WO2016165604A1 - 一种广谱的链球菌裂解酶及其应用 - Google Patents

一种广谱的链球菌裂解酶及其应用 Download PDF

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WO2016165604A1
WO2016165604A1 PCT/CN2016/079043 CN2016079043W WO2016165604A1 WO 2016165604 A1 WO2016165604 A1 WO 2016165604A1 CN 2016079043 W CN2016079043 W CN 2016079043W WO 2016165604 A1 WO2016165604 A1 WO 2016165604A1
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streptococcus
clyr
lyase
streptococcal
expression
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危宏平
杨航
王靖
余军平
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Wuhan Phagelux Bio-Tech Co Ltd
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Wuhan Phagelux Bio-Tech Co Ltd
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Priority to CA2981996A priority patent/CA2981996C/en
Priority to US15/564,063 priority patent/US9993532B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/51—Lyases (4)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
    • A23C9/1216—Other enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of microorganisms, and particularly relates to a lyase and a coding gene capable of killing Streptococcus, Enterococcus and Staphylococcus aureus, and its application in antibiotics.
  • Streptococcus is a type of spherical Gram-positive bacterium that causes many diseases in humans and animals. Streptococcus pneumoniae is the main pathogen of diseases such as lobar pneumonia, and it often causes diseases such as bronchial pneumonia in infants and young children. Streptococcus pyogenes is widely distributed in nature and is one of the most important pathogens in human bacterial infections. It can cause various purulent inflammations, including scarlet fever, erysipelas, neonatal sepsis, meningitis and streptococci allergies.
  • Group B streptococcus such as Streptococcus agalactiae
  • Streptococcus mutans is the main pathogen of caries, which is a serious threat to human oral health. At present, there is still no effective vaccine for the prevention and treatment of dental caries.
  • streptococcus can cause many important animal diseases that threaten the livestock industry.
  • Streptococcus suis is an acute, heat-borne disease that is common to humans and animals, usually caused by C, D, E, and L-group streptococci.
  • the number one disease cow mastitis in dairy farming is caused by four major pathogens such as Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactia and Staphylococcus aureus.
  • pathogens such as Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactia and Staphylococcus aureus.
  • Streptococci have a wide variety and can cause various diseases of humans and animals.
  • a phage lyase is a cell wall hydrolase that is expressed late in a double-stranded DNA phage infecting a host bacterium.
  • the lysing enzyme is usually 25kD to 40kD in size and consists of two independent functional domains, an N-terminal catalytic domain, and a C-terminal cell binding domain (CBD) that determines the cell binding site.
  • CBD C-terminal cell binding domain
  • lyase It provides theoretical feasibility for the control and treatment of clinically resistant bacteria. So far, there have been reports of natural lytic enzymes that act on streptococcus, which can kill certain types of streptococci better in vivo and in vitro. However, these lytic enzymes usually have a narrow spectrum of cleavage, most of which are difficult to express, or have low activity, and have a narrow pH range, generally at pH 5-8. There are currently no lyases that are effective in lysing Streptococcus mutans. Finding soluble, high-volume and broad-spectrum lyases is of great importance for the development of new anti-streptococcus drugs, in vitro control of streptococcal infections, and so on.
  • the technical problem to be solved by the present invention is to provide a lytic enzyme capable of killing various Streptococcus.
  • the lytic enzyme can kill streptococcus in vitro and in vivo, especially Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactia, Streptococcus mutans, Streptococcus equi, and the like
  • ClyR which encodes the gene ClyR.
  • the nucleic acid sequence of the gene encoding ClyR of the Streptococcus lyticase provided by the present invention is shown in SEQ. ID. NO. 1 in the Sequence Listing.
  • the protein sequence of the Streptococcus lyase ClyR provided by the present invention is shown in SEQ. ID. NO. 2 in the Sequence Listing.
  • the invention also provides a method for soluble expression of ClyR protein and purification, wherein the ClyR gene is cloned and then ligated into the expression vector pBAD24, and then the expression plasmid is transformed into E. coli BL21 (DE3) for expression; the expressed protein It was first purified by ion exchange and dialyzed against phosphate (PBS) buffer.
