CN111909264A - 一种人源化抗金黄色葡萄球菌生物活性肽及其编码基因与应用 - Google Patents
一种人源化抗金黄色葡萄球菌生物活性肽及其编码基因与应用 Download PDFInfo
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Abstract
本发明提供一种人源化的抗金黄色葡萄球菌生物活性肽及其编码基因与应用,该活性肽DNA序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示;生物活性肽是模拟万古霉素结构和功能的“β”类型人源化抗独特型基因工程抗体,该基因工程抗体经原核表达,纯化后的抗独特性抗体蛋白具有与万古霉素多克隆抗体F(ab)2片段结合的活性,具有特异性抑制金黄色葡萄球菌生长的活性;本发明不经动物免疫而采用体外高通量筛选和鉴定的方式获得可模拟万古霉素抗菌功能的抗独特型基因工程抗体小分子多肽,靶向性强,制备周期短,氨基酸序列小,适合体外大规模生产,且动物安全性更高,对食用农产品和食品中金黄色葡萄球菌的安全性生物防控具有重要的科学意义和实用价值。
Description
技术领域
本发明涉及抗独特性基因工程抗体创制技术领域,特别是一种人源化重链抗体基因改造的抗金黄色葡萄球菌生物活性肽及其编码基因。
背景技术
金黄色葡萄球菌(Staphylococcus aureus,S. aureus)是食品中最常见的食源性致病菌之一,其产生的肠毒素能引起严重的食物中毒,对金黄色葡萄球菌的防控不仅是食品、农产品质量安全监管和防腐保鲜的重要工作,同时也是生物医药领域研究的重大课题。抗生素自发现起,在人类疾病治疗上发挥着巨大作用,但近几十年来,由于滥用,其给人类带来的副作用及导致的病原菌耐药性问题日益凸显,严重威胁到了人类健康乃至生态安全。抗菌肽作为一类具有抗菌活性的生物小分子多肽,被普遍认为是对人类及生态安全的有望替代抗生素用于防控致病菌的新型抗菌类药物,具有重大挖掘潜力和应用价值,越来越受到科研工作者的青睐。
当前抗菌肽主流制备方法为从天然的动植物或微生物及海洋生物体内分离获得。据国际权威抗菌肽数据库(Antimicrobial Peptide Database,APD;http://aps.unmc.edu/AP/)最新收录信息显示:目前登记收录的各类抗菌肽数量已达2775种,其中2080种源于动物,339种源于植物,278种源于微生物,13 种源于真菌,8种源于原生生物,4种源于古生菌。尽管报道的抗菌肽数量很多,但能够实现商品化应用的药物却很少,美国FDA在过去数十年间只批准了十几种抗菌肽药品(Da-Costa J.P.等,Appliedmicrobiology and biotechnology,2015年05期)。究其原因,主要还是传统方法很难从生物体内获得具有高活性的抗菌肽,即便获得也极难避免天然小分子多肽制备困难、结构不稳定、易失活(因温度、pH变化)等缺陷(肖轶尘等,“生物信息学用于抗菌肽预测和分子设计的研究进展”,生物技术通报,2016年12期),而无法实现大规模生产应用。
1974 年, Jerne 首次提出“免疫网络学说”,认为在动物天然免疫系统中,采用抗体免疫机体后,通过级联反应能刺激免疫系统产生具有抗原独特型的抗体,其能模拟抗原表位的抗原决定簇,具有抗原类似的结构和功能,能发挥抗原部分特性功能。其中“β”型的抗独特型抗体具有抗原“内影像”效应,即具有与抗原相同的抗原决定簇,从而可以模拟特定抗原决定簇分子构象及乃至其生物活性的功能;随后由Nisonoff 和Lamoyi 进一步证实, 抗独特型抗体具有能替代抗原诱导特异性免疫应答的作用。
目前国内外多采用免疫动物的方式制备模拟抗原功能的“β”型抗独特型抗体,如:2009年,Hanoux等(“Polyclonal anti-idiotypic antibodies which mimic an epitopeof the human prion protein”,Molecular immunology,2009年06期)通过二次免疫获得了可模拟人类朊蛋白抗原表位的抗独特型多克隆抗体,能代替朊蛋白用于免疫治疗;2011年,Xu等(“Expression, characterization and therapeutic efficacy of chimericFab of anti-idiotypic antibody NP30 against Schistosoma japonicum”,Actatropica,2011年02期)获得鼠源吸血虫的抗独特型单链抗体NP-30可以模拟日本血吸虫肠道相关抗原(GAA),经小鼠体内实验,证实可用于治疗急性血吸虫病;2015年,Lan等(“Anti-idiotypic antibody: A new strategy for the development of a growth hormonereceptor antagonist”,The International Journal of Biochemistry & CellBiology,2015年68卷)借助免疫小鼠制备的生长激素抗独特型单克隆抗体能模拟生长激素与其受体结合,起到拮抗物的作用。
