CN105254736B - 源于缅甸蟒的cathelicidin家族广谱抗微生物肽Pb-CATH4及其基因、制备与应用 - Google Patents
源于缅甸蟒的cathelicidin家族广谱抗微生物肽Pb-CATH4及其基因、制备与应用 Download PDFInfo
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Abstract
本发明属于生物医学技术领域,涉及一种缅甸蟒蛇的cathelicidin家族广谱高效抗微生物肽Pb‑CATH4及其基因、制备方法和应用。Pb‑CATH4是直链多肽,含有29个氨基酸残基,理论等电点为12.54,分子量是3492.11Da。Pb‑CATH4的前体基因由465个核苷酸组成,其中编码成熟肽部分的为第376‑462位核苷酸。Pb‑CATH4富含碱性氨基酸,分子量小、结构简单,方便化学合成及基因工程制备。Pb‑CATH4具有极强的广谱抗微生物活性,对革兰氏阳性菌、革兰氏阴性菌和真菌均有活性,还具有独特的抗菌机理及不易产生耐药性等有益特点,应用前景广阔。
Description
技术领域
本发明属于生物医学技术领域,涉及一种源于缅甸蟒蛇(Python molurusbivittatus)的cathelicidin家族广谱抗微生物肽Pb-CATH4及其编码基因,氨基酸序列和其在化妆品、保健品、食品、饲料等日化添加剂及抗感染医药领域中的应用。
背景技术
Cathelicidin是一类由N端信号肽区域、中间保守cathelin结构域和C端高度特异的成熟肽区域构成的具有多功能的宿主抗菌肽家族,结构特点是:具有N端信号肽区域(30个残基左右)、中间保守cathelin结构域(99-114个残基)和C端高度特异的成熟肽区域(12-100个残基)。Cathelicidin具有广谱的抗菌活性,能快速、广谱地杀灭多种病原微生物,包括革兰阳性菌、革兰阴性菌、真菌、寄生虫和病毒等,特别是对许多临床耐药细菌同样具有作用,这引起了人们的注意。除此之外,cathelicidin还具有许多其他生物学活性,如对多种免疫细胞(中性粒细胞、单核细胞、肥大细胞和T细胞)具有趋化作用、诱导肥大细胞脱粒和组织胺释放、调节巨噬细胞转录、促进伤口愈合、诱导血管发生、诱导变异细胞系细胞凋亡和淋巴细胞活化等。这些优势使其在临床的应用有着良好的前景。
近年来由于抗生素的滥用,各种耐药及超级耐药菌层出不穷,对临床的抗感染治疗造成巨大威胁,因此新型抗病原微生物模板的研发迫在眉睫。Cathelicidins对许多临床耐药细菌有着很强的杀菌活性,对许多致病菌杀害作用的速度要远远大于传统抗生素的作用,体外抗微生物测试中,大多数Cathelicidin成熟肽可以在微摩尔和亚微摩尔的浓度下快速杀死广泛的微生物。最重要的是由于其特别的杀菌机制,不会引起耐药性。目前发现的Cathelicidins的杀菌机制有很多,常见的是通过破坏细菌细胞膜的完整性介导的即通过静电相互作用吸引并结合到带负电的细菌细胞膜表面,进一步在细菌细胞膜上形成跨膜的孔洞,导致细菌细胞内容物的外泄,从而导致细菌细胞的死亡。此外,抗菌肽还具有其他的杀菌机制,如抑制细菌细胞壁合成、改变细菌细胞质膜抑制隔膜形成、激活自溶素、抑制细胞内酶活性、抑制DNA、RNA和蛋白质的合成等。正是由于作用方式的不同,Cathelicidin介导的杀菌作用远远快于传统抗生素,并且不会产生耐药性。
