CN113388563A - 一种具有降糖作用的大肠杆菌Nissle 1917基因工程菌及其制备方法和应用 - Google Patents
一种具有降糖作用的大肠杆菌Nissle 1917基因工程菌及其制备方法和应用 Download PDFInfo
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- CN113388563A CN113388563A CN202110607168.9A CN202110607168A CN113388563A CN 113388563 A CN113388563 A CN 113388563A CN 202110607168 A CN202110607168 A CN 202110607168A CN 113388563 A CN113388563 A CN 113388563A
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- escherichia coli
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Abstract
本发明提供了一种具有降糖作用的大肠杆菌Nissle 1917基因工程菌及其制备方法和应用。本发明通过将来源于人体的胰高血糖素样肽基因序列插入大肠杆菌Nissle1917的基因组中构建了一种具有降糖功能的大肠杆菌Nissle 1917基因工程菌。该工程益生菌在体外可高效表达GLP‑1,回避了质粒过表达系统不稳定、易耐药等缺陷,同时具有良好的耐酸、耐胆盐和抗氧化能力,还能够明显降低胰腺炎症,改善血糖和糖耐。本发明的大肠杆菌Nissle 1917基因工程菌可用于制备具有降糖功能的食品或药品,对人体二型糖尿病方面的应用具有重要的现实意义及经济价值。
Description
技术领域
本发明属于基因工程技术领域,主要涉及一种具有降糖作用的大肠杆菌Nissle 1917基因工程菌及其制备方法和应用。
背景技术
糖尿病是一组以高血糖为特点的代谢性疾病。近年来,全球糖尿病患病率呈逐渐攀升趋势,2019年,中国患者数已达1.164亿,其中约90%的患者为二型糖尿病 (T2DM),成为全球糖尿病患病人数最多的国家。目前,针对T2DM的治疗药物如双胍类、格列奈类、磺脲类、α-糖苷酶抑制剂类和噻唑烷二酮类尽管有一定的治疗效果,但易造成低血糖和胃肠道不适等副作用。胰高血糖素样肽-1(GLP-1)类似物作为一类新型的T2DM治疗药物,因疗效显著、临床应用安全性高而备受青睐,但价格昂贵且需长期注射给药也限制了其应用。
益生菌作为对宿主有益的活性微生物,能够调节肠道菌群平衡从而增强肠上皮完整性、保护肠屏障、调节粘膜免疫系统、抑制致病菌生长等作用。
蛋白药物方面,肠道L细胞产生的胰高血糖素样肽-1(GLP-1)能减轻大脑对胰岛素的抵抗、增强其对胰岛素信号的敏感性,缓解PD、阿尔兹海默症(AD)等神经退行性疾病。尽管GLP-1来源于人体,疗效显著、副作用小。然而GLP-1的氨基酸序列中有可被二肽基肽酶IV(DPP-IV)识别的位点,易被体内DPP-IV降解,半衰期仅为几分钟,极大限制了其生理效能;此外,GLP-1反复注射导致的痛苦令许多患者望而却步。
发明内容
本发明的目的是解决现有技术的不足,本发明提供一种具有降血糖作用的大肠杆菌Nissle 1917基因工程菌,所述基因工程菌的核苷酸序列如SEQ ID NO.1所示。
一种具有降血糖作用的大肠杆菌Nissle 1917基因工程菌的制备方法,所述制备方法为:将GLP-1基因序列整合进大肠杆菌Nissle 1917的基因组中,所述基因工程菌的核苷酸序列如SEQ ID NO.1所示。
优选的,所述GLP-1基因是通过化学合成法合成的目的基因。
上述大肠杆菌Nissle 1917基因工程菌,经过验证,发现其可应用在发酵菌种、制备具有降糖功能的益生菌菌片以及制备治疗二型糖尿病药物等领域中。
本发明的有益效果为:本发明所述大肠杆菌Nissle 1917在胃酸抵抗能力,消化道胆盐耐受能力,抵抗胰腺炎症方面具有明显的优势。该工程菌能够显著逆转高脂饮食联合STZ诱导的二型糖尿病小鼠血糖,改善糖耐,降低胰腺炎症因子。本发明的大肠杆菌Nissle1917工程菌为具有降糖功能的益生菌产品开发提供了基础,可应用于降糖功能食品或药品的制备中。