噬菌体表面展示基因工程抗体技术是紧随基因工程抗体制备发展起来的外源基因与病毒核酸串联共表达的重组技术,是当前抗体创新研究和体外高通量靶向筛选研究的热点。1985年,美国科学家Simth等(“Filamentous fusion phage: novel expressionvectors that display cloned antigens on the virion surface”, Science,1985年05期)首次报道将外源多肽基因克隆到丝状噬菌体DNA上,经串联共表达,多肽展示到噬菌体衣壳表面,实现抗体表型与其基因型统一于一个噬菌体上,由此开创了噬菌体表面展示技术并获得了2018年度诺贝尔化学奖。噬菌体表面展示抗体技术有效避免了传统抗体在制备过程中需要动物免疫以及细胞杂交等繁冗操作过程,也摆脱了动物伦理上的舆论压力,同时基因工程抗体基因可以依托噬菌体和宿主菌进行持续表达,使抗体制备更方便、更省时、更省力(Pande等 “Phage display: concept, innovations, applications andfuture”, Biotechnol. Adv., 2010年06期);且还能将其基因转移克隆到更合适的载体或表达体系中进行优化表达(Ayyar等“Optimizing antibody expression: the nuts andbolts”, Methods (San Diego, Calif.), 2017年116卷),甚至还能借助抗体体外亲和成熟技术直接在分子水平上对基因工程抗体基因进行靶向成熟修饰和回溯淘筛(刘媛等“基因突变技术在抗体亲和力体外成熟中的应用”, 浙江大学学报农业与生命科学版),2016年01期)或者设计使噬菌体抗体自动进化(Badran等“Continuous evolution of Bacillusthuringiensis toxins overcomes insect resistance”, Nature,2016年533卷)等,极大提高了抗体制备效率和应用价值。
抗生素自发现起,在人类疾病治疗上发挥着巨大作用,但近几十年来,由于滥用,其存在的毒副作用日渐凸显,不仅危害人类健康,特别是耐药菌的大量报道,显示抗生素更是严重威胁生态安全;一些国家已经禁止将抗生素作为防腐保鲜剂用于食品、食用农产品中致病微生物防控。万古霉素(Vancomycin)是一种糖肽类大分子抗生素,能与S. aureus肽聚糖合成前体lipoII的D-Ala-D-Ala肽段末端结合,抑制细胞壁合成使菌体死亡,特别对防控超级耐药S. aureus具有显著效果,被视为人类的“最后一道防线”,随着滥用,近年也出现了抗药菌,“(Bisicchia P.等“Acquisition of Van-B-type vancomycin resistanceby Bacillus subtilis: the impact on gene expression, cell wall compositionand morphology”,Molecular microbiology,2011年01期;Yarlagadda V.等“AVancomycin Derivative with a Pyrophosphate-Binding Group: A Strategy toCombat Vancomycin-Resistant Bacteria”,Angewandte Chemie,2016年27期)。万古霉素尽管对金黄色葡萄球菌具有极强的特异性抑制活性,但是其作为抗生素的一种,同样不可避免存在抗生素的上述问题。目前针对可替代抗生素抗菌功能的多肽效应物,特别是模拟万古霉素抗菌活性的抗独特型基因工程抗体小分子多肽等相关材料及产品还未见报道。
发明内容
针对上述问题,本申请提供一种具有模拟万古霉素抗菌功能且安全性更高可作为食品防腐保鲜剂级别的抗菌生物活性肽,用于食品、食用农产品中金黄色葡萄球菌的防控,具有重要的科学意义和市场推广应用价值。
本发明是这样实现的:
本申请首先提供了一种人源化抗金黄色葡萄球菌生物活性肽,其氨基酸序列如SEQ IDNO.2所示。
其次,本申请提供了编码氨基酸序列如SEQ ID NO.2所示的人源化抗金黄色葡萄球菌生物活性肽的基因,其核苷酸序列如SEQ ID NO.1所示。
