J.萨姆布鲁克,D.W.拉塞尔(美)编著,黄培堂等译,科学出版社,北京,2002)进行。比如感受态细胞转化方法及感受态制备方法均参照《分子克隆实验指南》(第三版)第1章96页进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
主要培养基及缓冲液:
LB培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠。(固体培养基另加20g/L琼脂粉。)
2YT培养基:16g/L胰蛋白胨、10g/L酵母提取物、5g/L氯化钠,pH7.0,121℃高温高压灭菌20min。
pEcgRNA质粒由中国科学院分子植物科学卓越创新中心保存,任何单位和个人都可以获得该质粒及相关质粒和细菌用于验证本发明,但未经中国科学院分子植物科学卓越创新中心允许不得用作其他用途,包括开发利用、科学研究和教学。
实施例1敲除Nissle 1917基因组上的dapA和thyA基因
参考文献“Multigene editing in the Escherichia coli genome via theCRISPR-Cas9 system.Jiang,Yu,Chen,Biao,Duan,Chunlan,Sun,Bingbing,Yang,Junjie,Yang,Sheng,Kelly,R.M.,Applied and Environmental Microbiology,2015,81(7):2506-2514.”,采用CRISPR-Cas9系统敲除在Nissle 1917基因组上的dapA和thyA基因,包括下述步骤。
1.1构建质粒pTargetF-dapA
正向引物N20-dapA-F(5’-TAGTAGATCGCAGCCAGTACGGGA-3’)与反向引物N20-dapA-R(5’-AAACTCCCGTACTGGCTGCGATCT-3’)使用PCR退火配对,形成双链。pEcgRNA质粒(SEQ IDNO:14,其结构如图1所示。)用BsaI酶切,然后和前述退火双链用T4DNA连接酶连接。连接产物转化大肠杆菌DH5a,涂布含壮观霉素100μg/ml的LB平板,转化子测序验证正确,获得质粒pTargetF-dapA。
设计用于敲除dapA基因的供体DNA片段、正向引物dapA-F(5’-gacttttgaacagagtaagccatcaaatctccctaaactgggccatcctctgtgcaaac-3’)和反向引物dapA-R(5’-tgcttttaatgccataccaaacgtaccattgagacacttgtttgcacagaggatggccc-3’),两条引物使用PCR退火配对,获得片段ΔdapA。
将pCas质粒(Addgene 62225)转入大肠杆菌Nissle 1917菌株(购自NaturalTherapy Imports),然后再电转入构建的pTargetF-dapA质粒和片段ΔdapA,通过PCR验证和测序确定已将dapA基因敲除,获得Nissle 1917ΔdapA菌株。
接着,在Nissle 1917ΔdapA菌株上敲除thyA基因。
1.2构建质粒pTargetF-thyA
正向引物N20-thyA-F(5’-TAGTACCGGAACGCTTTCCATTTT-3’)与反向引物N20-thyA-R(5’-AAACAAAATGGAAAGCGTTCCGGT-3’)使用PCR退火配对,形成双链。pEcgRNA质粒用BsaI酶切,然后和前述退火双链用T4DNA连接酶连接。连接产物转化大肠杆菌DH5a,涂布含壮观霉素100μg/ml的LB平板,转化子测序验证正确,获得质粒pTargetF-thyA。
设计用于敲除thyA基因的供体DNA片段、正向引物thyA-F(5’-tatcgtcgcagcccacagcaacacgtttcctgaggaacctggaccggtggcgacacgca-3’)和反向引物thyA-R(5’-aattgcagatgagtttgatccatatggttgctgtagagatgcgtgtcgccaccggtcca-3’)使用PCR退火配对,获得片段ΔthyA。
将pCas质粒转入Nissle 1917ΔdapA菌株,然后再电转入pTargetF-thyA质粒和片段ΔthyA,通过PCR验证和测序确定已将thyA基因敲除,获得Nissle1917ΔdapAΔthyA菌株,简写为EcNΔDT。
实施例2构建菌株EcN-STING
在实施例1获得的菌株EcNΔDT的基因组上exo/cea位点插入四环素诱导的启动子抑制蛋白tetR和启动子Ptet转录的dacA基因,构建出产STING的菌株EcN-STING。包括下述步骤。
2.1构建质粒pTargetF-exo/cea
正向引物N20-exo/cea-F(5’-TAGTTATTGATATATTTACGTC-3’)与反向引物N20-exo/cea-R(5’-AAACGACGTAAATATATCAATA-3’),使用PCR退火配对形成双链。pEcgRNA质粒用BsaI酶切,然后和前述退火双链用T4DNA连接酶连接。连接产物转化大肠杆菌DH5a,涂布含壮观霉素100μg/ml的LB平板,转化子测序验证正确,获得质粒pTargetF-exo/cea。
2.2构建质粒pTarget-exo/cea-tetR-Ptet-dacA-rrnB-T1T2
pTargetF-exo/cea质粒用EcoRI/HindIII酶切得到片段A。PCR扩增获得exo/cea上游同源臂、tetR-Ptet片段、dacA片段、rrnB终止子片段和exo/cea下游同源臂,将上述5个片段和片段A采用全式金试剂盒DNA assembly Kit进行DNA assembly方法连接,获得质粒pTarget-exo/cea-tetR-Ptet-dacA-rrnB-T1T2,如图2所示。其中,
上述DNA片段的序列如下所示:
