CN116138341A - 一种含有重组菌的口香糖及配套牙膏 - Google Patents
一种含有重组菌的口香糖及配套牙膏 Download PDFInfo
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- CN116138341A CN116138341A CN202211636857.3A CN202211636857A CN116138341A CN 116138341 A CN116138341 A CN 116138341A CN 202211636857 A CN202211636857 A CN 202211636857A CN 116138341 A CN116138341 A CN 116138341A
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- chewing gum
- gene
- recombinant bacteria
- recombinant
- toothpaste
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Abstract
本发明提出了一种含有重组菌的口香糖,以及含有该重组菌诱导物的牙膏,所述含有重组菌中的口香糖中,重组菌的基因组上导入四环素感知基因和抗菌药物表达基因,而牙膏中含有重组菌诱导信号分子,具体为四环素信息分子。当患者通过咀嚼口香糖的方法,使重组菌定植于口腔中,通过使用牙膏,释放四环素信息分子,从而激活重组菌中的相对基因表达抗菌药物,达到治疗龋齿或预防龋齿的目的。选择夜间致病菌较为活跃的时期,释放抗菌药物,能够有效的抑制细菌合成生物膜,提高用药效率,实现智能给药,精准用药,避免过度用药。
Description
技术领域
本发明涉及食品技术领域,尤其涉及一种含有重组菌的口香糖及配套牙膏。
背景技术
口腔中含有丰度巨大的微生物,每种微生物均具有特定的功能,微生物种属之间、微生物与宿主之间据有关相互作用,形成动态稳定的状态,口腔内平衡被打破后,口腔疾病随之而来。龋齿就是由于口腔微生物失衡生长造成的典型口腔疾病之一。就目前主流学说认为,龋齿的发生机制主要与四种因素有关,具体为口腔细菌、口腔环境、宿主和时间,简单来说,致龋性食物糖紧紧贴于牙面,由涎液蛋白形成的获得性膜上,该生物膜能够牢固的附着于牙面,并给口腔细菌提供了充足的生长物质和生长环境,在适宜温度下,有足够的时间在菌斑深层产酸,侵袭牙齿,使之脱矿,并进而破坏有机质,产生龋洞。
对于龋齿的防治,常用的方法有以下几种;1、机械法,通过医学手段直接清楚牙齿表面的牙菌斑,同时应用密封剂对驱动进行密封,但医学手段往往给患者打来巨大的痛苦2、药物法,通常用于龋齿发生后,主要使用氟化物、酚类化合物等治疗龋,口腔给药时往往是以药片或缓释剂的形式进行服用,其有效成分能够在口腔中持续性的、大量的发挥作用,实际上忽略了口腔环境的变化,没有针对性地、区别性地对不同时间或不同机制进行给药,可能带来过度用药的不良后果,也不利于口腔环境的稳定与平衡;3、重组菌法,通过在口腔中引入重组菌来调整口腔中的生态平衡,改善口腔环境,在使用该方法时不得不考虑到重组菌在口腔中的定植时间,对于口腔环境来说,重组菌在口腔内的适应性不足,发挥作用的时间较短,难以避免治疗效果差、治疗周期增长的问题。
发明内容
不同时间段内,口腔细菌的活跃程度不同,尤其是在进食后和夜间睡眠的时间内,细菌活跃度最高。如链球菌属能够在生长过程中在牙齿表面产生生物被膜,牢固黏附在牙齿表面,在睡眠期间,口腔活动减少,唾液分泌减少,为细菌在牙齿表面生成生物被膜提供良好的时机。
现有技术中,对重组菌进行改造用以温度、pH等人体内部的环境因素实现诱导表达,由于人体内不可控,仍然不能解决重组菌对龋齿的治疗效果受到干扰较大的技术问题。因此,本发明设计将将诱导物和重组菌分离,并将两者分别融入到日常生活中的日化产品中。
在本发明中,提出了一种口香糖和牙膏配合使用的技术方案。重组菌被经过基因工程改造后,植入口香糖内,通过口香糖的日常咀嚼充分的附着在口腔内,并在一个较长的时间内存活于口腔。该菌在诱导后引发合成口腔治疗药物,而诱导物被存放在配套的另一种牙膏中,夜间刷牙之后将触发重组菌合成相关的抗菌药物。
本发明提供了一种含有重组菌的口香糖,以及含有该重组菌诱导物的牙膏。
本发明所述的一种含有重组菌的口香糖中,所述重组菌的基因组上导诱导物感知基因和抗菌药物表达基因;