第三,本申请还提供了含有人源化抗金黄色葡萄球菌生物活性肽基因SEQ ID NO:1的原核表达载体或工程菌(如大肠杆菌)。
第四,本申请还提供了氨基酸序列如SEQ ID NO.2所示的人源化抗金黄色葡萄球菌生物活性肽在(非医疗目的)杀灭金黄色葡萄球菌中的用途,特别是杀灭(非生物体内)环境、食品中金黄色葡萄球菌的应用。
第五,本申请还提供了氨基酸序列如SEQ ID NO.2所示的人源化抗金黄色葡萄球菌生物活性肽在制备食品保鲜剂中的应用。
本申请依托抗体“免疫网络学说”中“β”类型抗独特型抗体理论,首次设计以万古霉素多克隆抗体关键抗原结合区F(ab)2片段为包被靶点,并结合噬菌体展示基因工程抗体库技术,从商品化的大容量人源化噬菌体展示基因工程抗体库中进行体外高通量靶向筛选获得具有模拟万古霉素抗菌功能的“β”类型抗独特型生物活性肽。与现有技术相比,本申请获得的活性肽具有以下有益效果:
1、本发明锚定以万古霉素多克隆抗体F(ab)2片段作为包被靶点“抗原”,不经动物免疫而直接采用体外高通量靶向筛选和鉴定的方式,从人源化噬菌体展示单重链抗体库中获得可模拟万古霉素抗菌功能的抗独特型基因工程抗体小分子多肽,筛选过程靶向性强,制备周期短,氨基酸序列小,适合体外大规模生产。
2、本申请提供的抗菌生物活性肽来源于人类抗体重链片段,属于人源化的单重链抗体蛋白质材料,可自然降解,能高温变性失活,在应用过程中对动物安全性更高,特别是作为防腐保鲜剂用于食品、食用农产品中金黄色葡萄球菌防控上,其对人类不存在异源蛋白免疫排斥的问题,因此对食品、食用农产品中致病微生物安全防控具有极大应用价值。
3、本申请提供的抗菌生物活性肽经原核系统表达,纯化后的多肽蛋白能特异性对供试的金黄色葡萄球菌具有较强抑制活性,可替代万古霉素作为可食用级别的防腐保鲜剂用于食品中金黄色葡萄球菌的生物防控。
附图说明
图1万古霉素多克隆抗体F(ab)2片段制备效果图;
其中,泳道M为蛋白maker;1:万古霉素多克隆抗体血清;泳道2为经饱和硫酸铵沉淀法初级纯化的万古霉素多克隆抗体;泳道3为经Protein A柱纯化后的万古霉素多克隆抗体;泳道4为经β巯基乙醇变性处理后的万古霉素多克隆抗体;泳道5为经试剂盒酶切后获得的万古霉素多克隆抗体F(ab)2片段;泳道6为经β巯基乙醇变性处理后的万古霉素多克隆抗体F(ab)2片段。
图2 为万古霉素抗独特型噬菌体展示抗体富集效果示意图。
图3为2C12噬菌体展示抗菌生物活性肽与F(ab)2片段结合的ELISA检测结果示意图。
图4为2C12抗菌生物活性肽表达纯化后多肽蛋白SDS-PAGE电泳效果示意图;
其中,泳道M为蛋白maker,泳道1为未添加IPTG诱导的全细胞破碎液(阴性对照),泳道2为添加IPTG诱导表达后的全细胞破碎液,泳道3为过柱纯化后的2C12抗独特型抗菌生物活性肽蛋白。
图5为2C12抗菌生物活性肽对金黄色葡萄球菌抑菌效果图;
其中,图5A为供试菌种为金黄色葡萄球菌,图5A中,1:生理盐水,2:万古霉素,3:1F5抗独特型抗菌生物活性肽,4:2C12抗独特型抗菌生物活性肽;
图5B为供试菌种为大肠杆菌,图5B中,1:生理盐水;2:万古霉素;3:1F5抗独特型抗菌生物活性肽;4:2C12抗独特型抗菌生物活性肽。
具体实施方式
实施例中所涉及的试剂和培养基配方:
(1)2×TY 液体培养基:
在900 ml蒸馏水中加入16 g胰蛋白胨,10 g酵母提取物和5 g NaCl,搅拌混匀,用蒸馏水定容到1 L,置于高压灭菌锅中,121℃,20 min灭菌,冷却后,置于4℃保存备用。
(2)2×TY-AG液体培养基:
在2×TY的培养基中加入终浓度为100 μg/ml氨苄青霉素和质量比为1 %葡萄糖。
(5)TYE固体培养基:
在900 ml蒸馏水中加入15 g琼脂糖,8 g NaCl,10 g胰蛋白胨,5 g酵母提取物,用蒸馏水定容到1 L,置于高压灭菌锅中,121℃,20 min灭菌,冷却后,置于4℃保存备用。
(6)TYE-AG固体培养基:
在TYE固体培养基中加入终浓度为100 μg/ml氨苄青霉素和质量比为1 %葡萄糖。
(7)PBS溶液
称取NaCl 8 g,KCl 0.2 g,Na2HPO4·12H2O 2.9 g,KH2PO4 0.2 g,分别加入到蒸馏水中,充分溶解后,定容到1 L。
(8)PBST溶液
在PBS溶液中加入体积比为0.05%的吐温-20。
(9)PEG/NaCl溶液:
称取20 g PEG 8 000,14.61 g NaCl,加80 ml去离子水,定容到100 ml,置于高压灭菌锅中,121℃,20 min灭菌,冷却后,置于4℃保存备用。
(10)柠檬酸盐缓冲液(CPBS, 底物缓冲液,pH5.5):