exo/cea上游同源臂序列(exo/cea-L):
gcttcctgcagaccttcagccagagcagagaagctcttaccacccagcacaacccgcatccagctccagtggctccattccagacggaagtgatacaacttaccgacgatggtgacaactacacctttcagttcagtaaagtccgacaggcctcgcagaccgggctgatgtcgctggcggaacatgacctcctgctctgcactatactgtagcttccattcgcgaacccgccgttgcattgttcttcgaaggctgttgggatactggccgggatatttatcctgtagcatctccagcagagttgttggtgtcagagccggcctctctttcaacagaggaacaagcatgctgtcccacatagcttccagaggaagctttgcgtgtgcgccagtgccgaacactgttgttttcccactctcctttttcgatccgacgaccagaatggactgagataccagccttcatggccgagatatgcggtcggtcaggtggtatttgaaaccagactgaagcccacggtcgtcatcgggacgcaggccgccgccgtgcatggtatcagtgagcaggcactctgcggagcaccatcatggactgatgtggtgcggcaactgcgtcatgcaatcggggaccgaccagtaattatctttaatgcccggttcgacatccgcattctgaaaaagactgctgccgcacatagcgatccggctgactggctggaagaactgacggtatattgtgtgatggagctggctgcaggatattatggagcctccaaccgctatggcactatttcactggcctgtgctgccagccagaccggactgaactgggaagggcaggcacactcagcgatcgctgacgcacggatgacggcaggggtggtaaacgctattgctgcatatcatctggaactgctgcaggaacaggcacggctgaaaacctgactgcctggcctgtataccgcaatcattta(SEQ IDNO:9);
tetR-Ptet序列:
(虚线下划线序列为反向tetR终止子和末尾序列,加粗序列为反向tetR,单下划线序列为启动子Ptet,最后17位大写碱基序列为RBS(核糖体结合位点,ribosome bindingsite)):
dacA:
atggatttcagtaacatgtcaatcttgcattacttggctaatattgttgatattttagttgtttggtttgttatttataaagttattatgttgattcgtggtaccaaagctgttcaattattgaagggcatcttcatcatcatcgctgttaagttgttgagtggtttctttggcttacaaacagttgaatggattactgatcaaatgttaacgtggggctttttagctatcatcatcatcttccaaccagaattgcgtcgggcattagaaactttgggtcgtggcaatatttttacgcggtatggtagtcgtattgaacgggaacaacatcatttgatcgaaagtatcgaaaagtcaacacaatatatggctaagcgtcggattggcgctttgatttcagttgcacgtgataccggtatggatgattacatcgaaacaggcatcccattgaacgcaaagatcagttcacaattgttgatcaacatcttcatcccaaacactccattgcatgatggtgctgttatcatcaagggcaacgaaatcgcaagtgctgcatcatatttaccattgagtgattcaccatttttatcaaaggaattgggtacgcgtcatcgggctgcattaggcattagtgaagttacggattcaatcaccatcgttgttagtgaagaaaccggtggcatttcattaacaaaaggtggcgaattgtttcgggatgttagtgaagaagaattgcataagatcttgttgaaggaattggttacagttactgcaaaaaagccaagtatttttagtaagtggaagggtggcaagtcagaataa(SEQ IDNO:11);
rrnB-T1T2终止子序列:
gatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggccttttggatcctctagagtcgacctgcaggcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgt(SEQ ID NO:12);
exo/cea下游同源臂序列(exo/cea-R):