附图说明
图1所示为大肠杆菌Nissle 1917工程菌的GLP-1体外表达测定结果示意图;
图2所示为大肠杆菌Nissle 1917工程菌的耐酸测定结果示意图;
图3所示为大肠杆菌Nissle 1917工程菌的耐胆盐测定结果示意图;
图4所示为大肠杆菌Nissle 1917工程菌的抗氧化测定结果示意图;
图5所示为大肠杆菌Nissle 1917工程菌对高脂饮食联合STZ诱导的二型糖尿病小鼠血糖的影响;
图6所示为大肠杆菌Nissle 1917工程菌对高脂饮食联合STZ诱导的二型糖尿病小鼠血糖耐量的影响;
图7所示为大肠杆菌Nissle 1917工程菌对高脂饮食联合STZ诱导的二型糖尿病胰腺中TNF-αmRNA水平的影响;
图8所示为大肠杆菌Nissle 1917工程菌对高脂饮食联合STZ诱导的二型糖尿病胰腺中IL-1βmRNA水平的影响;
图9所示为实施例5的PCR条件。
具体实施方式
以下将结合实施例和附图对本发明的构思及产生的技术效果进行清楚、完整的描述,以充分地理解本发明的目的、方案和效果。
实施例1:大肠杆菌Nissle 1917工程菌的获取及鉴定
1、Nissle 1917的获取及鉴定
健康人体的粪便采用50%的甘油保存,以无菌磷酸盐缓冲液(PBS)做梯度稀释,选取3-5个合适梯度,吸取0.1mL于LB培养基中进行划线分离,每个稀释梯度做两个重复。将培养皿放入37℃培养箱中需氧培养48小时后挑选光滑、凸圆、边缘整齐、半透性白色菌落镜检。对疑似菌落按上述步骤在LB培养基中进行再次分离纯化,直到菌落完全纯化。将纯化的菌落连续传代3次,然后以50%甘油与菌液1:1混合保存于-80℃。
2、大肠杆菌Nissle 1917工程菌的构建
通过化学合成法合成GLP-1目的基因,核苷酸序列如SEQ ID NO.2所示,然后将其整合进大肠杆菌Nissle 1917的基因组中获得基因工程菌。该工程菌株的构建为实验室与杭州百赛生物科技有限公司合作完成。
2.1引物设计
1917attB-upF:GAAAGCCCAATCTTCACATCAATC
1917-attB-UP-R:CCTGTGTGAAATTGTTATCCGCTAAAAAAGCAGGCTTCAAC
Pelb-hglB-F:TGAAGCCTGCTTTTTTAGCGGATAACAATTTCACACAGGA
pelB-hglB-R:CGCTCAAGTTAGTATCCCAGTCACGACGTTGTAAAACGAC
1917-attb-down-F:ACAACGTCGTGACTGGGATACTAACTTGAGCGAAACGGG A
1917attb-downR:TTACGATGGCGATAATATTTCACC
1917attb-JD-UP-F:TGCGCCGCGACCAGAAACGATAT
1917attB-JDdown-R:CGTAGTACGCATCGGTACGCCAA
2.2修复同源臂扩增
基因组改造修复同源臂构建
以Nissle1917基因组DNA为模板用1917attb-UP-F/1917-attB-UP R和1917attB-downF/1917attB-down-R为模板扩增,扩增体系如下所示:
Pelb-hglB-F/pelB-hglB-R以之前构建好的pBSC-pelb-hglp-4质粒为模板扩增,扩增体系如下所示:
扩增好的片段经PCR产物纯化试剂盒回收后按照如下体系进行融合PCR扩增,扩增条件:94℃5min 2Cycle(94℃30sec、50℃30sec、72℃40sec),30cycle(9 4℃30sec、50℃30sec、72℃40sec)10℃hold on。
2.3Crisp整合
MG1655△lacZ菌株制备电转化感受态转化敲除质粒及修复同源臂涂布平板鉴定
整合鉴定1917attb-JD-UP-F:TGCGCCGCGACCAGAAACGAT
1917attB-JDdown-R:CGTAGTACGCATCGGTACGC
其中,插入成功为2579bp,插入不成功为1739bp。
3、大肠杆菌Nissle 1917工程菌的GLP-1体外表达测定结果