  • PBS phosphate
  • the invention proves the effect of killing various Streptococcus and Staphylococcus aureus strains by using ClyR in vitro, especially in Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococcus uberis, Streptococcus agalactiae, and colostrum Streptococcus, Streptococcus mutans, Streptococcus equi, and the like, as well as various Enterococcus and Staphylococcus aureus, demonstrate the high activity and broad spectrum of the lyase.
  • the protective effect of ClyR on experimental group animal mice infected with group B streptococci and the test of cytotoxicity were also preliminarily tested, and its potential for developing anti-streptococcal infection drugs was preliminarily confirmed.
  • ClyR can be used for killing various Streptococcus and Staphylococcus in vivo and in vitro
  • ClyR Can be used E. coli soluble expression; high enzyme activity, and can have higher activity in the range of pH 5-11; can broadly kill streptococcus, has the potential to be applied to anti-infective drugs in vivo.
  • Figure 1 shows the results of PCR amplification of the ClyR gene.
  • M is a standard molecular weight marker, and the arrow points to 750 bp.
  • R is a band that amplifies ClyR.
  • Figure 2 is a graph showing the time variation of ClyR killing S. dysgalactia standard strain ATCC 35666.
  • the solid squares show the trend of LogCFU over time after mixing with ClyR, and the open circles show the trend of OD 600 with time after mixing with S. dysgalactia and buffer.
  • the open triangle shows the trend of OD 600 over time after mixing with S. dysgalactia and ClyR.
  • FIG. 3 shows the results of ClyR enzyme activity affected by EDTA.
  • the S. lactis standard strain ATCC35666 was resuspended in PBS buffer of different EDTA, and the trend of OD 600 with time was detected after mixing with the same concentration of ClyR.
  • the ordinate indicates the relative enzyme activity of ClyR under different conditions.
  • Figure 4 shows the results of ClyR enzyme activity affected by pH.
  • the S. lactis standard strain ATCC35666 was resuspended in buffers of different pH and mixed with the same concentration of ClyR to detect the change trend of OD 600 with time.
  • the ordinate indicates the relative enzyme activity of ClyR under different conditions.
  • Figure 5 shows the results of ClyR enzyme activity affected by NaCl.
  • the S. dysgalactiae standard strain ATCC35666 was resuspended in a buffer of different NaCl and mixed with the same concentration of ClyR to detect the change trend of OD 600 with time.
  • the ordinate indicates the relative enzyme activity of ClyR under different conditions.
  • Figure 6 shows the results of ClyR killing Streptococcus in vitro and broad strains of different strains such as Staphylococcus.
  • the ordinate indicates the value of OD 600 reduction after incubation of different strains with ClyR for 15 min at 37 °C.
  • Figure 7 shows the results of killing Streptococcus in ClyR. After 20 mice in each group were injected with a lethal dose of Streptococcus agalactiae, the experimental group was intraperitoneally injected with 0.8 mg of ClyR after 3 hours. The control group was injected with an equal volume of PBS solution in the abdomen at 3 hours. The survival rate of each group of mice was observed every day.
  • Figure 8 shows the results of ClyR test for cytotoxicity. No significant cytotoxicity was observed in Caco-2 cells treated with ClyR at concentrations of 0.1, 0.5 and 1 mg/ml, respectively. Among them, ionomycin and mitomycin C are positive controls that are toxic to cells.
  • Figure 9 shows the results of ClyR killing streptococci in milk.
  • S. dysgalactiae ATCC 35666 was mixed with sterile milk and treated with different concentrations of ClyR for 1 hour and diluted plate counts.
  • the DNA sequence of the ClyR gene capable of expressing the lytic enzyme ClyR was synthesized in the entire sequence of Nanjing Kingsray Biotechnology Co., Ltd. The synthetic sequence was loaded into the pUC57 plasmid. Using the ClyR gene as a template, NcoI and XhoI were added to the ends of the target gene, and the primers were designed as follows:
  • the PCR amplification procedure was as follows: 1) 94 ° C, 5 min; 2) 94 ° C, 30 sec, 62 ° C, 45 sec, 72 ° C, 45 sec, 30 cycles; 72 ° C, 10 min; the product was recovered by gel electrophoresis, the electropherogram is shown in Figure 1.
