WO2014101322A1 - 提高细菌对血清敏感性的氨基酸 - Google Patents

提高细菌对血清敏感性的氨基酸 Download PDF

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WO2014101322A1
WO2014101322A1 PCT/CN2013/070429 CN2013070429W WO2014101322A1 WO 2014101322 A1 WO2014101322 A1 WO 2014101322A1 CN 2013070429 W CN2013070429 W CN 2013070429W WO 2014101322 A1 WO2014101322 A1 WO 2014101322A1
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serum
glycine
amino acid
bacteria
plasma
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彭宣宪
李惠
程志学
郭昌
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Sun Yat Sen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to a small molecular substance capable of improving the sensitivity of bacteria to serum.
  • the body is cleared by specific immunity and non-specific immunity.
  • Specific immunity is mainly cleared by antibodies in the blood
  • non-specific immunity is mainly cleared by the complement system and phagocytic cells.
  • the lytic system of the complement system is currently one of the main ways of clearing bacteria into the blood and mucous membranes of the body, and this effect is dependent on or independent of antibody.
  • Complement is a group of activated enzymatically active proteins present in human or vertebrate serum and tissue fluids, including 30 A variety of soluble proteins and membrane-bound proteins. It is widely involved in the body's microbial defense response and immune regulation, and can also mediate the invasive response of immunopathology. It is an effect system and an effect amplification system with important biological functions in the body.
  • pathogens have obvious serum resistance, that is, they are resistant to the bacteriolysis of complement, and are one of the important pathogenic features of pathogenic bacteria.
  • LPS And membrane (outer membrane) proteins lipopolysaccharide (LPS And membrane (outer membrane) proteins.
  • Bacteria express serum resistance by reducing the binding of membrane (outer membrane) proteins to complement and inactivating complement that has been bound to membrane (outer membrane) proteins. Therefore, eliminating the serum resistance of pathogenic bacteria is an important means to prevent infection of pathogenic bacteria, but so far has not been relevant.
  • a small molecular metabolite for improving the sensitivity of bacteria to serum the present invention can achieve the purpose of inhibiting pathogenic bacteria by using these small molecular substances.
  • the small molecule substance of the present invention belongs to an amino acid or a derivative thereof.
  • amino acid may be glycine, threonine or serine.
  • the amino acid is glycine (Glycine, Gly/glycine).
  • the invention discloses and protects the use of an amino acid or an amino acid derivative for the preparation of a medicament for enhancing serum or plasma or whole blood bactericidal action, wherein the amino acid is Glycine, threonine or serine.
  • the invention discloses and protects the use of amino acids or amino acid derivatives in the preparation of injectable bactericides, amino acids.
  • the invention discloses and protects the use of an amino acid in combination with serum in the preparation of a bactericidal drug, or an amino acid derivative, in combination with serum or plasma or whole blood for the preparation of a bactericidal drug; Glycine, threonine or serine.
  • the amino acid is glycine.
  • Metabolites of glycine and glycine derivatives such as glutathione, 5,10-methylenetetrahydrofolate, H-protein Aminomethyldihydrogen lysine, N1-(5-phosphate-D-ribosyl)glycinamide, L-2-amino-3-oxobutyric acid, L-cysteine.
  • the invention also protects a bactericidal pharmaceutical composition characterized by comprising an amino acid and serum/plasma/whole blood, said amino acid being Glycine, threonine or serine.
  • the ratio of serum to glycine is 1:3000 by weight.
  • the bacteria include Gram-negative bacteria and positive bacteria.
  • the bacterium is a Gram-negative and positive pathogen. More preferably, the bacterium is Staphylococcus aureus, retarded Edwardia, B Streptococcus, Pseudomonas aeruginosa, Escherichia coli or Vibrio.
  • the serum or plasma or whole blood is derived from a mammal, fish or bird.
  • the invention also discloses and protects a sterilization method comprising: amino acid and serum / plasma / Whole blood combination, or amino acid derivative and serum / plasma / whole blood; the amino acid is glycine, threonine or serine.
  • the method comprises: glycine and serum by weight 1:3000 The ratio is mixed to kill Gram-negative bacteria or positive bacteria; the sterilization time is 0.1 ⁇ 24 hours.
  • amino acid employed in the present invention is glycine.
  • the present invention analyzes all small molecule metabolites of Escherichia coli treated with human serum by metabolomics method, and finds that the content of glycine in Escherichia coli-resistant Escherichia coli is significantly decreased, suggesting that it can be used as an increase in serum. Sensitive small molecule metabolites.
  • the invention passes The effects of glycine addition on serum bactericidal effect by adding different concentrations of glycine, fixed glycine and different volumes of human serum, and immobilizing human serum and glycine at different time.
  • the control group in which only glycine was added had no effect on the growth of Escherichia coli, and when glycine was added to human serum, the bactericidal ability of the serum increased, and the ability thereof increased with the increase of the concentration of glycine and serum.
  • the effect increases as the action time is extended.
  • the same effect was obtained with plasma instead of serum, but serum or plasma with inactivated complement had no effect.
  • the invention adopts a sterilization experiment on Gram-positive bacteria Staphylococcus aureus and Streptococcus hemolyticus and Gram-negative bacteria such as Escherichia coli, Duchen Edwards, Pseudomonas aeruginosa and Vibrio, and the like It was demonstrated that the addition of glycine increased the sensitivity of all bacteria to human serum.
