CN111411054B - 一种表达抗菌肽基因的乳酸乳球菌 - Google Patents
一种表达抗菌肽基因的乳酸乳球菌 Download PDFInfo
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Abstract
本发明公开了一种表达抗菌肽基因的乳酸乳球菌,属于基因工程技术领域。本发明通过优化CRAMP蛋白的核苷酸序列,并将构建的表达CRAMP蛋白的乳酸乳球菌用于制备调节肠道菌群紊乱的疫苗,对肠道菌群的调节以及肠道免疫应答与维持有优势,全培养物可直接作为口服疫苗刺激小鼠并引起较强的细胞免疫应答,该重组乳酸乳球菌可以作为一种具有良好产业前景的新型口服疫苗产品,对减轻肠道炎症起到积极的作用,对促进肠道健康发展具有重要的实践意义。
Description
技术领域
本发明涉及一种表达抗菌肽基因的乳酸乳球菌,属于基因工程技术领域。
背景技术
抗菌肽是植物、无脊椎动物和脊椎动物(包括人类)等多种宿主的先天免疫和防御的主要成分。Cathelicidins是一类主要的抗菌肽,其特征在于保守的阴离子N-末端前体序列,称为cathelin。cathelin序列的保守性表明该家族的各种成员是从共同祖先基因的复制和修饰进化而来的。CRAMP(Cathelicidin-Related AntiMicrobial Peptide)含有34个氨基酸(GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ),对革兰氏阳性菌和革兰氏阴性菌具有强大的抗菌活性,但对人红细胞没有溶血活性。1mM的CRAMP能够直接导致大肠杆菌内膜的立即透化。抗CRAMP的抗血清在骨髓前体和中性粒细胞中显示出丰富的表达。与天蚕素A类似,50mM CRAMP未显示出对人红细胞的任何溶血活性。此外,研究发现CRAMP对一些致病真菌(Candia alicans和Aspergillus fumigatus)和肿瘤细胞具有强大的抗生素活性。体外研究证实CRAMP能显著抑制幽螺旋杆菌的增殖;而CRAMP缺失会导致小鼠肠胃病情加重,通过表达CRAMP的乳酸杆菌治疗患有肠胃病的CRAMP敲除小鼠则显著缓解效果。
表达于肠道的CRAMP由于肠道屏障的破坏导致CRAMP水平显著下降,导致不能发挥其免疫效果,调节肠道菌群平衡,因此,选择安全无毒,能够在肠道中存活并能表达CRAMP的载体系统,使得CRAMP能够在肠道发挥作用,对调节肠道菌群平衡具有重要意义。
乳酸乳球菌因其表面分子有高黏附性的特征,能使其成功地定植于动物机体的肠道内并成为在肠道内的优势菌群而发挥提高机体免疫力、促进营养物质吸收及维持肠道内菌群平衡等多种功能。所形成的生物学稳固屏障是维持肠道微生物平衡的重要保障。在表达外源基因方面上,乳酸乳球菌表达系统作为一种原核表达系统,有以下优势:(1)作为食品级细菌,作为活载体疫苗安全性更高;(2)外源基因能被表达在细胞内,也能被表达展示在细胞表面或被分泌到细胞外;(3)安全、无内毒素,无需纯化表达的外源蛋白,直接同菌体服用;(4)能定植在机体黏膜表面(属于共同黏膜免疫系统),接种黏膜某一位点便可诱导全身的黏膜免疫反应;最后的也是最重要的,这种免疫方式能帮机体获得的更长久免疫记忆力,以便于长期抵御病原体的侵犯。
发明内容
本发明所要解决的技术问题是克服现有技术中口服CRAMP易被消化道酶降低、无法实现肠道靶向递送CRAMP、最大化实现其局部免疫调节效果的缺陷,提供一种分泌表达CRAMP蛋白重组乳酸乳球菌及其应用。
本发明的第一个目的是提供一种乳酸乳球菌,其表达并分泌CRAMP蛋白;所述CRAMP蛋白为(a)或(b):
(a)如SEQ ID NO.2所示的CRAMP蛋白;
(b)在(a)的基础上对一个或几个氨基酸进行缺失、取代或增减且具有抑菌特性的由(a)衍生的蛋白质。
在一种实施方式中,所述乳酸乳球菌以乳酸乳球菌NZ9000为宿主。
在一种实施方式中,所述乳酸乳球菌以pMG36e或pNZ8148为载体。
在一种实施方式中,所述乳酸乳球菌还引入了Usp45信号肽促进CRAMP蛋白的表达。
在一种实施方式中,所述Usp45信号肽的核苷酸序列如SEQ ID NO.3所示。
本发明的第二个目的是提供一种构建上述任一所述重组乳酸乳球菌的方法,是将SEQ ID NO.1所示的编码CRAMP蛋白的基因与载体连接,再转化至乳酸乳球菌细胞中;所述载体为pMG36e或pNZ8148。
在一种实施方式中,所述pMG36e或pNZ8148上连接有usp45信号肽。
在一种实施方式中,所述方法包括如下步骤:
(1)合成SEQ ID NO.1所示的编码CRAMP蛋白的基因;
