WO2022252098A1 - 噬菌体制剂的制备方法、药物组合物以及应用 - Google Patents

噬菌体制剂的制备方法、药物组合物以及应用 Download PDF

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WO2022252098A1
WO2022252098A1 PCT/CN2021/097556 CN2021097556W WO2022252098A1 WO 2022252098 A1 WO2022252098 A1 WO 2022252098A1 CN 2021097556 W CN2021097556 W CN 2021097556W WO 2022252098 A1 WO2022252098 A1 WO 2022252098A1
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phage
solution
storage tank
concentration
preparation
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PCT/CN2021/097556
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French (fr)
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马迎飞
谭新
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中国科学院深圳先进技术研究院
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    • 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
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • 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
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

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  • the application relates to the field of biotechnology, in particular to a preparation method, pharmaceutical composition and application of a phage preparation.
  • the original liquid for preparing phage contains a large amount of toxic by-products, such as endotoxin, peptidoglycan, bacterial chromosomes and other unknown substances. Septic shock. This limits the large-scale clinical application of phages.
  • the embodiment of the present application provides a preparation method, pharmaceutical composition and application of a phage preparation to remove endotoxin in the phage preparation quickly, at low cost and with high efficiency.
  • a technical solution adopted by the present application is to provide a method for preparing a phage preparation, the method comprising: diafiltration of the phage mixed culture solution through a tangential flow system to obtain a phage stock solution; flow system, concentrate the phage original solution until the concentration is 7 to 10 times the concentration of the phage original solution, and obtain the phage concentrated solution; use cesium chloride solution to carry out cesium chloride density gradient centrifugation on the phage concentrated solution, and obtain the phage density gradient solution ; The 20KD membrane is used to dialyze the phage density gradient solution to obtain the phage preparation.
  • a pharmaceutical composition which includes: the phage preparation prepared by the aforementioned method; a pharmaceutically acceptable carrier, excipient or at least one of the diluents.
  • Another technical solution adopted by the present application is to provide an application in the preparation of Klebsiella pneumoniae phage preparations, Acinetobacter baumannii phage preparations, and Pseudomonas aeruginosa phage preparations as described above. .
  • the beneficial effect of the present application is: different from the situation of the prior art, the present application can remove endotoxins in phage preparations quickly, at low cost and efficiently, wherein the tangential flow system has the advantages of high throughput and reusability, Using tangential flow system filtration combined with tangential flow system concentration can realize rapid, efficient, and low-cost diafiltration of phage mixed culture solution and concentration of phage original solution; in addition, cesium chloride density gradient centrifugation combined with dialysis can be used to remove chloride Cesium can efficiently and quickly obtain high-purity phage preparations.
  • Fig. 1 is the schematic flow chart of an embodiment of the preparation method of the phage preparation of the present application
  • Fig. 2 is a schematic flow chart of step S10 in Fig. 1;
  • Fig. 3 is a schematic flow chart of step S20 in Fig. 1;
  • FIG. 4 is a schematic flow chart of step S30 in FIG. 1;
  • FIG. 5 is a schematic flow chart of step S40 in FIG. 1;
  • Figure 6 is a schematic flow diagram of another embodiment of the preparation method of the phage preparation of the present application.
  • Figure 7 is a schematic flow diagram of another embodiment of the preparation method of the phage preparation of the present application.