aatagcaatcccccagatacctaatgtagttccagcaagcacggccgggcagcttgttctgcctgcgttttcttcaattgagcagtagaccatttagctgtggcatgaatggctgcagaactttcactgttgctacctccagttccaccaccgctgccagagccactcccgtctggattatcattcaaaagagtaatgattacctgccccttatcatcataaggaacaccatctttatagtacgctacagctgtttccattataaaatcctctttgacattaaaaacaatcagttaaaaataagtactgcatatataattactggttttatatacagcataaaaattacgccgctgcgttttccctgtcaaccctgtggattttcatttttgtgaaaacgatcaaaaaacaactactcacaattcgacagtcccgccagataccgcaaaaccggccagacgttaccgttttccgggaccatgatatgagccccgttggggagggtatggagttgctgaaaatgacggtcagattgagttcaccgttttattgttacaggaggccagggcttgtctgctaccggtccggacgagagggataccgggaatttgaatccggttaactgagccggacatacggtaaataaggaatgcaggcagaacgggagtcactgcaggatgagaccggcactacagcagaaaaacaggtgattctggtacggctcagccactaaacacagttggaaactgatgataagatagtcagcggttatataactcactgagtaaaaagatataaaggtaaggagacactcatgagtcgttccggtagcacactgtatcttatcgccctgctgacagcggcaacggtcctgacagcctgcacgccaaagggcagtatggaacaacatacccggcattacgtttatgcatcagatgacggttttgatcctaact(SEQ ID NO:13)。
2.3整合方法:
参考文献“Multigene editing in the Escherichia coli genome via theCRISPR-Cas9 system.Jiang,Yu,Chen,Biao,Duan,Chunlan,Sun,Bingbing,Yang,Junjie,Yang,Sheng,Kelly,R.M.,Applied and Environmental Microbiology,2015,81(7):2506-2514.”,将pCas质粒电转入EcNΔDT菌株,然后再电转入pTarget-exo/cea-tetR-Ptet-dacA-rrnB-T1T2质粒,通过PCR验证和测序确定在exo/cea位点整合入tetR-Ptet-dacA-rrnB-T1T2片段,获得菌株EcN-STING,其基因型为Nissle 1917(ΔdapAΔthyA exo/cea::tetR-Ptet-dacA-rrnB-T1T2),或者表示为EcNΔdapAΔthyA exo/cea::tetR-Ptet-dacA-rrnB-T1T2。
实施例3菌株发酵及产物CDA检测
3.1发酵步骤:
(1)将菌种从-80℃保藏管接种至含2,6-二氨基庚二酸100μg/ml和胸腺嘧啶3mM的LB液体试管培养基,37℃,250rpm过夜培养16h左右;
(2)按照1%(v/v)接种量转接至20ml含2,6-二氨基庚二酸100μg/ml和胸腺嘧啶3mM的2YT培养基,装在250mL普通三角摇瓶中,37℃,250rpm培养1.5-2.0h,然后加入200ng/ml的ATC(脱水四环素)进行诱导4.5-5.0h;
(3)用PBS逐级稀释菌液,涂布含2,6-二氨基庚二酸100μg/ml和胸腺嘧啶3mM的LB固体平板,放于37℃培养箱培养过夜,计数。另外取样1ml,13000rpm离心10min,去上清,放于-20℃保存。
3.2检测CDA产量:
菌体处理方法:参考文献“An Essential Poison:Synthesis and Degradationof Cyclic Di-AMP in Bacillus subtilis.Jan Gundlach,Felix M.P.Mehne,et al.,JBacteriol 197:3265–3274.DOI:10.1128/JB.00564-15.”,包括下述步骤。
(1)将1.0ml菌液离心的菌体用500μl的10mM Tris-HCl,pH7.5缓冲溶液悬浮后,13000rpm离心10min,收集菌体;置于-80℃,冷冻10min;
(2)加入150μl溶菌酶溶液(10mM Tris-HCl溶液中含2mg/ml溶菌酶),悬浮沉淀物,摇床250rpm,30℃,30min;
(3)细胞裂解物放于-80℃,10min,再煮沸10min;
(4)加入800μl萃取混合液I(乙腈/甲醇1:1),漩涡震荡45秒,4℃冰浴15min;
(5)于13000rpm,4℃,离心10min,收集上清,放于新的2.0ml EP管;
(6)离心后的沉淀加入200μl萃取混合液II(乙腈/甲醇/水2:2:1),漩涡震荡45s,4℃冰浴15min;
(7)13000rpm,4℃,离心10min,收集上清,放于步骤(5)的2.0ml EP管中;
(8)离心后的沉淀再次加入200μl萃取混合液II(乙腈/甲醇/水2:2:1),漩涡震荡45s,4℃冰浴15min;
(9)13000rpm,4℃,离心10min,收集上清,放于步骤(5)的2.0ml EP管中;
(10)收集的所有上清液,置于-20℃孵育16h,取出离心13000rpm,4℃,离心20min;
(11)将上清转移至新的2.0ml EP管中,放于38℃,100rpm摇床烘干,烘干后用1.0ml无菌蒸馏水溶解。待HPLC检测。
3.3参考文献“牙龈卟啉单胞菌合成环二腺苷酸的高效液相色谱-串联质谱法定性分析。谭咏梅,杨小军,杜娟,赵望泓,陈晓丹,侯晋,《华西口腔医学杂志》,2016年第3期307-311.”中HPLC检测c-di-AMP的方法,进行菌株的CDA含量检测,结果见表1。
表1、菌株的CDA含量检测结果
菌株 | 不诱导CDA产量(μM) | 诱导后CDA产量(μM) |
Nissle 1917 | 0±0 | 0±0 |
EcNΔDT | 0±0 | 0±0 |
EcN-STING | 0.84±0 | 20.31±1.89 |