(2)将步骤(1)合成的基因连接至pMG36e中,获得重组质粒pMG36e-Usp45-CRAMP;采用电转化法将pMG36e-Usp45-CRAMP重组质粒导入乳酸乳球菌L.lactis NZ9000中,获得重组乳酸乳球菌L.lactis NZ9000/pMG36e-Usp45-CRAMP。
在一种实施方式中,所述方法包括如下步骤:
(1)合成SEQ ID NO.1所示的编码CRAMP蛋白的基因;
(2)将步骤(1)合成的基因连接至pNZ8148中,获得重组质粒pNZ8148-Usp45-CRAMP;采用电转化法将pNZ8148-Usp45-CRAMP重组质粒导入乳酸乳球菌L.lactis NZ9000中,获得重组乳酸乳球菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP。
在一种实施方式中,所述电转化法为:取L.lactis NZ9000的感受态细胞,加入重组质粒,混匀并转入电转化杯,电击后加入恢复培养基,冰浴后静置培养,平板筛选高拷贝转化子。
本发明的第三个目的是提供一种组合物,该组合物中含有所述乳酸乳球菌。
在一种实施方式中,所述乳酸乳球菌在组合物中的含量≥1×105CFU/mL或1×105CFU/g。
在一种实施方式中,所述组合物为药物,含有药学上可接受的载体。
本发明的第四个目的是提供所述重组乳酸乳球菌在制备疫苗中的应用。
在一种实施方式中,所述应用是培养所述重组乳酸乳球菌进行培养,再以乳酸乳球菌的全培养物作为口服疫苗。
在一种实施方式中,所述应用包括如下步骤:将重组乳酸乳球菌/pMG36e-Usp45-CRAMP接种于GM17液体培养基静置培养过夜,以一定比例转接于含GM17液体培养基,继续培养至细菌进入对数生长期,全培养物直接作为口服疫苗。
在一种实施方式中,所述静置培养的温度为28~30℃。
在一种实施方式中,所述转接是将L.lactisNZ9000/pMG36e-Usp45-CRAMP以体积比1:100的比例接种于GM17培养基。
在一种实施方式中,所述对数生长期的细菌培养液的OD值为0.4~0.6。
在一种实施方式中,所述应用包括如下步骤:将L.lactis NZ9000/pMG36e-Usp45-CRAMP重组表达菌以1:100的比例接种于含有GM17培养基,继续培养2~3h至细菌进入对数生长期(OD600=0.4~0.6);培养至重组菌的浓度达1012CFU/mL数量级,收集诱导后的全培养物作为口服疫苗。
本发明的第五个目的是提供一种预防急性结肠炎的口服疫苗,通过培养所述重组乳酸乳球菌,再以植物乳杆菌的全培养物作为口服疫苗或口服疫苗的主要成分制备而成。
在一种实施方式中,所述口服疫苗可采用灌胃或饲喂的方式使用。
本发明还要求保护所述乳酸乳球菌在制备可引入肠道的产品中的应用;所述产品具有如下至少一种功能:
(a)抑制肠道炎症;
(b)重塑肠黏膜屏障;
(c)改善肠黏膜通透性;
(d)预防和治疗肠道炎症以及肠道炎症引起的疾病。
本发明还要求保护所述乳酸乳球菌在制备预防或治疗炎症性肠病、腹泻或肠道稳态失衡引起的或相关的疾病的药物中的应用,所述肠道稳态失衡引起的或相关的疾病包括但不限于肝脏疾病、代谢内分泌疾病、循环系统疾病等,例如糖尿病、胰腺炎或代谢综合症。
本发明还要求保护所述乳酸乳球菌在制备预防或治疗急性结肠炎的药物中的应用。
有益效果:(1)本发明提供了一种分泌表达小鼠抗菌肽CRAMP蛋白重组乳酸乳球菌的制备方法,即采用能够调节肠道菌群且更强定植能力的乳酸乳球菌表达系统,并添加Usp45信号肽来进行CRAMP基因的分泌表达,使CRAMP蛋白的表达量可达40ng/μL;加上乳酸乳球菌作为益生菌的益生特性,使得此乳酸菌表达系统成为一个食品级表达系统,可以连同菌体一起服用。
(2)本发明将构建的表达CRAMP蛋白的乳酸乳球菌用于制备针对肠道菌群紊乱调节的疫苗,对肠道菌群的调节以及肠道免疫应答与维持上有优势,全培养物可直接作为口服疫苗刺激小鼠并引起较强的细胞免疫应答,该重组乳酸乳球菌可以作为一种具有良好产业前景的新型口服疫苗产品,对减轻肠道炎症起到积极的作用,对促进肠道健康发展具有重要的实践意义。
附图说明
图1为CRAMP和Usp45-CRAMP基因片段的PCR扩增结果;1为DL2000 DNA Marker;2-3为Usp45-CRAMP基因片段的PCR扩增;
图2为重组大肠杆菌E.coli MC1061/pMG36e-Usp45-CRAMP组的PCR鉴定结果,1为DL2000 DNA Marker,2为重组大肠杆菌E.coli MC1061/pMG36e-Usp45-CRAMP组的PCR鉴定,3为重组大肠杆菌E.coli MC1061/pNZ8148-Usp45-CRAMP组的PCR鉴定;