  • Figure 8a is the result of detecting the inflammatory index TNF- ⁇ in the blood of mice 1 hour, 6 hours, 24 hours, and 72 hours after the first dose in Example 3;
  • Figure 8b is the result of detecting the inflammatory index KC/GRO in the blood of mice 1 hour, 6 hours, 24 hours, and 72 hours after the first dose in Example 3;
  • Figure 8c is the result of detecting the inflammatory index IL-6 in the blood of mice 1 hour, 6 hours, 24 hours, and 72 hours after the first dose in Example 3;
  • Figure 8d is the result of detecting the inflammatory index IL-10 in the blood of mice 1 hour, 6 hours, 24 hours, and 72 hours after the first dose in Example 3;
  • Figure 8e is the detection results of the inflammatory index IL-4 in the blood of mice detected 1 hour, 6 hours, 24 hours, and 72 hours after the first dose in Example 3;
  • Figure 8f is the result of detecting the inflammatory index IL-2 in the blood of mice 1 hour, 6 hours, 24 hours, and 72 hours after the first dose in Example 3;
  • Figure 8g is the result of detecting the inflammatory index IL-1 ⁇ in the blood of mice 1 hour, 6 hours, 24 hours, and 72 hours after the first dose in Example 3;
  • Figure 8h is the result of detecting the inflammatory index IFN- ⁇ in the blood of mice 1 hour, 6 hours, 24 hours, and 72 hours after the first dose in Example 3;
  • Figure 8i is the result of detecting the inflammatory index IL-5 in the blood of mice 1 hour, 6 hours, 24 hours, and 72 hours after the first dose in Example 3;
  • Figure 9a is the result of detecting the white blood cell count in the mouse blood 24 hours after the second dose in Example 3;
  • Figure 9b is the result of detecting red blood cell counts in the blood of mice 24 hours after the second dose in Example 3;
  • Figure 9c is the result of detecting platelet count in mouse blood 24 hours after the second dose in Example 3.
  • Figure 9d is the result of detecting the amount of hemoglobin in the mouse blood 24 hours after the second dose in Example 3;
  • Figure 9e is the result of detecting the lymphocyte count in the mouse blood 24 hours after the second dose in Example 3.
  • Fig. 10 is the result of hematoxylin-eosin staining of mouse spleen pathological sections 24 hours after the second dose in Example 3.
  • FIG. 1 is a schematic flow chart of an embodiment of the preparation method of a phage preparation of the present application.
  • the method includes the following steps:
  • the tangential flow system has the advantages of high throughput and reusability, and the tangential flow system is used to filter and combine Tangential flow system concentration can realize fast, efficient and low-cost diafiltration of phage mixed culture solution and concentration of phage original solution; in addition, cesium chloride can be removed efficiently and quickly by cesium chloride density gradient centrifugation combined with dialysis Obtain highly pure phage preparations.
  • the tangential flow system in the above embodiment at least includes: a first storage tank, a sample injection tube, a return tube, a filtration tube, a peristaltic pump, a membrane bag device, and a second storage tank, and the peristaltic pump includes a roller , the sampling tube is clamped between the rollers to peristalize the liquid in the sampling tube, wherein one end of the sampling tube and one end of the return tube are placed in the first storage tank, and the filter tube is separately placed in the second storage tank.
  • FIG. 2 is a schematic flowchart of step S10 in FIG. 1 .
  • This step S10 comprises the following steps:
  • S12 Filtrate the supernatant through a tangential flow system, wherein the supernatant in the first storage tank enters the membrane package device from the injection tube, and the liquid flowing out from the return tube flows back into the first storage tank, and the filtrate Flow out from the filter tube into the second storage tank to obtain the original phage liquid.
  • the tangential flow system in the above embodiment includes: a first storage tank, a sampling tube, a return tube, a filtration tube, a peristaltic pump, a membrane bag device, a second storage tank, and a third storage tank
  • the peristaltic pump includes rollers, and the sampling tube is clamped between the rollers to peristalize the liquid in the sampling tube, wherein, one end of the sampling tube and one end of the return tube are placed in the first storage tank, and the filter tube is separately placed in the second storage tank. inside the tank.
  • FIG. 3 is a schematic flowchart of step S20 in FIG. 1 .
  • This step S20 comprises the following steps:
  • FIG. 4 is a schematic flowchart of step S30 in FIG. 1 .
  • This step S30 comprises the following steps:
  • S31 Provide a centrifuge tube, wherein, the bottom of the centrifuge tube is a CsCl solution of the first concentration, the middle part of the centrifuge tube is a CsCl solution of the second concentration, and the upper part of the centrifuge tube is a CsCl solution of the third concentration, and the first concentration is greater than the CsCl solution of the second concentration. Two concentrations, the second concentration is greater than the third concentration.
  • FIG. 5 is a schematic flowchart of step S40 in FIG. 1 .
  • This step S40 comprises the following steps:
  • the present application provides a preparation method of a phage preparation, as shown in FIG. 6 , which is a schematic flowchart of another embodiment of the preparation method of a phage preparation of the present application.
  • the above preset ratio is: the ratio between the number of phages and the number of host bacteria is: 1:10.