取C6H7O8(柠檬酸)21 g,Na2HPO4·12H2O 71.6 g,分别加入到蒸馏水中充分溶解后定容到1 L。
(11)四甲基联苯胺(TMB)溶液:
称取10 mg四甲基联苯胺溶于1 ml二甲基亚砜中,避光,置于4℃保存备用。
(12)底物显色溶液:
10 ml 配方成分:9.875 ml CPBS、100 μl TMB溶液、25 μl 体积比为20% H2O2。
(13)LB液体培养基
1L体系:称取10 g胰蛋白胨,5 g酵母提取物,10 gNaCl,以蒸馏水定容至1 L,置于高压灭菌锅中,121℃,20 min灭菌,冷却后,置于4℃保存备用。
(14)LB固体培养基
1 L体系:取10 g胰蛋白胨,5 g酵母提取物,10 gNaCl,15 g琼脂粉,以蒸馏水定容至1L置于高压灭菌锅中,121℃,20 min灭菌,冷却后,置于4℃保存备用。
(15)IPTG溶液
1ml体系:称取0.238 g (1mM) IPTG 溶于1 ml蒸馏水中,配置成浓度为1 mM/ml 的母液,于-20℃保存备用。
实施例中所涉及材料来源:
人源化噬菌体展示单域抗体库(Human domain antibody library)、E.coli TG1细菌和辅助噬菌体KM13购于英国Source BioScience公司;
pET26b 载体, E.coliBL21, Anti-M13-[HRP] Antibody, Anti-His-[HRP]Antibody, His-Trap HP 亲和纯化柱购于美国GE Healthcare 公司;
金黄色葡萄球菌(Staphylococcus aureus ATCC 27217)和大肠杆菌Escherichia coli O157:H7 CICC 21530均购于上海北诺生物科技有限公司;
万古霉素购于Sigma-Aldrich北京公司。
实施例中涉及的核苷酸和氨基酸序列:
2C12万古霉素抗独特型抗菌生物活性肽基因序列(SEQ ID NO:1)
atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60
cgtctctcct gtgcagcctc cggagatatg attagcgcaa gcgctatggc ctgggtccgc 120
caggctccag ggaagggtct agagtgggta tcaggcagca cggacgacga cggtagcaca 180
tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240
ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgagt 300
tcgtatgcga ttcggtcggt ggacgacaca gacgcggact tggcgttttg gggtcaggga 360
accctggtca ccgtctcgag cgcggccgca 390
2C12万古霉素抗独特型抗菌生物活性肽氨基酸序列(SEQ ID NO:2)
Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Met Ile Ser
20 25 30
Ala Ser Ala Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
35 40 45
Trp Val Ser Gly Ser Thr Asp Asp Asp Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Ser Ser Tyr Ala Ile Arg Ser Val Asp Asp Thr Asp Ala
100 105 110
Asp Leu Ala Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
115 120 125
Ala Ala
130
噬菌粒载体上游引物(SEQ ID NO.3)
pR2-F: 5'-AGGTGCAGCTGTTGGAGTCTG-3';
噬菌粒载体下游引物(SEQ ID NO.4):
pR2-R: 5'-TCGAGACGGTGACCAGGGT-3';
噬菌粒载体上游加酶切位点引物(SEQ ID NO.5)
pR2-F-NcoI: 5'-CATGCCATGGAGGTGCAGCTGTTGGAGTCTG-3';
噬菌粒载体上游加酶切位点引物(SEQ ID NO.6)
pR2-R-NotI: 5'-ATAAGAATGCGGCCGCTCGAGACGGTGACCAGGGT-3';
pET26b载体上游引物(SEQ ID NO.7)
