CN110951715B - 一种抗葡萄球菌的宽谱噬菌体编码裂解酶及制备方法和应用 - Google Patents
一种抗葡萄球菌的宽谱噬菌体编码裂解酶及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种抗葡萄球菌的宽谱噬菌体编码裂解酶及制备方法和应用,属于生物技术领域。所述裂解酶核苷酸序列如Seq ID NO.1所示,氨基酸序列如Seq ID NO.2所示;该裂解酶用于防治多种葡萄球菌,该抗葡萄球菌的宽谱噬菌体嵌合裂解酶可以单独或复配使用,或制备成酶制剂,用以防治由葡萄球菌引起的各种污染和感染,特别是奶牛养殖场中由葡萄球菌引起的奶牛乳房炎感染,具有极高的防治效果,且安全无毒副作用。
Description
技术领域
本发明涉及微生物技术领域,特别是涉及一种抗葡萄球菌的宽谱噬菌体编码裂解酶及制备方法和应用。
背景技术
葡萄球菌广泛分布于空气、饮水、地面、物体表面及饲料。人及畜禽的皮肤、黏膜、肠道、呼吸道及乳腺也有寄生。致病性葡萄球菌引起各种化脓性感染、败血症及脓毒败血症,污染食品时,可引起食物中毒。根据生化反应和产生色素不同,葡萄球菌属有20多种,其中金黄色葡萄球菌(Staphylococcu saureus)是一种重要的人兽共患病原菌,可以引起伤口感染、心内膜炎、骨髓炎、中毒性休克综合征等多种疾病。除金黄色葡萄球菌外其余种葡萄球菌可以称为凝固酶阴性葡萄球菌(Coagulase-negativestaphyloeocci,CNS),如:表皮葡萄球菌、模仿葡萄球菌、溶血葡萄球菌、缓慢葡萄球菌等,主要为条件致病菌,引起人和动物持续性感染。葡萄球菌也是感染奶牛乳腺炎的主要致病菌,引起牛奶中体细胞数(Somaticcellcount,SCC)升高,严重影响产奶量及牛奶质量,严重威胁食品安全。
金黄色葡萄球菌可引起奶牛乳腺炎,降低奶牛的产奶量和牛奶质量,从而给奶牛养殖行业带来巨大损失。目前,抗生素仍被视为控制奶牛乳腺炎的唯一方法。然而,抗生素的大量使用增强了病原菌的耐受性,尤其在畜禽养殖业中由于抗生素的滥用,导致金黄色葡萄球菌多重耐药性问题日趋严重,特别是对青霉素类药物具有严重的耐受性,最典型的代表是耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)。因此寻求一类不易诱导细菌产生耐受性,并且能够高效对抗由耐药金黄色葡萄球菌引起的奶牛乳腺炎的新型抗菌制剂尤为重要。
发明内容
本发明的目的是提供一种抗葡萄球菌的宽谱噬菌体编码裂解酶及其制备方法和应用,为控制由耐药性性金黄色葡萄球菌引致的奶牛乳腺炎提供新的抗菌制剂,以实现减量化使用抗生素,为生态养殖提供新策略。
为实现上述目的,本发明提供了如下方案:
本发明提供一种抗葡萄球菌的宽谱噬菌体编码裂解酶,其核苷酸序列如Seq IDNO.1所示,氨基酸序列如Seq ID NO.2所示。
进一步的,所述裂解酶具有酰胺酶催化结构域和SH3b结合结构域。
本发明还提供一种所述的裂解酶的制备方法,包括以下步骤:
(1)根据裂解酶的基因序列设计引物,并在两条引物的5’端插入pET28a连接接头,进行PCR扩增,反应结束后,将产物采用1%的琼脂糖凝胶电泳检测,并用胶回收试剂盒回收PCR扩增产物;其中,引物序列如下:
F:5’-GGTACCCTCGAGGGATCCATGGCTAAGACTCAAGCAGAA-3’;
R:5’-CTGCAGGTCGACAAGCTTTTAACTCTTGAATGTCCCCCA-3’;
PCR反应体系:引物F 2μL,引物R 2μL,噬菌体YNP1基因组2μL,PrimeSTAR MaxPremix(2×)25μL,去离子水19μL;
PCR反应条件:98℃ 3min;98℃ 10s,55℃ 15s,72℃ 15s,30个循环;72℃ 5min;
