CN114907451A - 一种人工合成的噬菌肽seyt1及其应用 - Google Patents
一种人工合成的噬菌肽seyt1及其应用 Download PDFInfo
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- CN114907451A CN114907451A CN202210542293.0A CN202210542293A CN114907451A CN 114907451 A CN114907451 A CN 114907451A CN 202210542293 A CN202210542293 A CN 202210542293A CN 114907451 A CN114907451 A CN 114907451A
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- seyt1
- seyt4
- bacteria
- bacteriophage
- peptides
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Abstract
本发明主要涉及抗感染领域,具体涉及一种人工合成的噬菌肽SEYT1及其应用。本发明提供的噬菌肽,为SEQ ID NO.1所示,该噬菌肽对***和革兰氏阴性细菌均有高效且广谱的杀菌活性。噬菌肽SEYT1在1h内对***和革兰氏阴性细菌均有良好的抑菌或杀菌作用,具有良好的温度耐受性和pH耐受性,能够在不同的温度和pH下稳定发挥抑菌或杀菌作用,对多种细菌具有有效防控。
Description
技术领域
本发明主要涉及抗感染领域,具体涉及一种人工合成的噬菌肽SEYT1及其应用。
背景技术
抗菌肽,又称宿主防御肽,是一大类广泛存在于各种生物体内的小型多肽,用于保护机体免受细菌、真菌、病毒等不同病原体的侵染。天然抗菌肽在结构和功能上显示出多样性,几乎在所有生物体中都有发现,最早可以追溯到20世纪30年代在土壤芽孢杆菌中提取到的一种抗菌剂。在此之后,研究人员陆续从原核生物和真核生物中发现了几种抗菌肽。至此,在动物、植物、细菌等中均发现抗菌肽。迄今为止,研究人员从青蛙皮肤中分离出了300多种不同的抗菌肽。其他已发现的抗菌肽来源有兔白细胞防御素、牛乳凝集铁蛋白、人白细胞溶酶体和女性生殖道低分子量抗菌肽等。
随着噬菌体的进化,出现了一些来源于噬菌体的、可以和细菌细胞中的关键蛋白相结合的多肽,它们会使细菌失活从而阻断细菌的关键代谢过程。这些来源于噬菌体且具有抗菌活性的多肽,简称噬菌肽。有研究通过对金黄色葡萄球菌噬菌体测序,确定了在金黄色葡萄球菌中表达后还能抑制细菌本身生长的新型多肽家族;另一项研究通过检查GenBank(http://www.ncbi.nlm.nih.gov)中的各种噬菌体裂解酶的基因组序列,寻找二级结构、电荷和疏水性类似于天然抗菌肽的肽序列,发现这些蛋白质至少含有一种抗菌肽类似物,其物理化学性质显示出强效抗菌活性;为了验证该结果的正确性,该研究选择了两个假单胞菌噬菌体裂解酶基因组序列(D3 ORF31和),来自这些基因组序列的抗菌肽类似物被验证可以通过使用类似于常规的抗菌肽杀菌机制来杀死细菌。
与来自动物、植物和微生物的抗菌肽一样,同属于多肽类抗菌剂的噬菌肽在食品、畜牧业、农业和环境中具有广泛的应用价值,本申请以从噬菌体中发现了一个新型的噬菌肽为研究对象,目前从噬菌体中发现的噬菌肽还相对较少。
发明内容
本发明的目的是提供一种人工合成的噬菌肽SEYT1,所述噬菌肽为SEQ ID NO.1所示。
本发明中的另一个目的是提供噬菌肽SEYT1在抑制细菌中的应用,特别是用于制备抑菌剂,可用于细菌导致的食品、环境、疾病等的细菌抑制。
为了达到以上目的,本发明采取了以下技术措施:
申请人提供了两条具有抑菌功能的噬菌肽,分别为SEYT1和SEYT4,SEYT1的氨基酸序列为:RKQNWTEMCNRITDWDMGGKYRGLTIRKQKEKALCLKPVQ,SEYT4的氨基酸序列为RKQNWTEMCNRITDWDMGGKYRGLTIRKQSQSRESQC。