参照Abcam公司人GLP-1ELISA检测试剂盒说明书体外检测工程菌发酵上清液中GLP-1的表达。
结果如图1所示,大肠杆菌Nissle 1917工程菌可高效表达GLP-1蛋白(88-104 pg/mL)。
实施例2:大肠杆菌Nissle 1917工程菌的耐酸实验
以1/100的体积接种大肠杆菌Nissle 1917和工程菌,在LB液体培养基中传代培养18h。8000rpm离心5min,保留沉淀弃上清,再用无菌PBS缓冲液洗涤2次,分别将其各分装于5个1.5mL离心管中,8000rpm离心5min,得到野生型大肠杆菌Niss le 1917和工程菌菌体各5份。然后分别加入预先配置好的pH为2.0、3.0、4.0、5.0、6.0和7.0的PBS缓冲液,置于37℃培养箱静置培养2h,培养结束后取0h和2h菌悬液倍比(10倍)稀释,通过平板计数法测定活菌数,并计算野生型大肠杆菌Nissle 1917和工程菌菌株存活率。
结果如图2所示,在耐酸实验中,在pH=4的PBS环境中培养2h,野生型大肠杆菌Nissle 1917和工程菌数量均能达到108CFU/mL以上,具有较强的耐酸能力。(E.coliNissle1917:野生型大肠杆菌,E.coli 1917-attb-pelB-GLP-1:工程菌组)
实施例3大肠杆菌Nissle 1917工程菌的耐胆盐实验
以1/100的体积接种大肠杆菌Nissle 1917和工程菌,在LB液体培养基中传代培养18h。8000rpm离心5min,保留沉淀弃上清,再用无菌PBS缓冲液洗涤2次,分别将其各分装于5个1.5mL离心管中,8000rpm离心5min,得到野生型大肠杆菌 Nissle 1917和工程菌菌体各5份。然后分别加入预先配置好的胆盐浓度为0%、0.1%、0.2%、0.3%、0.4%和0.5%的PBS缓冲液,置于37℃培养箱静置培养2h,培养结束后取0h和2h菌悬液倍比(10倍)稀释,通过平板计数法测定活菌数,并计算野生型大肠杆菌Nissle 1917和工程菌菌株存活率。
结果如图3所示,在耐胆盐实验中,工程菌和野生型大肠杆菌Nissle 1917均表现出良好的胆盐耐受性,在0.3%的胆盐浓度下培养2h,两株菌均能达到109CFU/mL 以上。(E.coliNissle1917:野生型大肠杆菌,E.coli 1917-attb-pelB-GLP-1:工程菌组)
实施例4:大肠杆菌Nissle 1917工程菌的抗氧化实验
(1)使用LB液体培养基分别培养野生型大肠杆菌Nissle 1917和工程菌,10000rpm离心10min沉淀菌体,吸取上清液置于冷藏冰箱保存备用。
(2)DPPH自由基清除能力的测定:
取1mL上清液与1mL配制好的DPPH自由基的甲醇溶液混合,震荡均匀后避光条件下室温反应30min,517nm波长处测其OD值(去离子水作为空白对照)。
DPPH自由基清除率:[1-A517(样品)/A517(空白)]×100%
(3)清除羟自由基的能力测定:
取1mL上清液加入玻璃试管中(样品管),空白管中加入1mL ddH2O,另分别加入1mL3mmol/L的水杨酸,1mL 1mmol/L的FeSO4,1mL3mmol/L的H2O2,混合均匀后37℃水浴锅中反应15min,用分光光度计在510nm波长处测定吸光度,羟自由基清除率(%)=[1-A510(样品)/A510(空白)]×100%。
(4)清除超氧自由基能力的测定:
配制Tris-HCl缓冲液(0.05mol/L,含1mmol/L Na2EDTA),取0.5mL上清液加到1号玻璃试管内,加入2mL的Tris-HCl溶液和1mL邻苯三酚溶液,2号管加0.5mL 的去离子水,2mL的Tris-HCl溶液和1mL邻苯三酚溶液,3号管加1.5mL的去离子水,2mL Tris-HCl溶液,4号管加1mL的去离子水,0.5mL细菌上清液和2mL Tris-HCl溶液,超氧自由基清除率(%)=[1-(A11-A10)/(A01-A00)]×100%,其中, A00:不含样品和邻苯三酚(3);A01:不含样品含邻苯三酚(2);A10:含样品不含邻苯三酚(4);A11:含样品和邻苯三酚(1)。
(5)对亚铁离子螯合能力的测定:
