CN101564534A - 一种防治口臭致病菌的特异性卵黄抗体制剂及其应用 - Google Patents
一种防治口臭致病菌的特异性卵黄抗体制剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种防治口臭致病菌的特异性卵黄抗体制剂及其应用,属于生物技术领域。一种防治口臭致病菌的特异性卵黄抗体制剂,其特征在于:涉及国际命名为Solobacterium moorei的口臭致病菌,该口臭致病菌作为免疫原,对蛋鸡进行初免和加强免疫,共计三次;免疫后,收集鸡蛋,分离并纯化特异性卵黄抗体,制备含特异性卵黄抗体的制剂。本发明的效果和益处是:本发明得到的特异性IgY对口臭的防治效果好;本发明得到的特异性IgY制剂不会影响口腔正常菌群,无毒副作用;本发明所述的方法易于规模化生产,且成本低廉,是一种安全的口腔局部防治制剂。
Description
技术领域
本发明属于生物技术领域,涉及一种防治口臭致病菌(Solobacterium moore)的特异性卵黄抗体(IgY)制剂及其应用,显著防治口臭病的新型制剂。
背景技术
口臭作为一种特殊症状,严重影响着人们的社会交往,甚至给患者造成心理障碍。大量研究已证实,对于不存在口腔疾患的口臭患者,其口臭主要由于口腔卫生不良造成舌苔的菌斑增多、增厚所引起,即舌苔中所隐藏的细菌成分是口臭VSCs的主要制造者。研究发现(Haraszthy V.I.et al.,Identification of oralbacterial species associated with halitosis.J.Am.Dent.Assoc.,2007,138:1113-1120;Haraszthy V.I.et al.,Characterization and prevalence of Solobacteriummoorei associated with oral halitosis.J.Breath Res.,2008,doi:10.1088/1752-7155/2/1/017002)口腔中一种名为S.moorei的细菌和口臭紧紧相关联,是引起口臭的主要致病菌。S.moorei寄生在口臭患者的舌头背面,分解食物残渣,并产生有腐臭味道的硫化物。对S.moorei的研究表明,其最初从人粪便中分离出来,为一种革兰氏阳性厌氧菌,无芽孢,是Solobacterium属目前唯一的一种细菌(Kageyama A.et al.,Characterization and prevalence of Solobacterium moorei associated with oralhalitosis.J.Breath Res.,2008,doi:10.1088/1752-7155/2/1/017002)。此外,S.moorei还可引起人的多种疾病,如牙根管感染(Rolph H.J.et al.,Molecular identificationof microorganisms from endodontic infections.J.Clin.Microbiol.,2001,39:3282-3289)、菌血症(Detry G.et al.,Septicemia due to Solobacterium moorei in apatient with multiple myeloma.Anaerobe,2006,12:160-162)以及与坏死杆菌病有关的血栓性静脉炎(Martin C.A.et al.,Femoral vein thrombophlebitis and septicpulmonary embolism due to a mixed anaerobic infection including Solobacteriummoorei:a case report.J.Med.Case Rep.,2007,1:40)等。
卵黄免疫球蛋白(Yolk Immunoglobulin,IgY),亦称卵黄抗体,是富集在禽类卵黄中的活性抗体分子,因其在抗菌、抗病毒方面的优良特性,近年来引起各国研究者的广泛关注。研究表明,口服IgY可为人或动物提供有效的被动免疫保护,防止由细菌或病毒引起的消化道或非消化道感染,如抗幽门螺杆菌(Helicobacter pylori)IgY能预防和治疗胃溃疡(Shin J.H.et al.,Use of eggyolk-derived immunoglobulin as an alternative to antibiotic treatment for control ofHelicobacter pylori infection.Clin.Diagn.Lab.Immunol.,2002,9(5):1061-1066),抗产肠毒素型大肠杆菌(Enterotoxigenic Escherichia coli)IgY可防治新生及断奶仔猪腹泻(Li X.Y.et al.,Chitosan-Alginate Microcapsules for Oral Delivery of EggYolk Immunoglobulin(IgY).J.Agric.Food Chem.,2007,55:2911-2917;尤建嵩等,鸡卵黄抗体在防治家畜疾病中的应用.