噬菌肽SEYT1为两亲性螺旋,其疏水性指数为0.202,疏水力矩为0.089,为净正电荷为+6的多肽;噬菌肽SEYT4为两亲性螺旋,其疏水性指数为0.109,疏水力矩为0.166,为净正电荷为+4的多肽。与SEYT1相比,SEYT4疏水性有所降低,两亲性有所增加。
噬菌肽SEYT1和/或SEYT4在抑制细菌中的应用,包括可利用该噬菌肽制备成抑菌的抑菌剂,或是用于食品抗菌剂,或是制备成治疗细菌感染的药物,或是制备成防治细菌导致的环境污染的试剂。
以上所述的应用中,优选的,所述的细菌包括但不限于:鲍曼不动杆菌(Acinetobacter baumannii),铜绿假单胞菌(Pseudomonas aeruginosa),粪肠球菌(Enterococcus faecalis)、屎肠球菌(Enterococcus faecium)、沙门氏菌(Salmonella)、大肠杆菌(Escherichia coli)、副溶血性弧菌(Vibrio parahaemolyticus)、阪崎克罗诺杆菌(Cronobacter sakazakii)、肺炎克雷伯菌(Klebsiella pneumoniae)、金黄色葡萄球菌(Staphylococcus aureus)、猪链球菌(Streptococcus suis)和/或白色念珠菌(CandidaAlbicans)。
与现有技术相比,本发明具有以下优点和有益效果:
(1)噬菌肽SEYT1和SEYT4具有广谱高效的杀菌活性,在1h内对常见的***和革兰氏阴性细菌均有良好的抑菌或杀菌作用;
(2)噬菌肽SEYT1和SEYT4具有良好的温度耐受性和pH耐受性,能够在不同的温度和pH下稳定发挥抑菌或杀菌作用;
(3)噬菌肽SEYT1和SEYT4可用于动物模型中猪链球菌、金黄色葡萄球菌和鲍曼不动杆菌的防控,对其具有良好的抑制作用。
(4)噬菌肽SEYT1和SEYT4不会对真核生物造成损害,也不会使哺乳动物的红细胞破裂。
附图说明
图1为噬菌肽SEYT1和SEYT4对***的杀菌谱。
图2为噬菌肽SEYT1和SEYT4对革兰氏阴性细菌的杀菌谱。
图3为噬菌肽SEYT1和SEYT4对猪链球菌LSM178、金黄色葡萄球菌LSA139、铜绿假单胞菌LPA3、沙门氏菌ATCC13311以及鲍曼不动杆菌LAB12的裂解曲线。
图4为温度对噬菌肽SEYT1和SEYT4杀菌活性的影响。
图5为pH对噬菌肽SEYT1和SEYT4杀菌活性的影响。
图6为噬菌肽SEYT1和SEYT4对动物养殖设备环境中或食品接触面上污染的金黄色葡萄球菌LSA139和LSA140及鲍曼不动杆菌LAB12和LAB13的抑制作用。
图7为噬菌肽SEYT1和SEYT4对真核生物的作用。
图8为噬菌肽SEYT1和SEYT4对哺乳动物红细胞的溶血作用。
图9为噬菌肽SEYT1和SEYT4在猪皮模型中的应用。
具体实施方式:
下面结合实施案例来进一步说明本发明,但本发明要求保护的范围并不局限于实施案例表述的范围。本发明所述技术方案,如未作说明,均为常规技术;所述试剂或材料,如未特别说明,均来源于商业渠道。本发明的噬菌肽SEYT1和SEYT4,可通过其氨基酸序列直接合成,或是利用本领域的常规方法,例如通过蛋白表达获得。
实施例1:
噬菌肽SEYT1和SEYT4对***的杀菌作用:
噬菌肽SEYT1的序列为SEQ ID NO.1所示,噬菌肽SEYT4为SEQ ID NO.2所示。
选择4株猪链球菌、4株金黄色葡萄球菌、2株屎肠球菌和3株粪肠球菌共13株菌株进行噬菌肽SEYT1和SEYT4对***的杀菌测定。
其中,4株猪链球菌分别命名为SC19,P1/7,LSM102,LSM178
4株金黄色葡萄球菌分别命名为LSA139,LSA140,LSA141,LSA144;
2株屎肠球菌分别命名为LEFM1,LEFM2;
3株粪肠球菌分别命名为LEFS1,LEFS2,LEFS8。