  • the present invention employs mice, rabbits, pigs, chickens, domestic pigeons, prawns and fish, etc.
  • the bactericidal effect of serum/plasma and glycine in different evolutionary levels on its main pathogens was found to be able to improve the bactericidal ability of different animal serums against its main pathogens.
  • the present invention further carried out in vivo experiments in mice and found that after adding glycine, the mice not only showed a significant improvement in scavenging ability against the model Escherichia coli but also on the clinically isolated pathogenic strains with complete serum resistance, indicating that glycine can be in vivo and in vivo. It works in the blood state.
  • the addition of glycine to serum can significantly improve the sensitivity of bacteria to serum, providing a new technical method for the treatment of serum resistant pathogen infection.
  • the invention discloses and protects glycine in improving bacterial pairs Application in serum sensitivity. It can be used to prepare drugs or fungicides to further enhance the sensitivity of bacteria to serum.
  • the invention discloses and protects a method for improving the sensitivity of bacteria to serum, characterized in that glycine is used as a medicine for improving the bactericidal action of serum.
  • the bacteria include, but are not limited to, Staphylococcus aureus, Streptococcus hemolyticus, Edwards deficient, Pseudomonas aeruginosa, Escherichia coli or Vibrio. Because these bacteria are common human and farm animal pathogens, among which Staphylococcus aureus and beta-hemolytic streptococcus are Gram-positive bacteria, Escherichia coli, Pseudomonas aeruginosa, E. sinensis, Vibrio parahaemolyticus And Vibrio alginolytic bacteria are Gram-negative bacteria. These bacteria are common pathogens, and their serum-resistant strains are common, and Escherichia coli is a model strain for studying bacterial serum resistance, so these bacteria are better representative bacteria for serum resistance.
  • glycine is used at a concentration of 0.05-1 g/kg body weight/day.
  • a new bactericide can be prepared, which contains glycine; or a preparation for improving the bactericidal action of serum on bacteria, the main component of which is glycine.
  • Figure 1 shows the independent component analysis (A) and data clustering analysis of serum-resistant Escherichia coli GC-MS (B) And metabolite content analysis (C).
  • Figure 2 shows the results of glycine on increasing bacterial sensitivity to serum.
  • A is different concentrations of glycine
  • B is different volume of serum
  • C For different times.
  • Figure 3 shows the effect of glycine on the sensitivity of various bacteria to serum.
  • A is the survival rate and B is the improvement factor.
  • Figure 4 shows the results of glycine enhancing the bactericidal ability of serum/plasma of different animals to its main pathogens.
  • a for the sterilization of Vibrio alginolyticus for shrimp and fish serum B is the result of sterilization of E. coli by chicken and domestic pigeon plasma, and C is the result of sterilization of E. coli by mouse, rabbit and pig plasma.
  • Figure 5 shows the results of glycine-enhanced bacterial clearance in mice.
  • a and B The number of bacteria and the survival rate in the blood of the mice were injected with or without injection of glycine.
  • Figure 6 shows the results of glycine-enhanced clearance of Escherichia coli in mice.
  • Glycine is a biomarker against Escherichia coli against serum bactericidal action
  • test samples A single colony of Escherichia coli K12 BW25113 was picked from LB plates and inoculated into 5 ml of LB medium, and cultured at 37 ° C with shaking at 200 rpm for 16 hours. The cells were inoculated in 200 ml of medium at a ratio of 1:100 (v/v), shaken at 37 ° C until the OD600 value was 1.0, and the cells were collected by centrifugation at 8000 rpm for 10 minutes at 4 ° C, and the cells were washed once with 0.85% physiological saline.
  • GC-MS sample preparation add 100 to the prepared sample Microliters of human serum (extracted from healthy human blood, naturally coagulated, centrifuged supernatant is serum), resuspended the cells and shaken at 37 ° C for 200 rpm for 1 hour. The control group was normal saline. 4°C8000rpm Centrifuge for 5 minutes to collect bacteria and remove residual serum or normal saline. Resuspend the cells with saline to give an OD600 of 1.0, and add 1 ml each to add cold methanol (Sigma) (containing 5 ⁇ l 0.1 mg/ml ribose pure (Sigma) as an internal standard for analysis).
  • human serum extracted from healthy human blood, naturally coagulated, centrifuged supernatant is serum
  • the control group was normal saline. 4°C8000rpm Centrifuge for 5 minutes to collect bacteria and remove residual serum or normal saline. Resuspend the cells with saline to give an OD600 of 1.0, and add
  • GC-MS analysis The sample first protects the carboxyl group by methylation, ie by containing 20 mg / with 40 ⁇ l /
  • the pyridine of mM methoxyamine hydrochloride was reacted at 37 ° C for 90 minutes, and then 80 ⁇ l of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA, Sigma-Aldrich) Derivatizes acidic protons by reaction at 37 ° C for 30 minutes.
  • MSTFA N-methyl-N-trimethylsilyltrifluoroacetamide
  • the initial temperature of the GC oven is maintained at 85 ° C for 5 minutes, followed by The rate of 15 ° C / min was increased to 330 ° C for another 5 minutes. 1 ml/min of helium is used as the carrier gas flow. MS operation is performed in the m/z 50-600 range.