  • the present application provides a preparation method of a phage preparation, as shown in FIG. 7 , which is a schematic flowchart of another embodiment of the preparation method of a phage preparation of the present application.
  • the method includes the following steps:
  • S80 Use the endotoxin detection Limulus kit to detect the endotoxin concentration of the phage preparation.
  • the present application provides a pharmaceutical composition, which includes: a phage preparation, and at least one of a pharmaceutically acceptable carrier, excipient or diluent.
  • the phage preparation is the phage preparation prepared by the preparation method of the above examples.
  • Klebsiella pneumoniae phage preparations Acinetobacter baumannii phage preparations, and Pseudomonas aeruginosa phage preparations prepared by the preparation methods in the above examples.
  • Step 1 Resuscitate the host bacteria.
  • the details are as follows: take out the Pseudomonas aeruginosa PAO1 strain and store it at -80°C, streak the LB plate without anti-bacteria, and pick a single colony to 10mL LB medium after 8 hours, and culture overnight (shaking table 37°C, shaking table speed 150rpm)) .
  • Step 2 Recover the phage.
  • the details are as follows: take 0.5mL bacterial liquid and add 20mL LB medium, cultivate for 2 hours (shaking table 37°C, shaking table speed 150 revolutions per minute (rpm), after that, sample phage PA39 from the phage library, add phage and The ratio of the number of host bacteria is 1:10, and continue to cultivate (shaking table 37°C, shaking table speed 150rpm). After the host bacteria are completely lysed, after about 4 hours, the mixed culture solution is centrifuged at a high speed (8000rpm, 10min), and the supernatant is taken Filtrate through a 0.22 ⁇ m filter membrane, and the filtrate immediately recovers the phage.
  • Step 3 Resuscitate the host bacteria.
  • Step 4 Expand the phage in 2L system.
  • the details are as follows: take 50mL of bacterial liquid and add 2L of LB medium, cultivate (shaking table at 37°C, shaking table speed at 150rpm)) for 2 hours, then add recovered phage PA39, the ratio of phage to host bacteria is 1:10 (different The ratio of the phage can be different), and the culture is continued (shaking table at 37° C., shaking table speed of 150 rpm) to obtain a phage mixed culture solution.
  • Step 5 Diafiltration of the phage mixed culture through a tangential flow system.
  • the centrifuge the phage mixed culture solution (wherein, the centrifugal speed is 8000rpm, 10min), and the supernatant is filtered through a tangential flow system (the filter membrane bag pore size of the tangential flow system is 0.22 ⁇ m).
  • the phage stock solution was serially diluted to 10 -8 using the LB liquid medium gradient, and the phage concentration of the phage stock solution was counted by the double-layer agar plate method, wherein the phage concentration was 4 ⁇ 10 9 PFU/mL.
  • Step 6 Concentrate the phage stock by tangential flow system.
  • the phage raw liquid is concentrated through a tangential flow system (the pore size of the ultrafiltration membrane bag is 100KDa).
  • a tangential flow system the pore size of the ultrafiltration membrane bag is 100KDa.
  • the remaining phage stock solution in the storage tank is the first concentrated solution.
  • the counting method is the same as above, wherein the phage concentration is 3 ⁇ 10 10 PFU/ mL.
  • Step 7 Purify the phage concentrate by cesium chloride density gradient centrifugation.
  • the details are as follows: add 20mL phage concentrate into a centrifuge tube (maximum volume 40mL), the bottom of the centrifuge tube is the CsCl solution of the first concentration (1.7g/L), and the middle part of the centrifuge tube is the CsCl solution of the second concentration (1.5g/L). CsCl solution, the upper part in the centrifuge tube is the third concentration (1.3g/L) CsCl solution.
  • concentration gradient first add 20mL of phage concentrate, then inhale 4mL of the third concentration (1.3g/L) of CsCl solution into the Pasteur pipette, touch its head to the bottom of the centrifuge tube, and slowly inject it.
  • Step 8 Centrifuge.
  • the details are as follows: use a HiMAC ultracentrifuge to centrifuge for 2 hours (4° C., 24000 rpm). After the centrifugation is completed, a white-brown layer can be seen between the CsCl solutions of different concentrations (if multiple layers are seen, each layer needs to be sampled separately), use a syringe needle to draw a needle at a position about 2mm below the phage plane Aspirate all the white-brown layers, and a centrifuge tube can aspirate about 3mL of phage density gradient solution.