分别培养上述菌株至对数期,取1mL培养至对数期的菌液于25℃,8000rpm/min离心10min,弃上清,用1mL 30mM HEPES(pH=7.4)缓冲液重悬菌体,同样条件下离心后弃上清,然后用1mL 30mM HEPES(pH=7.4)缓冲液重悬菌体,再用30mM HEPES(pH=7.4)缓冲液将其稀释至105CFU/mL。取50μL稀释后的菌液到96孔板中,实验组中加入50μL 100μg/mL的噬菌肽SEYT1或SEYT4,对照组中加入50μL 30mM HEPES(pH=7.4)缓冲液,置于37℃,200rpm条件下反应1h,将反应液稀释到合适的梯度,使用平板计数法测定细菌的数量。
结果如图1所示,噬菌肽SEYT1和SEYT4对4株猪链球菌(4/4,100%)、4株金黄色葡萄球菌(4/4,100%)、2株屎肠球菌(2/2,100%)和3株粪肠球菌(3/3,100%)均有较好的杀菌作用;噬菌肽SEYT4对4株猪链球菌(4/4,100%)、金黄色葡萄球菌(4/4,100%)、2株屎肠球菌(2/2,100%)和3株粪肠球菌(3/3,100%)均有良好的杀菌作用。
实施例2:
噬菌肽SEYT1和SEYT4对革兰氏阴性菌的杀菌作用
选择2株大肠杆菌、1株肺炎克雷伯菌、13株铜绿假单胞菌、4株沙门氏菌、22株鲍曼不动杆菌、2株副溶血性弧菌、1株阪崎克罗诺杆菌共45株菌株进行噬菌肽SEYT1对革兰氏阴性菌的杀菌谱测定。
其中,2株大肠杆菌分别命名为:LEC32,LEC33;
1株肺炎克雷伯菌命名为:LKP11;
13株铜绿假单胞菌分别命名为:
LPA1,LPA2,LPA3,LPA4,LPA5,LPA6,LPA7,LPA8,LPA9,LPA10,LPA11,LPA12,LPA13;
4株沙门氏菌分别命名为:
1)2株肠炎沙门氏菌ATCC13076,SGSC4901,
2)2株鼠伤寒沙门氏菌ATCC13311,ATCC14028;
22株鲍曼不动杆菌分别命名为:
LAB1,LAB2,LAB4,LAB5,LAB6,LAB7,LAB8,LAB9,LAB10,LAB11,LAB12,LAB13,LAB14,LAB15,LAB16,LAB17,LAB18,LAB19,LAB20,LAB21,LAB22,LAB23;
2株副溶血性弧菌分别命名为:LVP1,LVP78;
1株阪崎克罗诺杆菌命名为:LCS4。
具体方法见实施例1。
结果如图2所示,图2中的A-D显示,噬菌肽SEYT1和SEYT4对2株大肠杆菌(2/2,100%)、1株肺炎克雷伯菌(1/1,100%)、13株铜绿假单胞菌(13/13,100%)、4株沙门氏菌(4/4,100%)、19株鲍曼不动杆菌(19/22,86.37%)、2株副溶血性弧菌(1/2,50%)和1株阪崎克罗诺杆菌(1/1,100%)均有较好的杀菌作用;噬菌肽SEYT4对2株大肠杆菌(2/2,100%)、1株肺炎克雷伯菌(1/1,100%)、13株铜绿假单胞菌(9/13,69.23%)、4株沙门氏菌(3/4,75%)、19株鲍曼不动杆菌(19/22,86.37%)、2株副溶血弧菌(2/2,100%)和1株阪崎克罗诺杆菌(1/1,100%)均有较好的杀菌效果。
实施例3:
噬菌肽SEYT1和SEYT4对猪链球菌LSM178、金黄色葡萄球菌LSA139、铜绿假单胞菌LPA3、沙门氏菌ATCC13311以及鲍曼不动杆菌LAB12的裂解曲线。
分别培养上述菌株至对数期,取1mL培养至对数期的菌液于25℃,8000rpm/min离心10min,弃上清,用1mL 30mM HEPES(pH=7.4)缓冲液重悬菌体,同样条件下离心后弃上清,然后用1mL 30mM HEPES(pH=7.4)缓冲液重悬菌体,再用30mM HEPES(pH=7.4)缓冲液将其稀释至105CFU/mL。取50μL稀释后的菌液到96孔板中,实验组中分别加入50μL的噬菌肽SEYT1或SEYT4,对照组中加入50μL 30mM HEPES(pH=7.4)缓冲液,分别在0min、10min、20min、30min、40min、50min、60min吸取反应液并将其稀释到合适的梯度,使用平板计数法测定细菌的数量,完成后立即将96孔板放回37℃培养箱内振荡培养。
结果如图3中A-F所示,与不加噬菌肽的对照组相比,噬菌肽SEYT1和SEYT4对上述细菌均有高效快速的杀菌作用,最快可在10min内将细菌降低到检测限以下。