图3为重组乳酸乳球菌L.lactis NZ9000/pMG36e-Usp45-CRAMP组、L.lactisNZ9000/pNZ8148-Usp45-CRAMP组的PCR鉴定结果;1为DL2000 DNA Marker;2为L.lactisNZ9000/pMG36e-Usp45-CRAMP组中CRAMP的PCR鉴定;3为L.lactisNZ9000/pNZ8148-Usp45-CRAMP组的PCR鉴定;
图4为重组乳酸乳球菌中CRAMP的免疫印迹结果;1为蛋白Marker;2为L.lactisNZ9000/pMG36e-Usp45-CRAMP组上清中CRAMP的表达量;3为L.lactisNZ9000/pMG36e-Usp45-CRAMP组菌体中CRAMP的表达量;4为L.lactisNZ9000/pNZ8148-Usp45-CRAMP组上清中CRAMP的表达量;5为L.lactisNZ9000/pNZ8148-Usp45-CRAMP组菌体中CRAMP的表达量;
图5为重组植物乳杆菌CRAMP的ELISA结果;
图6为前人研究大肠杆菌中CRAMP的表达情况;1为大肠杆菌裂解液;2为大肠杆菌裂解液上清;3为大肠杆菌裂解液沉淀;4为洗脱柱上的GST-CRAMP洗脱缓冲液;5为蛋白Marker;
图7为结肠炎模型建立期间各组小鼠体重的变化情况;
图8为各组(A)小鼠结肠长度比较及(B)各组统计图;
图9为结肠炎临床指标评分;
图10为结肠组织学(A)病理学形态观察及(B)评分;
图11为qPCR测定肠道紧密连接蛋白(A)ZO-1、(B)ZO-2和(C)occludin的变化情况;
图12为qPCR测定炎症细胞因子(A)IL-6、(B)IL-1β、(C)TNF-α和(D)IL-10的表达情况;
图13为Western blot测定炎信号通路关键转录因子的磷酸化水平变化情况:(A)Western blot实验结果p-ERK、ERK、p-p38、p38、p-NF-kB和NF-kB条带图,(B)p-ERK/ERK灰度分析统计图,(C)p-p38/p38灰度分析统计图,(D)p-NF-kB/NF-kB灰度分析统计图。
具体实施方式
下面结合说明书附图和具体实施例,进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下例实施例中未注明具体条件的实验方法,通常按照本领域常规条件或按照制造厂商建议的条件。除非另行定义,文中所使用的所有专业与科学用语与本领域技术人员熟悉的意义相同。
小鼠体重、结肠长度、DAI评分参照文献《Curcumin Prevents the Developmentof Dextran Sulfate Sodium(DSS)-Induced Experimental Colitis》;
ZO-1、ZO-2、occludin、IL-10、IL-1β、TNF-α和IL-6的qPCR测定方法参照文献《Neutralization of IL-6and TNF-αameliorates intestinal permeability in DSS-induced colitis》的方法进行;
p-ERK、ERK、pp38、p38、p-NF-kB、NF-kB、CRAMP用Western Blot进行测定,方法参考文献《Dietary squalene supplementation improves DSS-induced acute colitis bydownregulating p38 MAPK and NFkB signaling pathways》。
实施例1重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP的构建
1、重组质粒pMG36e-Usp45-CRAMP的构建
(1)基因序列的密码子偏好性优化与合成:根据目的基因CRAMP基因的序列和表达载体pMG36e的特点,以及为达到高效分泌表达的目的而增加的信号肽序列Usp45,采用人工合成的方法将Usp45-CRAMP基因的228bp的密码子优化序列送公司进行合成。Xbal-Usp45-CRAMP-F为含有与pMG36e融合表达的酶切位点Xbal和信号肽Usp45-CRAMP的5'端最初一段序列的上游引物,Usp45-CRAMP-Sph1-R为信号肽Usp45-CRAMP基因反向引物。同时还设计了用于重组质粒的PCR检测和测序的引物pNZ1及pNZ2,是以pMG36e空质粒的MCS上游和下游7090bp左右的区域为依据设计的。优化合成的Usp45-CRAMP序列如SEQ ID NO:4所示;优化合成的Xbal-Usp45-CRAMP-F,Usp45-CRAMP-Sph1-R引物序列分别如SEQ ID NO:5~6所示。