  • Step 9 use the phage storage buffer as the dialysate, and use a 20KDa dialysis membrane to carry out repeated dialysis on the phage density gradient to obtain a purified phage preparation.
  • phage preservation buffer as the dialysate
  • 20KDa dialysis membrane to carry out multiple dialysis on the phage density gradient liquid
  • remove cesium chloride in the phage sample use 5L dialysate for each dialysis, every 4 Change the dialysate every hour and dialyze at least 3 times to finally obtain about 15 mL of purified phage preparation.
  • the phage concentration of the phage preparation was detected by double-layer agar plate method, wherein the phage concentration was 2 ⁇ 10 11 PFU/mL.
  • Step 10 Use the endotoxin detection Limulus kit to detect the endotoxin concentration of the phage preparation.
  • the endotoxin concentration of the phage preparation was detected by using the endotoxin detection Limulus kit (test tube quantitative chromogenic matrix method, Xiamen Limulus Reagent Biotechnology Co., Ltd., EC80545).
  • the detection kit utilizes the artificially synthesized chromogenic substrate and the azo reagent to make the rose bengal chromogenic reaction produced by Limulus reagent quantitatively detect endotoxin.
  • the detection range of the kit is 0.1-1EU/mL.
  • the experiment was carried out in strict accordance with the operation manual, and the endotoxin concentration of the phage stock solution prepared in the above step 5 was measured to be about 10,000 EU/mL, and the endotoxin content of the phage preparation prepared in the above step 9 was measured to be 500 EU/mL. After doubling dilution of the phage preparation prepared in the above step 9 to a phage concentration of 10 9 PFU/mL, the endotoxin content per ml of the diluted phage preparation was measured to be 2.5 EU.
  • Pseudomonas aeruginosa phage PA39, Acinetobacter baumannii phage AB6, Acinetobacter baumannii phage Ab_SZ3 and Klebsiella pneumoniae phage KPh were prepared by using the preparation method of the phage preparation of the present application, wherein the preparation steps can refer to the above-mentioned phage preparation Examples of preparation methods.
  • each phage concentration of each phage preparation Detect the endotoxin concentration of each phage original solution in the above-mentioned Example 2, the phage concentration of each phage preparation, the endotoxin concentration of each phage preparation, and after doubling dilution of the phage preparation to a phage concentration of 10 9 PFU/mL, each The endotoxin content of the diluted phage preparation in milliliters (hereinafter referred to as: the endotoxin content of 10 9 PFU unit dose).
  • the test results are shown in Table 1 below.
  • the U.S. Food and Drug Administration stipulates that the standard for endotoxin entering the blood is less than 5EU/Kg ⁇ h, taking a single dose of an adult (50Kg) as an example, that is, an acceptable dose within 1 hour
  • the maximum endotoxin content is 250EU, and in clinical application, the single-use dose of phage is generally recommended to be 10 9 PFU.
  • the phage preparation is diluted to a phage concentration of 10 9 PFU/mL , the endotoxin content per milliliter of the diluted phage preparation is lower than 250EU, which can be directly applied to clinical treatment, and is suitable for various application scenarios including nebulization, intravenous injection, intramuscular injection, external application, washing, etc.
  • the application has studied the influence of the Pseudomonas aeruginosa phage PA39 preparation prepared in Example 1 on the mouse body during intravenous injection.
  • mice were divided into four groups, 6 cases in each group, respectively vehicle control group, low dose group (2 ⁇ 10 8 PFU/Kg/), middle dose group (2 ⁇ 10 10 PFU/Kg), high dose group (2 ⁇ 10 10 PFU/Kg), high In the dose group (2 ⁇ 10 11 PFU/Kg), different concentrations of Pseudomonas aeruginosa phage PA39 preparations were injected into the mice through the tail vein, and the administration was divided into two doses, and the second dose was given 3 days after the first dose.
  • the inflammatory indicators in the blood of the mice were detected 1 hour, 6 hours, 24 hours, and 72 hours after the first dose.
  • some inflammatory indicators in the mice increased after 1 hour, and after 24 hours, the inflammatory indicators basically fell back to normal levels, and there was no obvious change in the inflammatory indicators in the low-dose group.