(2)将步骤(2)得到的扩增产物连接到质粒载体pET28a,构建裂解酶pET28a表达载体,方法如下:首先将pET28a进行酶切线性化处理,酶切体系:BamHⅠ1μL、HindⅢ1μL、pET28a3μL、10×Buffer 5μL、去离子水40μL,30℃ 15min,37℃ 20min酶切;随后将1.2.6.1回收的裂解酶基因连接至线性化的pET28a载体,连接体系为:2×Hieff Clone EnzymePremix 5μL、酶切后pET28a质粒2μL、回收的裂解酶基因1μL、去离子水2μL,50℃连接20min。转至感受态细胞,过夜培养,筛选得到阳性克隆;
(3)将阳性克隆接种于含有Kan的LB液体培养基中,摇床培养至对数期,然后加入IPTG,摇床过夜诱导表达,得到发酵液;
(4)将诱导后的发酵液进行破碎,用PBS重悬,重悬后的细菌采用超声破碎,破碎后的菌液离心处理,取上清,用0.45μm滤膜过滤后获得裂解酶LysYNP1粗提液。
(5)将获得的裂解酶LysYNP1粗提液,采用亲和层析进行纯化,获得纯化的裂解酶LysYNP1。
(6)将获得的纯化裂解酶,采用JY-1作为指数菌,测定酶活性效价,确定该酶裂菌活性单位为38μg/ml。
本发明还提供所述的裂解酶在抑制或杀灭原料乳内葡萄球菌中的应用。
本发明还提供所述的裂解酶在制备防治牛乳房炎药物中的应用。
本发明公开了以下技术效果:
本发明发明裂解酶具有高效裂菌特性,平板裂解实验证实该裂解酶能够高效杀死耐药菌株,可用于防治多种葡萄球菌,用以防治由葡萄球菌引起的各种污染和感染,特别是奶牛养殖场中由葡萄球菌引起的奶牛乳房炎感染,具有极高的防治效果,且安全无毒副作用。该抗葡萄球菌的宽谱噬菌体嵌合裂解酶可以单独或复配使用,或制备成酶制剂,用以防治由葡萄球菌引起的各种污染和感染。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为蛋白结构分析,其中A为软件预测裂解酶结构,B为葡萄球菌噬菌体裂解酶的结构;
图2为裂解酶LysYNP1蛋白表达的SDS-PAGE分析,M:蛋白质Marker;1:pET28a-LysYNP1诱导后上清;2:pET28a-LysYNP1诱导前;
图3为平板裂解实验检测LysYNP1粗提液的裂解活性;
图4为小鼠乳腺组织中细菌数量。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
本发明实施例中所述9株患乳腺炎的乳汁中分离的多重耐药金黄色葡萄球菌菌株为JY-1、JY-2、JY-3、JY-4、JY-5、JY-6、JY-7、JY-8、JY-9;4株患乳腺炎的乳汁中分离的其他多重耐药葡萄球菌为SH-1、SH-2、SH-3、SH-4;11株MRSA菌株为USA300、RN4220、PNB25、DL57-2、B52、05Q132、361、386、439、575、702、823、948;大肠杆菌Min27,均为上海交通大学农业与生物学院动物病原微生物学实验室提供;大肠杆菌实验菌株MC1061,无乳链球菌ATCC13813和猪链球菌HA9801均该实验室提供;BL21(DE3)感受态细胞购自北京全式金生物公司;pET28a(+)载体,由上海交通大学农业与生物学院动物病原微生物学实验室提供。
LysJD为上海交通大学农业与生物学院动物病原微生物学实验室提供的裂解酶,用于本发明的裂解酶对照实验。
本发明中所述SM缓冲液、PBS缓冲液、异丙基-β-D-硫代半乳糖苷(IPTG)、SDS-PAGE试剂盒购自生工生物工程(上海)股份有限公司;三氯甲烷(氯仿)购自国药集团化学试剂有限公司;2×PrimeSTAR Max Premix,购自宝日医生物技术(北京)有限公司;胶回收试剂盒,购自南京诺唯赞生物科技有限公司;HieffPlus One Step Cloning Kit一步法快速克隆试剂盒,购自上海翊圣生物科技有限公司。
本发明中120kv透射电镜购自Thermo Fisher Scientific,型号为Talos L120CG2、恒温摇床购自上海智诚、紫外分光光度计购自Bio-Rad。