中国饲料,2003,19:8-9;Yokoyama H.etal.,Passive protective effect of chicken egg yolk immunoglobulins againstexperimental enterotoxigenic Escherichia coli infection in neonatal piglets.Infect.Immun.,1992,60(3):998-1007),抗奶牛乳腺炎IgY能明显抑制奶牛乳腺炎致病菌的体外增殖(Zhen Y.H.et al.,Charcterization of specific egg yolk immunoglobulin(IgY)against mastitis-causing Escherichia coli.Vet.Microbiol.,2008,130(1-2):126-133;王宇等,抗奶牛乳腺炎特异性卵黄抗体的制备及活性研究.辽宁师范大学学报,2006,29:90-91)并减轻乳腺炎症状(Stadelman W.J.et al.,Theincredibly functional egg.Poult.Sci.,1999,78(6):807-811)。
有关研究已证实,特异性IgY对口腔中变异链球菌(Streptoccus mutans)引起的龋齿具有显著的预防效果,其最大的特点就是专一性强,无毒副作用,容易生产且成本低廉,是一种安全的口腔局部预防制剂(Hamada S.et al.,Oralpassive immunization against dental caries in Rats by use of hen-egg-yolk antibodiesspecific for cell-associated glucosyltransferase of Streptoccus mutans.Infect.Immun.,1991,59(11):4161-4167)。
本发明研制了抗口臭主要致病菌S.moorei特异性IgY,代替抗菌制剂,将之以不同的剂型(漱口液、口腔喷剂、牙膏、含片和口香糖)使用,有针对性地消除口臭,从而为口臭的预防和治疗提供一种安全有效的新方法。
发明内容
本发明要解决的技术问题是提供一种防治口臭致病菌Solobacterium moorei的特异性IgY制剂、制备方法及其应用。
本发明的技术方案是:
涉及的S.moorei的国际命名为Solobacterium moorei,把口臭致病菌S.moorei作为免疫原,对蛋鸡进行注射免疫(初免和加强免疫,共计三次);免疫后,收集鸡蛋,分离并纯化特异性卵黄抗体(IgY);制备含特异性卵黄抗体(IgY)的制剂。
本发明的效果和益处是:
(1)本发明得到的特异性IgY对口臭的防治效果好;
(2)本发明得到的特异性IgY专一性强;
(3)本发明得到的特异性IgY制剂不会影响口腔正常菌群,无毒副作用,安全可靠;
(4)本发明所述的方法易于规模化生产,且成本低廉,是一种安全的口腔局部防治制剂。
具体实施方式
以下结合技术方案详细叙述本发明的实施例。
实施例1
抗S.moorei特异性IgY的制备方法,其中:
A、菌体种类:S.moorei
菌体的来源和培养:S.moorei,包括标准的S.moorei(JCM 10645,其16SrRNA序列见序列表),和来自口臭患者舌苔部位分离的S.moorei,经肉汤培养基分离培养和扩增培养,并经鉴定确认为S.moorei。
B、免疫原制备
将37℃厌氧条件下肉汤培养的S.moorei菌液于4℃8000r/min离心15min,收集菌体,用生理盐水稀释并调节菌体浓度至108-1012cfu/ml,加入体积分数0.3%甲醛,37℃灭活24h,不时摇动,经无菌检验合格后分别加入等量弗氏完全佐剂和弗氏不完全佐剂,充分乳化后制成S.moorei灭活疫苗。
C、免疫
初免为弗氏完全佐剂疫苗,二、三免为弗氏不完全佐剂疫苗。初免每只鸡胸肌注射4点,颈部皮下注射1点,每点0.2ml。初免后2周进行二免,方法同初免,每点免疫剂量增加至0.3ml。二免后2周进行三免,每点免疫剂量增加至0.4ml。
