CN114410670A - 一种溶菌酶及其在口腔护理用品中的应用 - Google Patents
一种溶菌酶及其在口腔护理用品中的应用 Download PDFInfo
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- CN114410670A CN114410670A CN202210112680.0A CN202210112680A CN114410670A CN 114410670 A CN114410670 A CN 114410670A CN 202210112680 A CN202210112680 A CN 202210112680A CN 114410670 A CN114410670 A CN 114410670A
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Abstract
本发明公开了一种溶菌酶及其在口腔护理用品中的应用。所述溶菌酶基因的DNA序列如SEQ ID NO.1所示，所述溶菌酶基因所编码的蛋白的氨基酸序列如SEQ ID NO.2所示。本发明所述的溶菌酶基因编码的溶菌酶具有抑制口腔微生物的作用，可用于制备口腔护理用品，如牙膏、漱口水等。
Description
技术领域
本发明涉及口腔医学领域，具体涉及一种溶菌酶及其在口腔护理用品中的应用。
背景技术
溶菌酶(lysozyme)又称胞壁质酶(muramidase)或N-乙酰胞壁质聚糖水解酶(N-acetylmuramide glycanohydrlase)，是一种能水解细菌中黏多糖的碱性酶。溶菌酶主要通过破坏细胞壁中的N-乙酰胞壁酸和N-乙酰氨基葡萄糖之间的β-1,4糖苷键，使细胞壁不溶性黏多糖分解成可溶性糖肽，导致细胞壁破裂内容物逸出而使细菌溶解。溶菌酶还可与带负电荷的病毒蛋白直接结合，与DNA、RNA、脱辅基蛋白形成复合体，使病毒失活。该酶广泛存在于人体多种组织中，鸟类和家禽的蛋清、哺乳动物的泪、唾液、血浆、乳汁等液体，以及微生物也含此酶，其中以蛋清含量最为丰富。其中根据其来源的不同，可以将其分为四类，分别是植物溶菌酶、动物溶菌酶、微生物溶菌酶以及蛋清溶菌酶。溶菌酶对革兰氏阳性菌和革兰氏阴性菌均有良好的分解作用，常用于有害微生物的防治。
龋病，是在以细菌为主的多种因素影响下，引起牙体硬组织局部微生态破坏、有机酸产生和矿物质流失的一种慢性进行性破坏性疾病，主要包括牙菌斑生物膜、食物以及牙所处的微生态环境等。牙菌斑微生物与龋病发生密切相关，随着龋病的发展，牙菌斑微生物内细菌的比例可不断变化。龋病是多种微生物在特殊的微生态环境下共同作用的结果，致龋微生物需符合以下条件：具有较强的表面黏附力、产酸力强、耐酸力强、能合成细胞内外多糖。目前公认的致龋菌主要有链球菌属、乳杆菌属、放线菌属等。有研究发现变异链球菌Streptococcus mutans(S.mutans)可以造成啮齿类动物和灵长类动物实验室龋，并大量报道了其致龋的相关证据，认为S.mutans与人类龋病具有密切的相关性。
溶菌酶在口腔生物医学中已经得到了大量的研究应用。Tenovuo等的临床研究表明，将溶菌酶添加入牙膏中有显著抑菌效果，从而有效预防龋齿。有文献报道溶菌酶能够破坏S.mutans细胞壁完整性从而达到抗龋的效果。
发明内容
本发明的目的在于提供一种溶菌酶及其在口腔护理用品中的应用。
为了实现上述目的，本发明采用的技术方案如下：
一种溶菌酶基因，其DNA序列如SEQ ID NO.1所示。
所述的溶菌酶基因所编码的蛋白，其氨基酸序列如SEQ ID NO.2所示。
本发明所述的溶菌酶基因，是从福建福州鼓楼区收集的一份来自环境的水样品中，通过宏基因组学技术，用试剂盒提取宏基因组DNA，并直接对提取好的DNA进行PCR扩增得到。
本发明所述的溶菌酶基因编码的溶菌酶具有抑制口腔微生物的作用，可用于制备口腔护理用品，如牙膏、漱口水等。
附图说明
图1为实施例1中的PCR扩增产物；其中，左道为takara 250bp DNA laddermarker，右道为PCR扩增产物。
图2为SEQ ID NO.2所示蛋白序列的Conserved domains。
图3为实施例3中对照组和溶菌酶组OD值。
具体实施方式
以下结合附图和实施例对本发明做进一步说明。以下未注明具体条件的实验方法，按照本领域常规实验条件或者制造厂商所建议的条件。
实施例1
本发明所述溶菌酶基因的来源，是从福建福州鼓楼区收集的一份来自环境的水样品中，用0.22微米的微孔滤膜过滤，截留其中的微生物，取出滤膜，用试剂盒(Mobio牌，