由表1可见,本发明构建的工程菌EcN-STING能够产出CDA,达到了发明目的。原始菌Nissle 1917和中间菌EcNΔDT无论是否进行ATC(脱水四环素)进行诱导,都不生产CDA,也印证了c-di-AMP(CDA)是菌株在表达基因dacA时的代谢产物。
下面考察产c-di-AMP的工程菌EcN-STING是否具有抗肿瘤作用。
实施例4考察工程菌EcN-STING对人源THP1-Dual细胞中I型干扰素通路(ISG)和NF-kB通路的激活效应
下文中可将工程菌EcN-STING称为“益生菌”,考察其对人源THP1-Dual报告细胞中I型干扰素通路(ISG)和NF-kB通路的激活影响,实验方法如下。
细胞实验菌液制备方法:参照实施例3,对应菌液培养后5000rpm,10min离心,用生理盐水洗涤一遍后,重悬在1ml无菌的生理盐水中,备用。
检测方法及原理:采用人源的THP1-Dual-ISG细胞(InvivoGen)作为实验对象,该细胞内转有含ISG和NF-kB的报告系统,报告系统可分别诱导下游荧光素酶和碱性磷酸酶的表达。荧光素酶含量可通过检测luminal(鲁米诺)的读值来测定,碱性磷酸酶的分泌可通过显色反应测定OD650可反应其含量。当细胞加入益生菌后,若激活STING,则可促进ISG和NF-kB的表达,进而促进下游的荧光素酶分泌增加和碱性磷酸化显色反应的吸光度增加。
1.实验步骤:
(1)加细胞:THP1-Dual细胞计数,调整细胞浓度至5×105/ml,每孔加入200μl的细胞进行孵育。
(2)加细菌(中间菌EcNΔDT和工程益生菌EcN-STING):96孔细胞培养板中待细胞贴壁后每孔加入5MOI或25MOI细菌,对照组为不加菌液的细胞,各设3个重复孔。孵育24h小时进行检测。其中MOI为侵染复数或称感染复数,即吸附于THP1-Dual细胞上的细菌数与培养中的THP1-Dual细胞数之比。
(3)检测显色反应:24小时后,每孔取20μl的培养液至新的96孔板中,加入显色液Quanti-lucia 50μl。立即测定荧光值。Quanti-blue显色:20μL细胞上清加入到180μL的quanti-blue中,37℃培养箱中放置1h后测定OD650的读值。实验重复3次。
(4)实验结果以倍数改变(Fold Change)为标准来评价细菌诱导I型干扰素通路和NF-kB通路分泌的能力,按下式计算:
Fold Change=菌株组发光值/对照组发光值(Ratio to control),其中倍数改变(Fold Change)的平均值(Average)≥2倍视为有效激活干扰素通路和NF-kB通路。
实验分组如下:
2、实验结果
中间菌株EcNΔDT和工程益生菌EcN-STING对人源THP1-Dual报告细胞中I型干扰素通路(ISG)的激活结果见表2。
表2:菌株对人源THP1-Dual细胞中I型干扰素通路的激活影响
中间菌株EcNΔDT和益生菌EcN-STING对NF-kB通路的激活结果见表3。
表3:菌株对人源THP1-Dual细胞中NF-kB通路的激活影响
细胞水平的上述实验结果显示,本发明的重组益生菌EcN-STING在人源THP1-Dual细胞中具有诱导I型干扰素通路和NF-kB通路的活性。
实施例5考察工程菌EcN-STING对鼠源Raw-lucia细胞中I型干扰素通路(ISG)的激活效应
考察工程菌EcN-STING对鼠源Raw-lucia细胞中I型干扰素通路(ISG)通路激活影响的实验方法如下。
检测方法及原理:采用鼠源的Raw-lucia细胞(InvivoGen),该细胞内转有含ISG的报告系统。报告系统可诱导ISG启动子的活化并产生荧光素酶,该酶存在于细胞上清并能够通过荧光素酶检测试剂QUANTI-LucTM检测定量。当细胞加入化合物后,若激活STING,则可促进ISG的表达,进而促进下游的荧光素酶分泌增加。
1.实验步骤:
(1)加细胞:Raw-lucia细胞计数,调整细胞浓度至2.5×105/ml,每孔加入200μl的细胞进行孵育。
(2)加细菌(中间菌EcNΔDT和益生菌即工程菌EcN-STING):96孔细胞培养板中待细胞贴壁后每孔加入5/25/100MOI细菌,对照组为不加菌液的细胞,各设3个重复孔。孵育24h小时进行检测。
(3)检测显色反应:24小时后,每孔取20μl的培养液至新的底部透光96孔板中,加入荧光素酶检测试剂QUANTI-LucTM50μl,立即测定荧光值。实验重复3次。
实验结果以倍数改变(Fold Change)为标准来评价细菌诱导I型干扰素通路和NF-kB通路分泌的能力,按下式计算:
Fold Change=菌株组发光值/对照组发光值(Ratio to control),其中倍数改变(Fold Change)的平均值(Average)≥2倍视为有效激活干扰素通路和NF-kB通路。
实验分组:
2、实验结果
中间菌株EcNΔDT和益生菌EcN-STING对鼠源Raw-lucia细胞中I型干扰素通路(ISG)的激活结果见表4。
表4:菌株对鼠源Raw-lucia细胞中I型干扰素通路的激活影响
细胞水平的上述实验结果显示,本发明的重组益生菌EcN-STING在鼠源Raw-lucia细胞中具有诱导I型干扰素通路分泌的功能。
实施例6考察工程菌EcN-STING对淋巴瘤的抑制作用
考察了工程菌EcN-STING(益生菌)在小鼠淋巴瘤细胞A20皮下移植瘤中的抗肿瘤作用活性。
1.实验方案包括下述步骤。
(1)将菌种冻存于PBS+15%甘油培养基,并用平板计数;
(2)实验前解冻对应菌种,加入PBS重悬洗涤两遍后,用PBS重悬到合适的菌浓;
(3)动物模型建立:小鼠淋巴瘤细胞A20,每只Balb/C小鼠腋下皮下各注射细胞(2*105个)。观察小鼠肿瘤生长情况,待肿瘤体积长到约50~100mm3时,将A20小鼠随机分组,每组9只。