实施例4:
温度对噬菌肽SEYT1和SEYT4杀菌活性的影响
分别培养金黄色葡萄球菌LSA140和鲍曼不动杆菌LAB12至对数期,取1mL培养至对数期的菌液于25℃,8000rpm/min离心10min,弃上清,用1mL 30mM HEPES(pH=7.4)缓冲液重悬菌体,同样条件下离心后弃上清,然后用1mL 30mM HEPES(pH=7.4)缓冲液重悬菌体,再用30mM HEPES(pH=7.4)缓冲液将其稀释至105CFU/mL。取50μL稀释后的菌液到96孔板中,实验组中加入50μL 100μg/mL的SEYT1和SEYT4,对照组中加入50μL 30mM HEPES(pH=7.4)缓冲液,分别在4℃、25℃和37℃下反应1h,随后吸取反应液并将其稀释到合适的梯度,使用平板计数法测细菌的数量。
噬菌肽SEYT1和SEYT4对温度的耐受性如图4所示,在不同温度下,噬菌肽SEYT1和SEYT4的活性未受到影响,对金黄色葡萄球菌LSA140和鲍曼不动杆菌LAB12均有良好的杀菌效果。
实施例5:
pH对噬菌肽SEYT1和SEYT4杀菌活性的影响
不同pH缓冲液的配制
pH=5:0.1M acetate缓冲液,称取770.8mg acetate溶于80mL无菌水中,充分溶解后用稀醋酸或稀氢氧化铵溶液进行pH调整,最后用水定容至100mL,用0.22μm滤膜过滤除菌后保存备用,使用时按照一定比例稀释至所需浓度即可;
pH=6:0.1M MES缓冲液,称取MES 2.1325g溶于80ml无菌水中,充分溶解后用KOH溶液调pH,最后用水定容至100mL,用0.22μm滤膜过滤除菌后保存备用,使用时稀释至所需浓度即可;
pH=7和pH=8:0.1M HEPES缓冲液,分别称取2.383g HEPES和2.603g HEPESsodium salt溶于80mL无菌水中,充分溶解后定容至100mL,二者互调,配制pH=7和pH=8的缓冲液,用0.22μm滤膜过滤除菌后保存备用,使用时稀释至所需浓度即可;
pH=9:0.1M CHES缓冲液,称取CHES 2.0729g溶于80mL无菌水中,充分溶解后用KOH溶液调pH,最后用水定容至100mL,用0.22μm滤膜过滤除菌后保存备用,使用时稀释至所需浓度即可;
pH=10:0.1M CAPS缓冲液,称取CAPS 2.213g溶于80mL无菌水中,充分溶解后用KOH溶液调pH,最后用水定容至100mL,用0.22μm滤膜过滤除菌后保存备用,使用时稀释至所需浓度即可。
分别培养金黄色葡萄球菌LSA140和鲍曼不动杆菌LAB12至对数期,取1mL培养至对数期的菌液于25℃,8000rpm/min离心10min,弃上清,用1mL对应pH的缓冲液重悬菌体,同样条件下离心后弃上清重悬菌体后,用对应pH的缓冲液将其稀释至105CFU/mL。取50μL稀释后的菌液到96孔板中,实验组中加入50μL 100μg/mL的SEYT1或SEYT4,对照组中加入50μL对应pH的缓冲液,置于37℃,200rpm条件下反应1h,将反应液稀释至合适梯度,使用平板计数法测细菌的数量。
噬菌肽SEYT1和SEYT4对pH的耐受性如图5中A和B所示,噬菌肽SEYT1和SEYT4具有良好的pH耐受性,在pH为5至10时仍可保持抑菌或杀菌活性。
实施例6:
噬菌肽SEYT1和SEYT4对动物养殖环境设备中或厨房用具上污染的金黄色葡萄球菌LSA139和LSA140及鲍曼不动杆菌LAB12和LAB13的抑制作用
使用大小约为2cm×2cm的不锈钢、硅胶和塑料切片模拟动物在养殖过程中接触到的器具如围栏、料槽等或厨房用具如砧板、菜刀等。将上述细菌培养至对数期后稀释至105CFU/mL,取100μL稀释后的菌液滴加到切片表面,并在无菌条件下干燥。随后,将每个区域用100μL 200μg/mL噬菌肽SEYT1或SEYT4处理,同时用100μL 30mM HEPES(pH=7.4)作对照,于室温下放置1h。随后用HEPES浸湿的棉签在水平,垂直和对角线方向上擦拭10次,然后将棉签放入装有500μL 30mM HEPES的EP管中,使用台式涡旋混合器以最大速度搅拌2min以恢复细菌。将细菌悬浮液稀释至合适梯度,使用平板计数法测定细菌的数量。