(2)Usp45-CRAMP基因片段的PCR扩增:以含优化合成的Usp45-CRAMP基因为模板,加入高保真DNA聚合酶KOD-Plus-(1.0U/μL)1μL,0.3μM的引物Xbal-Usp45-CRAMP-F,Usp45-CRAMP-Sph1-R各1.5μL,模板1.5μL,25mM MgSO4 2μL,2mM dNTPs 5μL,10×Buffer forKOD-Plus-5μL,用ddH2O补至50μL,PCR反应程序为:94℃预变性5min;94℃变性30s,55℃退火30s,72℃延伸1min,35个循环;72℃后延伸10min。PCR反应完成,将产物进行1.0%琼脂糖凝胶观察与回收,可见大小约228bp的扩增条带,与预期结果一致(如图1),回收的产物将作为连接模板用于获得添加Usp45-CRAMP序列的完整片段。
(3)重组质粒pMG36e-Usp45-CRAMP的构建:将步骤(2)中回收的PCR产物用Sph1、Xbal进行双酶切处理,胶回收大小约228bp的条带;以同样的方法对pMG36e空质粒进行双酶切,胶回收大小约3600bp的条带。分别取4μL双酶切后胶回收的Usp45-CRAMP基因片段和1μL双酶切后胶回收的pMG36e空质粒,将Usp45-CRAMP和pMG36e按照摩尔比6:1添加,并加入10×ligation buffer 2μL,T4 DNA Ligase(350U/μL)1μL,用ddH2O补至20μL,混匀后置于4℃条件下连接过夜,将连接产物转化E.coli MC1061感受态细胞,在含有5μg/mL红霉素(Erythromycin,Er)的LB琼脂培养板中,37℃培养两天,然后挑取单个菌落进行PCR鉴定。PCR鉴定以待检菌落为模板,加入高保真DNA聚合酶KOD-Plus-(1.0U/ul)1μL,0.3μM的引物Xbal-Usp45-CRAMP-F,Usp45-CRAMP-Sph1-R各1.5μL,模板1.5μL,25mM MgSO4 2μL,2mMdNTPs 5μL,10x Buffer for KOD-Plus-5μL,用ddH20补至50μL,PCR反应程序为:94℃预变性5min;94℃变性30s,55℃退火30s,72℃延伸1min,35个循环;72℃后延伸10min。PCR反应完成,将产物进行1.0%琼脂糖凝胶观察与回收,可见大小约228bp的扩增条带,与预期结果一致(如图2),对检测阳性的菌液用质粒DNA抽提试剂盒进行质粒抽提,并进行双酶切鉴定和测序测定,即为获得了重组质粒pMG36e-Usp45-CRAMP。
(4)乳酸乳球菌电转化感受态细胞的制备:将冻存的L.lactis NZ9000乳酸乳球菌划GM17平板复苏,挑取单个菌落在GM17液体培养辈中30℃培养过夜,以1:100的比例接入50mL新的GM17液体培养基30℃培养,监测OD500至0.3-0.4,迅速置冰上冷却,4℃6000×g离心20min,弃上清;用50mL预冷的0.5M蔗糖、10%甘油溶液重悬菌体,4℃6000×g离心20min,弃上清;用25mL预冷的0.5M蔗糖、10%甘油、50mM EDTA溶液重悬菌体,4℃6000×g离心15min,弃上清;再用15mL预冷的0.5M蔗糖、10%甘油溶液重悬菌体,4℃6000×g离心15min,弃上清;最后用500μL预冷的0.5M蔗糖、10%甘油溶液重悬菌体,即为乳酸乳球菌感受态细胞,50μL每管分装,-80℃保存备用。
(5)乳酸乳球菌的电击转化及转化子的PCR鉴定:各取50μL L.lactis NZ9000感受态细胞,冰浴上融解,加入1μL步骤1构建的重组质粒pMG36e-Usp45-CRAMP,轻轻混匀;分别将以上混合物转入冰预冷的2mm电激杯,迅速给予一个单脉冲,参数设置为2kV,25F,200Q,电击后立即轻柔加入1mL冰预冷的恢复培养基GM17培养基,再分别将菌液全部吸入一灭菌离心管,盖紧管盖,冰浴5min后30℃静置培养2h;将含质粒pMG36e-Usp45-CRAMP的菌液分为10μL、l00μL、900μL均匀涂布于含有5ug/mL红霉素的GM17平板,30℃静置培养1-2天。挑取单个菌落,取菌落进行PCR鉴定,具体操作过程如前述步骤(2)所述,区别在于将模板换成待检重组乳酸乳球菌菌液,PCR产物用1%琼脂糖凝胶电泳进行检测,可见约228bp的扩增条带(图3),阳性重组表达菌命名为L.lactis NZ9000/pMG36e-Usp45-CRAMP。
实施例2重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP的构建
(1)基因序列的密码子偏好性优化与合成:根据目的基因CRAMP基因的序列和表达载体pNZ8148的特点,以及为达到高效分泌表达的目的而增加的信号肽序列Usp45,采用人工合成的方法将Usp45-CRAMP基因的228bp的密码子优化序列送公司进行合成。Sph1-Usp45-CRAMP-F为含有与pNZ8148融合表达的酶切位点Xbal和信号肽Usp45-CRAMP的5'端最初一段序列的上游引物,Usp45-CRAMP-Xbal-R为信号肽Usp45-CRAMP基因反向引物。优化合成的Usp45-CRAMP序列如SEQ ID NO:4所示;优化合成的Sph1-Usp45-CRAMP-F,Usp45-CRAMP-Xbal-R引物序列分别如SEQ ID NO:7~8所示。