(4)给药方式:瘤内注射菌株(100μL含5*107cfu每只),腹腔注射ATC诱导剂(60μl-180μg/ml)。第1/4/7天瘤内注射菌株,注射前4h需要ATC诱导的组别腹腔注射ATC诱导剂,不需要诱导组注射用于ATC的稀释液(PBS)。
(5)分组:
序号 | 菌株编号 | 注射菌液前4h | 注射菌液 | 小鼠数量 |
1 | 对照组 | 腹腔注射PBS 60μl/小鼠 | 瘤内注射PBS 100μl/小鼠 | 9 |
3 | EcN-STING | 腹腔注射ATC 60μl/小鼠 | 瘤内注射菌液100μl/小鼠 | 9 |
(6)实验观察和数据收集:肿瘤细胞接种后,常规监测肿瘤生长及体重增加或降低情况,小鼠活跃状态。开始给药后,每周测量两次小鼠的体重和肿瘤的大小。肿瘤体积计算公式:肿瘤体积(mm3)=1/2×(a×b2)(其中a表示长径,b表示短径)。
2.实验结果:
实验结果如图3所示。
结论:动物体内抗肿瘤实验结果见图3,采用工程益生菌EcN-STING治疗的小鼠中A20皮下移植瘤体积明显小于对照,说明本发明的工程益生菌(EcN-STING)在小鼠A20皮下移植瘤模型中具有显著的抗肿瘤活性。
3.免疫记忆效应:
瘤子完全消退的小鼠留存3个月后,重新接种A20细胞。同时设置对照组(未接种A20的小鼠)。每周两次记录小鼠瘤体积。肿瘤生长曲线参见图4。
结论:重新接种A20细胞的小鼠体内不再生成皮下移植瘤,说明本发明的益生菌EcN-STING治愈的肿瘤具有免疫记忆效应。
实施例7考察工程菌EcN-STING对黑色素瘤的抑制作用
考察了工程菌EcN-STING在小鼠黑色素瘤B16F10皮下移植瘤中的抗肿瘤作用活性。
1.实验方案包括下述步骤。
(1)动物模型建立:C57小鼠黑色素瘤B16F10,每只小鼠腋下皮下各注射细胞(5*104)。观察小鼠肿瘤生长情况,待肿瘤体积长到约50~100mm3时,将B16F10小鼠随机分组,每组5只。
(2)第1、4、7天三次瘤内注射菌株100μL,注射给菌前4小时腹腔注射60μL诱导剂
(3)分组:
序号 | 菌株编号 | 剂量 | 注射菌液前4h |
1 | 对照组 | 腹腔注射溶剂60μl/小鼠 | |
2 | EcN△DT | 1×10<sup>8</sup> | 腹腔注射ATC 60μl/小鼠 |
3 | EcN-STING | 1×10<sup>8</sup> | 腹腔注射ATC 60μl/小鼠 |
4 | EcN-STING | 1×10<sup>9</sup> | 腹腔注射ATC 60μl/小鼠 |
(4)实验观察和数据收集:肿瘤细胞接种后,常规监测肿瘤生长及体重增加或降低情况,小鼠活跃状态。开始给药后,每周测量两次小鼠的体重和肿瘤的大小。肿瘤体积计算公式:肿瘤体积(mm3)=1/2×(a×b2)(其中a表示长径,b表示短径)。
2.实验结果:
结论:癌细胞B16F10的动物实验结果见图5,本发明的益生菌(EcN-STING)在B16F10皮下移植瘤模型中具有显著的抗肿瘤活性,呈剂量依赖效应,而对照组和中间菌EcN△DT无显著药效。
实施例8考察工程菌EcN-STING对结肠癌的抑制作用
考察了工程菌EcN-STING在小鼠结肠癌MC38皮下移植瘤中的口服给药抗肿瘤作用活性。
1.实验方案包括下述步骤。
(1)动物模型建立:小鼠结肠癌细胞MC38,每只小鼠腋下皮下各注射细胞(2.5*104)。观察小鼠肿瘤生长情况,待肿瘤体积长到约50~100mm3时,将小鼠随机分为4组,每组5只。
(2)第1、4、7、10天四次灌胃给菌,注射给菌前4小时腹腔注射160μl诱导剂(ATC浓度500μg/ml)。
(3)分组:
编号 | 剂量 | 注射菌液前4h |
对照组 | 0 | 腹腔注射ATC160μl/小鼠 |
EcNΔDT | 5×10<sup>10</sup> | 腹腔注射ATC 160μl/小鼠 |
EcN-STING | 5×10<sup>10</sup> | 腹腔注射ATC 160μl/小鼠 |
(4)实验观察和数据收集:癌细胞接种后,常规监测肿瘤生长及体重增加或降低情况,小鼠活跃状态。开始给药后,每周测量两次小鼠的体重和肿瘤的大小。肿瘤体积计算公式:肿瘤体积(mm3)=1/2×(a×b2)(其中a表示长径,b表示短径)。
2.实验结果:
结论:癌MC38皮下移植瘤的动物实验结果见图6,采用工程益生菌EcN-STING治疗的小鼠中MC38皮下移植瘤体积明显小于对照,说明本发明的益生菌EcN-STING在MC38皮下移植瘤模型中具有显著的抗肿瘤活性,而对照组和中间菌EcNΔDT无显著药效。
以上实验表明,本发明的工程益生菌EcN-STING无论是通过瘤内注射给药或口服给药,都可抑制小鼠体内因移植A20、B16F10、MC38等癌细胞而产生的多种肿瘤,具有广谱的抗肿瘤活性,极具开发应用潜力。
序列表
<110> 中国科学院分子植物科学卓越创新中心
中国科学院上海药物研究所
<120> 具有肿瘤抑制效应的产STING激动剂的工程益生菌
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acacctttca gttcagtaaa gtccgacagg cctcgcagac cgggctgatg tcgctggcgg 180
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gttgttggtg tcagagccgg cctctctttc aacagaggaa caagcatgct gtcccacata 360
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tttttcgatc cgacgaccag aatggactga gataccagcc ttcatggccg agatatgcgg 480
tcggtcaggt ggtatttgaa accagactga agcccacggt cgtcatcggg acgcaggccg 540