优选地,所述诱导物感知基因为四环素感知基因;
优选地,所述四环素感知基因为pVanA启动子基因、TetR基因和TRE基因;
优选地,所述四环素感知基因为PR基因、TetR基因、TRE基因和cI基因;
优选地,所述重组菌的基因组构建于载体pET28a质粒上;
优选地,所述重组菌的起始菌为E.coli Nissle 1917、植物乳杆菌(Lactiplantibacillus plantarum)、唾液乳杆菌(Lactobacillus salivarius)、发酵乳杆菌CECT5716((Lactobacillus fermentum)和罗伊氏乳杆菌(Lactobacillus reuteri)中的至少一种;
优选地,所述重组菌表达的抗菌药物种类包括糖苷酶、抗菌肽LL-37、防御素、抗菌肽bactenecin、富组蛋白中的至少一种;
在优选的实施方案中,本发明还提供了一种含有上述重组菌的口香糖,所述口香糖的原料包括以下组分,按重量百分比组成:重组菌冻干菌粉0.1-5%,柠檬酸0.1-1%,甘油1-10%,木糖醇50-70%,胶基30-40%;
本发明还提供一种含有重组菌的口香糖的制备方法,具体包括以下步骤:
(1)重组菌活化:将重组菌接种于LB液体培养基中,在37℃恒温培养箱中培养24-48h;
(2)重组菌冻干粉的制备:5000g-10000g高速离心分离10-20min,分离得到的菌体使用无菌生理盐水洗涤1-2次,将菌体置于真空冷冻干燥机中,设置参数-40℃,4Pa,冷冻干燥48-72h;收集冻干粉置于-70℃冰箱备用;
(3)口香糖胶基的预处理:将胶基置于50-60℃恒温烘箱内,保湿软化2-4h;
(4)口香糖混合料:将软化后的口香糖胶基降温至40-50℃,将其加入搅拌机中,准确称取重组菌冻干菌粉、木糖醇、柠檬酸、甘油保持搅拌温度40-50℃,搅拌10-15min,使其充分搅拌均匀;
(5)延压切割:将混合物投入切面机中挤压,反复挤压制成组织结构紧密的口香糖,切割成1cm×3cm规格
(6)老化:将切好的块状口香糖盛放于托盘上,置于温度20℃,相对湿度30%~60%的条件下老化24h;
(7)包装:将口香糖用涂蜡锡箔纸包装,置于老化条件下贮藏。
本发明还提供一种与上述含有重组菌口香糖配合使用的牙膏,所述牙膏的原料包括以下组分,按重量份组成:诱导物信息分子0.2-0.8份,碳酸钙25-40份,氢氧化铝5-10份,二氧化硅5-10份,山梨糖醇10-15份,十二烷基硫酸钠1-6份,羧甲基纤维素钠0.5-2份,甘油5-10份,去离子水25-35份;
优选地,所述诱导物信息分子为四环素诱导物信息分子;
所述与重组菌口香糖配合使用的牙膏的制备方法,具体包括以下步骤:
a.摩擦剂的制备:将碳酸钙、氢氧化铝、二氧化硅分别用粉碎机粉碎,在加入混合机中混合均匀;
b.甜味剂的制备:将山梨糖醇用去离子水溶解;
c.牙膏膏体的制备:将甘油、摩擦剂、甜味剂、四环素信息分子、十二烷基硫酸钠、羧甲基纤维素钠,在真空条件下以7500-8000rpm/min高速搅拌10-15min,制得膏体;
d.包装:将膏体用复合软管包装。
本发明还提供一种配合使用的含有上述重组菌的口香糖和含有上述诱导物的牙膏进行口腔治疗或保健的方法,其特征在于,使用者预先咀嚼口香糖对口腔进行不定期的重组菌植入,再通过含有诱导物的牙膏激发药效,实现特定时间的精确释药。
有益效果:
本发明中使用了两样日常物品——口香糖和牙膏,而不需要使用者额外使用药物。口香糖日常被咀嚼,可以有效地将口香糖的物质在口腔内充分的进行分散,其咀嚼和粘附的特性,可以将重组菌充分分散到牙缝的深处。但口香糖本身不具备控制药物的能力,它只能在空间上进行有效的分散。牙膏一般在夜间使用,由于刷牙的时间有限,不如口香糖能全天候多次使用以充分植入重组菌。在引入基因工程改造菌后,将两者组合,在白天通过咀嚼口香糖使重组菌充分的附着到口腔内。由于缺少诱导物,白天重组菌并不工作并不合成抗菌药物以节省生物资源。夜间刷牙的时候,牙膏中所附带的少量诱导物触发口腔护理药物合成,实现精准的夜间保护,同时避免白天过度用药。两者组合之后,优势互补同时解决各自缺点,并且通过这种组合提高了工程改造菌的口腔护理效率而没有增加使用者额外的负担,产生了这两种日用品常规不具有的效果。