pET26b-T7-F: 5'-TAATACGACTCACTATAGGG-3';
pET26b载体下游引物(SEQ ID NO.8)
pET26b-T7-R :5'-GCTAGTTATTGCTCAGCGG-3'。
实施例1 制备万古霉素多克隆抗体F(ab)2片段
取发明人前期制备的经纯化的万古霉素多克隆抗体F(ab)2片段(制备及鉴定过程详见,徐重新等, Establishment of an ultrasensitive indirect competitive time-resolved fluoroimmunoassay for vancomycin determination, Food andAgricultural Immunology,2019年01期),按照Thermo Scientific™公司Pierce™ F(ab')2 Preparation Kit附录的说明书操作步骤进行酶切并纯化获得万古霉素多克隆抗体F(ab)2片段,酶切及纯化效果见图1,测得最后收集获得的万古霉素多克隆抗体F(ab)2片段蛋白浓度是800 μg /ml。
实施例2淘筛具模拟万古霉素抗菌功能的抗独特型基因工程抗体
(1)取500 μl 冻存的Human domain antibody library菌液加入到200 ml 2×TY-AG液体培养基中,37℃恒温培养到OD600为0.4,接着加入200 μl 滴度为~1012 pfu/ml的KM13辅助噬菌体进行侵染,随即在37℃中孵育30 min,然后以3300 g离心10 min,弃上清液,用100ml 2×TY-AK液体培养基重悬沉淀物;最后30 ℃恒温培养过夜。次日取出培养体系,以3300g离心30 min,收集上清液并加入25 ml PEG/NaCl溶液,冰浴1 h,再以3300 g离心30 min,最后用5 ml PBS重悬沉淀。重悬液以11600 g离心10min,上清液即为扩增的噬菌体抗体库。
(2)取步骤1获得的扩增的噬菌体抗体库进行4 轮富集淘筛:第1轮富集淘筛,吸取4 ml浓度为80 μg /ml的万古霉素多克隆抗体F(ab)2片段溶液加入无菌的细胞培养瓶中,在4 ℃冰箱中静置包被过夜;次日,每次以1 ml PBS溶液清洗细胞培养瓶,重复洗涤3次,然后加入1 ml步骤(1)获得扩增的噬菌体抗体库与4 ml 3%的MPBS溶液混匀的混合物,在室温下缓慢摇动1 h,再静置1 h,倾去培养瓶中液体;以每次1ml PBST溶液洗瓶,重复洗涤20次;最后加入1 ml浓度为10 mg/ml的胰蛋白酶(Trypsin)溶液洗脱特异性结合的噬菌体抗体,洗脱液即为第1轮富集淘筛的噬菌体抗体;第2、3、4轮富集淘筛中万古霉素多克隆抗体F(ab)2片段包被抗原的浓度分别为60、30、10 μg/ml,所投入富集淘筛的噬菌体抗体库为前一轮富集淘筛获得的噬菌体抗体库,富集淘筛方法与第1轮相同,各轮次富集效果如图2所示;
取20 μl第4轮富集淘筛获得的噬菌体抗体库溶液侵染1 ml 处于对数生长期的E.coliTG1细菌,37℃孵育1 h后,涂布于TYE-AG固体培养基上,接着37℃培养过夜。次日,随机挑取单菌落,接种到含有100 μl/孔2×TY-AG液体培养基的96孔板中,37℃培养过夜;接着从板孔中吸出2.5 μl菌液转接到新的含有100 μl/孔2×TY-AG液体培养基的96孔板中,37℃孵育2 h;随即每孔加入25 μl滴度为~1012 pfu/ml的KM13辅助噬菌体,30℃孵育1.5 h,1800 g离心10min,用200 μl 2×TY-AK液体培养基重悬沉淀后30℃培养过夜;次日以1800 g离心30 min,分别取上清液用于ELISA分析。
(3)万古霉素多克隆抗体F(ab)2片段溶液,按照100 μl/孔加入到96孔板中,4 ℃包被过夜;次日,每孔分别加入100 μl步骤(2)获得的上清液,阴性对照加100 μl 2×TY-AK液体培养基,37℃水浴2 h;每孔用250 μl PBST洗板后,每孔加入100 μl 1:3000稀释的Anti-M13-[HRP] Antibody,37℃孵育2 h;每孔加入100 μl底物显色溶液,每孔加入100 μl底物显色溶液,室温下避光显色反应15 min,最后每孔加入50 μl浓度为2 M的H2SO4快速终止反应,用酶标仪测定OD450值。以此评价步骤(3)中筛选到的阳性抗独特型克隆噬菌体基因工程抗体对万古霉素多克隆抗体F(ab)2片段的结合活性,即溶液OD450值/阴性对照OD450值大于3.0,判定为阳性,与该溶液对应的步骤(2)中的上清液即为筛选到的含有与万古霉素多克隆抗体F(ab)2片段结合活性的抗独特型噬菌体基因工程抗体上清液。