关于抗菌肽药物,目前cathelicidin的研究热点主要集中在消炎、抗感染和抗真菌等方面,应用方式可以是局部的也可以是系统的,剂型可以是口服也可以外用。尤其由于阳离子抗菌肽的表达与皮炎、侵入性烧伤脓血症、肿瘤病毒引起的肉赘等之间的病理联系,这些抗菌肽对这些疾病的局部治疗有较好的开发前景。目前有部分抗菌肽药物已进入临床试验阶段,例如Pexiganan是爪蟾抗菌肽Magainin的类似物,目前已作为治疗足部感染药物进入临床试验阶段,它也是第一个进行商业开发的抗菌肽;Hlf1-11是人乳铁蛋白前11个氨基酸残基组成的抗菌肽,通过Ⅰ/Ⅱ期临床试验研究表明Hlf1-1通过静脉给药时是安全的、耐受性良好的;来源于猪protegrin的IB-367用于治疗肿瘤患者口腔溃疡,已进入临床III期试验等。Cathelicidin的应用不仅限于医药领域,在农业、畜牧业和日化用品领域,cathelicidin也存在巨大的应用潜能。
2002年,世界卫生组织(WHO)发表公告指出“在牲畜养殖过程中,停止以往惯用的饲料添加剂抗生素的做法,将在不危害动物和农民利益的前提下,可减少对人类健康的威胁。抗生素作为饲料添加剂应用于动物生产中,对畜牧业的发展起了重要的作用,但是其在动物体内和动物产品中的残留,以及病原菌产生的抗药性问题,对人类的健康和环境产生了负面的影响。并且完全使我国出口肉类,海产品等受到了限制,从而影响收益。寻找新型、安全的抗菌剂代替抗生素,已成为当前国内外饲料学科的一项重要内容。抗菌肽具有广谱的抗菌作用,对畜禽具有促生长和治疗疾病的功能,是无毒、无害、无残留的绿色产品,有望成为抗生素的替代品,在畜牧生产上发挥重要的作用。目前在畜禽生产,水产养殖等均有应有。
缅甸蟒蛇(Python molurus bivittatus),是蛇亚目蟒科蟒属印度蟒的亚种之一,以体型巨大著称,平均身长至高可达7米,体重可达91公斤,也是世界上最巨型的六种蛇类之一。缅甸蟒属于东南亚地区的本土品种,是一种夜行性蛇类,多居于热带森林里,在中国多见于广东、广西、福建、云南、海南、贵州等地。目前关于缅甸蟒蛇cathelicidin宿主防御肽的研究和应用还没有报道。
发明内容
本发明提供一种在亚微摩尔剂量下即具有很强的抗微生物活性的来源于缅甸蟒蛇的一种抗微生物肽,Pb-CATH4及其基因、氨基酸序列和应用。本发明的目的是基于上述理论研究和现有技术基础,提供一种具有强抗菌活性的缅甸蟒蛇抗微生物肽Pb-CATH4及其应用,着重医药制备,化妆品、保健品、食品和新型饲料添加剂等中的应用。
为了实现本发明的目的,本发明提供了如下技术方案:
一种源于缅甸蟒的广谱高效抗微生物肽Pb-CATH4,所述的广谱抗微生物肽Pb-CATH4是一种直链多肽,含有29个氨基酸残基,分子量是3492.11Da,理论等电点(pI)为12.54,含有9个碱性氨基酸残基及17个疏水性氨基酸残基。
其序列为:苏氨酸-精氨酸-丝氨酸-精氨酸-色氨酸-精氨酸-精氨酸-苯丙氨酸-异亮氨酸-精氨酸-甘氨酸-丙氨酸-甘氨酸-精氨酸-苯丙氨酸-丙氨酸-精氨酸-精氨酸-酪氨酸-甘氨酸-色氨酸-精氨酸-异亮氨酸-丙氨酸-亮氨酸-甘氨酸-亮氨酸-缬氨酸-甘氨酸。
基因测序结果表明编码缅甸蟒蛇抗菌肽Pb-CATH4前体cathelicidin的基因由465个核苷酸组成,自5’端至3’端序列为:
编码缅甸蟒蛇成熟肽Pb-CATH4的为376位到462位的核苷酸序列,其编码的氨基酸序列为:Thr1-Arg2-Ser3-Arg4-Trp5-Arg6-Arg7-Phe8-Ile9-Arg10-Gly11-Ala12-Gly13-Arg14-Phe15-Ala16-Arg17-Arg18-Tyr19-Gly20-Trp21-Arg22-Ile23-Ala24-Leu25-Gly26-Leu27-Val28-Gly29。