实施例1裂解酶LysYNP1的表达
裂解酶LysYNP1的诱导表达
根据裂解酶基因序列设计引物,其核苷酸序列如Seq ID NO.1所示,预测该基因表达的蛋白同时具有酰胺酶催化结构域和SH3b结合结构域(见图1),命名为LysYNP1。由图1可知,该裂解酶基因具有酰胺酶催化域和SH3b结合结构域,因为具有酰胺酶催化域的裂解酶的活性较高,而具有SH3b结合域的裂解酶一般会呈现相对较宽的裂解谱,因此该裂解酶为控制由耐药性金黄色葡萄球菌引致的奶牛乳腺炎提供新的抗菌制剂,实现了减量化使用抗生素,为生态养殖提供了新策略。并在两条引物的5’端插入pET28a连接接头(如下划线所示),引物序列如下:
F:5’-GGTACCCTCGAGGGATCCATGGCTAAGACTCAAGCAGAA-3’;
R:5’-CTGCAGGTCGACAAGCTTTTAACTCTTGAATGTCCCCCA-3’;
PCR反应体系:引物F 2μL,引物R 2μL,噬菌体YNP1基因组2μL,PrimeSTAR MaxPremix(2×)25μL,去离子水19μL。
PCR反应条件:98℃ 3min;98℃ 10s,55℃15s,72℃15s,30个循环;72℃5min。反应结束后,将产物采用1%的琼脂糖凝胶电泳检测,并用胶回收试剂盒回收PCR产物。
表达载体的构建
利用HieffPlus One Step Cloning Kit一步法快速克隆试剂盒构建裂解酶pET28a表达载体,方法如下:首先将pET28a进行酶切线性化处理,酶切体系:BamHⅠ1μL、HindⅢ1μL、pET28a3μL、10×Buffer 5μL、去离子水40μL,30℃ 15min,37℃ 20min酶切;随后将1.2.6.1回收的裂解酶基因连接至线性化的pET28a载体,连接体系为:2×Hieff CloneEnzyme Premix 5μL、酶切后pET28a质粒2μL、回收的裂解酶基因1μL、去离子水2μL,50℃连接20min。将连接产物转化至BL21感受态细胞,涂板,过夜培养,并将筛选到的阳性克隆送去测序。将比对成功的质粒,转化到大肠杆菌BL21。
裂解酶LysYNP1的表达
将构建好的裂解酶表达菌株按1:100的比例接种于含有Kan的LB液体培养基中,在37℃摇床200rpm振荡培养至对数期(OD约0.6)。然后加入IPTG至终浓度为1mmol,25℃120rpm摇床过夜诱导表达。并于诱导前和诱导后各取菌液1mL,诱导后的1mL菌液进行破碎,破碎后的沉淀用1mL的PBS重悬,分别取诱导前菌液、诱导后上清、诱导后沉淀各50μL,煮沸后进行10%SDS-PAGE电泳分析。剩余菌液于4℃ 5000rpm离心20min,弃去上清液,25mLPBS重悬后反复洗三次,最后用25mL PBS重悬,重悬后的细菌采用超声破碎,破碎后的菌液在4℃条件下8000rpm离心20min取上清用0.45μm滤膜过滤后获得裂解酶LysYNP1粗提液,保存于4℃条件下。
取表达菌株培养至OD600达到0.6时加入诱导剂IPTG至终浓度为1mmol/L,25℃诱导12h,进行SDS-PAGE电泳检测融合蛋白表达情况。结果显示,诱导后上清在55kD处有一条明显的蛋白条带,诱导后沉淀、未加诱导剂组和空载体组均未出现条带(见图2),裂解酶的氨基酸序列如Seq ID NO.2所示。
裂解酶LysYNP1的活性检测与裂菌谱测定
以15株金黄色葡萄球菌为指示菌,采用平板裂解实验检测LysYNP1的活性以及裂菌谱。A、B孔分别加入90μ LLysYNP1和LysJD,C孔加入等量的PBS为阴性对照。结果显示A、B孔能够形成透亮的裂菌圈,C孔不能形成裂菌圈(见图3)。裂解酶能够裂解多株MRSA菌株以及患乳腺炎的乳汁中分离的多重耐药金黄色葡萄球菌,裂菌谱表1所示,结果显示,LysYNP1的裂菌谱比LysJD更广。
表1裂解酶的裂菌谱
表注:+:裂解性;-:不裂解.