取0.5mL上清液加到玻璃管内,加入0.1mL 2mol/L的FeSO4,0.1mL 1%的维生素C和1mL 0.2mol/L的NaOH溶液,混匀后37℃反应20min,继续加入10%的三氯乙酸,冷冻离心机4℃,6000rpm 10min离心沉淀蛋白。取0.4mL的上清,加入4mL邻二氮菲。空白对照中加入0.5mL的ddH20,其他相同。室温反应10min 后536nm处测定吸光度,Fe2+螯合能力=(A空白-A样品)/A空白×100%。
(6)还原活性的测定:
取1mL细菌上清液,加入1mL 0.2mol/L PBS缓冲液和1mL 1%的铁氰化钾,混匀后50℃反应20min后加入1mL 10%的三氯乙酸,6000rpm 10min离心沉淀反应物。取1mL的混合液,加入4mL的ddH20和0.4mL 0.1%的FeCl3溶液,室温反应10min。空白对照管加1mL的ddH20,其他加的试剂和处理情况相同。静置后700nm 波长处测吸光度。
结果如图4所示,对两株菌的抗氧化性进行评价,抗氧化试验结果表明,E.coli1917-attb-pelB-GLP-1与Nissle 1917均对DPPH和-OH的清除率很高,没有显著差异(95.4%vs 95.2%,81.3%vs 78.6%)。大肠杆菌1917-attb-pelB-GLP-1和Nissle 1917的超氧自由基清除率和Fe2+螯合率较低。
实施例5:大肠杆菌Nissle 1917工程菌对高脂饮食联合STZ诱导的二型糖尿病小鼠血糖的影响
1、二型糖尿病小鼠模型的建立
48只8周龄C57BL/6雄性小鼠购自湖南斯莱克景达实验动物公司。以正常饲料适应性喂养一周后测量血糖体重,并给予高脂饲料(购自江苏协同生物)喂养9周。随机选取其中12只小鼠作为对照组,其余小鼠连续5天腹腔注射30mg/kg STZ(溶于0.1mol/L的柠檬酸缓冲液,pH=4.5)。每隔3天监测小鼠血糖,当空腹血糖高于11.1 mmol/L时纳入研究,并将糖尿病小鼠随机分成模型组,大肠杆菌Nissle 1917治疗组和工程菌治疗组。
2、实验分组及治疗
a.对照组(C组,n=12):灌胃100μL细菌包被液(0.9%的生理盐水中含有0.01%的明胶),每天1次,持续治疗9周;
b.模型组(M组,n=12):灌胃100μL细菌包被液,每天1次,持续治疗9周;
c.大肠杆菌Nissle 1917治疗组(EcN组,n=12):灌胃100μL以包被液重悬大肠杆菌Nissle 1917,菌液浓度为1010CFU/mL,每天1次,持续治疗9周;
d.工程菌治疗组(EcN-G组,n=12):灌胃100μL以包被液重悬工程菌,菌液浓度为1010CFU/mL,每天1次,持续治疗9周。
实验结束后解剖小鼠,取胰腺、肝脏、肠道等器官。
3、实验动物血糖的变化情况
造模成功后开始治疗,检测血糖前小鼠禁食12h,并通过尾静脉取血检测小鼠血糖,每周检测一次血糖观察治疗效果。
结果如图5所示,工程菌治疗小鼠血糖在第6周开始呈现较为显著的下降趋势,第9周时,工程菌组血糖与模型组相比显著降低,而模型组小鼠血糖一直维持在较高水平。(C:正常对照组,M:模型组,EcN:野生型大肠杆菌Nissle 1917组,EcN-GLP-1:工程菌组)
4、实验动物葡萄糖耐量试验(GTT)
葡萄糖耐量试验被用来检测小鼠胰岛β细胞功能和机体对血糖的调节能力。在实验结束前一周进行。实验前小鼠禁食16h,再根据体重腹腔注射葡萄糖(2g/kg),然后在注射后不同时间点(0,30,60,90,120min)尾静脉取血检测血糖变化。
结果如图6所示,糖耐试验结果表明,与模型组相比,工程菌治疗显著提高了实验动物的血糖耐量。葡萄糖曲线下面积(AUC)分析也有类似的结果,与工程菌组相比,模型组具有最大的AUC值。(C:正常对照组,M:模型组,EcN:野生型大肠杆菌Nissle 1917组,EcN-GLP-1:工程菌组)
5、实验动物胰腺组织中炎症因子测定
(1)腺组织RNA的提取