本发明利用夜间为口腔防护的重点时间。使用基因改造后的重组菌引入口腔治疗药物是一种有效的方法,但是该方法受到其重组菌定植效率和给药效果的影响。首先,重组菌在持续合成治疗药物时将降低存活能力,然后进一步影响定值效果。其次,重组菌需要尽可能进入牙齿的缝隙深处以有效的发挥作用。尤其对于糖苷酶一类的通过分解口腔致病菌的生物膜物质来保护口腔的物质,它们对于生物膜的破坏,需要尽可能的接触牙齿的最深处。
在优选的实施方案中,本发明将能够感知四环素的感知基因和抗菌药物表达基因导入目标菌株,通过感知环境中是否含有四环素,而选择性表达抗菌药物。本发明所制备的口香糖中含有该重组菌,能够通过咀嚼口香糖将重组菌定植在口腔环境中。在一般情况下,口腔环境中不存在四环素信号分子,因此,重组菌内的感知基因没有受到激活,抗菌药物表达受到抑制;当使用本申请所制备的具有重组菌信号分子的牙膏进行刷牙时,牙膏中的四环素信息分子释放到口腔环境中,能够激活重组菌中的四环素感知基因,下游抗菌药物基因能够表达合成抗菌药物,释放到口腔环境中。本发明所述含有重组菌信号分子的牙膏多用于夜间睡前清洁,此时,口腔中的重组菌充分感受到信号分子,启动预定程序抗菌药物,由于夜间是抑制口腔致病菌形成生物膜的重要时段,在此间释放抗菌药物能够有效地抑制细菌合成生物膜,提高用药效率,实现智能给药,精准用药,避免过度用药。
附图说明
图1为本发明实施例1所述重组质粒结构图。
图2为本发明实施例2所述重组质粒结构图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。此外,下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为具体公开了该范围的上限和下限以及它们之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
实施例1
本实施方案包括制备一套含有基因工程改造的重组菌的口香糖,和含有对应该重组菌诱导物的牙膏的组合物的方法。
所述重组菌质粒上具有四环素感知基因和抗菌药物表达基因;所述四环素感知基因依次包括pVanA启动区,TetR基因,TRE基因;
所述抗菌药物表达基因为DspB基因,所表达抗菌药物为β-N-乙酰氨基葡萄糖苷酶;
其中pVanA启动区域为序列SEQ ID NO:9,DspB为序列SEQ ID NO:10;
所述重组菌质粒的基因从上游至下游依次为pVanA启动子,TetR基因,TRE基因,DspB基因。
在该序列中,TetR基因和TRE基因构成四环素诱导控制系统,TRE基因具有TetO操纵子重复排序的特定结构,pVanA启动子基因是固定表达TetR基因的启动子。
不存在四环素诱导时,pVanA固定表达TetR基因,TetR基因表达出的TetR蛋白默认与TRE结构进行结合,从而阻碍TRE基因下游基因DspB的表达,此时,该重组菌质粒不能合成β-N-乙酰氨基葡萄糖苷酶。
存在四环素诱导时,四环素与TetR基因表达的TetR蛋白进行结合,而不与TRE结构进行结合,TRE基因失去阻碍,能够顺利表达其下游DspB基因,合成β-N-乙酰氨基葡萄糖苷酶,达到抑菌作用。本发明中所重组菌的起始菌为E.coli Nissle 1917,以质粒pET28a载体,将四环素感应基因与抗菌药物表达基因与载体片段连接,得到重组质粒。
可使用不同方法获得质粒,例如全合成或无缝克隆方法等。
使用全合成法,可以使用重叠PCR方法,一般可由试剂公司直接完成,所述重组菌目标质粒的完整序列如SEQ ID NO:1所示。
使用无缝克隆法,将重组基因目标序列作为PCR扩增模板,所述重组基因目标序列包括四环素感知基因和抗菌药物表达基因的完整序列片段,进行PCR扩增后,通过GibsonAssembly方法上述基因插入质粒;所述插入目标基因片段的序列如SEQ ID NO:2所示;
根据载体和插入序列设计引物序列:V-F和V-R用来扩增载体骨架;F-F和F-R包含和插入片段序列和载体重叠序列,用于扩增插入片段,15~25bp的重叠区用于GibsonAssembly。
V-F/V-R做引物,pET28a作为模板通过PCR的方式扩增载体骨架。F-F/F-R做引物,包含合成序列的质粒作为模板,同样通过PCR的方式扩增插入片段。使用高保真酶2x预混液,扩增温度根据引物Tm值调整(Tm值下2~3度),扩增时间根据扩增长度和酶扩增速度而调整。琼脂糖凝胶电泳分析扩增产物长度是否正确,扩增正确的PCR产物通过凝胶回收试剂盒回收扩增产物。对凝胶回收的扩增产物进行定量测量,评估浓度、纯度。
所述在载体上插入目标基因序列所使用的引物详细信息如下:
引物名称 | 引物序列 |