  • Spectral convolution and calibration are performed using AMDIS and internal standards. All samples perform retention time (RT) correction, then RT For reference, the remaining spectra are queried and assembled to generate a file containing information on the abundance of each metabolite in all samples. Metabolites in GC-MS spectra using NIST MS search 2.0 software National Institute of Standards and Technology (NIST) The database is compared to obtain an identification. The derived data matrix is corrected using the total peak area. Use retention time and m/z The results of the partitioning gave the peak intensity normalized to each substance in each sample, and the produced documents were used for further statistical analysis.
  • Glycine improves the sensitivity of Escherichia coli to serum
  • Example 1.1 Prepare experimental bacterial samples according to Example 1.1, add glycine to a final concentration of 0, 5, 10, 30, 50 After incubating with 100 mM, shaking at 200 ° C for 1 hour at 37 ° C, 100 ⁇ l of human serum was added, and physiological saline was used as a control. 2 hours of centrifugation, 1 The cells were resuspended in milliliters of saline and viable counted using LB solid plates.
  • the survival rate calculation formula is: the number of viable bacteria after treatment with different concentrations of glycine / the number of viable bacteria after physiological saline control treatment ⁇ 100%.
  • Figure 2A The survival rate of Escherichia coli after adding different concentrations of glycine to human serum.
  • the prepared bacterial samples were divided into 4 groups: 100 mM glycine group, 100 ⁇ l human serum group, 100 ⁇ l human serum and 100 mM glycine group, saline control group, shaking culture at 37 ° C 200 rpm, respectively, centrifuged at 0.5, 1, 2, 3 and 4 hours, using 1
  • the bacteria were suspended in milliliters of saline and viable counted using LB solid plates.
  • the survival rate is calculated as: the number of viable cells at different times after adding serum and / or glycine treatment / the number of viable bacteria at different times in the saline control group ⁇ 100%.
  • Figure 2C is 3 The survival rate of the experimental group at different times can be seen from the figure.
  • glycine itself has no effect on the growth of E. coli; serum itself has a certain bactericidal effect on bacteria, and after adding glycine to human serum, the bactericidal ability of serum is significantly increased, and its bactericidal ability increases with the concentration of glycine. Enhanced; after the addition of glycine, the bactericidal rate increases as the amount of human serum increases.
  • Glycine enhances the sensitivity of bacteria, including Gram-negative and positive bacteria, to serum
  • Example 1.1 Escherichia coli K12 BW25113, Escherichia coli Y17 Clinical bacteria, type B hemolytic streptococcus, Vibrio alginolyticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa and Staphylococcus aureus), respectively, collected 3 ml of each bacterium with an OD600 of 1.0. Then divided into 4 groups: add 100 microliters saline control group, 100-150 mM glycine group, 125-175 microliter human serum group, and 125-175 microliter human serum plus 100-150 mM Glycine group.
  • the optimal bactericidal effect of different types of bacteria is slightly different from the amount of serum added to glycine. After incubating at 37 ° C for 200 hours with shaking at 200 rpm, the cells were centrifuged, and the bacteria were suspended in 1 ml of physiological saline. OD value (OD600nm).
  • Figure 3A shows the OD values of each of the three treatments
  • Figure 3B Comparison of the bactericidal folds after treatment with glycine for each bacterial in serum treatment and serum.
  • the bactericidal effect of serum on different bacteria is different, for Escherichia coli K12 BW25113 Type B hemolytic streptococcus and Pseudomonas aeruginosa have a certain bactericidal effect, but on other bacteria, it has a growth-promoting effect; but after adding glycine, the serum has a significant bactericidal effect on all bacteria, and its bactericidal effect is enhanced. in More than 1 time, up to 4.13 times.
  • Glycine enhances the bactericidal effect of serum/plasma of different evolutionary species on its main pathogens
  • Example 1.1 Escherichia coli K12 BW25113, Escherichia coli Y17 Clinical bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa and Staphylococcus aureus, respectively, collected 3 ml of each bacterium with an OD600 of 1.0, and then divided into 2 groups: 100 Microliters of different animal serum/plasma groups and 100 ⁇ l of different animal serum/plasma plus 100 mM glycine group. At the same time, 100 mM glycine was added as a control.
  • Figure 4 shows the main pathogens of different evolutionary levels of animals in serum / OD values after plasma treatment and serum/plasma addition of glycine.
  • Glycine can improve the clearance of Escherichia coli in mice
  • mice purchased from the Experimental Animal Center of the East Campus of Sun Yat-sen University
  • mice were randomly divided into 3 groups, a saline control group and two concentrations (3.55 and 6.5 mg/mouse) of the glycine administration group.
  • Intravenous administration according to 3.25 and 6.5 mg glycine / time / only, two doses of 12 hours, continuous administration of 6 times; 12 hours after stopping the drug, intravenous injection of Escherichia coli K12 BW25113 and the large intestine Escherichia coli Y17 (clinical isolate), dose of 1 ⁇ 10 6 bacteria / bacteria, 48 hours after the mouse tail vein blood, gradient dilution plate count, detection of bacterial clearance.