D、抗S.moorei特异性IgY的制备
三免后收集鸡蛋,先用清水洗净蛋表污物,再用0.5%新洁尔灭溶液浸泡消毒20min,晾干。使用卵黄分离器分离卵黄,去除卵清蛋白和卵黄膜,收集卵黄,加6倍于卵黄体积的蒸馏水,用0.1M盐酸调pH至5.0,搅匀后4℃放置过夜,4℃10000r/min离心25min,取上清,经直径0.45μm滤膜过滤,收集的滤液即为WSF(水溶性组分,Water Soluble Fraction)。在WSF中缓慢加入(NH4)2SO4至饱和度为50%,混匀,待完全溶解后置4℃过夜。4℃10000r/min离心15min,弃上清,用去离子水重新悬浮沉淀至原体积,再加入Na2SO4至饱和度为14%,混匀,置室温过夜。25℃10000r/min离心15min,弃上清,用去离子水重新悬浮沉淀至原体积,即为IgY的提取液。经凝胶过滤纯化,经SDS-PAGE测得IgY的纯度达90%以上,用单向琼脂扩散法测得IgY含量超过33%,采用ELISA(酶联免疫吸附实验)测得特异性IgY效价达1∶200000。
实施例2
抗S.moorei特异性IgY的漱口液:
抗S.moorei特异性IgY提取物1.0%
甘露醇5.0%
氯己定0.001%
蒸馏水补充至100%
经微孔滤膜过滤除菌,无菌灌装入消毒的漱口液罐中。
实施例3
抗S.moorei特异性IgY的口腔喷剂:
抗S.moorei特异性IgY提取物0.90%
甘露醇5.0%
氯己定0.001%
蒸馏水补充至100%
经微孔滤膜过滤除菌,无菌灌装入消毒的喷剂罐中。
实施例4
抗S.moorei特异性IgY的牙膏:
抗S.moorei特异性IgY提取物1.0%
药用辅料99%
经微孔滤膜过滤除菌,无菌加入到牙膏生产线中。
实施例5
抗S.moorei特异性IgY的含片:
抗S.moorei特异性IgY提取物0.90%
山梨醇5%
阿斯巴甜0.2%
药用辅料93.9%
将上述药片药物低温制粒,然后压成不同形状的含片,每片2克。
实施例6
抗S.moorei特异性IgY的口香糖:
抗S.moorei特异性IgY提取物0.80%
山梨醇5%
药用辅料94.2%
经微孔滤膜过滤除菌,无菌加入到口香糖生产线中。
实施例7
S.moorei特异性IgY制剂的临床实验
(1)实验期限
应按受检产品的功效强度来确定,可为数小时至数周。
(2)研究对象
受试者为符合纳入及排除标准的患有口源性口臭的男性和女性。
一般每组实验全过程的人数应大于等于10人。
(3)检查指标或采用指数,可同时采用以下两种评价方法:
1、感官评价:由训练有素的人员利用有效计分指标评价受试者的口气。可采用0-5的等级评价口臭程度(0:觉察不到气味;1:勉强可以觉察到气味,若有若无;2:轻微但可明确觉察;3:中等臭味;4:重度臭味;5:不能忍受的恶臭)。
2、仪器测量
分析仪器测量(口气测量仪Halimeter)可将口腔气体中挥发性硫化物的浓度以数值的形式表现出来,作为衡量口气水平的指标。
(4)效果评价:
按医学统计方法,对实验组和对照组分别统计实验前后口气值均数的变化和仪器读数均值变化情况。下表为抗S.moorei特异性IgY抑制口臭效果:
病人数:60人,均患四级(重度臭味)口臭。随机分为六组,包括五个实验组和一个对照组,每组10人。
第一组:使用1ml抗S.moorei特异性IgY的漱口液漱口,含漱三分钟,每天三次,疗程一个月,评价治疗效果。
第二组:取1ml抗S.moorei特异性IgY的口腔喷剂喷于口腔中,每天三次,疗程一个月,评价治疗效果。
第三组:使用抗S.moorei特异性IgY的牙膏刷牙,每天三次,疗程一个月,评价治疗效果。
第四组:口含抗S.moorei特异性IgY的含片,每天数次,疗程一个月,评价治疗效果。
第四组:咀嚼抗S.moorei特异性IgY的口香糖,每天数次,疗程一个月,评价治疗效果。
对照组:不使用特异性IgY制剂。
序列表
5’-agagtttgat cctggctcag tatgaactct gccggcgtgc ctaatacttg caagtcgaac gctgaagatc
tagcttgcta gatcgaagga gtgccgaacg ggtgagtaat acataagcaa cctacccacg aagactggga
taatctctgg aaacggggac taataccgga taggtaatcg gaaggcatct tctggttatt aaaggttaaa
aacactggtg gatgggctta tggcgcatta gttagttggt gaggtaacgg cccaccaaga cgatgatgcg