缅甸蟒蛇抗菌肽Pb-CATH4基因的克隆包括:缅甸蟒蛇肺总RNA提取,mRNA纯化,mRNA反转录及cDNA文库构建,设计引物,利用PCR方法筛选缅甸蟒蛇抗菌肽Pb-CATH4基因。5’端引物分别为P1(5'-ATGACGGGTGTTTGGGCACTG-3')和P2(5'-GCTGAAGTTCACCATCAAGGAGAC-3'),PCR另一扩增引物为CLONTECH公司CreatorTM SMART TMcDNA Library Construction Kit中的3’PCR Primer引物,其序列为5’–ATTCTAGAGGCCGAGGCGGCCGACATG–3’。获得阳性单克隆进行基因核苷酸序列测定。
Pb-CATH4的化学制备方法:根据编码缅甸蟒蛇cathelicidin抗微生物肽基因推断的成熟肽Pb-CATH4氨基酸序列,用自动多肽合成仪(433A,Applied Biosystems)合成其全序列。通过HPLC反相柱层析脱盐纯化,并确定其纯度大于95%。用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)测定其分子量,等电聚焦电泳测定等电点,用自动氨基酸测序仪测定氨基酸序列结构。
缅甸蟒蛇抗菌肽Pb-CATH4基因可以作为基因工程制备缅甸蟒蛇抗菌肽Pb-CATH4。所述广谱高效抗微生物肽Pb-CATH4,具有极强的广谱抗微生物活性,作为制备抗病原微生物感染药物或化妆品、保健品、食品、饲料等日化中的添加剂被应用。合成的Pb-CATH4抗菌肽可以溶于灭菌超纯水,用于药理活性检测。
本发明基因克隆得到编码缅甸蟒蛇cathelicidin抗微生物肽的基因,通过化学合成方法得到成熟肽Pb-CATH4。该抗微生物肽富含碱性氨基酸,具有很强的抗菌活性,抗菌实验显示其对多种临床耐药菌也有较好的杀灭作用。另外,其还具有很低细胞毒性,无溶血活性,结构简单,不含有二硫键及环状结构,方便化学合成及基因工程制备。
附图说明
附图是Pb-CATH4对E.coil ATCC25922的杀菌动力学图。
具体实施方式
下面结合技术方案详细叙述本发明的具体实施例,但本发明的内容并不局限于此。
严格参照试剂盒说明书,采用RNeasy AxyPrepTM Multisource Total RNAMiniprep Kit(Qiagen,union city,CA,USA)提取缅甸蟒肺组织总RNA,然后利用CreatorTMSMART TM cDNA Library Construction Kit建库试剂盒构建缅甸蟒蛇肺组织cDNA文库。利用试剂盒中PowerScript Reverse Transcriptase反转录合成cDNA第一链,引物为:
正向SMART Ⅳ Oligonucleotide引物:
5'–AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG–3'
反向CDSⅢ/3'PCR引物:5’–ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N–3’(N=A,C,G,or T;N-1=A,G,or C).
利用Advantage DNA Polymerase合成cDNA第二链,引物为:正向5'–AAGCAGTGGTATCAACGCAGAGT-3',反向引物同为CDSⅢ/3'PCR Primer。.