  • Figures 5A and B show the number of bacteria and bacterial survival in vivo after injection of two glycine concentrations in mouse serum. From this result, it can be seen that when the mice were injected with glycine, the number of bacteria in the mice was significantly reduced. For E. coli K12 BW25113, the clearance rate was 89.32% at 3.25 mg and 96.85% at 6.5 mg. For E. coli Y17 clinical bacteria, the clearance rate was 32.95% at 3.25 mg and 52.53% at 6.5 mg. These results indicate that glycine enhances the clearance of bacteria by bacteria, and as the amount of addition increases, the clearance is enhanced.
  • Threonine increases the sensitivity of Escherichia coli to serum
  • the prepared bacterial samples were divided into 4 groups: 100 mM threonine group, 100 ⁇ l human serum group, 100 ⁇ L human serum and 100 mM suglycine group, saline control group, shaking culture at 37 ° C 200 rpm, centrifuged at 2 hours, and the body weight was suspended with 1 ml of physiological saline to determine the OD value (OD600). ).
  • the survival rate was calculated as: OD value after treatment with serum and / or threonine / OD value of saline control group ⁇ 100%.
  • Figure 6 is 3 The survival rate of the experimental groups at different times can be seen from the figure. There is no bactericidal effect when only threonine is added. When only serum is added, there is a certain bactericidal effect. When threonine is added to the serum, the bactericidal effect is significantly increased. Increased by 1.5 Times.

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Abstract

甘氨酸、苏氨酸和丝氨酸能够提高细菌对血清、血浆或全血的敏感性，可作为药物提高血清、血浆或全血的杀菌作用。

Description

一种提高细菌对血清 敏感性的物质
技术领域
本发明属于医药技术领域，具体涉及一种能提高细菌对血清敏感性的小分子物质。
背景技术
细菌进入机体后，机体通过特异性免疫和非特异性免疫来清除。特异性免疫主要是通过血液中抗体来清除，非特异性免疫主要是通过补体系统和吞噬细胞来清除。
补体系统溶菌是目前已知的清除进入机体血液和粘膜中细菌的主要方式之一，此作用依赖或不依赖抗体的辅助。补体是存在于人或脊椎动物血清与组织液中的一组经活化后具有酶活性的蛋白质，包括 30 多种可溶性蛋白和膜结合蛋白。其广泛参与机体微生物防御反应以及免疫调节，也可介导免疫病理的损伤性反应，是体内具有重要生物学作用的效应系统和效应放大系统。
然而，许多病原菌具有明显的血清抗性，即能够抵抗补体的溶菌作用，是病原菌的重要病原特征之一。近一个世纪的研究表明 , 细菌抵抗血清补体溶菌主要归于脂多糖（ LPS ）和膜（外膜）蛋白。细菌通过减少膜（外膜）蛋白与补体的结合以及灭活已经与膜（外膜）蛋白结合了的补体而表现血清抗性。由此，消除病原菌血清抗性是防治病原菌感染的重要手段，但迄今为止尚未见有关 通过抑制病原菌血清抗性以提高血清杀菌目的的研究报道。因此，发现新的能增加补体对细菌溶解的物质，对防治病原菌的危害，保障人们身体健康具有十分重要的意义。
发明内容
本发明的目的在于提供一种小分子物质在提高细菌对血清敏感性方面的应用。作为一种提高细菌对血清敏感性的小分子代谢物，本发明通过这些小分子物质可达到抑制病原菌的目的。