  • mice were dissected. There were no macroscopic morphological changes in skin, coat, lips, mouth, heart, liver, spleen, lung, thymus, kidney, adrenal gland, pancreas, gastrointestinal tract, testis and epididymis, uterus and ovary in low, medium and high dose groups .
  • the tangential flow system has the advantages of high throughput and reusability, and the tangential flow system is used to filter and combine Tangential flow system concentration can realize fast, efficient and low-cost diafiltration of phage mixed culture solution and concentration of phage original solution; in addition, cesium chloride can be removed efficiently and quickly by cesium chloride density gradient centrifugation combined with dialysis Obtain highly pure phage preparations.

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Abstract

一种噬菌体制剂的制备方法、药物组合物以及应用，该方法包括：通过切向流系统，将噬菌体混合培养液渗滤，得到噬菌体原始液；通过切向流系统，将噬菌体原始液浓缩，直到浓度为噬菌体原始液的浓度的7～10倍，得到噬菌体浓缩液；采用氯化铯溶液对噬菌体浓缩液进行氯化铯密度梯度离心，得到噬菌体密度梯度液；采用20KD膜对噬菌体密度梯度液进行透析，得到噬菌体制剂。通过上述方式，能够快速、低成本、高效地去除噬菌体制剂中的内毒素。

Description

噬菌体制剂的制备方法、药物组合物以及应用 【技术领域】
本申请涉及生物技术领域，特别是涉及一种噬菌体制剂的制备方法、药物组合物以及应用。
【背景技术】
在制备噬菌体的原始液中由于细菌的裂解，含有大量的毒副产物，如内毒素、肽聚糖、细菌染色体和其他不明物质等杂质，这些杂质在进入人体后会引起机体严重的炎症反应甚至败血症性休克。这限制了噬菌体在临床的大规模应用。
本申请发明人在长期研发过程中，发现已有多种方法可去除噬菌体原始液中的杂质，在各大实验室常用的有：使用聚乙二醇富集噬菌体，随后再用氯仿去除引入的聚乙二醇；直接使用正辛醇萃取内毒素；或者使用层析(阴离子交换柱)柱纯化噬菌体等方法。但这些方法引入了有机溶剂，对人体健康是有潜在风险的，并且这些方法也存在噬菌体浓度不稳定，内毒素去除效果不佳等问题。
【发明内容】
本申请实施例提供了一种噬菌体制剂的制备方法、药物组合物以及应用，以快速、低成本、高效地去除噬菌体制剂中的内毒素。
为解决上述技术问题，本申请采用的一个技术方案是：提供一种噬菌体制剂的制备方法，该方法包括：通过切向流系统，将噬菌体混合培养液渗滤，得到噬菌体原始液；通过切向流系统，将噬菌体原始液浓缩，直到浓度为噬菌体原始液的浓度的7～10倍，得到噬菌体浓缩液；采用氯化铯溶液对噬菌体浓缩液进行氯化铯密度梯度离心，得到噬菌体密度梯度液；采用20KD膜对噬菌体密度梯度液进行透析，得到噬菌体制剂。
为解决上述技术问题，本申请采用的另一个技术方案是：提供一种药物组合物，该药物组合物包括：如前述的方法制备得到的噬菌体制剂；药学上可接受的载体、赋形剂或稀释剂中的至少一种。
为解决上述技术问题，本申请采用的又一个技术方案是：提供一种如前述的方法制备肺炎克雷伯菌噬菌体制剂、鲍曼不动杆菌噬菌体制剂、铜绿假单胞菌噬菌体制剂中的应用。
本申请的有益效果是：区别于现有技术的情况，本申请能够快速、低成本、高效地去除噬菌体制剂中的内毒素，其中，切向流系统具有高通量、可反复使用的优点，采用切向流系统过滤结合切向流系统浓缩，可以实现快速、高效、低成本的噬菌体混合培养液的渗滤和噬菌体原始液的浓缩；此外，通过氯化铯密度梯度离心结合透析去除氯化铯，可高 效、快速地获得高纯度的噬菌体制剂。
【附图说明】
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述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申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。其中：