ccgccgtgca tggtatcagt gagcaggcac tctgcggagc accatcatgg actgatgtgg 600
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ttccaaccag aattgcgtcg ggcattagaa actttgggtc gtggcaatat ttttacgcgg 300
tatggtagtc gtattgaacg ggaacaacat catttgatcg aaagtatcga aaagtcaaca 360
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agtgaagaag aattgcataa gatcttgttg aaggaattgg ttacagttac tgcaaaaaag 780
ccaagtattt ttagtaagtg gaagggtggc aagtcagaat aa 822
<210> 12
<211> 323
<212> DNA
<213> 人工序列()
<400> 12
gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg 60
aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct 120
cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg 180
gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct 240
gacggatggc cttttggatc ctctagagtc gacctgcagg catgcaagct tggcgtaatc 300
atggtcatag ctgtttcctg tgt 323
<210> 13
<211> 956
<212> DNA
<213> 人工序列()
<400> 13
aatagcaatc ccccagatac ctaatgtagt tccagcaagc acggccgggc agcttgttct 60
gcctgcgttt tcttcaattg agcagtagac catttagctg tggcatgaat ggctgcagaa 120
ctttcactgt tgctacctcc agttccacca ccgctgccag agccactccc gtctggatta 180
tcattcaaaa gagtaatgat tacctgcccc ttatcatcat aaggaacacc atctttatag 240
tacgctacag ctgtttccat tataaaatcc tctttgacat taaaaacaat cagttaaaaa 300
taagtactgc atatataatt actggtttta tatacagcat aaaaattacg ccgctgcgtt 360
ttccctgtca accctgtgga ttttcatttt tgtgaaaacg atcaaaaaac aactactcac 420
aattcgacag tcccgccaga taccgcaaaa ccggccagac gttaccgttt tccgggacca 480
tgatatgagc cccgttgggg agggtatgga gttgctgaaa atgacggtca gattgagttc 540
accgttttat tgttacagga ggccagggct tgtctgctac cggtccggac gagagggata 600
ccgggaattt gaatccggtt aactgagccg gacatacggt aaataaggaa tgcaggcaga 660
acgggagtca ctgcaggatg agaccggcac tacagcagaa aaacaggtga ttctggtacg 720
gctcagccac taaacacagt tggaaactga tgataagata gtcagcggtt atataactca 780
ctgagtaaaa agatataaag gtaaggagac actcatgagt cgttccggta gcacactgta 840
tcttatcgcc ctgctgacag cggcaacggt cctgacagcc tgcacgccaa agggcagtat 900
ggaacaacat acccggcatt acgtttatgc atcagatgac ggttttgatc ctaact 956
<210> 14
<211> 3106
<212> DNA
<213> 人工序列()
<400> 14
tcgagttcat gtgcagctcc atcagcaaaa ggggatgata agtttatcac caccgactat 60
ttgcaacagt gccgttgatc gtgctatgat cgactgatgt catcagcggt ggagtgcaat 120
gtcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc 180
atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat 240
ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat 300
gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag 360
agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg 420
cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca 480
ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga 540
gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa 600
caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg 660
gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc 720
ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag 780
tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg 840
gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag 900
gtagtcggca aataagatgc cgctcgccag tcgattggct gagctcatga agttcctatt 960
ccgaagttcc gcgaacgcgt aaaggatcta ggtgaagatc ctttttgata atctcatgac 1020
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1080
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1140
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1200
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1260
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1320
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1380
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1440
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1500
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1560
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1620
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1680
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1740
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1800
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1860
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatgctg 1920
gatccttgac agctagctca gtcctaggta taatactagt tgagacccag gtcgaccgac 1980
agccttccaa atgttcttct caaacggaat cgtcgtatcc agcctactcg ctattgtcct 2040
caatgccgta ttaaatcata aaaagaaata agaaaaagag gtgcgagcct cttttttgtg 2100
tgacaaaata aaaacatcta cctattcata tacgctagtg tcatagtcct gaaaatcatc 2160
tgcatcaaga acaatttcac aactcttata cttttctctt acaagtcgtt cggcttcatc 2220
tggattttca gcctctatac ttactaaacg tgataaagtt tctgtaattt ctactgtatc 2280
gacctgcaga ctggctgtgt ataagggagc ctgacattta tattccccag aacatcaggt 2340
taatggcgtt tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa 2400
cggagaccgg cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat 2460
gcaccaccgg gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg 2520
ggatcaccat ccgtcgcccg ggcgtgtcaa taatatcact ctgtacatcc acaaacagac 2580
gataacggct ctctctttta taggtgtaaa ccttaaactg catttcacca gtccctgttc 2640
tcgtcagcaa aagagccgtt catttcaata aaccgggcga cctcagccat cccttcctga 2700
ttttccgctt tccagcgttc ggcacgcaga cgacgggctt cattctgcat ggttgtgctt 2760