本发明所述的小分子物质，属于氨基酸或其衍生物。
更具体的说，所述的氨基酸可以是甘氨酸，苏氨酸，丝氨酸。
优选地，所述的氨基酸为甘氨酸（Glycine，Gly/ 甘氨酸）。
由此，发明公开并保护了氨基酸或氨基酸衍生物在制备提高血清或血浆或全血杀菌作用药物方面的应用，所述的氨基酸为 甘氨酸，苏氨酸或丝氨酸 。
同时，发明公开并保护了氨基酸或氨基酸衍生物在制备注射用杀菌剂方面的应用，氨基酸。
同时，发明公开并保护了氨基酸与血清联用在制备杀菌药物方面的应用，或氨基酸衍生物，与血清或血浆或全血联用在制备杀菌药物方面的应用；所述的氨基酸为 甘氨酸，苏氨酸或丝氨酸 。
优选地，所述的氨基酸为甘氨酸。
所述的甘氨酸的代谢产物和甘氨酸衍生物，如谷胱甘肽、 5,10 - 亚甲基四氢叶酸、 H- 蛋白 - 氨甲基二氢脂赖氨酸 、N1-( 5-磷酸-D-核糖基)甘氨酰胺、L-2-氨基-3-氧代丁酸、L-半胱氨。
发明同时保护了一种杀菌药物组合物，其特征在于含有氨基酸和血清 / 血浆 / 全血，所述的氨基酸为 甘氨酸，苏氨酸或丝氨酸 。
优选地，所述的血清和甘氨酸的比例以重量计为 1:3000 。
所述的细菌包括革兰氏阴性菌和阳性菌。优选的，所述的细菌为革兰氏阴性和阳性病原菌。更优选地，所述的细菌为金黄色葡萄球菌、迟钝爱德华菌、 B 链球菌、绿脓杆菌、大肠杆菌或弧菌。
优选地，所述的血清或血浆或全血来源于哺乳动物、鱼类或禽类。
本发明同时公开并保护了一种杀菌方法，该方法包括：将氨基酸和血清 / 血浆 / 全血联用，或氨基酸衍生物和血清 / 血浆 / 全血联用；所述的氨基酸为 甘氨酸，苏氨酸或丝氨酸 。
优选地，所述的方法包括：将甘氨酸和血清以重量计为 1:3000 的比例混合，用于杀灭革兰氏阴性菌或阳性菌；杀菌所用时间为 0.1~24 小时。
更优选地，本发明所采用的氨基酸为甘氨酸。
本发明通过代谢组学的方法，对用人血清处理的大肠埃希菌的所有小分子代谢物进行分析，发现甘氨酸在耐人血清的大肠埃希菌中含量明显下降，提示其可以作为提高对血清敏感性的小分子代谢物质。
本发明通过 固定人血清量添加不同浓度的甘氨酸、固定甘氨酸浓添加不同体积的人血清，以及固定人血清和甘氨酸在不同作用时间的三种情况下，研究甘氨酸添加对血清杀菌作用的影响。结果发现，仅添加甘氨酸的对照组对大肠杆菌生长没有影响，而在人血清中加入甘氨酸后，血清的杀菌能力增加，且其能力随甘氨酸和血清浓度的增加而增强。同时，在一定范围内，随作用时间延长而作用增加。此外，以血浆代替血清也获得同样效果，但采用灭活补体的血清或血浆则无任何效果。
本发明通过对革兰氏阳性菌的金黄色葡萄球菌和乙型溶血性链球菌以及革兰氏阴性菌的大肠杆菌、迟钝爱德华菌、绿脓杆菌、弧菌等多种细菌的杀菌实验，结果证明添加甘氨酸能提高所有细菌对人血清的敏感性。
本发明通过采用 小鼠、兔、猪、鸡、家鸽、 对 虾和鱼等 不同进化层次物种动物血清/血浆与甘氨酸合用对其主要病原菌的杀菌作用，发现甘氨酸能提高不同动物血清对其主要病原菌的杀菌能力。
本发明进一步进行了小鼠体内实验，发现添加甘氨酸后，小鼠不仅对模式大肠杆菌而且对具有完全血清抗性的临床分离病原性菌株也表现明显提高的清除能力，说明甘氨酸可以在体内和全血状态下起作用。
综上所述，在血清中添加甘氨酸能够明显提高细菌对血清的敏感性，为血清抗性病原菌感染的治疗提供了一种崭新的技术方法。
由此，发明公开并保护了甘氨酸在提高细菌对 血清敏感性方面的应用。其可用于制备药物或杀菌剂，进一步增强细菌对血清的敏感性。
同时，发明公开并保护了一种提高细菌对血清敏感性的方法，其特征在于采用甘氨酸作为一种提高血清杀菌作用的药物。
所述的细菌包括但不限于为金黄色葡萄球菌、乙型溶血性链球菌、迟钝爱德华菌、绿脓杆菌、大肠杆菌或弧菌。因为这些细菌是常见人类和养殖动物致病菌，其中金黄色葡萄球菌和乙型溶血性链球菌为革兰氏阳性菌，大肠埃希氏菌、绿脓杆菌、迟钝爱德华菌、副溶血弧菌和溶藻弧菌为革兰氏阴性菌。这些细菌为常见的病原菌，且常见其血清抗性菌株，同时大肠埃希氏菌是研究细菌血清抗性的模式菌，故这些细菌为血清抗性的较好代表菌。
应用上述方法来提高细菌对血清的敏感性时，甘氨酸的使用浓度为 0.05-1g/ 公斤体重 / 天。
通过本发明所公开的内容，还可制备出一种新的杀菌剂，该杀菌剂含有甘氨酸；或者一种提高血清对细菌杀菌作用的制剂，其主要成分为甘氨酸。
附图说明
图 1 为耐血清大肠埃希氏菌 GC-MS 的独立成分分析（ A ）、数据聚类分析（ B ）及代谢物含量分析（ C ）。
图 2 为甘氨酸对提高细菌对血清敏感性的结果。 A 为不同浓度甘氨酸、 B 为不同体积血清， C 为不同时间。
图 3 为甘氨酸可提高多种细菌对血清敏感性的结果。 A 为生存率， B 为提高倍数。
图 4 为甘氨酸提高不同动物血清 / 血浆对其主要病原菌杀菌能力的结果。 A 为虾和鱼血清对溶藻弧菌杀菌的结果， B 为鸡和家鸽血浆对大肠杆菌杀菌的结果， C 为小鼠、兔和猪血浆对大肠杆菌杀菌的结果。
图 5 为甘氨酸提高小鼠对体内细菌清除的结果。 A 和 B 依次为小鼠注射或不注射甘氨酸血液中细菌数量和生存率。
图 6 为甘氨酸提高小鼠对大肠埃希菌的清除的结果。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例 1