V-F | CACCACCACCACCACCACTGAGATC |
F-F | TGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCCGGGA |
V-R | TGTAAGTTAGCTCACTCATTAGGCACCGGG |
F-R | CAGTGGTGGTGGTGGTGGTGTCACTCATCCCCATTCGTCTTATGA |
混合纯化后的载体片段、插入序列片段(摩尔比1:1~1:3,载体片段总量~50ng,片段用量根据和载体的长度比来调整)和Gibson Assembly 2x预混液(使用过通用生物、生工的对应产品),反应体系总体积10ul,50℃30min,结束后置于冰上等待转化。
电转感受态细菌的制备:
A.接种Nissle1917于含有5ml新鲜灭菌的LB培养基的试管中,37℃震荡培养过夜;
B.取上述过夜培养的菌液1ml至含有100ml新鲜灭菌LB培养基的250ml锥形瓶中,37℃震荡培养;
C.检测OD600,当OD600在0.5~0.8之间时,将培养物置于冰上15min;
D.将冰上冷却的培养物转移到冰上预冷的50ml无菌离心管中(注意配平),4℃,4000g离心15min,弃净上清;
E.30ml提前冰浴的灭菌水重悬菌体沉淀,4℃,4000g离心15min,弃净上清,重复该步骤一次;
F.30ml高压灭菌并提前冰浴的10%甘油重悬菌体沉淀,4℃,4000g离心15min,弃净上清;
G.取5ml上述10%甘油重悬菌体沉淀,以100ul等份分装入无菌EP管中,液氮冷冻,-80℃长期存放。
所述重组质粒导入感受态细胞的方法为电转化法,具体包括以下步骤:
I.冰上解冻电转感受态细胞,将Gibson assembly加入到电转感受态中,冰上孵育5~10min;
II.转移DNA/感受态混合物至冰上预冷的电击杯中;
III.将电击杯放入电转仪上,启动点击程序;
IV.电击结束后,立刻向感受态细胞中加入约0.5~1ml新鲜LB培养基,无菌条件下转移至无菌EP管中。37℃震荡培养约50min;
V.离心收集菌体沉淀,保留部分液体培养基,重悬菌体,涂布到卡纳(pET28a)抗性的固体LB平板上;37℃培养过夜。
为了保证重组菌中质粒序列是否与目标一致,进行克隆鉴定进行验证,具体包括以下步骤:
a)分装含有卡纳抗性的LB培养基至无菌EP管中,挑取37℃培养过夜平板上的克隆至EP管中,37℃震荡培养,至能看到较为明显的浑浊;
b)F-F/F-R做引物,菌液做模板,进行PCR扩增(低保真酶预混液),并设置阴性和阳性对照;
c)琼脂糖凝胶鉴定克隆的扩增信号是否和阳性对照一致;
d)对于PCR鉴定正确的克隆,吸取菌液样品或扩增后抽取的质粒样品,进一步进行测序鉴定;
e)对于测序正确的克隆,备份菌液样品和质粒样品。
在所述重组质粒中,插入目的基因序列包括pVanA基因序列、TetR基因序列、TRE基因序列和DspB基因序列;
所述pVanA序列为:
ATTGGATCCAATTGACAGCTAGCTCAGTCCTAGGTACCATTGGATCCAA TAAGGAGGAAAAAAAA;
所述四环素操纵区域(TetR基因序列、TRE基因序列)序列为:
ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGC GCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGA;
所述DspB基因序列为:
ATGAATTGTTGCGTAAAAGGCAATTCCATATATCCGCAAAAAACAAGTACCAAGCAGACCGGATTAATGCTGGACATCGCCCGACATTTTTATTCACCCGAGGTGATTAAATCCTTTATTGATACCATCAGCCTTTCCGGCGGTAATTTTCTGCACCTGCATTTTTCCGACCATGAAAACTATGCGATAGAAAGCCATTTACTTAATCAACGTGCGGAAAATGCCGTGCAGGGCAAAGACGGTATTTATATTAATCCTTATACCGGAAAGCCATTCTTGAATTATCGGCAACTTGACGATATCAAAGCCTATGCTAAGGCAAAAGGCATTGAGTTGATTCCCGAACTTGACAGCCCGAATCACATGACGGCGATCTTTAAACTGGTGCAAAAAGACAGAGGGGTCAAGTACCTTCAAGGATTAAAATCACGCCAGGTAGATGATGAAATTGATATTACTAATGCTGACAGTATTACTTTTATGCAATCTTTAATGAGTGAGGTTATTGATATTTTTGGCGACACGAGTCAGCATTTTCATATTGGTGGCGATGAATTTGGTTATTCTGTGGAAAGTAATCATGAGTTTATTACGTATGCCAATAAACTATCCTACT