  • the gene size of ClyR is 759 bp, which is consistent with the size of the designed lyase.
  • the expression strain BL21(DE3)/pB-ClyR was induced to express at a low temperature with 0.2% L-arabinose. After the cells were collected, the cells were sonicated, and the supernatant was precipitated with 33% ammonium sulfate. The precipitate was dissolved in PBS and dialyzed against PBS overnight. The dialysate has obvious bactericidal activity.
  • the S. lactis overnight culture was collected, collected by centrifugation, washed once with PBS and then dissolved in PBS.
  • a certain amount of ClyR was mixed with the above-mentioned bacterial liquid, and the change of the absorption value of the mixed solution at 600 nm and the number of viable bacteria were monitored by a microplate reader.
  • a mixture of buffer and Streptococcus dysgalactia was used as a negative control.
  • the resulting cracking curve is shown in Figure 2. The results show that ClyR can rapidly cleave the ATCC 35666 strain resulting in a rapid decrease in absorbance at 600 nm.
  • the S. lactis standard strain ATCC 35666 overnight culture was collected by centrifugation, washed once with PBS and then dissolved in PBS. Then, the bacterial suspension was aliquoted into several portions, and separately mixed with different concentrations of EDTA. A certain amount of ClyR was mixed with all the above-mentioned bacterial liquids, and the change of the absorption value of the mixed solution at 600 nm was monitored by a microplate reader. At the same time, a mixture of buffer and Streptococcus dysgalactia was used as a negative control.
  • the OD 600 reduction value is defined as a maximum of the other reduced the OD 600 value is compared thereto, to obtain the relative activity values.
  • the resulting cracking curve is shown in Figure 3. The results showed that the ClyR enzyme activity was less affected by EDTA.
  • the S. lactis standard strain ATCC 35666 overnight culture was collected by centrifugation, washed once with PBS and then dissolved in buffers of different pH values.
  • a certain amount of ClyR was mixed with all the above-mentioned bacterial liquids, and the change of the absorption value of the mixed solution at 600 nm was monitored by a microplate reader.
  • a mixture of buffer and Streptococcus dysgalactia was used as a negative control.
  • the OD 600 reduction value is defined as a maximum of the other reduced the OD 600 value is compared thereto, to obtain the relative activity values.
  • the resulting cracking curve is shown in Figure 4. The results showed that ClyR had better activity in the range of pH 5-11.
  • the S. lactis standard strain ATCC 35666 overnight culture was collected by centrifugation, washed once with PBS and then dissolved in PBS. Then, the bacterial suspension was aliquoted into several portions, and different concentrations of NaCl were separately added and mixed. A certain amount of ClyR was mixed with all the above-mentioned bacterial liquids, and the change of the absorption value of the mixed solution at 600 nm was monitored by a microplate reader. At the same time, a mixture of buffer and Streptococcus dysgalactia was used as a negative control.
  • the OD 600 reduction value is defined as a maximum of the other reduced the OD 600 value is compared thereto, to obtain the relative activity values.
  • the resulting cracking curve is shown in Figure 5. The results showed that the ClyR enzyme activity was less affected by NaCl.
  • Example 6 Validation of broad spectrum of different strains such as Streptococcus and Staphylococcus in ClyR in vitro.
  • ClyR can quickly kill a variety of streptococcus (especially Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococcus uberis, Streptococcus agalactiae, Streptococcus mutans, Streptococcus mutans, Streptococcus equi, etc.)
  • streptococcus especially Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococcus uberis, Streptococcus agalactiae, Streptococcus mutans, Streptococcus mutans, Streptococcus equi, etc.
  • Example 7 Verification of the killing of Streptococcus in ClyR in vivo.
  • mice used in the experiment were 6-week-old BALB/c female mice weighing approximately 20 to 22 grams.
  • Experimental mice 40 rats were intraperitoneally injected with 6 x 10 8 Streptococcus agalactiae strains. After 3 h, the mice were divided into two groups of 20 each.
  • the mice in the experimental group were intraperitoneally injected with 800 ⁇ g of ClyR, and the control group was injected with PBS buffer.
  • the survival rate of the mice was observed every day, and the results obtained are shown in Fig. 7.