甘氨酸是大肠埃希菌抵抗血清杀菌作用的生物标记物
1. 试验样本的准备：从 LB 平板上挑取大肠埃希菌起始株 (Escherichia coli K12 BW25113) 单菌落接种于 5 毫升 LB 培养基中， 37℃ 200rpm 振荡培养 16 小时。按 1 ： 100 （ v/v ）的比例接种于 200 毫升培养基中， 37℃ 振荡培养至 OD600 值为 1.0 ， 4℃ 8000rpm 离心 10 分钟收集菌体，并用 0.85% 生理盐水洗涤菌体 1 次。
2. GC-MS 样品的制备和数据处理
GC-MS 样品制备：向准备好的样本中分别加入 100 微升人血清（抽取健康人血液，自然凝固，离心上清即为血清），重悬菌体后置于 37 ℃ 200rpm 振荡培养 1 小时，对照组为生理盐水。 4℃8000rpm 离心 5 分钟收菌，去除残余血清或生理盐水。用生理盐水重悬菌体，使 OD600 都为 1.0 ，各取 1 毫升加入冷甲醇 (Sigma)( 包含 5 微升 0.1 毫克 / 毫升核糖纯 (Sigma) 作为分析的内标 ) 。在 10W 强度设置下超声 3 分钟使细胞破碎， 4℃12000rpm 离心 10 分钟，取 500 微升上清转移至新的 1.5 毫升离心管并进行真空干燥 (LABCONCO) 。干燥后的样品用于 GC/MS 分析。每个样本生物学重复 3 次。
GC-MS 分析：样本首先通过甲基化保护羧基，即通过与 40 微升包含 20 毫克 / 毫升甲氧胺盐酸盐的吡啶在 37℃ 反应 90 分钟，然后加入 80 微升 N- 甲基 -N- 三甲基硅基三氟乙酰胺 (MSTFA, Sigma-Aldrich) 在 37℃ 反应 30 分钟使酸性质子衍生化。 1 微升衍生化过的样本注入 30 米 ×250 微米内径 ×0.25 微米 DBS-MS 柱，采用不分流进样和 DSQ II （ Thermo Scientific ）进行分析。 GC 烘箱的初始温度在 85℃ 维持 5 分钟，接着以 15℃ /min 的速率提高到 330℃ ，再维持 5 分钟。 1 毫升 / 分钟氦气作为载气流量。 MS 操作在 m/z 50-600 范围内进行。
数据处理：谱卷积和校准使用 AMDIS 的和内标进行。所有样本执行保留时间（ RT ）校正，然后 RT 作为参考，对剩余谱进行查询，组装生成一个包含所有样品中每个代谢物丰度信息的文件。使用 NIST MS search 2.0 软件，将 GC-MS 谱中代谢物与 National Institute of Standards and Technology (NIST) 数据库进行比对，从而获得鉴定。导出的数据矩阵使用总峰面积进行校正。使用保留时间和 m/z 区配结果赋予每个样本中每个物质标准化过的峰强度，生产的文件用于进一步的统计学分析。
3. 数据分析。
通过 GC-MS 进行鉴定生理盐水对照组与用 100 微升人血清和 1 毫升人血清处理后的大肠埃希菌代谢产物，通过代谢物的热图和等级聚类分析，发现 3 个重复样品各自聚在一起且血清处理组聚在一起（图 1A ）。进一步独立成分分析，发现 IC01 可很好的解释人血清的效应（图 1B ），通过对所有代谢产物分析，发现甘氨酸（ Glycine ）在人血清处理样品中明显下降（图 1C ），提示甘氨酸可能是大肠埃希菌抵抗血清杀菌作用的生物靶标物质。
实施例 2
甘氨酸可提高大肠埃希菌对血清的敏感性
为了解甘氨酸是否有助于血清的杀菌作用，分别进行了在同样含量人血清中添加不同浓度的甘氨酸、在不同体积的人血清中添加相同浓度的甘氨酸，以及在不同作用时间等三种情况下的试验研究。
1. 甘氨酸促进血清杀菌作用具有浓度梯度效应
按照实例 1.1 制备实验用细菌样本，添加甘氨酸使其终浓度分别 0 、 5 、 10 、 30 、 50 和 100mM ， 37℃ 200rpm 振荡培养 1 小时后，再添加 100 微升人血清处理，同时以生理盐水为对照。 2 小时离心收菌，用 1 毫升生理盐水重悬菌体，用 LB 固体平板进行活菌计数。存活率计算公式为：加入不同浓度甘氨酸处理后活菌数 / 生理盐水对照处理后活菌数× 100% 。图 2A 为在人血清中加入不同浓度甘氨酸后，大肠埃希菌的生存率。从这个结果可以看出，加入 5mM 甘氨酸后，血清对大肠埃希菌的杀菌作用提高了 1.36 倍，随着加入甘氨酸浓度的提高，杀菌作用逐渐增强， 100mM 提高幅度最大，可达约 86 倍。
2. 甘氨酸促进血清杀菌作用随血清体积增大而效果加强
取制备好的细菌样本，先添加 100mM 甘氨酸， 37 ℃ 200rpm 振荡培养 1 小时，然后分别添加 0 、 20 、 40 、 60 、 80 和 100 微升人血清，同时以不添加甘氨酸只添加不同体积人血清为对照。 37℃ 200rpm 振荡培养 2 小时后离心收菌，用 1 毫升生理盐水将菌体重悬后，用平板进行活菌计数。存活率计算公式为：加入不同体积人血清和甘氨酸处理后活菌数 / 只加人血清处理活菌数× 100% 。计数结果（图 2B ）说明，当添加了甘氨酸后，血清对大肠埃希菌的杀菌作用都比不添加甘氨酸有提高，随着添加血清量的增加，杀菌作用显著增加，当添加 100 微升人血清时，可增加 86 倍。
3. 甘氨酸促进血清杀菌作用具有时间效应