设计两条特异性正向引物(P1、P2)和一条反向非特异性通用引物(3'PCRPrimer),以缅甸蟒蛇肺cDNA为模板,采用半巢式PCR的方法扩增cathelicidin的cDNA。
正向P1:5'-ATGACGGGTGTTTGGGCACTG-3';
正向P2:5'-GCTGAAGTTCACCATCAAGGAGAC-3';
反向非特异性通用引物为3'PCR Primer,其序列为:
5‘-ATTCTAGAGGCCGAGGCGGCCGACATG-3’。
所获阳性单克隆进行基因核苷酸序列测定,pMD19-T Vecter测序通用引物:
正向M13F:5'-CGCCAGGGTTTTCCCAGTCACGAC-3';
反向M13R:5'-GAGCGGATAACAATTTCACACAGG-3';
具体步骤如下:
第一步、缅甸蟒蛇肺总RNA提取(以下实验所用器具和试剂均经过DEPC水处理,无RNase):
A.从一岁龄大小的雄性缅甸蟒蛇各种新鲜组织中取下绿豆大小个小块,分别放于标记好的冻存管中,迅速置于液氮中保存备用;
B.将保存在液氮中的组织材料取出,放入预冷的研钵内,迅速充分研磨,期间不断向研钵内加入少许液氮;将大约30mg的组织粉末转移至预冷的1.5ml离心管中,向其中分别加入400μl Buffer R-I(裂解液,RNA Miniprep Kit提供),用21-25号针头的注射器反复抽吸8-10次,转入1.5ml离心管中,加入150μl Buffer R-Ⅱ,涡旋振荡15-30s,4℃,12000rpm离心5min;
C.取上清转移至新的1.5ml离心管中,加入250ul异丙醇,迅速吸打混匀;将混合液分别转移至2ml离心管中(试剂盒内提供),4℃,6000rpm离心1min;弃废液,将制备管置回到2ml离心管中,然后加入500μl Buffer W1A,4℃,12000rpm离心1min;弃废液,加入700μlBufferW2A,4℃,12000rpm离心1min;弃废液,加入700μl Buffer W2A,4℃,12000rpm离心1min;弃废液,空管12000rpm离心1min;将离心吸附柱转移至新的1.5ml离心管中,然后直接向吸附膜上滴加70-100μl Buffer TE,室温放置1min,4℃,12000rpm离心1min洗脱得到总RNA;
第二步、缅甸蟒蛇肺组织cDNA文库的构建
第一链的合成(mRNA反转录):
A.在新的0.2ml PCR管(无DNase和RNase)中配制下列混合液:
混合均匀,短暂离心;PCR仪中72℃保温2min后,然后冰浴2min;短暂离心后,在上述管中配制如下反转录反应液:
混合均匀后,短暂离心;在PCR仪中完成以下程序:
42℃,90min;68℃,10min;冰浴终止反应。cDNA保存于-80℃。
第二链的合成:
轻弹管壁,短暂离心,放入提前预热的PCR仪中,反应条件为:95℃,1min,21cycles(95℃,15s;65℃,30s;68℃,6min)。取5μl的产物电泳检测分析。
第三步、半巢式PCR进行缅甸蟒蛇cathelicidin的基因克隆筛选
引物使用前先12000rpm离心5min,然后根据标明的摩尔数加入相应体积的ddH2O溶解至20μM的浓度。合成的肺组织cDNA稀释20倍作为模板,以P1和3'PCR Primer为引物,进行第一次PCR扩增。在0.2ml PCR管中加入下列试剂:
混匀后,短暂离心。PCR条件为:94℃变性5min;28个循环:94℃变性30s,55.5℃退火30s,72℃延伸50s;72℃延伸10min;4℃保存。反应结束后,取5μl产物于1%琼脂糖凝胶电泳检测目的条带。
取上步PCR产物1μl用ddH2O稀释100倍作为模板,以P2和3'PCR Primer为引物,进行第二次PCR扩增。在0.2ml PCR管中先后加入下列试剂:
PCR条件为:94℃变性5min;28个循环:94℃变性30s,56℃退火30s,72℃延伸50s;72℃延伸10min;4℃保存。反应结束后,取5μl,1%琼脂糖凝胶电泳检测目的条带。
扩增完成后用胶回收试剂盒(天根生物)进行目的片段回收。将回收的目的DNA片段与测序载体pMD19-T Vecter连接,转化进CaCl2法制备好的DH5α感受态细胞。取100μl转化菌液均匀涂布在含有100μg/ml氨苄青霉素(Amp)的LB琼脂培养基平板上;表面晾干后,放在37℃恒温培养箱中倒置培养12-16h。挑取单菌落用M13引物PCR检测插入片段大小。挑取阳性菌落,摇菌提取质粒,使用Applied Biosystems DNA sequencer,model ABI PRISM377进行核苷酸测序。
第四步、缅甸蟒蛇cathelicidin的基因序列测定和结果:
编码其前体cathelicidin的基因由465个核苷酸组成,自5’端至3’端序列为:
缅甸蟒蛇cathelicidin编码区的cDNA核苷酸的序列表为:序列长度为465个碱基,序列类型:核酸,链数:单链,拓扑学:直链状,序列种类:cDNA,来源:缅甸蟒蛇肺。
编码缅甸蟒蛇cathelicidin成熟肽Pb-CATH4为376位到462位的核苷酸序列,其编码的氨基酸序列为:Thr1-Arg2-Ser3-Arg4-Trp5-Arg6-Arg7-Phe8-Ile9-Arg10-Gly11-Ala12-Gly13-Arg14-Phe15-Ala16-Arg17-Arg18-Tyr19-Gly20-Trp21-Arg22-Ile23-Ala24-Leu25-Gly26-Leu27-Val28-Gly29。
Pb-CATH4的化学制备方法:
Ⅰ、根据编码缅甸蟒蛇cathelicidin基因推断的成熟肽Pb-CATH4氨基酸序列,用自动多肽合成仪(Applied Biosystems)合成其全序列,通过HPLC反相柱层析脱盐纯化,并确定其纯度大于95%。
Ⅱ、分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF),等电聚焦电泳测定等电点,用自动氨基酸测序仪测定氨基酸序列结构。合成的Pb-CATH4抗菌肽可以溶于灭菌超纯水,用于药理活性检测。
缅甸蟒蛇抗微生物肽Pb-CATH4的药理实验:
1.Pb-CATH4抗菌活性检测:
将化学合成的Pb-CATH4以2mg/ml的浓度溶解在无菌超纯水中;用接种环挑取新活化的微生物,然后均匀涂布在新的LB琼脂板上;将直径0.5cm的圆形无菌滤纸片放在上述琼脂板上,然后向纸片上滴加10μl Pb-CATH4样品溶液;放入37℃恒温培养箱中培养12-24h;观察抑菌圈形成与否,将对Pb-CATH4敏感的菌株记录下来。
2.Pb-CATH4对敏感菌株最小抑菌浓度(Minimum Inhibitory Concentration,MIC)的测定。
该实验以无菌的LB液体培养基作阴性对照,观察有细菌生长孔的浓度与无细菌生长的孔的浓度,取平均值即为最小抑菌浓度。
挑取新活化的微生物,接种至无菌液体LB培养基,37℃恒温振荡器中200rpm培养10-16h至对数生长期;用紫外分光光度计测菌液600nm处的吸光值,吸光值为1时,浓度大约为109cfu/ml,将菌液用无菌液体LB培养基稀释至2×105cfu/ml,冰上待用;用二倍稀释法在96微孔板上用无菌LB培养基配制浓度梯度为200μg/ml、100μg/ml、50μg/ml、25μg/ml、12.5μg/ml、6.25μg/ml、3.13μg/ml、1.56μg/ml、0.78μg/ml、0.39μg/ml、0.20μg/ml的Pb-CATH4样品溶液,每孔50μl;每孔加入50μl上述稀释菌液;在恒温振荡器中37℃,100rpm振荡培养10-16h;使用酶标仪测600nm吸光值或肉眼观察;上述实验重复3次。