通过上述筛选和鉴定,申请人筛选到一种与万古霉素多克隆抗体F(ab)2片段具有较强结合活性且能模拟万古霉素抗金黄色葡萄球菌的抗独特型基因工程抗体生物活性肽,将其自命名为2C12,其所对应步骤(2)的上清液对万古霉素多克隆抗体F(ab)2片段结合的ELISA检测效果如附图3所示。
以Sanger测序法,分别设计上游引物SEQ ID NO.3和下游引物SEQ ID NO.4,测定单域抗体2C12(申请人自命名)核苷酸序列如SEQ ID NO.1所示,核苷酸翻译后的氨基酸序列如SEQ ID NO.2所示。
实施例3 2C12的万古霉素独特型抗菌生物活性肽表达及抗菌活性测试
(1)基因克隆
以实施例1中筛选到的2C12单克隆噬菌体菌液为模板,分别设计上游引物SEQ ID NO.5和下游引物SEQ ID NO.6(下划线为酶切位点和保护碱基),进行PCR扩增:
PCR反应体系(20 μl):2×Trq Mix,10 μl;10 mM的上游引物,1 μl;10 mM的下游引物,1 μl;处于对数生长期浓度为108 cfu/ml的菌液模板,1 μl;无菌的ddH2O补足至20 μl;
反应条件:95℃10min,(94℃ 1min,56℃ 1min,72℃ 1min)× 35个循环, 72℃10min。
将反应获得的2C12抗独特型基因工程抗体基因产物以PCR产物纯化试剂盒纯化(步骤参见Sigma公司PCR产物纯化试剂盒说明书),获得的纯化的2C12抗独特型基因工程抗体基因,置于-20℃中保存备用。
另取含有pET26b质粒的E.coliBL21菌种,在LB液体培养基(含终浓度为50 μg/ml卡那霉素)中培养过夜,次日提取pET26b质粒(步骤参照Sigma公司质粒提取试剂盒说明书步骤执行)并于-20℃中保存备用。
分别将上述扩增纯化的纯化的2C12抗独特型抗菌生物活性肽和提取的pET26b质粒分别同时以NotI和NcoI核酸内切酶进行双酶切过夜(50 μl体系:10×NEB Buffer,5μl;100×BSA,1 μl,扩增的基因或质粒载体,30 μl,NotI和NcoI,各2.5 μl;最后以无菌的蒸馏水补足至50 μl。置酶切体系于37℃恒温水浴锅中酶切过夜,次日,将体系置于80℃恒温金属浴中进行热失活25 min(NotI和NcoI核酸内切酶在80℃热处理20 min均可变性失活)。
取热失活后的酶切体系进行酶连,构建pET26b-万古霉素抗独特型抗菌生物活性肽重组质粒:
20 μl反应体系:酶切载体pET26b,4 μl;酶切万古霉素抗独特型抗菌生物活性肽基因,10 μl;10×T4 ligBuffer ,2 μl;T4 ligase,1 μl;最后以无菌的蒸馏水补足至20 μl;
置混合体系于16℃冰箱中进行酶连过夜。次日,将酶连体系化转到E.coli BL21感受态细胞中,菌液涂布在LB固体培养基上(含终浓度为50 μg/ml卡那霉素),培养过夜后,挑单菌落做PCR和测序鉴定(引物为上游引物SEQ ID NO.7、下游引物SEQ ID NO.8),具体化转步骤参照南京师范大学毕业论文“人源化苏云金芽孢杆菌Bt(Cry1B)毒素蛋白单链抗体的筛选、表达及生物学活性测定”第3章35页,徐重新,2013)操作,利用PCR扩增和序列比对分析双重鉴定,含2C12抗独特型抗菌生物活性肽基因条带大小基因片段和相同核酸序列,即为阳性克隆。
(2)2C12万古霉素抗独特型抗菌生物活性肽可溶性表达及纯化
取步骤(1)中鉴定为阳性的单菌落(即含有实施例1中2C12万古霉素抗独特型抗菌生物活性肽基因片段),在LB液体培养基(含终浓度为50 μg/ml卡那霉素)中25℃ 250rpm震荡培养至OD600 nm为0.6,然后加入IPTG(终浓度为0.8 mM)诱导表达12 h;次日,取菌液,以3300g离心 20 min,收集沉淀细胞,用细胞破碎仪破碎细胞,然后5000 g离心20 min取上清液,过His-Trap HP affinity chromatography 纯化收集抗体蛋白(纯化方法参照论文“人源化抗Cry1B毒蛋白单链抗体的原核表达及生物学活性测定”徐重新等,南京农业大学学报,2013年3期)。以SDS-PAGE鉴定蛋白纯度(~15 kDa),以超微量分光光度计测得收集纯化蛋白浓度为568.69 μg/ml。
对上述蛋白进行电泳检测结果如图4所示,图4中泳道M:蛋白marker,泳道1为未添加IPTG诱导的全细胞破碎液(阴性对照),泳道2为添加IPTG诱导表达后的全细胞破碎液,泳道3为过柱纯化后的2C12抗独特型抗菌生物活性肽蛋白。
在实际应用中,也可以根据SEQ ID NO.2公开的氨基酸序列人工合成活性肽2C12后加以应用。
(3)2C12的万古霉素独特型抗菌生物活性肽抗菌实验