将制备好的细菌样本分成 4 组：添加 100mM 甘氨酸组、 100 微升人血清组、 100 微升人血清和 100mM 甘氨酸组、生理盐水对照组， 37℃ 200rpm 振荡培养，分别在 0.5 、 1 、 2 、 3 和 4 小时离心收菌，用 1 毫升生理盐水将菌体重悬，用 LB 固体平板进行活菌计数。存活率计算公式为：加入血清和 / 或甘氨酸处理后不同时间活菌数 / 加生理盐水对照组不同时间活菌数× 100% 。图 2C 为 3 个实验组在不同时间的生存率，从图可见，只加入甘氨酸没有杀菌效果，当只加入血清时会有一定的杀菌效果，而当在血清中加入甘氨酸后，杀菌效果明显增强，在 0.5 小时后增加了 2 倍， 1 小时猛增到 77 倍， 4 小时高达 120 倍。
综上所述，甘氨酸本身对于大肠杆菌生长没有影响；血清本身对细菌有一定的杀菌作用，而在人血清中加入甘氨酸后，血清的杀菌能力显著增加，且其杀菌能力随甘氨酸浓度的增强而增强；在添加甘氨酸后，随人血清量的增加而杀菌率增加。
实施例 3
甘氨酸提高细菌包括革兰氏阴性菌和阳性菌对血清的敏感性
按照实例 1.1 方法培养多种细菌（大肠杆菌 K12 BW25113 ，大肠杆菌 Y17 临床菌，乙型溶血性链球菌，溶藻弧菌，副溶血弧菌，绿脓杆菌和金黄色葡萄球菌），分别收集 3 毫升 OD600 为 1.0 的每种细菌。然后分别分为 4 组：添加 100 微升生理盐水对照组、 100-150mM 甘氨酸组、 125-175 微升人血清组和 125-175 微升人血清加 100-150mM 甘氨酸组。不同种类细菌最佳杀菌作用加入的血清量和甘氨酸略有不同。 37℃ 200rpm 振荡培养 4 小时后离心收菌，用 1 毫升生理盐水将菌体重悬，测定 OD 值（ OD600nm ）。图 3A 为每种菌在三种处理后的 OD 值，图 3B 为每种细菌在血清处理和血清中加入甘氨酸处理后的杀菌倍数比较。从图可以看出，血清对不同细菌的杀菌效果是不一样的，对大肠杆菌 K12 BW25113 、乙型溶血性链球菌和绿脓杆菌具有一定的杀菌作用，而对其他几种细菌反而有促进生长作用；但是在添加甘氨酸后，血清对所有细菌均具有显著杀菌作用，其增强杀菌作用都在 1 倍以上，最高可达 4.13 倍。
实施例 4
甘氨酸提高不同进化层次物种血清 / 血浆对其主要病原菌的杀菌效果
按照实例 1.1 方法培养多种细菌（大肠杆菌 K12 BW25113 ，大肠杆菌 Y17 临床菌，溶藻弧菌，副溶血弧菌，绿脓杆菌和金黄色葡萄球菌），分别收集 3 毫升 OD600 为 1.0 的每种细菌 , 然后分别分为 2 组：添加 100 微升不同动物血清 / 血浆组和 100 微升不同动物血清 / 血浆加 100mM 甘氨酸组。同时以添加 100mM 甘氨酸为对照。 37℃ 200rpm 振荡培养， 4 小时后离心收菌，用 1 毫升生理盐水将菌体重悬，测定 OD 值（ OD600nm ）。图 4 为不同进化层次动物的主要病原菌在血清 / 血浆处理和血清 / 血浆中加入甘氨酸处理后的 OD 值。从图可以看出，虽然不同进化层次动物的血清 / 血浆对其主要病原菌有一定的杀菌作用，但在添加甘氨酸后，血清 / 血浆对细菌的杀菌作用显著增强，其杀菌作用增加最低的为 1.5 倍，最高的可高达近 10 倍，这些结果表明甘氨酸可以明显提高不同进化动物的血清 / 血浆对其主要病原菌的杀菌作用。
实施例 5
甘氨酸可以提高小鼠对大肠埃希菌的清除作用
将 6 周龄小鼠共 15 只（购买于中山大学东校区实验动物中心）随机分为 3 组，生理盐水对照组和 2 种浓度（ 3.25 和 6.5 毫克 / 只）的甘氨酸给药处理组。采用静脉注射给药的方式，按照 3.25 和 6.5 毫克甘氨酸 / 次 / 只，两次给药时间间隔 12 小时，连续给药 6 次；停药 12 小时后静脉分别注射大肠埃希菌 K12 BW25113 和大肠埃希菌 Y17 （临床分离菌株），剂量为 1×106 个细菌 / 只的细菌， 48 小时后小鼠尾静脉取血，梯度稀释后平板计数，检测细菌被清除情况。图 5A 和 B 是小鼠血清中注射 2 种甘氨酸浓度后细菌数量和体内细菌生存率。从此结果可以看出，当给小鼠注射甘氨酸后，小鼠体内细菌数量明显减少。对于大肠杆菌 K12 BW25113 来说，注射 3.25 毫克时清除率为 89.32% ， 6.5 毫克时清除率达到 96.85% 。对于大肠杆菌 Y17 临床菌来说，注射 3.25 毫克时清除率为 32.95% ， 6.5 毫克时清除率达到 52.53% 。这些结果表明甘氨酸提高了血清对细菌的清除作用，而且随着加入量的增加，清除作用加强。
实施例 6
苏氨酸可提高大肠埃希菌对血清的敏感性
将制备好的细菌样本分成 4 组：添加 100mM 苏氨酸组、 100 微升人血清组、 100 微升人血清和 100mM 苏甘氨酸组、生理盐水对照组， 37℃ 200rpm 振荡培养，在 2 小时离心收菌，用 1 毫升生理盐水将菌体重悬，测定 OD 值（ OD600 ）。存活率计算公式为：加入血清和 / 或苏氨酸处理后 OD 值 / 加生理盐水对照组 OD 值× 100% 。图 6 为 3 个实验组在不同时间的存活率，从图可见，只加入苏氨酸没有杀菌效果，当只加入血清时会有一定的杀菌效果，而当在血清中加入苏氨酸时，杀菌效果明显增增加了 1.5 倍。