(2)Usp45-CRAMP基因片段的PCR扩增:以含优化合成的Usp45-CRAMP基因为模板,加入高保真DNA聚合酶KOD-Plus-(1.0U/ul)1μL,0.3μM的引物Sph1-Usp45-CRAMP-F,Usp45-CRAMP-Xbal-R各1.5μL,模板1.5μL,25mM MgSO4 2μL,2mM dNTPs 5μL,10×Buffer forKOD-Plus-5μL,用ddH2O补至50μL,PCR反应程序为:94℃预变性5min;94℃变性30s,55℃退火30s,72℃延伸1min,35个循环;72℃后延伸10min。PCR反应完成,将产物进行1.0%琼脂糖凝胶观察与回收,可见大小约228bp的扩增条带,与预期结果一致(如图1),回收的产物将作为连接模板用于获得添加Usp45-CRAMP序列的完整片段。
(3)重组质粒pNZ8148-Usp45-CRAMP的构建:将步骤(2)中回收的PCR产物用Sph1、Xbal进行双酶切处理,胶回收大小约228bp的条带;以同样的方法对pNZ8148空质粒进行双酶切,胶回收大小约3100bp的条带。分别取4μL双酶切后胶回收的Usp45-CRAMP基因片段和1μL双酶切后胶回收的pMG36e空质粒,将Usp45-CRAMP和pMG36e按照摩尔比6:1添加,并加入10×ligation buffer 2μL,T4 DNA Ligase(350U/μL)1μL,用ddH2O补至20μL,混匀后置于4℃条件下连接过夜,将连接产物转化E.coli MC1061感受态细胞,在含有5μg/mL氯霉素(Chloramphenicol,Ch)的LB琼脂培养板中,37℃培养两天,然后挑取单个菌落进行PCR鉴定。PCR鉴定以待检菌落为模板,加入高保真DNA聚合酶KOD-Plus-(1.0U/ul)1μL,0.3μM的引物Sph1-Usp45-CRAMP-F,Usp45-CRAMP-Xbal-R各1.5μL,模板1.5μL,25mM MgSO4 2μL,2mMdNTPs 5μL,10x Buffer for KOD-Plus-5μL,用ddH20补至50μL,PCR反应程序为:94℃预变性5min;94℃变性30s,55℃退火30s,72℃延伸1min,35个循环;72℃后延伸10min。PCR反应完成,将产物进行1.0%琼脂糖凝胶观察与回收,可见大小约228bp的扩增条带,与预期结果一致(如图2),对检测阳性的菌液用质粒DNA抽提试剂盒进行质粒抽提,并进行双酶切鉴定和测序测定,即为获得了重组质粒pMG36e-Usp45-CRAMP。
(4)乳酸乳球菌电转化感受态细胞的制备:将冻存的L.lactis NZ9000乳酸乳球菌划GM17平板复苏,挑取单个菌落在GM17液体培养辈中30℃培养过夜,以1:100的比例接入50mL新的GM17液体培养基30℃培养,监测OD500至0.3-0.4,迅速置冰上冷却,4℃6000×g离心20min,弃上清;用50mL预冷的0.5M蔗糖、10%甘油溶液重悬菌体,4℃6000×g离心20min,弃上清;用25mL预冷的0.5M蔗糖、10%甘油、50mM EDTA溶液重悬菌体,4℃6000×g离心15min,弃上清;再用15mL预冷的0.5M蔗糖、10%甘油溶液重悬菌体,4℃6000×g离心15min,弃上清;最后用500μL预冷的0.5M蔗糖、10%甘油溶液重悬菌体,即为乳酸乳球菌感受态细胞,50μL每管分装,-80℃保存备用。
(2)乳酸乳球菌的电击转化及转化子的PCR鉴定:各取50μL L.lactis NZ9000感受态细胞,冰浴上融解,加入1μL重组质粒pNZ8148-Usp45-CRAMP重组质粒,轻轻混匀;分别将以上混合物转入冰预冷的2mm电激杯,迅速给予一个单脉冲,参数设置为2kV,25F,200Q,电击后立即轻柔加入1mL冰预冷的恢复培养基GM17培养基,再分别将菌液全部吸入一灭菌离心管,盖紧管盖,冰浴5min后30℃静置培养2h;将含质粒pNZ8148-Usp45-CRAMP的菌液分为10μL、l00μL、900μL均匀涂布于含有5ug/mL氯霉素的M17平板,30℃静置培养1-2天。挑取单个菌落,取菌落进行PCR鉴定,具体操作过程如步骤(2)所述,区别在于将模板换成待检重组乳酸乳球菌菌液,PCR产物用1%琼脂糖凝胶电泳进行检测,可见约228bp的扩增条带(图3),阳性重组表达菌命名为L.lactis NZ9000/pNZ8148-Usp45-CRAMP。