3.1)药敏纸片扩散法测抑菌圈:取金黄色葡萄球菌(Staphylococcus aureus ATCC27217)和大肠杆菌(Escherichia coli O157:H7 CICC 21530)在LB液体培养基中培养至对数生长期,将它们的菌液浓度定量到108 cfu/ml,然后各吸取200 μl涂布到LB固体培养基平板上,置于无菌操作台中静置晾干备用。取孔径为6 mm的无菌圆形滤纸片放在实施例2中蛋白浓度定量到500 μg/ml的2C12万古霉素独特型抗菌生物活性肽蛋白溶液中(阳性对照为50 μg/ml的万古霉素;阴性对照为0.9%的生理盐水;平行实验对照为与2C12同期制备的但与万古霉素F(ab)2的ELISA结合活性相对较弱的独特型抗菌生物活性肽,自命名为1F5),浸泡10 min,取出滤纸,晾干后分别贴到上述涂布有金黄色葡萄球菌和大肠杆菌的平板上,随即置于37℃恒温培养箱中倒置培养过夜,次日取出培养板,测得2C12抗独特型抗菌生物活性肽对供试的金黄色葡萄球菌的抑菌圈为8 cm (万古霉素为16 cm、1F5抗独特型抗菌生物活性肽为6 cm、生理盐水为0 cm),而对供试大肠杆菌为0 cm,没有抑菌活性(万古霉素、1F5抗独特型抗菌生物活性肽、生理盐水同样为0 cm,没有抑菌活性);测试效果见附图5;
由图5可知,上述实施例获得的抗菌生物活性肽具有模拟万古霉素抗菌功能,其抗菌活性尽管无法与万古霉素相比,在医药应用中不具备优势;但是因其人源化基因工程抗体的抗菌生物活性肽属性,对人类不产生免疫排斥反应和其他毒副作用,因此可作为食品防腐保鲜剂用于食品、食用农产品中金黄色葡萄球菌防控,具有广泛的应用价值。
3.2)二倍稀释法测最低抑菌浓度(Minimum inhibitory concentration, MIC)。取2 ml实施例2中蛋白浓度定量到500 μg/ml的2C12万古霉素独特型抗菌生物活性肽蛋白溶液用含有4 μl菌液浓度定量到108 cfu/ml处于对数生长期的金黄色葡萄球菌或大肠杆菌的LB培养基混合物按照二倍稀释法进行连续稀释8个梯度,然后将各稀释体系置于37℃培养箱中培养过夜。次日,测得2C12万古霉素独特型抗菌生物活性肽对供试的金黄色葡萄球菌的MIC值为125 μg/ml,对供试大肠杆菌没有抑菌活性。
以上实施例验证了实施例2获得的人源化抗金黄色葡萄球菌生物活性肽2C12可应用于杀灭金黄色葡萄球菌。在具体应用中,可以将该人源化抗金黄色葡萄球菌生物活性肽溶液稀释至500 μg/ml,喷洒于环境中,或用作食品保鲜剂,以杀灭金黄色葡萄球菌。
实施例4 2C12万古霉素抗独特型抗菌生物活性肽在猪肉防腐保鲜中的应用
(1)取100 g超市购买的生鲜猪瘦肉,以研钵研磨成匀质状态,然后在高压灭菌锅中以121 ℃灭菌20 min,冷却后备用。取1 g上述灭菌后的猪肉匀质物与1 ml定量浓度为500 μg/ml的2C12万古霉素独特型抗菌生物活性肽蛋白溶液充分混匀,在4℃中静置30 min后,向混合物中加入1μl菌液浓度定量到108 cfu/ml的处于对数生长期的金黄色葡萄球菌,充分混匀后置于37℃恒温培养箱中培养过夜。
(2)次日取样品参照《食品安全国家标准 食品微生物学检验 金黄色葡萄球菌检验》(GB4789.10—2016)中“第二法 金黄色葡萄球菌平板计数法”步骤进行金黄色葡萄球菌菌落数测定。
结果显示供样品中金黄色葡萄球菌菌落数为0,说明供试的2C12万古霉素抗独特型抗菌生物活性肽能完全抑制或杀灭猪肉中的金黄色葡萄球菌,可作为防腐保鲜剂用于食品、食用农产品中金黄色葡萄球菌防控。
序列表
<110> 江苏省农业科学院
<120> 一种人源化抗金黄色葡萄球菌生物活性肽及其编码基因与应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 390
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60
cgtctctcct gtgcagcctc cggagatatg attagcgcaa gcgctatggc ctgggtccgc 120
caggctccag ggaagggtct agagtgggta tcaggcagca cggacgacga cggtagcaca 180
tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240
ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgagt 300
tcgtatgcga ttcggtcggt ggacgacaca gacgcggact tggcgttttg gggtcaggga 360
accctggtca ccgtctcgag cgcggccgca 390