将胰腺组织在液氮中磨成粉末后,再以50-100mg组织加入1mL Rizol液研磨,注意样品总体积不能超过所用Trizol体积的10%,反复用枪吹打或剧烈振荡以裂解细胞,在室温15~30℃下放置5min;以每1mL Trizol液加入0.2mL的比例加入氯仿,盖紧离心管,用手剧烈摇荡离心管15s,在室温下放置2~3min;12000rpm,4℃,离心15min;取上层水相于一新的离心管,按每1mL Trizol液加0.5mL异丙醇的比例加入异丙醇,室温放置10min;12000rpm,4℃,离心15min;弃去上清液,按每 1mL Trizol液加入1mL的75%乙醇进行洗涤,涡旋混匀,12000rpm,4℃,离心15min,重复两次;弃去上清液,让沉淀的RNA在室温下自然干燥或真空干燥5-10min,注意不要干燥过分,否则会使RNA失去溶解性,导致A260/280<1.6;加适量 RNase-freewater溶解RNA沉淀,用枪头反复吸取混匀;65℃水浴15min,保存于-80℃备用;紫外吸收法测定RNA浓度和纯度。
(2)Real Time PCR:
根据TAKARA逆转录及实时定量荧光试剂盒说明书进行RNA的实时定量荧光测定。PCR条件如图9所示,引物如表1所示:
表1引物序列
(3)结果如图7-8所示,与对照组小鼠相比,模型组小鼠胰腺组织中炎症因子 TNF-α和IL-1β表达水平显著增加,而大肠杆菌Nissle 1917工程菌逆转了高脂饮食联合STZ诱导的二型糖尿病小鼠胰腺组织中炎性因子的表达水平。(C:正常对照组, M:模型组,EcN:野生型大肠杆菌Nissle 1917组,EcN-GLP-1:工程菌组)
尽管本发明的描述已经相当详尽且特别对几个所述实施例进行了描述,但其并非旨在局限于任何这些细节或实施例或任何特殊实施例,而是应当将其视作是通过参考所附权利要求考虑到现有技术为这些权利要求提供广义的可能性解释,从而有效地涵盖本发明的预定范围。此外,上文以发明人可预见的实施例对本发明进行描述,其目的是为了提供有用的描述,而那些目前尚未预见的对本发明的非实质性改动仍可代表本发明的等效改动。
序列表
<110> 南昌大学
<120>一种具有降糖作用的大肠杆菌Nissle 1917基因工程菌及其制备方法和应用
<130> 2021
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1889
<211> 1889
<212> DNA
<213> 大肠杆菌Nissle 1917(大肠杆菌)
<400> 1889
gaaagcccaa tcttcacatc aatcggtttt tcacccgtac cgtacagagt aattccaccc 60
ggagcggcag ggacatacac cgttccctga tactcaccag gcatcacggc aatatactgg 120
cgcttgttgg tacgcttgat aattgccgca tctaccgccg cctgaatcgt ggtatgcgtt 180
acaccttgag tacccgccgg gccgacaaca aagtcaggtt gcgcaggcag ggtaatcggg 240
gaaggattcc acgctgccgc acctggtgtc agggatgcaa aatagtgttg agcatcgaaa 300
ttctgcgctt cttttgccga cagaatcggg cgcgaagagg taccaggcgc ggtttgatca 360
gaaggacgtt gatcgggcgg tgttgagcta caggcggtca gcgtcacgcc aaaagccaat 420
gccagcgcca gacgggaaac tgaaaatgtg ttcacaggtt gctccgggct atgaaataga 480
aaaatgaatc cgttgaagcc tgcttttttc aggaaacagc tatgaccatg attacgccaa 540
gcttgatctc tccttcacag attcccaatc tcttgttaaa taacgaaaaa gcatcaatta 600
aaacggcggc atgtctttct atattccagc aatgttttat aggggacata ttgatgaaga 660
tgggtatcac cttagtaaaa aaaaagaatt gctataagct gctctttttt gttcgtgata 720