Claims (10)

  1. 氨基酸或其衍生物在制备提高血清或血浆或全血杀菌作用药物方面的应用，所述的氨基酸为 甘氨酸，苏氨酸或丝氨酸。
  2. 氨基酸或其衍生物在制备注射用杀菌剂方面的应用，所述的氨基酸为 甘氨酸，苏氨酸或丝氨酸。
  3. 氨基酸或其衍生物，与血清或血浆或全血联用在制备杀菌药物方面的应用，所述的氨基酸为 甘氨酸，苏氨酸或丝氨酸。
  4. 一种杀菌药物组合物，其特征在于含有氨基酸和血清 / 血浆 / 全血，所述的氨基酸为 甘氨酸，苏氨酸或丝氨酸。
  5. 如权利要求 4 所述的组合物，其特征在于所述的血清和甘氨酸的比例以重量计为 1:1-3000 。
  6. 如权利要求 1-4 任一所述的应用或组合物，其特征在于所述的细菌为革兰氏染色阴性或阳性的病原菌；所述的氨基酸为甘氨酸。
  7. 如权利要求 5 任一所述的应用，其特征在于所述的细菌为金黄色葡萄球菌、迟钝爱德华菌、溶血性链球菌、绿脓杆菌、大肠杆菌或弧菌。
  8. 如权利要求 1 、 3 或 4 任一所述的应用或组合物，其特征在于所述的血清或血浆或全血来源于哺乳动物、鱼类或禽类。
  9. 一种杀菌方法，其特征在于：将氨基酸和血清 / 血浆 / 全血联用，或氨基酸衍生物和血清 / 血浆 / 全血联用，所述的氨基酸为 甘氨酸，苏氨酸或丝氨酸 。
  10. 如权利要求 9 所述的杀菌方法，其特征在于：将血清和氨基酸以重量计 1:1-3000 的比例混合，用于杀灭革兰氏阴性菌或阳性菌；杀菌所用时间为 0.1~24 小时。
PCT/CN2013/070429 2012-12-31 2013-01-14 提高细菌对血清敏感性的氨基酸 Ceased WO2014101322A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101827612A (zh) * 2007-09-07 2010-09-08 联合治疗公司 针对革兰氏阴性细菌具有选择性杀菌活性的缓冲液及其使用方法
CN102791262A (zh) * 2010-01-08 2012-11-21 哈佛大学校长及研究员协会 用于处理生物薄膜的d-氨基酸

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3674863A (en) * 1969-12-08 1972-07-04 Parke Davis & Co Polyvalent immunizing agents and methods for their production
US20100297081A1 (en) * 2007-12-18 2010-11-25 Huang Yanshan Pharmaceutical formulation containing recombinant human serum albumin-interferon alpha fusion protein
US8945540B2 (en) * 2008-05-09 2015-02-03 Exoxemis, Inc. Compositions for enhancing the antibacterial activity of myeloperoxidase and methods of use thereof
CN101647808A (zh) * 2008-08-14 2010-02-17 洛阳惠中兽药有限公司 一种治疗畜禽呼吸道疾病药物组合物及其制备方法和用途
EP2440251A4 (en) * 2009-06-09 2013-01-16 Defyrus Inc INTERFERON ADMINISTRATION FOR PROPHYLAXIS AGAINST PATHOGEN INFECTION OR TREATMENT OF PATHOGEN INFECTION

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101827612A (zh) * 2007-09-07 2010-09-08 联合治疗公司 针对革兰氏阴性细菌具有选择性杀菌活性的缓冲液及其使用方法
CN102791262A (zh) * 2010-01-08 2012-11-21 哈佛大学校长及研究员协会 用于处理生物薄膜的d-氨基酸

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Lack of bactericidal activity against E. coli in serum of hepatic cirrhosis patients", FOREIGN MEDICAL INFORMATION, 1980, pages 71 *
LIU, TAOTAO ET AL.: "Changes in Phospholipase A2 in Blood of Rat Bacterial Infection Model", CHINESE JOURNAL OF NOSOCOMIOLOGY, vol. 13, no. 1, 2003, pages 10 - 12 *

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