<210> 2
<211> 130
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Met Ile Ser
20 25 30
Ala Ser Ala Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
35 40 45
Trp Val Ser Gly Ser Thr Asp Asp Asp Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Ser Ser Tyr Ala Ile Arg Ser Val Asp Asp Thr Asp Ala
100 105 110
Asp Leu Ala Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
115 120 125
Ala Ala
130
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aggtgcagct gttggagtct g 21
<210> 4
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tcgagacggt gaccagggt 19
<210> 5
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
catgccatgg aggtgcagct gttggagtct g 31
<210> 6
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ataagaatgc ggccgctcga gacggtgacc agggt 35
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
taatacgact cactataggg 20
<210> 8
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gctagttatt gctcagcgg 19
Claims (6)
1.一种人源化抗金黄色葡萄球菌生物活性肽,其氨基酸序列如SEQ ID NO.2所示。
2.编码如权利要求1所述人源化抗金黄色葡萄球菌生物活性肽的基因序列,其核苷酸序列如SEQ ID NO.1所示。
3.如权利要求1所述人源化抗金黄色葡萄球菌生物活性肽在杀灭环境、食品中金黄色葡萄球菌中的应用。
4.如权利要求1所述人源化抗金黄色葡萄球菌生物活性肽在制备食品保鲜剂中的应用。
5.含有如权利要求2所述人源化抗金黄色葡萄球菌生物活性肽编码基因序列的原核表达载体。
6.含有如权利要求2所述人源化抗金黄色葡萄球菌生物活性肽编码基因序列的工程菌。
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CN109021109A (zh) * | 2018-07-20 | 2018-12-18 | 上海交通大学 | 一种牛源性抗金黄色葡萄球菌融合抗体scFv-Fc及其制备方法 |
CN110498854A (zh) * | 2019-09-28 | 2019-11-26 | 中国人民解放军陆军军医大学 | 一种抗金黄色葡萄球菌肠毒素b的抗体及其应用 |
-
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103773775A (zh) * | 2014-01-26 | 2014-05-07 | 江苏省农业科学院 | 一种人源抗虫基因及其编码的抗Cry1B毒素独特型单链抗体与应用 |
CN109021109A (zh) * | 2018-07-20 | 2018-12-18 | 上海交通大学 | 一种牛源性抗金黄色葡萄球菌融合抗体scFv-Fc及其制备方法 |
CN110498854A (zh) * | 2019-09-28 | 2019-11-26 | 中国人民解放军陆军军医大学 | 一种抗金黄色葡萄球菌肠毒素b的抗体及其应用 |
Non-Patent Citations (2)
Title |
---|
SOPHIE M LEHAR等: "Novel antibody-antibiotic conjugate eliminates intracellular S. aureus", 《NATURE》 * |
郭迎迎等: "肉制品中金黄色葡萄球菌的检测方法", 《吉林农业》 * |
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