本发明提供一种抗葡萄球菌的宽谱噬菌++体编码裂解酶,通过平板裂解实验,发现8-12小时内该裂解酶可以形成半透明的菌圈,并且随着作用时间的增加裂菌圈逐渐扩大且透明清晰。同时发现该裂解酶的裂菌谱比其噬菌体自身以及实验室制备保存的能够高效裂菌的裂解酶LysJD的裂菌谱更广,裂解酶具有特异性、高效快速、安全性高、不易诱导细菌产生耐药性等优势,因此该裂解酶可用来制备治疗奶牛乳腺炎的新型抗菌药物。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 王中华 严亚贤 吴丽飞 王兆飞 陈冈
<120> 一种抗葡萄球菌的宽谱噬菌体编码裂解酶及制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1491
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggctaaga ctcaagcaga aataaataaa cgtttagacg cttatgcaaa aggaacagta 60
gatagtcctt atagagttaa gaaagcaaca agttatgacc catcatttgg tgtgatggaa 120
gcaggagcaa ttgatgtaga taaatattat cacgctcagt gtcaagacct tattacagac 180
tatgtattat ggttaacaga taataaagtt agaacttggg gtaatgctaa agaccaaatc 240
aaacaaaatt atggtactgg atttaaaata caccaaaata aaccttctac agtacctaaa 300
aaaggttgga ttgcagtatt tacttcaggt agctatacac agtggggtca cataggtatt 360
gtatatgatg gaggtaatac ttcaacattt actattttag agcaaaactg gaacggttac 420
gctaataaaa aacctacaaa acgtgttgac aactactatg gattaactca ctttatagag 480
atacctgtaa aagcaggaac tactattaaa aaagaaacag ctaagaaaag ttcaagtaaa 540
acacctgcac ctaaaaagaa agcaacatta aaagtttcta agaatcatat taactataca 600
atggataaac gtggtaagaa acctgaaggc atggtaatac acaacgatgc gggtcgttct 660
acaggacaac aatacgagaa ttcattagct aaagcaggtt atgctagata tgctaatggt 720
attgctcact actacggttc tgaaggttat atatgggaag caatagatgc taagaatcaa 780
attgcttggc atacaggaga tggaacagga gcaaactcag gtaactttag atttgcaggt 840
attgaagtct gtcaatcaat gagtgctagt gatgctcaat tccttaaaaa tgaacaagca 900
gtattccaat tcacagcaga gaaattcaaa gaatggggtc ttacacctaa ccgtaaaact 960
gtaagattgc atatggaatt cgttcctact gcatgtcctc accgttctat ggttcttcat 1020
acaggattta atccagtaac acaaggaaga ccatcacaag caataatgaa taaactaaaa 1080
gattatttca ttaaacaaat taaaaactac atgagtaacg gaacttcaag ctctactgta 1140
gttaaaaaag ataaaacaag tagtgcaagt acaccggcaa ctagaccagt tacaggttct 1200
tggaaaaaga accaatacgg aacttggtac aaaccggaat ctgcaacatt tgttaatggt 1260
aaccaaccta tagtaactag aataggttct ccattcctaa atgctccagt aggaggtaac 1320
ctccctgcag gtgctacaat tgtatatgac gaagtttgta tccaagcagg tcatatttgg 1380
ataggttata atgcttacaa cggtaacaga gtatattgtc ctgttagaac ttgtcaaggt 1440
gttccacctt cacatgtacc tggagttgcc tgggggacat tcaagagtta a 1491
<210> 2
<211> 496
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala
1 5 10 15
Lys Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser Tyr
20 25 30
Asp Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Val Asp Lys