tactgataat aaattgaatt ttcacacttc tggaaaaagg agatatacca tggaagagct 780
cggtaccatg aaatacctgc tgccgaccgc tgctgctggt ctgctgctcc tcgctgccca 840
gccggcgatg gccatggata tcggaattaa ttcggatccg atgcatgatg aatttgaacg 900
tcatgctgaa ggtactttta cgagtgatgt tagttcatat ttagaaggtc aagctgcaaa 960
ggaatttatt gcatggttgg ttaagggtcg gggttaactc gagggctgtt ttggcggatg 1020
agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca 1080
gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt 1140
gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca 1200
ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt 1260
tgtcggtgaa cgctctcctg agtaggacaa atgaattcac tggccgtcgt tttacatact 1320
aacttgagcg aaacgggaag gtaaaaagac aaaaagttgt ttttaatacc tttaagtgat 1380
accagatggc attgcgccat ctggcagagt gattaactaa acatcgcagt aatcgaggca 1440
ctcgccagag agtgaaaatg aacgttaaac ccgaccatcg cgccgctggc accttcatcg 1500
acatcaatac gttctacatc cagcgcgtga acggtaaaaa tgtagcgatg ggtttcgcct 1560
ttcggcggcg ctgcgccatc gtacccggtt ttaccaaagt cggtacgcgt ctgcaaaacg 1620
ccgtctggca tagctaccag accagagcca aacccttgcg gtaatacgcg ggtatcagcg 1680
ggtaaattaa caactaccca gtgccaccag ccggagccgg ttggcgcatc cgggtcatag 1740
caggtgacaa caaaactttt cgttcccaca ggaacatcat cccacgccag atgcggtgaa 1800
atattatcgc catcgtaacc catgccgtta aagacatgac gatgcggcag cttatcgcca 1860
tcgcgcagat cgttactgat gagtttcat 1889
Claims (6)
1.一种具有降血糖作用的大肠杆菌Nissle 1917基因工程菌,其特征在于:所述基因工程菌的核苷酸序列如SEQ ID NO.1所示。
2.一种具有降血糖作用的大肠杆菌Nissle 1917基因工程菌的制备方法,其特征在于:所述制备方法为:将GLP-1基因序列整合进大肠杆菌Nissle 1917的基因组中,所述基因工程菌的核苷酸序列如SEQ ID NO.1所示。
3.根据权利要求2所述的制备方法,其特征在于:所述GLP-1基因通过化学合成法合成。
4.一种如权利要求1所述的基因工程菌,其特征在于:所述基因工程菌在发酵菌种中的应用。
5.一种如权利要求1所述的基因工程菌,其特征在于:所述基因工程菌在制备具有降糖功能的益生菌菌片中的应用。
6.一种如权利要求1所述的基因工程菌,其特征在于:所述基因工程菌在制备治疗二型糖尿病药物中的应用。
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