CN106188265A - 一种抗微生物肽Cm‑CATH2及其基因、制备方法和应用 - Google Patents
一种抗微生物肽Cm‑CATH2及其基因、制备方法和应用 Download PDFInfo
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- CN106188265A CN106188265A CN201610585747.7A CN201610585747A CN106188265A CN 106188265 A CN106188265 A CN 106188265A CN 201610585747 A CN201610585747 A CN 201610585747A CN 106188265 A CN106188265 A CN 106188265A
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Abstract
一种抗微生物肽Cm‑CATH2及其制备方法和应用,Cm‑CATH2是一种直链多肽,含有33个氨基酸残基,理论等电点为12.96,分子量是4089.97Da,净电荷为+12。编码抗微生物肽Cm‑CATH2前体cathelicidin的基因由486个核苷酸组成。Cm‑CATH2对革兰氏阴性菌、革兰氏阳性菌、真菌均有很强的抗菌活性,包括多种临床耐药菌,并且最小抑菌浓度很低,杀菌效果快速且持久;其不含有二硫键及环状结构,方便化学合成及基因工程制备;还具有低溶血、低细胞毒、不易产生耐药性等特点,可替代抗生素制备抗病原微生物感染,消炎等临床药物,以及化妆品、保健品、食品、饲料等日化中的添加剂被应用,有很好的应用前景。
Description
技术领域
本发明提供一种来源于绿海龟(C.mydas)的cathelicidin家族广谱抗微生物肽Cm-CATH2及其制备方法和在抗病原微生物感染、消炎等临床药物、化妆品、保健品、食品、饲料的添加剂等中的应用,属于生物医学技术领域。
背景技术
Cathelicidin是一类多功能的抗菌肽,在哺乳动物、鸟类、爬行动物、两栖动物、以及鱼类中均发现其存在。自首次从牛中性粒白细胞中发现Bac5后,越来越多的Cathelicidin抗菌肽得以鉴定。Cathelicidin通常以前体的形式合成,包括N端的大约30个氨基酸残基的信号肽,保守的cathelin结构域及C端高度特异的成熟肽区域,三个部分组成。Cathelicidin前体到达作用部位后,在水解酶的作用下迅速释放cathelin结构域和成熟肽,发挥抗菌、免疫调节的作用。大量的抗菌肽活性研究显示,Cathelicidin是一种高效广谱的抗菌类药物,甚至对临床上分离的高耐药性细菌也具有良好的抑制效果,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,因此被视为理想的设计新型抗生素的模板。除此之外,Cathelicidin还具有抗病毒、抗寄生虫、抗肿瘤癌等多种功能,而且绝大多数抗菌肽对哺乳动物细胞无毒性作用;还具有对多种免疫细胞具有趋化作用、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生物活性,因此Cathelicidin在医药、畜牧业、食品和化妆品等领域具有很好的应用前景。
目前抗菌肽的应用主要集中于医药领域的开发,并且取得了一些令人满意的成果,已经有许多新型药物逐步迈入医药市场。比如,来自爪蟾抗菌肽Magainin的Pexiganan,由Genaera公司开发,目前以外用霜剂的形式用来治疗脓疱病和糖尿病患者足底溃烂等,已经进入临床III期试验阶段,它也是第一个进行商业开发的抗菌肽;Daptomycin是一种阴离子抗菌肽,由Cubit Pharmaceuticals公司研发,2003年9月由美国食品与药物管理局(FDA)批准上市;来自猪白细胞的Iseganan,由Intrabiotics公司开发,用药途径是口服液或喷雾剂,用来治疗抗肿瘤治疗引发的口腔炎和囊肿性纤维化病人肺感染,目前也处于临床III期试验阶段;来自于牛的MBI-肽,由Micrologix生物技术公司,以外用乳膏的形式用来治疗尿道相关的血流感染,处于III期临床;来自人唾液的Histatin衍生肽,做成口腔洗剂,用来治疗牙根炎和口腔感染(II-III期临床)、口腔念珠菌感染(II期临床)和慢性绿脓杆菌感染(I期临床)。可见,抗菌肽在医药领域有着很好的应用前景。