TTTTAGAGAAAAAAGGGTTGAAAACCCGAATGTGGAATGACGGATTAATTAAAAATACTTTTGAGCAAATCAACCCGAATATTGAAATTACTTATTGGAGCTATGATGGCGATACGCAGGACAAAAATGAAGCTGCCGAGCGCCGTGATATGCGGGTCAGTTTGCCGGAGTTGCTGGCGAAAGGCTTTACTGTCCTGAACTATAATTCCTATTATCTTTACATTGTTCCGAAAGCTTCACCAACCTTCTCGCAAGATGCCGCCTTTGCCGCCAAAGATGTTATAAAAAATTGGGATCTTGGTGTTTGGGATGGACGAAACACCAAAAACCGCGTACAAAATACTCATGAAATAGCCGGCGCAGCATTATCGATCTGGGGAGAAGATGCAAAAGCGCTGAAAGACGAAACAATTCAGAAAAACACGAAAAGTTTATTGGAAGCGGTGATTCATAAGACGAATGGGGATGAGTGA;
所述插入合成序列为:
TTAATTGCGTTGCGCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCATTGGATCCAATTGACAGCTAGCTCAGTCCTAGGTACCATTGGATCCAATAAGGAGGAAAAAAAAATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACA ACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGATTTGTTTAACTTTAAGAAGGAGATATACCATGAATTGTTGCGTAAAAGGCAATTCCATATATCCGCAAAAAACAAGTACCAAGCAGACCGGATTAATGCTGGACATCGCCCGACATTTTTATTCACCCGAGGTGATTAAATCCTTTATTGATACCATCAGCCTTTCCGGCGGTAATTTTCTGCACCTGCATTTTTCCGACCATGAAAACTATGCGATAGAAAGCCATTTACTTAATCAACGTGCGGAAAATGCCGTGCAGGGCAAAGACGGTATTTATATTAATCCTTATACCGGAAAGCCATTCTTGAATTATCGGCAACTTGACGATATCAAAGCCTATGCTAAGGCAAAAGGCATTGAGTTGA TTCCCGAACTTGACAGCCCGAATCACATGACGGCGATCTTTAAACTGGTGCAAAAAGACAGAGGGGTCAAGTACCTTCAAGGATTAAAATCACGCCAGGTAGATGATGAAATTGATATTACTAATGCTGACAGTATTACTTTTATGCAATCTTTAATGAGTGAGGTTATTGATATTTTTGGCGACACGAGTCAGCATTTTCATATTGGTGGCGATGAATTTGGTTATTCTGTGGAAAGTAATCATGAGTTTATTACGTATGCCAATAAACTATCCTACTTTTTAGAGAAAAAAGGGTTGAAAACCCGAATGTGGAATGACGGATTAATTAAAAATACTTTTGAGCAAATCAACCCGAATATTGAAATTACTTATTGGAGCTATGATGGCGATACGCAGGACAAAAATGAAGCTGCCGAGCGCCGTGATATGCGGGTCAGTTTGCCGGAGTTGCTGGCGAAAGGCTTTACTGTCCTGAACTATAATTCCTATTATCTTTACATTGTTCCGAAAGCTTCACCAACCTTCTCGCAAGATGCCGCCTTTGCCGCCAAAGATGTTATAAAAAATTGGGATCTTGGTGTTTGGGATGGACGAAACACCAAAAACCGCGTACAAAATACTCATGAAATAGCCGGCGCAGCATTATCGATCTGGGGAGAAGATGCAAAAGCGCTGAAAGACGAAACAATTCAGAAAAACACGAAAAGTTTATTGGAAGCGGTGATTCATAAGACGAATGGGGATGAGTGA。