accagaccgg agatattgac atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa 2820
ctgtcactgt aatacgctgc ttcatagcac acctcttttt gacatacttc gggtatacat 2880
atcagtatat attcttatac cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc 2940
ttttagtaag ccggatccct gggtctcggt tttagagcta gaaatagcaa gttaaaataa 3000
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaattctct 3060
agagtcgacc tgcagaagct tagatctatt accctgttat ccctac 3106
Claims (10)
1.一种产STING激动剂的工程益生菌,其是大肠杆菌Nissle 1917的衍生菌,基因型为Nissle 1917(ΔdapA ΔthyA exo/cea::tetR-Ptet-dacA-rrnB-T1T2)。
2.如权利要求1所述的工程益生菌,其特征在于,所述STING激动剂是环二腺苷酸c-di-AMP。
3.一种构建如权利要求1或2所述工程益生菌的方法,其特征在于,包括以下步骤:
A.以大肠杆菌Nissle 1917为底盘菌,敲除dapA基因和thyA基因,获得Nissle 1917 ΔdapA ΔthyA菌株;
B.对于步骤A中获得的Nissle 1917 ΔdapA ΔthyA菌株,在其基因组的exo/cea位点插入四环素诱导的启动子抑制蛋白tetR和启动子Ptet转录的dacA基因,得到菌株Nissle1917 ΔdapA ΔthyA exo/cea::tetR-Ptet-dacA-rrnB-T1T2。
4.如权利要求3所述的方法,其特征在于,步骤A包括下述步骤:
A-1.构建质粒pTargetF-dapA;
A-2.设计敲除dapA基因的供体DNA片段、引物dapA-F(SEQ ID NO:1)和dapA-R(SEQ IDNO:2),两条引物使用PCR退火配对获得片段ΔdapA;
A-3.将pCas质粒转入Nissle 1917菌株,然后转入构建的pTargetF-dapA质粒和片段ΔdapA,通过PCR验证和测序确定已将dapA基因敲除,获得Nissle 1917 ΔdapA菌株;
A-4.构建质粒pTargetF-thyA;
A-5.设计敲除thyA基因的供体DNA片段、引物thyA-F(SEQ ID NO:3)和thyA-R(SEQ IDNO:4),两条引物使用PCR退火配对,获得片段ΔthyA;
A-6.将pCas质粒转入步骤A-3中获得的Nissle 1917 ΔdapA菌株,然后再转入pTargetF-thyA质粒和片段ΔthyA,通过PCR验证和测序确定已将thyA基因敲除,获得Nissle 1917 ΔdapA ΔthyA菌株。
5.如权利要求3所述的方法,其特征在于,步骤B包括下述步骤:
B-1.构建质粒pTargetF-exo/cea;
B-2.将步骤B-1中得到的pTargetF-exo/cea质粒用EcoRI/HindIII酶切得到片段A;PCR扩增获得exo/cea上游同源臂、tetR-Ptet片段、dacA片段、rrnB终止子片段和exo/cea下游同源臂,将上述5个片段和所述片段A进行连接,获得质粒pTarget-exo/cea-tetR-Ptet-dacA-rrnB-T1T2;
B-3.将pCas质粒转入步骤A中获得的Nissle 1917 ΔdapA ΔthyA菌株,然后转入步骤B-2中得到的pTarget-exo/cea-tetR-Ptet-dacA-rrnB-T1T2质粒,通过PCR验证和测序确定在exo/cea位点整合入tetR-Ptet-dacA-rrnB-T1T2片段,获得目的菌株Nissle 1917 ΔdapA ΔthyA exo/cea::tetR-Ptet-dacA-rrnB-T1T2。
6.如权利要求4所述的方法,其特征在于,所述质粒pTargetF-dapA的构建包括下述步骤:引物N20-dapA-F(SEQ ID NO:5)与N20-dapA-R(SEQ ID NO:6)使用PCR退火配对,pEcgRNA质粒(SEQ ID NO:14)用BsaI酶切,然后和前述退火配对所得的双链用T4DNA连接酶连接,获得质粒pTargetF-dapA。
7.如权利要求5所述的方法,其特征在于,所述质粒pTargetF-exo/cea的构建包括下述步骤:引物N20-exo/cea-F(SEQ ID NO:7)与N20-exo/cea-R(SEQ ID NO:8)使用PCR退火配对;将pEcgRNA质粒用BsaI酶切,然后和前述退火配对所得的双链用T4DNA连接酶连接,获得质粒pTargetF-exo/cea。
8.如权利要求1或2所述的工程益生菌在制备免疫应答相关疾病和/或STING活性相关疾病治疗药物中的应用。
9.如权利要求8所述的应用,其特征在于,所述免疫应答相关疾病和/或STING活性相关疾病选自下组:结肠癌、乳腺癌、肺癌、黑色素瘤、肝癌、胃癌、宫颈癌、卵巢癌、纤维肉瘤和鳞状细胞癌、脑癌、脑癌、脊椎癌、头颈部癌、白血病和血癌、皮肤癌、生殖系统癌、胃肠系统癌、肝和胆管癌、肾癌和膀胱癌、骨癌、肺癌、恶性间皮瘤、肉瘤、淋巴瘤、腺癌、甲状腺癌、心脏肿瘤、生殖细胞肿瘤、恶性神经内分泌肿瘤、中线束癌和不明原发性癌症,优选选自下组:结肠癌、淋巴瘤、黑色素瘤。
10.如权利要求8或9所述的应用,其特征在于,所述工程益生菌通过口服或瘤内注射方式给药。
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