抗菌肽的应用不局限于医药领域,动物饲料、化妆品、保健品、食品等中也有很好的应用。抗生素作饲料添加剂的使用早已引起了争议,世界卫生组织发表公告指出“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。美国的R&D报道,抗茵肽可作为饲料防霉剂;温刘发等应用含抗茵肽的柞蚕免疫血淋巴粉添加于断奶仔猪饲料中,饲喂试验表明,柞蚕抗茵肽可减轻断奶仔猪的腹泻。他们又将抗茵肽的发酵制剂制作为饲料添加剂喂粤黄鸡,研究表明该制剂可促进小鸡生长和减少排泄物氮含量。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,其成分为易消化吸收的氨基酸,可作为畜禽饲料添加剂取代或部分取代饲喂动物所用的抗生素,将会减少抗生素对动物体的危害,避免抗生素残留对人类的伤害。另外,化妆品、保健品、食品中会添加一些添加剂,比如防腐剂,抗氧化剂等,为了抑制食品微生物生长和繁殖,延长食品的保存时间,但是这些添加剂往往被擅自滥用,给人身体带来巨大伤害,特别是有致癌的副作用,所以可以用抗菌肽替代防腐剂和抗氧化剂。
绿蠵龟(C.mydas)又名绿海龟,是各种海龟中体形较大的一种,和其他海龟一样,除了上岸产卵外,终其一生都在大洋中渡过。它广泛分布于热带及亚热带之海域,并产卵於温度达25C以上的沙滩。觅食区多为海草丰富的浅水区,为草食性动物,是海龟里唯一摄食较多海藻的种类。目前关于绿海龟cathelicidin宿主防御肽的研究和应用还没有报道。
发明内容
本发明提供一种来源于绿海龟(C.mydas)的cathelicidin家族广谱抗微生物肽Cm-CATH2及其编码基因和制备方法,主要应用于制备抗病原微生物感染、消炎等临床药物及在化妆品、保健品、食品、饲料中作为添加剂。
为了实现本发明的目的,本发明提供了如下技术方案:
抗微生物肽Cm-CATH2的分离纯化方法:
选取绿海龟脾脏组织,洗涤,匀浆,获得脾脏组织蛋白粗提物。随后,Sephadex G-50凝胶过滤层析,用自动部分收集器收集3mL/管,220nm检测并收集各峰检测抗菌活性,冻干备用。然后经反相高压液相层析(RP-HPLC)进行梯度洗脱,收集各多肽样品峰,用灭菌去离子水溶解,检测抗菌活性。最后对得到多肽样品进行一级结构的解析,包括采用电喷雾四级杆飞行时间串联质谱法(ESI-Q-TOF-MS,Biosystems/MDS Sciex多伦多,加拿大)测定分子量。利用等电聚焦电泳测定其等电点,用Edman降解法确定其多肽氨基酸序列组成。多肽全序列一级结构为:精氨酸-精氨酸-丝氨酸-精氨酸-苯丙氨酸-甘氨酸-精氨酸-苯丙氨酸-苯丙 氨酸-赖氨酸-赖氨酸-缬氨酸-精氨酸-赖氨酸-谷氨酰胺-亮氨酸-甘氨酸-精氨酸-缬氨酸-亮氨酸-精氨酸-组氨酸-丝氨酸-精氨酸-异亮氨酸-苏氨酸-缬氨酸-甘氨酸-甘氨酸-精氨酸-甲硫氨酸-精氨酸-苯丙氨酸。
绿海龟Cm-CATH2基因的克隆包括:
绿海龟肝脏总RNA提取,mRNA纯化,mRNA反转录及cDNA文库构建,设计引物,利用PCR方法筛选绿海龟抗菌肽Cm-CATH2基因。获得阳性单克隆进行基因核苷酸序列测定。基因测序结果表明编码抗微生物肽Cm-CATH2前体cathelicidin的基因由486个核苷酸组成,自5’端至3’端序列为:
其中385到483位是编码抗微生物肽Cm-CATH2的核苷酸序列,其编码氨基酸序列为:精氨酸1-精氨酸2-丝氨酸3-精氨酸4-苯丙氨酸5-甘氨酸6-精氨酸7-苯丙氨酸8-苯丙氨酸9-赖氨酸 10-赖氨酸11-缬氨酸12-精氨酸13-赖氨酸14-谷氨酰胺15-亮氨酸16-甘氨酸17-精氨酸18-缬氨酸19-亮氨酸20-精氨酸21-组氨酸22-丝氨酸23-精氨酸24-异亮氨酸25-苏氨酸26-缬氨酸27-甘氨酸28-甘氨酸29-精氨酸30-甲硫氨酸31-精氨酸32-苯丙氨酸33。
抗微生物肽Cm-CATH2的合成,包括以下步骤:
(1)根据基因编码的Cm-CATH2氨基酸序列,用自动多肽合成仪合成其全序列;
(2)通过HPLC反相柱层析脱盐纯化,并确定其纯度大于97%;
(3)用基质辅助激光解析电离飞行时间质谱测定其分子量;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。
所述抗微生物肽Cm-CATH2可应用在制备抗病原微生物感染、消炎等临床药物及在化妆品、保健品、食品、饲料等的添加剂中。