实施例3含CRAMP基因的分泌型重组乳酸乳球菌体外诱导表达
将实施例1构建的重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP重组表达菌以1:100的比例分别接种于含有5ug/mL红霉素的GM17液体培养基,将实施例2构建的重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP以1:100的体积比接种于含有5ug/mL氯霉素的GM17液体培养基,30℃静置培养过夜;将过夜培养物以1:50的比例接种于10mL含有对应抗生素的液体培养基,继续培养约2.5h至细菌进入对数生长期(OD500=0.4~0.6),向L.lactis NZ9000/pNZ8148-Usp45-CRAMP培养体系中加入40ng/mL的乳酸链球菌素(nisin)诱导4h,4℃10000rpm离心5min,收集培养上清,经SDS-PAGE电泳并进行Western Blot分析,结果显示,L.lactis NZ9000/pNZ8148-Usp45-CRAMP和L.lactis NZ9000/pMG36e-Usp45-CRAMP培养上清中检测到17KDa的目的条带(如图4),表明目的基因已经得到分泌表达。
实施例4乳酸乳球菌制备疫苗中的应用
L.lactis NZ9000/pNZ8148-Usp45-CRAMP和L.lactis NZ9000/pMG36e-Usp45-CRAMP重组乳酸乳球菌口服疫苗的制备:将实施例1构建的重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP以1:100的体积比分别接种于5ug/mL红霉素GM17液体培养基,将实施例2构建的重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP以1:100的体积比分别接种于5ug/mL氯霉素的GM17液体培养基,30℃静置培养过夜,将过夜培养物以1:100的比例接种于10mL含有相应抗生素的GM17液体培养基,继续培养约2.5h至细菌进入对数生长期(采用梯度稀释涂板测定重组菌的浓度达1012CFU/mL数量级),此时的全培养物直接作为口服疫苗使用,或离心收集菌体,将菌体作为口服疫苗的主要成分。
实施例5乳酸乳球菌在预防急性结肠炎中的应用
将实施例4制备的含重组乳酸乳球菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP和L.lactis NZ9000/pMG36e-Usp45-CRAMP全培养物的口服疫苗用于预防急性结肠炎。将84只6~8周龄周龄的雄性Balb/c小鼠随机分为6组饲养,每组5只,第1组为生理盐水对照,第2组急性结肠炎模型,第3组L.lactis NZ9000/pMG36e组,第4组L.lactis NZ9000/pNZ8148组,第5组L.lactis NZ9000/pMG36e-Usp45-CRAMP组,第6组L.lactis NZ9000/pNZ8148-Usp45-CRAMP组(即口服疫苗)。预饲一周后,采用灌胃的方式在3%DSS饮水7天后进行口服免疫,连续免疫4天,剂量为160μL/只。然后连续10天处死小鼠,对肠道屏障及炎症相关因子测定。结果显示(图6-图13):
(1)各组小鼠第10天的小鼠比第7天平均体重相比:第1组增重1.084g,第2组下降2.19688g,第3组下降1.984g,第4组下降1.658g,第5组增重0.948g,第6组增重0.732g;
(2)各组第10天的平均结肠长度为:第1组为9.66厘米,第2组为5.32厘米,第3组为6.43厘米,第4组为6.41厘米,第5组为6.88厘米,第6组为6.86厘米;
(3)各组在第10天时DAI评分结果为:第1组0.2,第2组为7.2,第3组为6.6,第4组为6.4,第5组为4.2,第6组4.0;
(4)各组结肠形态学评分结果为:第1组0.2,第2组为3.8,第3组为3.0,第4组为3.4,第5组为2.4,第6组2.4;
(5)各组结肠紧密连接蛋白变化情况为:第2组与第1组相比:ZO-1(p<0.01)、ZO-2(p<0.0001)和occludin(p<0.0001)表达量显著下降;第5组与第2组相比:ZO-1(p<0.05)、ZO-2(p<0.05)和occludin(p<0.05)表达量显著增加;第6组与第2组相比:ZO-1(p<0.05)、ZO-2(p<0.05)和occludin(p<0.05)表达量显著增加;可见,口服疫苗可使ZO-1、ZO-2和occludin的表达量相对于结肠炎组恢复约50%;