  • the results showed that ClyR can effectively kill streptococci in vivo, thereby increasing the survival rate of mice.
  • Caco-2 cells were seeded into 96-well plates at a concentration of 5 ⁇ 10 3 per well. After 24 hours of culture, a certain concentration of ClyR (0.1-1 mg/mL) and ionomycin (15 mg/mL) were added to the well plates. And mitomycin C (15 mg/mL). Continue to train for 24 hours. After the completion of the culture, the staining agent WST-8 was added to the well plate, and after standing, the absorbance at 450 nm was read on a microplate reader. The results obtained are shown in Figure 8. The results showed that high concentrations of ClyR were not cytotoxic.
  • Example 9 ClyR was used for verification of streptococci clearance in milk.
  • the Streptococcus overnight culture was collected by centrifugation, washed once with PBS and then dissolved in sterile pure milk. Different concentrations of ClyR were mixed with the above bacterial solution, and the mixed reaction solution was allowed to stand at 37 degrees for 1 hour, and then the number of viable cells was calculated by diluting the plate. The identification results obtained by the test are shown in Figure 9. The results show that the enzyme can effectively remove streptococci in milk.

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Abstract

提供一种能杀灭多种链球菌、肠球菌和金黄色葡萄球菌的裂解酶ClyR及其应用。

Description

一种广谱的链球菌裂解酶及其应用 技术领域
本发明属于微生物领域，具体涉及一种能杀灭链球菌、肠球菌和金黄色葡萄球菌的裂解酶和编码基因，以及其在抗生素方面的应用。
背景技术
链球菌是一类球形的革兰氏阳性细菌，能引起人和动物的很多疾病。肺炎链球菌是大叶性肺炎等疾病的主要致病菌，更常在婴幼儿引起支气管肺炎等疾病。化脓链球菌广泛分布于自然界，是人类细菌感染中最重要的病原之一，能引起各种化脓性炎症，包括猩红热，丹毒，新生儿败血症，脑膜炎以及链球菌变态反应等疾病。B族链球菌，如无乳链球菌等，是围产期感染的重要病原菌之一，严重威胁新生儿的健康。变形链球菌是龋齿的主要致病菌，严重威胁人类口腔健康。目前龋齿的预防与治疗还缺乏有效的疫苗。此外，链球菌还能引起许多重要的动物疾病，严重威胁畜牧业。猪链球菌病是一种人畜共患的急性、热性传染病，通常由C、D、E及L群链球菌引起。而奶牛养殖业的头号疾病奶牛乳腺炎，是由无乳链球菌、乳房链球菌、停乳链球菌以及金黄色葡萄球菌等四种主要的致病菌引起的，目前尚缺乏有效的疫苗和应对策略。由上可知，链球菌种类繁多，能引起人和动物的多种疾病。