35 40 45
Tyr Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp
50 55 60
Leu Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile
65 70 75 80
Lys Gln Asn Tyr Gly Thr Gly Phe Lys Ile His Gln Asn Lys Pro Ser
85 90 95
Thr Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr
100 105 110
Thr Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser
115 120 125
Thr Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys
130 135 140
Pro Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu
145 150 155 160
Ile Pro Val Lys Ala Gly Thr Thr Ile Lys Lys Glu Thr Ala Lys Lys
165 170 175
Ser Ser Ser Lys Thr Pro Ala Pro Lys Lys Lys Ala Thr Leu Lys Val
180 185 190
Ser Lys Asn His Ile Asn Tyr Thr Met Asp Lys Arg Gly Lys Lys Pro
195 200 205
Glu Gly Met Val Ile His Asn Asp Ala Gly Arg Ser Thr Gly Gln Gln
210 215 220
Tyr Glu Asn Ser Leu Ala Lys Ala Gly Tyr Ala Arg Tyr Ala Asn Gly
225 230 235 240
Ile Ala His Tyr Tyr Gly Ser Glu Gly Tyr Ile Trp Glu Ala Ile Asp
245 250 255
Ala Lys Asn Gln Ile Ala Trp His Thr Gly Asp Gly Thr Gly Ala Asn
260 265 270
Ser Gly Asn Phe Arg Phe Ala Gly Ile Glu Val Cys Gln Ser Met Ser
275 280 285
Ala Ser Asp Ala Gln Phe Leu Lys Asn Glu Gln Ala Val Phe Gln Phe
290 295 300
Thr Ala Glu Lys Phe Lys Glu Trp Gly Leu Thr Pro Asn Arg Lys Thr
305 310 315 320
Val Arg Leu His Met Glu Phe Val Pro Thr Ala Cys Pro His Arg Ser
325 330 335
Met Val Leu His Thr Gly Phe Asn Pro Val Thr Gln Gly Arg Pro Ser
340 345 350
Gln Ala Ile Met Asn Lys Leu Lys Asp Tyr Phe Ile Lys Gln Ile Lys
355 360 365
Asn Tyr Met Ser Asn Gly Thr Ser Ser Ser Thr Val Val Lys Lys Asp
370 375 380
Lys Thr Ser Ser Ala Ser Thr Pro Ala Thr Arg Pro Val Thr Gly Ser
385 390 395 400
Trp Lys Lys Asn Gln Tyr Gly Thr Trp Tyr Lys Pro Glu Ser Ala Thr
405 410 415
Phe Val Asn Gly Asn Gln Pro Ile Val Thr Arg Ile Gly Ser Pro Phe
420 425 430
Leu Asn Ala Pro Val Gly Gly Asn Leu Pro Ala Gly Ala Thr Ile Val
435 440 445
Tyr Asp Glu Val Cys Ile Gln Ala Gly His Ile Trp Ile Gly Tyr Asn
450 455 460
Ala Tyr Asn Gly Asn Arg Val Tyr Cys Pro Val Arg Thr Cys Gln Gly
465 470 475 480
Val Pro Pro Ser His Val Pro Gly Val Ala Trp Gly Thr Phe Lys Ser
485 490 495
Claims (3)
1.一种抗葡萄球菌的宽谱噬菌体编码裂解酶,其特征在于,其核苷酸序列如Seq IDNO.1所示,氨基酸序列如Seq ID NO.2所示 。
2.一种如权利要求1所述的宽谱噬菌体编码裂解酶在抑制或杀灭原料乳内葡萄球菌中的应用。
3.一种如权利要求1所述的宽谱噬菌体编码裂解酶在制备防治牛乳房炎药物中的应用。
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