所述含有基因工程改造的重组菌的口香糖的原料包括以下成分,按重量百分比组成:重组菌冻干菌粉1%,柠檬酸0.5%,甘油3%,木糖醇58.5%,胶基37%;
所述重组菌冻干菌粉中含有本实施例所述的重组菌;
所述口香糖的制备方法,具体包括以下步骤:
(1)重组菌活化:将重组菌接种于LB液体培养基中,在37℃恒温培养箱中培养36h;
(2)重组菌冻干粉的制备:8000g高速离心分离15min,分离得到的菌体使用无菌生理盐水洗涤2次,将菌体置于真空冷冻干燥机中,设置参数-40℃,4Pa,冷冻干燥48-72h;收集冻干粉置于-70℃冰箱备用;
(3)口香糖胶基的预处理:将胶基置于60℃恒温烘箱内,保湿软化3h;
(4)口香糖混合料:将软化后的口香糖胶基降温至40℃,将其加入搅拌机中,准确称取重组菌冻干菌粉,木糖醇,柠檬酸,甘油保持搅拌温度40℃,搅拌10-15min,使其充分搅拌均匀;
(5)延压切割:将混合物投入切面机中挤压,反复挤压制成组织结构紧密的口香糖,切割成1cm×3cm规格
(6)老化:将切好的块状口香糖盛放于托盘上,置于温度20℃,相对湿度40%的条件下老化24h;
(7)包装:将口香糖用涂蜡锡箔纸包装,置于老化条件下贮藏。
所述含有重组菌口香糖配合使用的牙膏,所述牙膏的原料包括以下组分,按重量份组成:四环素信息分子0.6份,碳酸钙30份,氢氧化铝8份,二氧化硅8份,山梨糖醇12份,十二烷基硫酸钠3份,羧甲基纤维素钠1份,甘油7份,去离子水31.4份;
所述与重组菌口香糖配合使用的牙膏的制备方法,具体包括以下步骤:
1.摩擦剂的制备:将碳酸钙、氢氧化铝、二氧化硅分别用粉碎机粉碎,在加入混合机中混合均匀;
2.甜味剂的制备:将山梨糖醇用去离子水溶解;
3.牙膏膏体的制备:将甘油、摩擦剂、甜味剂、四环素信息分子、十二烷基硫酸钠、羧甲基纤维素钠,在真空条件下以8000rpm/min高速搅拌15min,制得膏体;
4.包装:将膏体用复合软管包装。
实施例2
本实施方案包括制备一套含有基因工程改造的重组菌的口香糖,和含有对应该重组菌诱导物的牙膏的组合物的方法。相比实施例1,本方案的重组菌在被牙膏诱导后可以持续释放抗菌药物。
所述重组菌的质粒上具有四环素感知基因和抗菌药物表达基因;
所述四环素感知基因依次包括PR启动子,TerT基因,TRE基因,cI基因;
所述抗菌药物表达基因为DspB基因,所表达抗菌药物为β-N-乙酰氨基葡萄糖苷酶;
所述重组菌质粒上的基因从上游至下游依次为PR启动子,TetR基因,TRE基因,cI基因,DspB基因。
在该序列中,TetR基因和TRE基因构成四环素诱导控制系统,TRE基因具有TetO操纵子重复排序的特定结构,cI基因和PR启动子组成了记忆诱导效果的结构。
其中PR启动区域为序列SEQ ID NO:7,cI区域为序列SEQ ID NO:8。
不存在四环素诱导时,PR启动子固定表达TetR基因,TetR基因表达出的TetR蛋白默认与TRE结构进行结合,从而阻碍TRE基因下游基因DspB的表达,此时,该重组菌质粒不能合成β-N-乙酰氨基葡萄糖苷酶。
存在四环素诱导时,四环素与TetR蛋白结合,TRE基因能够同时启动其下游cI基因和DspB基因同时表达,cI基因表达合成阻碍PR启动子的蛋白,使PR下游基因TetR基因表达失效,后续的TRE基因不再受到TetR蛋白的阻碍,能够持续进行表达,不断合成β-N-乙酰氨基葡萄糖苷酶。
本发明所述重组菌可通过不同方法获得质粒,具体可通过基因序列全合成方和无缝克隆方法。
使用全合成法获得质粒,具体使用重叠PCR方法,试剂公司可直接提供完整序列的重组菌目标质粒,所述重组据质粒的完整序列如SEQ ID NO:4所示。
使用无缝克隆法,将重组基因目标序列作为PCR扩增模板,所述重组基因目标序列包括四环素感知基因和抗菌药物表达基因的完整序列片段,进行PCR扩增后,通过GibsonAssembly方法上述基因插入质粒;
根据载体和插入序列设计引物序列:V-F和V-R用来扩增载体骨架;F-F和F-R包含和插入片段序列和载体重叠序列,用于扩增插入片段,15~25bp的重叠区用于GibsonAssembly。