噬菌体裂解酶是双链DNA噬菌体感染宿主细菌后晚期表达的一种细胞壁水解酶。裂解酶大小通常为25kD～40kD，在结构上由两个独立的功能域构成，N-端的催化功能域，和一个决定细胞结合位点的C-端细胞结合功能域(CBD)，二者之间由一个小片段链接。序列分析表明，同一类裂解酶的催化域高度保守，而细胞结合域可变，这为构建新的嵌合裂解酶提供了可能。裂解酶具有很高的特异性，只能特异性的识别和裂解特定种类的细菌。此外，裂解酶作用位点很保守，加上噬菌体与细菌共同进化的特异性，宿主菌很难对其产生抗性。裂解酶的这些特点，为 其用于临床上耐药细菌的控制和治疗提供了理论的可行性。到目前为止，已有一些能作用于链球菌的天然裂解酶被报道，这些酶可以在体内、体外较好地杀特定种类的链球菌。但是这些裂解酶通常具有较窄的裂解谱，大多比较难于可溶性表达，或者活性不高，作用pH范围较窄，一般在pH 5-8。目前尚没有能有效裂解变形链球菌的裂解酶报道出来。寻找可溶、大量表达以及广谱的裂解酶对于开发新的抗链球菌的药物，体外控制链球菌的感染等等都具有非常重要的意义。
发明内容
本发明所要解决的技术问题是提供一种能杀灭多种链球菌的裂解酶。该裂解酶能在体外、体内杀灭链球菌，特别是肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌、马链球菌等以及多种肠球菌和金黄色葡萄球菌。为了叙述方便，我们命名为ClyR，其编码基因ClyR。
本发明所提供的链球菌裂解酶的编码基因ClyR的核酸序列如序列表中SEQ.ID.NO.1所示。
本发明所提供的链球菌裂解酶ClyR的蛋白序列如序列表中SEQ.ID.NO.2所示。
本发明还提供一种可溶表达ClyR蛋白质及纯化的方法，其步骤为将ClyR基因克隆后连入表达载体pBAD24中，然后将表达质粒转化大肠杆菌BL21(DE3)中进行表达；所表达的蛋白质先经过离子交换纯化，再用磷酸盐(PBS)缓冲液透析处理。
本发明通过体外实验证实了采用ClyR体外杀灭多种链球菌以及金黄色葡萄球菌株的效果，特别是肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌、马链球菌等以及多种肠球菌和金黄色葡萄球菌，证明了该裂解酶的高活性与广谱性。还初步试验了ClyR对实验动物模型小鼠感染B族链球菌后的保护效果，以及对细胞毒性的测试，初步证实了其用于开发抗链球菌感染药物的潜能。
本发明有以下的有益效果和优点：
本发明ClyR可用于体内、体外杀灭多种链球菌和葡萄球菌，ClyR 能采用大肠杆菌可溶性表达；酶活性高，且能在pH5-11的范围内都具有较高活性；能广谱杀灭链球菌，具有应用于体内抗感染药物的潜能等优点。
附图说明
图1为ClyR基因PCR扩增结果。图中M为标准分子量marker，箭头所指处为750bp。R为扩增ClyR的条带。
图2为ClyR杀灭停乳链球菌标准菌株ATCC 35666的时间变化结果。实心方块所示为停乳链球菌与ClyR混合后LogCFU随时间的变化趋势，空心圆圈所示为停乳链球菌与缓冲液混合后OD600随时间的变化趋势。空心三角所示为停乳链球菌与ClyR混合后OD600随时间的变化趋势。
图3为ClyR酶活受EDTA影响的结果。将停乳链球菌标准菌株ATCC35666重悬在不同EDTA的PBS缓冲液中，与相同浓度的ClyR混合后检测OD600随时间的变化趋势。纵坐标表示ClyR在不同条件下的相对酶活。
图4为ClyR酶活受pH影响的结果。将停乳链球菌标准菌株ATCC35666重悬在不同pH的缓冲液中，与相同浓度的ClyR混合后检测OD600随时间的变化趋势。纵坐标表示ClyR在不同条件下的相对酶活。
图5为ClyR酶活受NaCl影响的结果。将停乳链球菌标准菌株ATCC35666重悬在不同NaCl的缓冲液中，与相同浓度的ClyR混合后检测OD600随时间的变化趋势。纵坐标表示ClyR在不同条件下的相对酶活。
图6为ClyR体外杀灭链球菌以及葡萄球菌等不同菌株广谱性的结果。纵坐标表示将不同菌株与ClyR混合后37℃孵育15min，OD600降低的数值。
图7为ClyR体内杀灭链球菌的结果。每组20只小鼠，分别注射致死剂量的无乳链球菌后，实验组于3小时后腹腔注射0.8mg ClyR。对照组于3小时候腹部注射的等体积的PBS溶液。每天观测各组小鼠的存活率。