噬菌肽SEYT1和SEYT4在接触面上的杀菌作用如图6所示:与对照组相比,SEYT1可分别将各接触面上污染的金黄色葡萄球菌LSA139的菌量降低约90.92%、95.13%和100%;可分别将各接触面上污染的金黄色葡萄球菌LSA140的菌量降低约100%、99.92%和100%;可分别将各接触面上污染的鲍曼不动杆菌LAB12的菌量降低约99.97%、99.99%和100%;可分别将各接触面上污染的鲍曼不动杆菌LAB13的菌量降低约100%、100%和100%;SEYT4可分别将各接触面上污染的金黄色葡萄球菌LSA139的降低约63.08%、75.04%和100%;可分别将各接触面上污染的金黄色葡萄球菌LSA140的降低约99.79%、84.40%和100%;可分别将各接触面上污染的鲍曼不动杆菌LAB12的降低约99.14%、90.45%和99.88%;可分别将各接触面上污染的鲍曼不动杆菌LAB13的降低约99.99%、93.30%和100%。
以上结果表明,噬菌肽SEYT1和SEYT4可有效抑制动物养殖环境或食品接触面上的病原菌。
实施例7:
噬菌肽SEYT1和SEYT4对真核生物的作用
选择真菌中的白色念珠菌进行噬菌肽SEYT1和SEYT4对真核生物的作用的测定。
其中,2株白色念珠菌分别命名为LCA1和LCA4。
具体方法见实施例1。
实验结果如图7所示,噬菌肽SEYT1和SEYT4最多可将白色念珠菌LCA1和LCA4的菌量降低约77.78%,由此可见,噬菌肽对真菌的杀菌作用并不明显,即不会对真核细胞造成损害。
实施例8:
噬菌肽SEYT1和SEYT4对哺乳动物红细胞的溶血作用
取C57BL/6小鼠新鲜血液1mL,于4℃,3500rpm/min下离心10min,弃上清用PBS洗涤2-3次,以除去血清和其他杂质,得到100%红细胞后用PBS将其稀释至4%(v/v)备用;取100μL稀释后的红细胞与不同浓度的噬菌肽SEYT1和SEYT4混匀后孵育,同时分别以PBS和Triton X-100为阴性对照和阳性对照,于37℃下孵育1h。随即将混合物离心,吸取上清液于96孔板中,用酶标仪测定其在540nm处的吸光值,将引起5%溶血活性对应的肽的浓度定义为最小溶血浓度(Minimal hemolytic concentration,MHC)。
噬菌肽SEYT1和SEYT4对哺乳动物红细胞的溶血作用如图8所示:与对照组相比,噬菌肽SEYT1和SEYT4不会裂解红细胞,即不会使红细胞发生溶血现象。
实施例9:
噬菌肽SEYT1和SEYT4在猪皮模型中的应用
解冻的猪皮尽量去除油脂部分后用胶带剥离以去除上部死层,然后将其切成1cm×1cm大小的切片,将切片置于75%酒精中浸泡20min-30min进行消毒杀菌,然后用无菌水冲洗并浸泡20min-30min以去除多余油脂和残留酒精,随后将其取出置于无菌培养皿中于通风橱风干备用。
将猪链球菌SC19和P1/7、金黄色葡萄球菌LSA139和LSA140以及鲍曼不动杆菌LAB12和LAB13,培养至对数期后稀释至105CFU/mL,吸取5μL稀释后的菌液涂抹于猪皮表面,将其置于通风橱中感染1h,随后将5μL 500μg/mL噬菌肽SEYT1或SEYT4涂抹于猪皮表面进行治疗,同时以5μL 30mM HEPES(pH=7.4)作对照,治疗时间为1h;将用噬菌肽治疗后的猪皮切片转移至装有500μL 30mM HEPES(pH=7.4)缓冲液的EP管中,对其进行研磨以恢复细菌。将细菌悬浮液转移至96孔板中,并将其稀释至合适梯度,使用平板计数法测定细菌的数量。
噬菌肽SEYT1和SEYT4在猪皮模型中的杀菌作用如图9所示:与对照组相比,SEYT1可分别将猪皮上污染的猪链球菌SC19和P1/7降低约82.55%、84.03%;可分别将猪皮上污染的金黄色葡萄球菌LSA140降低约36.39%,对LSA139无杀菌作用;可分别将猪皮上污染的鲍曼不动杆菌LAB12和LAB13降低约30.22%和70.25%;SEYT4可分别将猪皮上污染的猪链球菌SC19和P1/7降低约76.60%和76.05%;可分别将猪皮上污染的金黄色葡萄球菌LSA139和LSA140降低约35.39%和8.92%;可分别将猪皮上污染的鲍曼不动杆菌LAB12和LAB13降低约36.97%和74.46%。