tagccggcct gagagggtga acggccacat tgggactgag acacggccca gactctacgg gaggcagcag
tagggaattt tcggcaatag gggcaaccct gaccgagcaa cgccgcgtga gtgaagacgg ccttcgggtt
gtaaaagctc ttgtttgtaa gggaagaacg gtagatagag aatatctaag tgacggtacc ttaccagaaa
gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcgacc gttatccgga attattggcc
gtaaagggtg cgtaggcggc ctgttaagta agtggttaaa ttgttgggct caacccaatc cagccactta
aactggcagg ctagagtatt ggagaggcaa gtggaattcc atgtgtagcg gtaaaatgcg tagatatatg
gaggaacacc agtggcgaag gcggcttgct agccaaagac tgacgctcat gcacgaaacc gtggggagcc
aataggatta gataccctag tagtccacgc cgtaaccgat gaatactaag tattggggaa actcagtgct
gcactaacgc aataagtatt ccgcctgtgg agtatgcacg caagtgtgaa acataaagga attgacgggg
gcccgcacaa gcggtggagt atgtggttta attcgacgca acgcgaagaa ccttaccagg ccttgacatc
ccttgcaaag ctgtagagat acagtagagg ttatcaagga gacaggtggt tgcatggtgt cgtcagctcg
tgtcgtgaga tgttgggtta agtccggcaa cgagcgcaac ccttgccttc agttaccagc atttagttgg
ggactctgga gggactgccg gtgataaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta
tggcctgggc tacacacgta ctacaatggc tgttacaacg tgcagcgacc tagcgatagg aagcgaatca
ctaaaagaca gtctcagttc ggattgaagt ctgcaactcg acttcatgaa gctggaatcg ctagtaatcg
cggatcagaa tgccgcggtg aatacgttct cgggccttgt acacaccgcc cgtcatacca tgagagctgg
taatacccga ggccggtggc ctaaccgcaa ggaaggagcc gtcgaaggta ggactagtga ttggggtcaa
gtcgtaacaa ggtaacc-3’
Claims (4)
1、一种防治口臭致病菌的特异性卵黄抗体制剂,其特征在于:涉及国际命名为Solobacterium moorei的口臭致病菌,该口臭致病菌作为免疫原,对蛋鸡进行初免和加强免疫,共计三次;免疫后,收集鸡蛋,分离并纯化特异性卵黄抗体,制备含特异性卵黄抗体的制剂。
2、权利要求1所述的一种防治口臭致病菌的特异性卵黄抗体制剂的制备方法,其特征如下:
A菌体种类:Solobacterium moorei
B免疫原制备:Solobacterium moorei菌液经处理后与弗氏完全佐剂和弗氏不完全佐剂混合,充分乳化,制成弗式完全佐剂疫苗和弗式不完全佐剂疫苗;
C免疫:初免为弗氏完全佐剂疫苗,二、三免为弗式不完全佐剂疫苗;初免每只鸡胸肌注射2到6点,颈部皮下注射1到3点,每点0.1ml-0.3ml;初免后2周进行二免,方法同初免,每点免疫剂量增加至0.2ml-0.4ml;二免后2周进行三免,每点免疫剂量增加至0.3ml-0.5ml;
D、特异性卵黄抗体的制备:定期收集免疫鸡蛋的蛋黄,运用水稀释法、二次盐析法、凝胶层析等技术分离纯化特异性卵黄抗体。
3、一种防治口臭致病菌的特异性卵黄抗体制剂的应用,其特征在于:将0.02-80%的特异性卵黄抗体配以99.98-20%的药用辅料;所述的制剂采用权利要求1所述的制剂。
4、一种防治口臭致病菌的特异性卵黄抗体制剂的应用,其特征在于:制剂用于漱口液、口腔喷剂、牙膏、含片或口香糖中;所述的制剂采用权利要求1所述的制剂。
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