图8为ClyR对于细胞毒性的测试结果。分别用浓度为0.1,0.5和1mg/ml的ClyR作用于Caco-2细胞没有观察到明显的细胞毒性。其中离子霉素和丝裂霉素C为对细胞有毒性的阳性对照。
图9为ClyR在牛奶中杀灭链球菌的结果。停乳链球菌ATCC 35666与无菌牛奶混合后用不同浓度的ClyR处理1小时后稀释平板计数。
具体实施方式
本发明通过以下实验例进一步说明本发明，但本发明并不受限于此。
通过对葡萄球菌噬菌体裂解酶的催化域和细胞结构域氨基酸序列的分析，我们设计和人工合成一种新的链球菌裂解酶ClyR。
下列实施例中所用的方法如无特别说明均是常规的实验方法。实验中所用到的引物均由上海英骏公司提供。测序均在上海英骏公司完成。
实施施1 能特异杀灭链球菌的裂解酶的构建
(1)在南京金斯瑞生物技术有限公司全序列合成能表达裂解酶ClyR的ClyR基因DNA序列。合成序列装入pUC57质粒中。以ClyR基因为模板，在目的基因的两端分别加入NcoI和XhoI的酶切位点，引物设计如下：
正向引物：5-TATACCATGGGCATGGCAGCAAATCTGG-3(SEQ.ID.No.3)
NcoI
反向引物：
5-ATATCTCGAGTTTGAAGGTACCCCATGCGTTG-3(SEQ.ID.No.4)
XhoI
以2μl基因为模板，各加入引物1μg进行PCR扩增，PCR扩增程序如下：1)94℃，5min；2)94℃，30sec，62℃，45sec，72℃，45sec，30个循环；3)72℃，10min；将产物进行凝胶电泳回收，电泳图谱如图1，ClyR的基因大小为759bp，与所设计的裂解酶的大小一致。
(2)将ClyR基因连入表达质粒pBAD24中得到重组载体pB-ClyR，然后将其转化大肠杆菌BL21(DE3)。
(3)ClyR的表达纯化。
将表达菌株BL21(DE3)/pB-ClyR用0.2％的L-阿拉伯糖低温诱导表达。收集菌体后超声破碎，取上清用33％硫酸铵沉淀，将沉淀溶于PBS后于PBS中透析过夜。透析液即有明显的杀菌活性。
透析后的粗提液，或者是超声后的上清，过HiTrap Q Sepharose FF column(GE Healthcare)，收集柱流出物。将柱流出物过HiTrap SP Sepharose FF column，然后用1M的NaCl梯度洗脱，分段收集洗脱峰。将有活性的各管混合后于PBS中透析过夜，即为纯化后的酶液。
实施例2 ClyR杀灭停乳链球菌标准菌株ATCC 35666的验证
将停乳链球菌过夜培养物，离心收集后用PBS洗涤一次然后溶于PBS中。取一定量的ClyR与上述菌液混合，同时用酶标仪监测混合液在600nm吸收值的变化以及活菌数目的变化。同时，用缓冲液与停乳链球菌的混合液作为负对照。最后得到的裂解曲线见附图2。结果显示ClyR能快速的裂解ATCC 35666菌株从而导致在600nm吸收值快速降低。
实施例3 ClyR酶活受EDTA影响结果的验证
将停乳链球菌标准菌株ATCC 35666过夜培养物，离心收集后用PBS洗涤一次然后溶于PBS中。然后将菌悬液等分为多份，分别加入不同浓度的EDTA混匀。取一定量的ClyR与所有上述菌液混合，同时用酶标仪监测混合液在600nm吸收值的变化。同时，用缓冲液与停乳链球菌的混合液作为负对照。检测结束后计算每组与空白组相比较OD600降低的数值，将OD600降低最多的值定义为1，其它组OD600的降低值与之作比较，得到相对酶活数值。最后得到的裂解曲线见附图3。结果显示ClyR酶活受EDTA影响较小。
实施例4、ClyR酶活受pH影响结果的验证。
将停乳链球菌标准菌株ATCC 35666过夜培养物，离心收集后用PBS洗涤一次然后溶于不同pH值的缓冲液中。取一定量的ClyR与所有上述菌液混合，同时用酶标仪监测混合液在600nm吸收值的变化。同时，用缓冲液与停乳链球菌的混合液作为负对照。检测结束后计算每组与空白组相比较OD600降低的数值，将OD600降低最多的值定义为1，其它组OD600的降低值与之作比较，得到相对酶活数值。最后得到的裂解曲线见附图4。 结果显示ClyR在pH5-11的范围内具有较好的活性。
实施例5 ClyR酶活受NaCl影响结果的验证