表1 Pb-CATH4的最小抑菌浓度(MIC)
*MIC:最小抑菌浓度;ND:抑菌圈实验检测无抑菌活性;IS:临床分离菌株。以上结果为三次独立重复实验平均值。
由表1可见,Pb-CATH4对测试所用微生物具有广谱的抗菌活性,其MIC值大都在2.68-10.74μM之间,抗菌活性明显强于传统使用的抗生素氨苄青霉素(MIC值大都在12.62–201.9μM之间)。Pb-CATH4不仅对革兰氏阳性菌(G+)和革兰氏阴性菌(G-)具有抗菌活性,还对测试用的所有真菌都有抗菌活性,特别是对几种常见的临床分离的致病菌株如肺克杆菌,铜绿假单胞菌,表皮葡萄球菌以及白色念珠菌等具有强的杀菌作用。其中,Pb-CATH4对痢疾杆菌最为敏感,最小抑菌浓度仅为1.34μM。
3.Pb-CATH4的杀菌动力学检测。
活化菌株:将大肠杆菌ATCC25922划线接种到LB固体培养基平板上,置于37℃培养箱中倒置培养至单菌落长出。用接种环挑取单菌落接种到LB液体培养基中,37℃振荡过夜培养至对数生长期。用新鲜的LB液体培养基将菌液稀释至1×105CFU/ml的浓度,备用。
将Pb-CATH4或者美罗培南加入到稀释好的菌液中,使其终浓度为5倍MIC(阴性对照用相应体积的灭菌双蒸水)。加入样品的菌液迅速放入37℃振荡培养箱中培养,分别于0min、10min、20min、30min、45min、60min、90min及120min时取10μl菌液用灭菌的LB液体培养基稀释100倍,然后取100ul稀释后的菌液涂LB固体培养基,37℃过夜培养,计算菌落数。
由附图可知,Pb-CATH4杀菌迅速,在10分钟内就能够杀死全部大肠杆菌ATCC25922细胞;而阳性对照美罗培南作用相对较慢,需要90分钟才能全部杀死大肠杆菌ATCC25922。美罗培南是人工合成的广谱碳青霉烯类抗生素,通过抑制细菌细胞壁的合成而产生抗菌作用。与美罗培南相比,Pb-CATH4迅速的杀菌能力表明其作用机制不可能是抑制细菌细胞壁的合成,也不可能是抑制细菌细胞内核酸、蛋白质的合成或抑制细菌某些蛋白酶的活性,因为这需要一定的作用时间才能导致细菌的死亡,从而可以看出Pb-CATH4具有不同于传统抗生素的独特的杀菌机理,也正因为这点而不易引起菌体耐药性的产生,显示了Pb-CATH4的良好的抗菌药用前景。
Claims (4)
1.一种源于缅甸蟒的cathelicidin家族抗微生物肽Pb-CATH4,其特征在于,所述的cathelicidin家族抗微生物肽Pb-CATH4是直链多肽,含有29个氨基酸残基,理论等电点为12.54,分子量是3492.11Da,其序列为:苏氨酸-精氨酸-丝氨酸-精氨酸-色氨酸-精氨酸-精氨酸-苯丙氨酸-异亮氨酸-精氨酸-甘氨酸-丙氨酸-甘氨酸-精氨酸-苯丙氨酸-丙氨酸-精氨酸-精氨酸-酪氨酸-甘氨酸-色氨酸-精氨酸-异亮氨酸-丙氨酸-亮氨酸-甘氨酸-亮氨酸-缬氨酸-甘氨酸。
2.按权利要求1所述cathelicidin家族抗微生物肽Pb-CATH4的编码基因,其特征在于,核苷酸序列为:
Acaagaagtcgctggagaagattcataaggggagcgggacgcttcgccaggagatatggatggcgcattgcattgggcctagtgggataa。
3.权利要求1所述cathelicidin家族抗微生物肽Pb-CATH4的应用,其特征在于,用于药理活性检测,或用于制备抗病原微生物感染药物,或用于化妆品、保健品、食品、饲料的添加剂。
4.权利要求2所述cathelicidin家族抗微生物肽Pb-CATH4编码基因的应用,其特征在于,根据编码基因重组表达Pb-CATH4。
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