DNA Isolation Kit，14900-50-NF，USA)提取滤膜所截留的全部微生物的宏基因组DNA，并直接对提取好的宏基因组DNA进行PCR扩增得到。
PCR体系如下：
上游引物：5’ATggCCACAACCATgATGGAGATAATCATG 3’
下游引物：5’gTTATgCTAgTCTAgTCACTAAGCCAAAAACCA 3’
PCR程序设定如下：95℃预变性2min；(95℃30s，56℃30s，72℃45s)，并设置35个循环；最后72℃10min。
利用琼脂糖凝胶电泳鉴定PCR扩增产物，结果如图1所示：
使用Omega公司胶回收试剂盒，货号D2500-01，对PCR产物进行胶回收。胶回收的PCR产物作为后续in fusion连接反应的底物，用于连接到表达载体上。
本实施例所得PCR产物使用一代测序技术，对其DNA序列进行鉴定，所用测序引物为上述上游和下游引物，得到本发明所述溶菌酶基因的DNA序列，如SEQ ID NO.1所示；其对应的蛋白质序列，如SEQ ID NO.2所示。
通过NCBI BLAST程序，对SEQ ID NO.2蛋白质序列进行检索，发现与其同源性最接近的为来自Psychrobacter属的溶菌酶序列，氨基酸相似率仅为57.34％，因此，可判断SEQID NO.2是一条全新的溶菌酶序列。通过NCBI分析SEQ ID NO.2的Conserved domains，结果如图2所示，发现含有典型的溶菌酶相关结构域。
实施例2
使用无细胞蛋白合成系统(Takara code 3281)对上述目的基因进行蛋白表达。
使用NcoI和XbaI这两种限制性内切酶，对购得的pT7-IRES质粒进行双酶切，反应体系如下：
10X FastDigest buffer 2μl；
质粒DNA 1μg；
NcoI 1μl；
Xba1 1μl；
补水到20μl。
放置于37℃水浴锅保温30分钟，之后加热到85度保温5分钟，灭活内切酶。
使用Omega公司胶回收试剂盒，货号D2500-01，对上述双酶切产物进行胶回收。胶回收的双酶切质粒回收产物，作为载体，用于插入目的基因。
In-Fusion连接反应
将上述反应体系在50℃水浴锅中保温15min，然后置于冰上，即可构建出表达载体。
取上述连接产物10μl，加入到大肠杆菌NEB-10beta感受态细胞中，混匀，冰浴30min，42℃热激45s，冰浴2min，涂LB平板(带有氨苄青霉素抗性)。将平板在37度培养箱培养16个小时，平板上形成单克隆菌落。
挑取单克隆菌落10个(编号1-10)，分别至10个各有10μl无菌水的Ep管中，吹打混匀，各取1μl做模板进行菌落PCR，验证目的基因是否连接到质粒载体上。
PCR体系：
PCR程序设定如下：94℃预变性10min；(94℃变性30s，56℃退火30s，72℃延伸1min)，并设置30个循环；最后72℃延伸5min。PCR产物电泳检测，通过目的条带的有无和大小，判断目的基因是否成功连接于载体，进而筛选出阳性克隆，并提取重组质粒备用。
1)以下无细胞蛋白合成系统试剂盒(Takara code 3281)中的试剂在冰上完全溶解后混匀，分取至反应管中，使用移液枪轻轻吸打混合均匀反应液。
Cell Lysate 9μl；
Mixture-1 6μl；
Mixture-2 1μl；
2)室温静置10分钟；
3)以下试剂在冰上完全溶解后混匀，添加至上述反应管中。全部试剂添加后，使用移液枪轻轻吸打混合均匀反应液。
Mixture-3 2μl；
质粒1μl(实验组为插入了溶菌酶基因的重组质粒，对照组为pT7-IRES质粒)；
T7 RNA Polymerase 1μl；
4)32℃反应6小时。
实施例3
抑菌活性检测
S.mutans标准菌株(ATCC 25175)从ATCC细菌库购买，存储在-80℃环境中。每次使用前复苏，用灭菌后的金属接种环进行BHI琼脂平板分区划线培养，于37℃恒温培养箱有氧条件下培养24h，观察S.mutans克隆生长情况。S.mutans纯化培养24h，生长状况良好，菌落散在点状分布，用无菌接种环刮取平板上的新鲜单菌落加入BHI培养液摇瓶中培养，于37℃恒温摇床培养16h，转速200r/min。
在96孔细胞培养板中，每孔放置100μl的脑心浸液培养基，并在每孔接种上述已经摇瓶培养好的S.mutans菌液5μl。将实施例2的实验组和对照组的无细胞蛋白合成产物，按照每孔5μl用量，分别添加到96孔细胞培养板的每个孔里。之后将培养板过夜培养。第二天目测培养液浑浊程度。
结果显示，添加对照组无细胞蛋白合成产物的孔，S.mutans正常生长，孔内液体浑浊。而添加实验组无细胞蛋白合成产物的孔，S.mutans受到了抑制，孔内液体仍然是澄清的。因此可说明实验组中合成的溶菌酶，具有抑制S.mutans生长的生物学功能和作用。