本发明的有益效果在于:通过分离纯化及Edman降解的方法确定其成熟肽氨基酸组成;通过基因克隆得到了编码绿海龟cathelicidins抗微生物肽基因;再通过化学合成方法得到成熟 肽Cm-CATH2。该抗微生物肽富含碱性氨基酸,具有很强的广谱抗菌活性,抗菌实验显示其对多种临床耐药菌也有较好的杀灭作用。此外,其结构简单,不含有二硫键及环状结构,方便化学合成及基因工程制备;还具有低溶血、低细胞毒、不易产生耐药性等特点。故可替代抗生素制备抗病原微生物感染,消炎等临床药物,还可作为化妆品、保健品、食品、动物饲料等中的添加剂,有很好的应用前景。
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。
实施例1 抗微生物肽Cm-CATH2的分离纯化:
(1)从动物园获取濒临死亡的绿海龟新鲜完整脾脏组织,用生理盐水少许洗涤脾脏组织表面。匀浆后,用少量生理盐水溶解,按PS液与正丁醇1:50(V/V)比例,把PS与正丁醇在室温下搅拌60min,13000r/min,20min离心两次,然后将沉淀冻干。随后,第一步SephadexG-50凝胶过滤层析:0.9g冻干粉用10ml 0.1M磷酸盐(Na2HPO4-NaH2PO4,pH 6.0)缓冲液溶解,12000rpm离心10min,取上清液上样于已平衡好的Sephadex G-50凝胶排阻色谱柱(1.6cm x 90cm,Amersham Bioscience),用同样缓冲液洗脱,流速3mL/10min,用自动部分收集器收集3mL/管,220nm检测并收集各峰检测抗菌活性,冻干备用。
(2)反相高压液相层析(RP-HPLC):将Sephadex G-50凝胶排阻色谱分离得到的活性成分的峰重新溶解于纯水中,4℃,12000rpm离心15min,取上清液,用0.45μm滤膜过滤,收集滤液上样于经含1‰三氟乙酸的超纯水充分平衡的C18反相柱(Hypersil BDS C18,30cmx 0.46cm)用乙腈(含1‰三氟乙酸)构成的洗脱系统进行梯度洗脱,215nm检测多肽浓度。收集得到的各峰,冻干浓缩,用灭菌的去离子水重新溶解并进行抗菌活性检测。
(3)活性多肽一级结构分析。纯化的Cm-CATH2采用电喷雾四级杆飞行时间串联质谱法(ESI-Q-TOF-MS,Biosystems/MDS Sciex多伦多,加拿大)测定分子量。利用等电聚焦电泳测定其等电点,用Edman降解法确定了其氨基酸序列组成为Cm-CATH2:RRSRFGRFFKKVRKQLGRVLRHSRITVGGRMRF。
实施例2 Cm-CATH2前体基因的克隆及基因测序
第一步、绿海龟肝脏总RNA提取(以下实验所用器具和试剂均经过处理,无RNase):
A.从新鲜宰杀的绿海龟各种新鲜组织上分别剪下1g左右的小块,分别放入液氮预冷的细胞冻存管中,然后迅速放入液氮中保存;
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-П,涡旋振荡15-30s,4℃,12000rpm离心5min;
C.将离心后的上清转移至新的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离心吸附柱,室温,6000rpm离心1min;弃废液,然后加入500μl BufferW1A,4℃,12000rpm离心1min;弃废液,加入700μl BufferW2A,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,12000rpm离心1min洗脱得到总RNA;
第二步、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5min后,然后42℃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;4℃,保存。cDNA保存于-80℃。
第二链的合成:
第三步、采用半巢式PCR进行绿海龟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。以合成的肝脏cDNA为模板,以P1和In-Fusion SMARTer CDS为引物,进行第一次PCR扩增。在0.2ml PCR管中加入下列试剂(总体积20μl):
混匀后,短暂离心。PCR条件为:94℃变性5min;25个循环:94℃变性30s,60℃退火30s,72℃延伸1min;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加入99μl ddH2O稀释100倍作为模板,以P2和CDSⅢ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(总体积20μl):