(6)各组结肠炎症因子变化情况为:第2组与第1组相比:IL-6(p<0.0001)、IL-1β(p<0.0001)、TNF-α(p<0.0001)显著上升,IL-10(p<0.0001)显著下降;第5组与第2组相比:IL-6(p<0.05)、IL-1β(p<0.05)、TNF-α(p<0.05)显著下降,IL-10(p<0.05)显著上升;第6组与第2组相比:IL-6(p<0.05)、IL-1β(p<0.05)、TNF-α(p<0.01)显著下降,IL-10(p<0.05)显著上升;可见,口服疫苗可使炎症因子IL-6、IL-1β、TNF-α水平相对于结肠炎模型组降低30~50%,并使IL-10升高至少一倍;
(7)各组结肠关键转录因子蛋白水平变化为:第2组与第1组相比:p-ERK/ERK(p<0.01)、p-p38/p38(p<0.01)和p-NF-kB/NF-kB(p<0.01)显著增加;第5组和第2组相比:p-p38/p38(p<0.05)和p-NF-kB/NF-kB(p<0.05)显著降低,p-ERK/ERK(p>0.05)无显著差异性;第6组和第2组相比:p-ERK/ERK(p<0.05)、p-p38/p38(p<0.05)和p-NF-kB/NF-kB(p<0.05)显著降低。
以上结果表明,应用含L.lactis NZ9000/pMG36e-Usp45-CRAMP或L.lactisNZ9000/pNZ8148-Usp45-CRAMP的口服疫苗能够很好的恢复肠道屏障,减轻炎症细胞浸润,抑制炎症细胞因子分泌,具有很好的恢复效果。
对比例1含CRAMP基因的分泌型重组乳酸乳球菌
以现有技术中含CRAMP基因的分泌型重组乳酸乳球菌作为对照,其表达的CRAMP基因(GGACTTCTCCGCAAAGGTGGGGAGAAGATTGGTGAAAAGCTTAAGAAAATTGGCCAGAAAATTAAGAATTTTTTTCAGAAACTTGTACCTCAGCCAGAG)未经过密码子优化,不能促进Usp45信号肽和CRAMP基因在胞内自剪切作用,不能促进CRAMP在细胞外分泌,上清中分泌的CRAMP蛋白含量低,表达产物约为1.5ng/μL。
经ELISA检测比较对比例1与实施例1~2构建的重组菌表达CRAMP的能力,结果显示(图5),重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP和重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP菌体的CRAMP蛋白表达量约为20ng/μL,比现有技术相比(1.5ng/μL)高13倍,重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP分泌至胞外的蛋白量约为40ng/μL,比对比例的表达量(1.5ng/μL)高27倍,重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP分泌至胞外的CRAMP蛋白表达量约为60ng/μL,比对比例1(1.5ng/μL)高40倍。
SEQUENCE LISTING
<110> 江南大学
<120> 一种表达小鼠抗菌肽基因的乳酸乳球菌
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 102
<212> DNA
<213> 人工序列
<400> 1
ggtctgctgc gtaaaggcgg cgagaagatc ggcgagaagc tgaagaagat cggccagaag 60
atcaagaact tcttccagaa actggtgccg cagccggaat aa 102
<210> 2
<211> 34
<212> PRT
<213> 人工序列
<400> 2
Gly Leu Leu Arg Lys Gly Gly Glu Lys Ile Gly Glu Lys Leu Lys Lys
1 5 10 15
Ile Gly Gln Lys Ile Lys Asn Phe Phe Gln Lys Leu Val Pro Gln Pro
20 25 30
Glu Gln
<210> 3
<211> 114
<212> DNA
<213> 人工序列
<400> 3
atgaaaaaaa aaatcatcag cgcgattctg atgagcaccg ttattctgag tgccgccgcc 60
ccactgagtg gcgtttatgc cgacaccaac agcgatatcg ccaaacaaga tgcc 114
<210> 4
<211> 228
<212> DNA
<213> 人工序列
<400> 4
gcatgcatga aaaaaaaaat catcagcgcg attctgatga gcaccgttat tctgagtgcc 60