实施例4
参考目前市售牙膏中主流的二氧化硅配方体系作为验证配方(溶菌酶0.45％，低聚果糖4.55％，山梨醇30.00％，丙二醇4.00％，PEG-400 2.00％，二氧化硅25.00％，十二烷基硫酸钠0.5％，月桂酰肌氨酸钠0.50％，椰油酰胺丙基甜菜碱0.50％，羧甲基纤维素钠0.70％，黄原胶0.30％，三氯蔗糖0.15％，香精1.00％，水)，为尽量避免其他原料的影响，配方中不添加任何其他功能剂和防腐剂。
选取新鲜拔除的牛切牙，自颈部截除牙根后，于水冷却下片切冠部牙釉质，并将其制成长、宽均为4mm，厚3mm的釉质块。用环氧树脂分别包埋每个釉质块，暴露釉质块的唇面后，进行高温高压消毒灭菌，共制备20个釉质块包埋试样，将试样置于24孔板中，每孔放置400μl的脑心浸液培养基，并在每孔接种上述已经摇瓶培养好的S.mutans菌液20μl。37℃恒温摇床培养24h。将试样分为2组，每组10个试样，其中一组用上述含有溶菌酶的牙膏涂抹，另外一组用不含溶菌酶的对照牙膏涂抹，37℃静置15分钟，去除牙膏，用无菌PBS漂洗试样，并重新放置于新的24孔板，每孔加入400μl的脑心浸液培养基，37℃恒温摇床培养24h，观察。
结果显示，肉眼可见所有对照牙膏涂抹组的培养物均浑浊，而溶菌酶牙膏涂抹组的培养物澄清或轻微浑浊。每孔取100μl于96孔板中，利用酶标仪中测试波长为600nm下的OD值，进行统计分析，结果如图3。由此可得出结论，含有本发明的溶菌酶的牙膏，可更彻底地杀灭附着在釉质块包埋试样表面的S.mutans。因此，本发明的溶菌酶可作为商用牙膏中的抑菌除菌用添加剂。
综上，本发明所述的溶菌酶基因编码的溶菌酶具有抑制口腔微生物的作用，可用于制备口腔护理用品，如牙膏、漱口水等。
序列表
<110> 福建医科大学附属口腔医院
<120> 一种溶菌酶及其在口腔护理用品中的应用
<130> 2022
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 669
<212> DNA
<213> 人工序列(artificial series)
<400> 1
atggagataa tcatgacaga gttagaattg ttcgattggc tacgcactaa acaagccaat 60
aaaaagttaa cacaaaccat ggtcgatggg gtcaatgaat tgttggcatt gatgtctatc 120
gaagacttaa aagaatcctt gcaaaaaatc aatggttggg atgataacac cgcaaacaaa 180
gcgttgagtt tttcccaaaa aggcattgag atgctgtgtg cctttgaagg gtttgagcct 240
gcgccatacc ttgacgcggt aaaaaaacca accatcggat atggcaccac ttactacgtc 300
aatgcagatg gcacgcgaac gaatgtcagt atgaaagaca aaccaatcac taaggcgcag 360
gcgcttgcta tcaagcaaaa cgtgattaac catgactttg ccccggcggt caatctgatg 420
tttgccgatg aaattgccag tggcaaaatc aaacaaaacc aatttgacgc cctgatcagc 480
cttgcttata acatcggtat taaagggctt aaaggttcta gcgtctatcg ctatatcaag 540
caaggtaatt ttaaggcggc tgccgatgcg ttcttggcat ggaataaagg gcgggtgaat 600
ggcaagctcg ttgtgctagg cggacttagt aagcgcagag ccaaagagcg tgaatggttt 660
ttggcttag 669
<210> 2
<211> 222
<212> PRT
<213> 人工序列(artificial series)
<400> 2
Met Glu Ile Ile Met Thr Glu Leu Glu Leu Phe Asp Trp Leu Arg Thr
1 5 10 15
Lys Gln Ala Asn Lys Lys Leu Thr Gln Thr Met Val Asp Gly Val Asn
20 25 30
Glu Leu Leu Ala Leu Met Ser Ile Glu Asp Leu Lys Glu Ser Leu Gln