PCR条件为:94℃变性5min;25个循环:94℃变性30s,58℃退火30s,72℃延伸1min;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-MgCl2法制备好的DH5α感受态细胞。取100μl转化菌液均匀涂布在含有100μlg/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 ABIPRISM 377进行核苷酸测序。
第四步、绿海龟cathelicidin的基因序列测定和结果:
编码绿海龟cathelicidin抗微生物肽Cm-CATH2为第385到483位核苷酸,其氨基酸序列为:精氨酸1-精氨酸2-丝氨酸3-精氨酸4-苯丙氨酸5-甘氨酸6-精氨酸7-苯丙氨酸8-苯丙氨酸9-赖氨酸10-赖氨酸11-缬氨酸12-精氨酸13-赖氨酸14-谷氨酰胺15-亮氨酸16-甘氨酸17-精氨酸18-缬氨酸19-亮氨酸20-精氨酸21-组氨酸22-丝氨酸23-精氨酸24-异亮氨酸25-苏氨酸26-缬氨酸27-甘氨酸28-甘氨酸29-精氨酸30-甲硫氨酸31-精氨酸32-苯丙氨酸33。
实施例3 Cm-CATH2的化学合成方法:
(1)根据编码绿海龟cathelicidin基因推断的成熟肽Cm-CATH2氨基酸序列,用自动多肽合成仪(Applied Biosystems)合成其全序列。
(2)通过HPLC反相C18柱层析对合成多肽进行脱盐纯化:过程中使用柱子是4.6×250nm,Venusil XBP-C4;溶剂A是0.1%trifluoroacetic溶于100%乙腈,溶剂B是0.1%trifluoroacetic溶于100%水;梯度设为:0.01min(A 15%,B 85%),25min(A40%,B60%),25.1min(A 100%,B 0%),30min(stop);流速为1.0ml/min,波长为220nm,体积为5μl,结果测得合成的多肽序列纯度大于97%。
(3)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):首先将Cm-CATH2样品分散在基质分子中并形成晶体,然后用激光照射晶体,基质从激光中吸收能量,样品解吸附,基质-样品之间发生电荷转移使得样品分子电离,电离的样品在电场作用下飞过真空的飞行管,根据到达检测器的飞行时间不同而被检测,即通过离子的质量电荷之比(M/Z)与离子的飞行时间成正比来分析离子,并测得样品分子的分子量,得到Cm-CATH2样品的分子量是4089.97Da。
(4)等电聚焦电泳测定等电点:配胶:胶液组成为载体两性电解质,凝胶贮液,蒸馏水,混合样品,染色物质等,灌胶,加电极液,电泳,样品收集进行检测,得到Cm-CATH2等电点。
(5)用自动氨基酸测序仪测定氨基酸序列结构。合成的Cm-CATH2抗菌肽可以溶于灭菌超纯水或PBS,用于药理活性检测。
实施例4 绿海龟抗微生物肽Cm-CATH2的药理实验:
(1)Cm-CATH2抗菌活性检测:
将化学合成的CM-CATH2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 CM-CATH2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Cm-CATH2敏感的菌株记录下来。
(2)Cm-CATH2对敏感菌株最小抑菌浓度(Minimum Inhibitory Concentration,MIC)的测定
该实验以无菌液体LB作阴性对照,最小抑菌浓度的定义为肉眼可以观察到的完全抑制微生物生长的最低多肽浓度,或者是光吸收值不高于阴性对照5%的最低浓度。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光波处的吸光值,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106cfu/ml,冰上待用;在96微孔板上配制浓度梯度为100μg/ml、50μg/ml、25μg/ml、12.5μg/ml、6.25μg/ml、3.13μg/ml、1.56μg/ml、0.78μg/ml、的经0.22μm孔径膜过滤的Cm-CATH2样品溶液(即24.4501、12.2250、6.1125、3.0563、1.5281、0.7641、0.3820、0.1910μM),每孔50μl;每孔加入50μl上述稀释菌液,充分混匀后,在37℃培养箱中培养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,取平均值。