序列表
<110> 华中农业大学
<120> 一种人工合成的噬菌肽SEYT1及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Arg Lys Gln Asn Trp Thr Glu Met Cys Asn Arg Ile Thr Asp Trp Asp
1 5 10 15
Met Gly Gly Lys Tyr Arg Gly Leu Thr Ile Arg Lys Gln Lys Glu Lys
20 25 30
Ala Leu Cys Leu Lys Pro Val Gln
35 40
<210> 2
<211> 37
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Arg Lys Gln Asn Trp Thr Glu Met Cys Asn Arg Ile Thr Asp Trp Asp
1 5 10 15
Met Gly Gly Lys Tyr Arg Gly Leu Thr Ile Arg Lys Gln Ser Gln Ser
20 25 30
Arg Glu Ser Gln Cys
35
Claims (5)
1.一种人工合成的噬菌肽,所述噬菌肽为SEQ ID NO.1所示。
2.权利要求1所述的噬菌肽在制备抑菌剂中的应用。
3.权利要求1所述的噬菌肽在制备治疗或预防细菌感染引起的疾病的药物中的应用。
4.权利要求1所述的噬菌肽在制备食品抑菌剂中的应用。
5.权利要求1所述的噬菌肽在制备防治细菌导致的环境污染的试剂中的应用。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140178353A1 (en) * | 2012-12-26 | 2014-06-26 | Yeda Research And Development Co., Ltd. | Bacterial anti-phage defense systems |
CN112175928A (zh) * | 2020-10-13 | 2021-01-05 | 华中农业大学 | 一种源于沙门氏菌噬菌体基因编码的蛋白在作为革兰氏阴性菌裂解酶中的应用 |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140178353A1 (en) * | 2012-12-26 | 2014-06-26 | Yeda Research And Development Co., Ltd. | Bacterial anti-phage defense systems |
CN112175928A (zh) * | 2020-10-13 | 2021-01-05 | 华中农业大学 | 一种源于沙门氏菌噬菌体基因编码的蛋白在作为革兰氏阴性菌裂解酶中的应用 |
Non-Patent Citations (3)
Title |
---|
WEAVER, L.H.等: ""glycoside hydrolase family protein [Vibrio vulnificus]"", 《GENBANK》, pages 224639360 * |
ZHANG, J.等: ""glycoside hydrolase family protein, partial [Vibrio vulnificus]"", 《GENBANK》, pages 0777073 * |
杨甜 等: ""噬菌肽SEYT4对食品链中常见致病菌的清除效果"", 《华中农业大学学报》, pages 1 - 8 * |
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