35 40 45
Lys Ile Asn Gly Trp Asp Asp Asn Thr Ala Asn Lys Ala Leu Ser Phe
50 55 60
Ser Gln Lys Gly Ile Glu Met Leu Cys Ala Phe Glu Gly Phe Glu Pro
65 70 75 80
Ala Pro Tyr Leu Asp Ala Val Lys Lys Pro Thr Ile Gly Tyr Gly Thr
85 90 95
Thr Tyr Tyr Val Asn Ala Asp Gly Thr Arg Thr Asn Val Ser Met Lys
100 105 110
Asp Lys Pro Ile Thr Lys Ala Gln Ala Leu Ala Ile Lys Gln Asn Val
115 120 125
Ile Asn His Asp Phe Ala Pro Ala Val Asn Leu Met Phe Ala Asp Glu
130 135 140
Ile Ala Ser Gly Lys Ile Lys Gln Asn Gln Phe Asp Ala Leu Ile Ser
145 150 155 160
Leu Ala Tyr Asn Ile Gly Ile Lys Gly Leu Lys Gly Ser Ser Val Tyr
165 170 175
Arg Tyr Ile Lys Gln Gly Asn Phe Lys Ala Ala Ala Asp Ala Phe Leu
180 185 190
Ala Trp Asn Lys Gly Arg Val Asn Gly Lys Leu Val Val Leu Gly Gly
195 200 205
Leu Ser Lys Arg Arg Ala Lys Glu Arg Glu Trp Phe Leu Ala
210 215 220
Claims (4)
1. 一种溶菌酶基因，其特征在于：所述基因的DNA序列如SEQ ID NO.1所示。
2.如权利要求1所述的溶菌酶基因所编码的蛋白，其特征在于：所述蛋白的氨基酸序列如SEQ ID NO.2所示。
3.如权利要求1所述的溶菌酶在口腔护理用品中的应用。
4.根据权利要求3所述的应用，其特征在于：所述口腔护理用品为牙膏或漱口水。
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Citations (4)
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|---|---|---|---|---|
| WO2002048188A1 (en) * | 2000-12-14 | 2002-06-20 | The Council Of The Queensland Institute Of Medical Research | Plasmodium aldolase polypeptides and nucleic acids |
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| CN111971058A (zh) * | 2017-12-12 | 2020-11-20 | 康特拉费克特公司 | 对铜绿假单胞菌具有细菌活性的溶素及其衍生物的鉴定 |
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| WO2002048188A1 (en) * | 2000-12-14 | 2002-06-20 | The Council Of The Queensland Institute Of Medical Research | Plasmodium aldolase polypeptides and nucleic acids |
| CN101892252A (zh) * | 2010-02-26 | 2010-11-24 | 广西大学 | 一种编码溶菌酶的基因及其应用 |
| CN111971058A (zh) * | 2017-12-12 | 2020-11-20 | 康特拉费克特公司 | 对铜绿假单胞菌具有细菌活性的溶素及其衍生物的鉴定 |
| CN108018276A (zh) * | 2018-01-12 | 2018-05-11 | 中国科学院南海海洋研究所 | 一种深海细菌角蛋白酶及其编码基因、酶蛋白生产工程菌和应用 |
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