表1.Cm-CATH2的最小抑菌浓度(MIC)
*MIC:最小抑菌浓度,浓度值为3次重复实验的平均值;ND:2mg/ml剂量纸片抑菌试验无明显活性;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,Cm-CATH2对测试的微生物具有广谱的抗菌活性,特别是对几种常见病原菌株大肠杆菌,痢疾杆菌,金黄色葡糖球菌等。在40种实验检测的菌株(包括标准菌株和临床分离的耐药菌株)中,Cm-CATH2对绝大多数革兰氏阴性细菌表现出极强的抗菌活性,其中对大肠杆菌和痢疾杆菌抗性最强,MIC值仅为1.1462μM;革兰氏阳性菌中,对金黄色葡萄球菌和屎肠球菌最为敏感,最小抑菌浓度分别为1.1462μM和0.5731μM。还对部分真菌也有很强的抗菌活性。Cm-CATH2对临床分离耐药细菌具有极强的活性,如铜绿假单孢杆菌、大肠杆菌等。在微生物耐药性日益严重的今天,Cm-CATH2对耐药菌极强的抗菌活性及其独特的杀菌机制,使其成为新型抗生素药物开发的优良模板。
(3)Cm-CATH2对大肠杆菌的杀菌动力学:
大肠杆菌ATCC25922接种到LB固体培养基平板上,37℃培养箱中倒置培养至菌落长出。用接种环挑取单菌落接种到LB液体培养基中,37℃振荡培养箱中培养到对数生长期。用新 鲜的LB液体培养基将菌液稀释至1×106CFU/ml,将样品加入稀释好的菌液中,使其终浓度为5×MIC(阴性对照加入相应体积的灭菌超纯水)。加入样品的菌液迅速放入37℃振荡培养箱中,150rpm振荡培养,分别于0min、5min、10min、20min、30min、45min、60min、90min、120min取10μl菌液用灭菌的生理盐水稀释1×103倍,取50μl涂布LB固体平板。平板放入37℃培养箱中倒置培养16h,菌落计数。
表2.Cm-CATH2对大肠杆菌的杀菌动力学
由表2可知,Cm-CATH2平均在20分钟左右就能够杀死全部大肠杆菌ATCC25922细胞,而阳性对照美罗培南需要90多分钟才能全部杀死大肠杆菌ATCC25922,结果证明了Cm-CATH2不仅有很强的杀菌活性,并且其杀菌速度也要远超过抗生素药物美罗培南;不仅如此,Cm-CATH2对细菌的作用是致死性的,大肠杆菌经Cm-CATH2作用后全部死亡,2h后无细菌恢复生长,显示了Cm-CATH2的良好和持久的杀菌作用。
Claims (4)
1.一种抗微生物肽Cm-CATH2,其特征在于,Cm-CATH2是一种直链多肽,含有33个氨基酸残基,理论等电点为12.96,分子量是4089.97Da,净电荷为+12,其全序列一级结构为:精氨酸-精氨酸-丝氨酸-精氨酸-苯丙氨酸-甘氨酸-精氨酸-苯丙氨酸-苯丙氨酸-赖氨酸-赖氨酸-缬氨酸-精氨酸-赖氨酸-谷氨酰胺-亮氨酸-甘氨酸-精氨酸-缬氨酸-亮氨酸-精氨酸-组氨酸-丝氨酸-精氨酸-异亮氨酸-苏氨酸-缬氨酸-甘氨酸-甘氨酸-精氨酸-甲硫氨酸-精氨酸-苯丙氨酸(RRSRFGRFFKKVRKQLGRVLRHSRITVGGRMRF);
编码抗微生物肽Cm-CATH2前体cathelicidin的基因由486个核苷酸组成,自5’端至3’端序列为:
其中385到483位是编码抗微生物肽Cm-CATH2的核苷酸序列。
2.权利要求1所述一种抗微生物肽Cm-CATH2的制备方法,其特征在于:根据编码Cm-CATH2抗微生物肽基因推断的成熟肽基酸序列,用自动多肽合成仪合成其全序列;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。
3.权利要求1所述一种抗微生物肽Cm-CATH2的应用,其特征在于:所述抗微生物肽Cm-CATH2,具有很强的广谱抗菌活性,不易引起耐药性,对许多临床分离的耐药菌株也有很强的杀伤活性,可用于制备抗病原微生物感染以及消炎用的临床药物;或作为动物饲料中抗生素的替代品;或作为化妆品、保健品、食品中的添加剂以替代传统防腐剂。
4.根据权利要求3所述一种抗微生物肽Cm-CATH2基因的应用,其特征在于,据其编码合成的Cm-CATH2抗微生物肽具有很强的抗微生物作用,溶于灭菌超纯水,用于药理活性检测。
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