将停乳链球菌标准菌株ATCC 35666过夜培养物，离心收集后用PBS洗涤一次然后溶于PBS中。然后将菌悬液等分为多份，分别加入不同浓度的NaCl混匀。取一定量的ClyR与所有上述菌液混合，同时用酶标仪监测混合液在600nm吸收值的变化。同时，用缓冲液与停乳链球菌的混合液作为负对照。检测结束后计算每组与空白组相比较OD600降低的数值，将OD600降低最多的值定义为1，其它组OD600的降低值与之作比较，得到相对酶活数值。最后得到的裂解曲线见附图5。结果显示ClyR酶活受NaCl影响较小。
实施例6、ClyR体外杀灭链球菌以及葡萄球菌等不同菌株广谱性的验证。
将多种链球菌、金黄色葡萄球菌以及其它参照菌株过夜培养，离心收集后用PBS洗涤一次然后溶于PBS中。取一定量的ClyR与上述菌液混合，同时用酶标仪监测混合液在600nm吸收值的变化15min。用OD600降低的数值表示不同菌株的裂解效果。同时，用缓冲液与菌液的混合液作为负对照。试验得到的杀灭效果见附图6。结果显示ClyR能快速的杀灭多种链球菌(特别是肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌、马链球菌等)以及葡萄球菌而对其它的测试菌株没有裂解作用。
实施例7、ClyR体内杀灭链球菌的验证。
实验中所用小鼠为6周大的BALB/c雌性鼠，重约20到22克。在实验小鼠(40只)腹腔注射6×108无乳链球菌菌株。3h后，将小鼠分为两组，每组20只。实验组小鼠腹腔注射ClyR 800μg，对照组则注射PBS缓冲液。每天观测小鼠的存活率，所得到的结果见附图7。结果显示ClyR能有效的杀灭体内的链球菌，从而提高小鼠的存活率。
实施例8、ClyR无细胞毒性的验证。
将Caco-2细胞以每孔5×103的浓度接种到96孔板中，培养24小时后，向孔板中加入一定浓度的ClyR(0.1-1mg/mL)以及离子霉素(15mg/mL)和丝裂霉素C(15mg/mL)。继续培养24小时。培养结束后，向孔板中加入染色剂WST-8，静置后在酶标仪上读取450nm吸光值。所得到的结果见附图8。结果显示高浓度的ClyR无细胞毒性。
实施例9、ClyR用于牛奶中链球菌清除的验证。
将链球菌过夜培养物离心收集后用PBS洗涤一次然后溶于无菌纯牛奶中。取不同浓度的ClyR与上述菌液混合，混匀后的反应液置于37度作用1小时，然后稀释平板计算其中的活菌数。试验得到的鉴定结果见附图9。结果显示该酶能有效的清除牛奶中的链球菌。

Claims (10)

  1. 一种链球菌裂解酶ClyR，其蛋白序列如SEQ.ID.NO.2所示。
  2. 编码权利要求1所述的链球菌裂解酶的基因ClyR，其核酸序列如SEQ.ID.NO.1所示。
  3. 权利要求1所述的链球菌裂解酶ClyR可溶表达及纯化的方法，其特征为：将ClyR基因克隆后连入表达载体pBAD24中，然后将表达质粒转化大肠杆菌BL21(DE3)中进行表达；所表达的蛋白质先经过离子交换纯化，再用磷酸盐缓冲液透析处理。
  4. 权利要求1所述的链球菌裂解酶ClyR在动物和人体外杀灭链球菌、肠球菌以及金黄色葡萄球菌株的用途。
  5. 根据权利要求4所述的用途，其特征在于，所述链球菌为肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌或马链球菌。
  6. 根据权利要求4所述的用途，其特征在于，权利要求1所述的链球菌裂解酶ClyR在牛奶中杀灭链球菌的用途。
  7. 权利要求1所述的链球菌裂解酶ClyR在制备抗链球菌感染药物上的用途。
  8. 根据权利要求7所述的用途，其特征在于，权利要求1所述的链球菌裂解酶ClyR在制备预防或治疗奶牛乳腺炎药物上的用途。
  9. 根据权利要求7所述的用途，其特征在于，权利要求1所述的链球菌裂解酶ClyR在制备预防或治疗龋齿药物上的用途。
  10. 一种抗生素，其中含有权利要求1所述的链球菌裂解酶ClyR。
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EP3312275A1 (en) 2018-04-25
CN104726439B (zh) 2017-11-14
EP3312275B1 (en) 2022-04-27

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