gccgccccac tgagtggcgt ttatgccgac accaacagcg atatcgccaa acaagatgcc 120
ggtctgctgc gtaaaggcgg cgagaagatc ggcgagaagc tgaagaagat cggccagaag 180
atcaagaact tcttccagaa actggtgccg cagccggaat aatctaga 228
<210> 5
<211> 40
<212> DNA
<213> 人工序列
<400> 5
tctagaatga aaaaaaaaat catcagcgcg attctgatga 40
<210> 6
<211> 40
<212> DNA
<213> 人工序列
<400> 6
gcatgcttat tccggctgcg gcaccagttt ctggaagaag 40
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<211> 40
<212> DNA
<213> 人工序列
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gcatgcatga aaaaaaaaat catcagcgcg attctgatga 40
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<212> DNA
<213> 人工序列
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tctagattat tccggctgcg gcaccagttt ctggaagaag 40
Claims (13)
1.一种重组乳酸乳球菌,其特征在于,表达并分泌CRAMP蛋白;所述CRAMP蛋白的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的重组乳酸乳球菌,其特征在于,以乳酸乳球菌NZ9000为宿主。
3.根据权利要求1或2所述的重组乳酸乳球菌,其特征在于,以pMG36e或pNZ8148为载体。
4.根据权利要求1或2所述的重组乳酸乳球菌,其特征在于,以Usp45信号肽促进CRAMP蛋白的表达;所述Usp45信号肽的核苷酸序列如SEQ ID NO.3所示。
5.根据权利要求3所述的重组乳酸乳球菌,其特征在于,以Usp45信号肽促进CRAMP蛋白的表达;所述Usp45信号肽的核苷酸序列如SEQ ID NO.3所示。
6.一种构建权利要求1~5任一所述重组乳酸乳球菌的方法,其特征在于,将编码SEQID NO.1所示CRAMP蛋白的基因与载体连接,再转化至乳酸乳球菌细胞中;所述载体为pMG36e或pNZ8148。
7.一种组合物,其特征在于,含有权利要求1~5任一所述的重组乳酸乳球菌;所述重组乳酸乳球菌的含量≥1×105CFU/mL或1×105CFU/g。
8.一种口服疫苗,其特征在于,含有权利要求1~5任一所述的重组乳酸乳球菌或其全培养物;所述全培养物按如下步骤制备:将所述重组乳酸乳球菌接种于GM17液体培养基静置培养过夜,以一定比例转接于GM17液体培养基,继续培养至细菌进入对数生长期,全培养物直接作为口服疫苗。
9.根据权利要求8所述的口服疫苗,其特征在于,所述静置培养的温度为28~30℃。
10.根据权利要求8所述的口服疫苗,其特征在于,所述转接是将所述的重组乳酸乳球菌以体积比1:100的比例接种于GM17液体培养基。
11.根据权利要求8所述的口服疫苗,其特征在于,所述对数生长期的细菌培养液的OD值为0.4~0.6。
12.权利要求1~5任一所述的重组乳酸乳球菌在制备可引入肠道的功能性食品或药物中的应用,其特征在于,具有如下至少一种功能:
(a)抑制肠道炎症;
(b)重塑肠黏膜屏障;
(c)改善肠黏膜通透性;
(d)预防或治疗肠道炎症以及肠道炎症引起的疾病;
(e)降低炎症因子IL-6、IL-1β、TNF-α水平。
13.权利要求1~5任一所述的重组乳酸乳球菌在制备发酵食品中的应用。
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Title |
---|
Cramp 转基因小鼠的构建及鉴定;石桂英 等;《中国比较医学杂志》;20100930;第12-15页,参见全文 * |
Identification of CRAMP,a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse;Gallo RL 等;《J Biol Chem》;19971231;第13088-13093页,参见全文 * |
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