V-F/V-R做引物,pET28a作为模板通过PCR的方式扩增载体骨架。F-F/F-R做引物,包含合成序列的质粒作为模板,同样通过PCR的方式扩增插入片段。使用高保真酶2x预混液,扩增温度根据引物Tm值调整(Tm值下2~3度),扩增时间根据扩增长度和酶扩增速度而调整。琼脂糖凝胶电泳分析扩增产物长度是否正确,扩增正确的PCR产物通过凝胶回收试剂盒回收扩增产物。对凝胶回收的扩增产物进行定量测量,评估浓度、纯度。
所述在载体上插入目标基因序列所使用的引物详细信息与实施例1引入目标基因的引物相同;区别在于,其中的合成序列使用SEQ ID NO:5。
在所述重组质粒中,插入目的基因序列包括PR启动子序列、TetR基因序列、TRE基因序列、cI基因序列和DspB基因序列;
其中,PR启动子序列为:
ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTT GACTATTTTACCTCTGGCGGTGATAATGGTTGCATGTACTAAGGAGGTTGT;
所述cI基因序列为:
ATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTTGAGGACG CACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGA;
所述插入合成序列为:
TTAATTGCGTTGCGCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCG GCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGCATGTACTAAGGAGGTTGTATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGATTTGTTTAACTTTAAGAAGGAGATATACCATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTG GCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGAATGAATTGTTGCGTAAAAGGCAATTCCATATATCCGCAAAAAACAAGTACCAAGCAGACCGGATTAATGCTGGACATCGCCCGACATTTTTATTCACCCGAGGTGATTAAATCCTTTATTGATACCATCAGCCTTTCCGGCGGTAATTTTCTGCACCTGCATTTTTCCGACCATGAAAACTATGCGATAGAAAGCCATTTACTTAATCAACGTGCGGAAAATGCCGTGCAGGGCAAAGACGGTATTTATATTAATCCTTATACCGGAAAGCCATTCTTGAATTATCGGCAACTTGACGATATCAAAGCCTATGCTAAGGCAAAAGGCATTGAGTTGATTCCCGAACTTGACAGCCCGAATCACATGACGGCGATCTTTAAACTGGTGCAAAAAGACAGAGGGGTCAAGTACCTTCAAGGATTAAAATCACGCCAGGTAGATGATGAAATTGATATTACTAATGCTGACAGTATTACTTTTATGCAATCTTTAATGAGTGAGGTTATTGATATTTTTGGCGACACGAGTCAGCATTTTCATATTGGTGGCGA TGAATTTGGTTATTCTGTGGAAAGTAATCATGAGTTTATTACGTATGCCAATAAACTATCCTACTTTTTAGAGAAAAAAGGGTTGAAAACCCGAATGTGGAATGACGGATTAATTAAAAATACTTTTGAGCAAATCAACCCGAATATTGAAATTACTTATTGGAGCTATGATGGCGATACGCAGGACAAAAATGAAGCTGCCGAGCGCCGTGATATGCGGGTCAGTTTGCCGGAGTTGCTGGCGAAAGGCTTTACTGTCCTGAACTATAATTCCTATTATCTTTACATTGTTCCGAAAGCTTCACCAACCTTCTCGCAAGATGCCGCCTTTGCCGCCAAAGATGTTATAAAAAATTGGGATCTTGGTGTTTGGGATGGACGAAACACCAAAAACCGCGTACAAAATACTCATGAAATAGCCGGCGCAGCATTATCGATCTGGGGAGAAGATGCAAAAGCGCTGAAAGACGAAACAATTCAGAAAAACACGAAAAGTTTATTGGAAGCGGTGATTCATAAGACGAATGGGGATGAGTGA。
所述含有重组菌口香糖的原料与实施例1所述口香糖原料相同,其区别在于,所述重组菌冻干菌粉中含有本实施例所述的重组菌;
所述口香糖的制备方法与实施例1所述制备方法相同;
所述含有重组菌口香糖配合使用的牙膏,其制备原料与实施例1相同所述与与重组菌口香糖配合使用的牙膏的制备方法与实施例1相同。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (11)
1.一种含有重组菌的口香糖,其特征在于,所述重组菌的基因组上导入了诱导物感知基因和抗菌药物表达基因。
2.根据权利要求1所述的口香糖,其特征在于,所述诱导物感知基因为四环素感知基因。
3.根据权利要求2所述的口香糖,其特征在于,所述四环素感知基因为pVanA启动子基因、TetR基因和TRE基因。
4.根据权利要求2所述的口香糖,其特征在于,所述四环素感知基因为PR基因、TetR基因、TRE基因和cI基因。
5.根据权利要求1所述的口香糖,其特征在于,所述重组菌的基因组构建于载体pET28a质粒上。
6.根据权利要求1所述的口香糖,其特征在于,所述重组菌的起始菌为E.coliNissle1917、植物乳杆菌、唾液乳杆菌、发酵乳杆菌CECT5716和罗伊氏乳杆菌中的至少一种。
7.根据权利要求1所述的口香糖,其特征在于,所述重组菌表达的抗菌药物种类包括糖苷酶、抗菌肽LL-37、防御素、抗菌肽bactenecin、富组蛋白中的至少一种。
8.根据权利要求1所述的口香糖,其特征在于,所述口香糖的原料包括以下组分,按重量百分比组成:重组菌冻干菌粉0.1-5%,柠檬酸0.1-1%,甘油1-10%,木糖醇50-70%,胶基30-40%;
所述含有重组菌的口香糖的制备方法,具体包括以下步骤:
(1)重组菌活化:将重组菌接种于LB液体培养基中,在37℃恒温培养箱中培养24-48h;
(2)重组菌冻干粉的制备:5000g-10000g高速离心分离10-20min,分离得到的菌体使用无菌生理盐水洗涤1-2次,将菌体置于真空冷冻干燥机中,设置参数-40℃,4Pa,冷冻干燥48-72h;收集冻干粉置于-70℃冰箱备用;
(3)口香糖胶基的预处理:将胶基置于50-60℃恒温烘箱内,保湿软化2-4h;
(4)口香糖混合料:将软化后的口香糖胶基降温至40-50℃,将其加入搅拌机中,准确称取重组菌冻干菌粉、木糖醇、柠檬酸、甘油保持搅拌温度40-50℃,搅拌10-15min,使其充分搅拌均匀;
(5)延压切割:将混合物投入切面机中挤压,反复挤压制成组织结构紧密的口香糖,切割成1cm×3cm规格;
(6)老化:将切好的块状口香糖盛放于托盘上,置于温度20℃,相对湿度30%~60%的条件下老化24h;
(7)包装:将口香糖用涂蜡锡箔纸包装,置于老化条件下贮藏。
9.一种与权利要求1-8任一项所述含有重组菌的口香糖配合使用的牙膏,其特征在于,所述牙膏的原料包括以下组分,按重量份组成:诱导物信息分子0.2-0.8份,碳酸钙25-40份,氢氧化铝5-10份,二氧化硅5-10份,山梨糖醇10-15份,十二烷基硫酸钠1-6份,羧甲基纤维素钠0.5-2份,甘油5-10份,去离子水25-35份;
所述与含有重组菌的口香糖配合使用的牙膏的制备方法,具体包括以下步骤:
1.摩擦剂的制备:将碳酸钙、氢氧化铝、二氧化硅分别用粉碎机粉碎,在加入混合机中混合均匀;
2.甜味剂的制备:将山梨糖醇用去离子水溶解;
3.牙膏膏体的制备:将甘油、摩擦剂、甜味剂、四环素信息分子、十二烷基硫酸钠、羧甲基纤维素钠,在真空条件下以7500-8000rpm/min高速搅拌10-15min,制得膏体;
4.包装:将膏体用复合软管包装。
10.根据权利要求9所述的牙膏,其特征在于,所述诱导物信息分子为四环素诱导物信息分子。
11.一种配合使用的如权利要求1-8任一项所述含有重组菌的口香糖和如权利要求9-10任一项所述含有诱导物的牙膏进行口腔治疗或保健的方法,其特征在于,使用者预先咀嚼口香糖对口腔进行不定期的重组菌植入,再通过含有诱导物的牙膏激发药效,实现特定时间的精确释药。
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