图1为本申请噬菌体制剂的制备方法一实施例的流程示意图；
图2为图1中步骤S10的流程示意图；
图3为图1中步骤S20的流程示意图；
图4为图1中步骤S30的流程示意图；
图5为图1中步骤S40的流程示意图；
图6为本申请噬菌体制剂的制备方法另一实施例的流程示意图；
图7为本申请噬菌体制剂的制备方法又一实施例的流程示意图；
图8a是实施例3中首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎性指标TNF-α的结果；
图8b是实施例3中首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎性指标KC/GRO的结果；
图8c是实施例3中首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎性指标IL-6的结果；
图8d是实施例3中首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎性指标IL-10的结果；
图8e是实施例3中首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎性指标IL-4的检测结果；
图8f是实施例3中首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎性指标IL-2的结果；
图8g是实施例3中首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎性指标IL-1β的结果；
图8h是实施例3中首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎性指标IFN-γ的结果；
图8i是实施例3中首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎性指标IL-5的结果；
图9a是实施例3中第二剂后24小时检测小鼠血液中白细胞计数的结果；
图9b是实施例3中第二剂后24小时检测小鼠血液中红细胞计数的结果；
图9c是实施例3中第二剂后24小时检测小鼠血液中血小板计数的结果；
图9d是实施例3中第二剂后24小时检测小鼠血液中血红蛋白量的结果；
图9e是实施例3中第二剂后24小时检测小鼠血液中淋巴细胞计数的结果；
图10是实施例3中第二剂后24小时，小鼠脾脏病理切片的苏木精-伊红染色结果。
【具体实施方式】
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性的劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
本申请提供一种噬菌体制剂的制备方法，如图1所示，图1为本申请噬菌体制剂的制备方法一实施例的流程示意图。该方法包括以下步骤：
S10：通过切向流系统，将噬菌体混合培养液渗滤，得到噬菌体原始液。
S20：通过切向流系统，将噬菌体原始液浓缩，直到浓度为噬菌体原始液的浓度的7～10倍，得到噬菌体浓缩液。
S30：采用氯化铯溶液对噬菌体浓缩液进行氯化铯密度梯度离心，得到噬菌体密度梯度液。
S40：采用20KD膜对噬菌体密度梯度液进行透析，得到噬菌体制剂。
区别于现有技术的情况，本申请能够快速、低成本、高效地去除噬菌体制剂中的内毒素，其中，切向流系统具有高通量、可反复使用的优点，采用切向流系统过滤结合切向流系统浓缩，可以实现快速、高效、低成本的噬菌体混合培养液的渗滤和噬菌体原始液的浓缩；此外，通过氯化铯密度梯度离心结合透析去除氯化铯，可高效、快速地获得高纯度的噬菌体制剂。
在一实施例中，上述实施例中的切向流系统至少包括：第一储罐、进样管、回流管、滤出管、蠕动泵、膜包装置、第二储罐，蠕动泵包括滚轮，进样管夹在滚轮之间以蠕动进样管中的液体，其中，进样管的一端和回流管的一端置于第一储罐中，滤出管单独放入第二储罐内。
如图2所示，图2为图1中步骤S10的流程示意图。该步骤S10包括以下步骤：
S11：待噬菌体混合培养液中的宿主菌裂解完全，高速离心噬菌体混合培养液，得到上清液。
S12：通过切向流系统过滤上清液，其中，将第一储罐内的上清液从进样管进入膜包装置，从回流管流出的液体回流到第一储罐中，滤出液从滤出管流出至第二储罐内，得到噬菌体原始液。
S13：使用LB液体培养基梯度稀释噬菌体原始液，通过双层琼脂平板法对噬菌体原始液的噬菌体浓度进行计数。
在一实施例中，上述实施例中的切向流系统包括：第一储罐、进样管、回流管、滤出管、蠕动泵、膜包装置、第二储罐、第三储罐，蠕动泵包括滚轮，进样管夹在滚轮之间以蠕动进样管中的液体，其中，进样管的一端和回流管的一端置于第一储罐中，滤出管单独放入第二储罐内。
如图3所示，图3为图1中步骤S20的流程示意图。该步骤S20包括以下步骤：
S21：将第一储罐内的噬菌体原始液从进样管进入膜包装置，从回流管流出的液体回流到第一储罐中，第一滤出液从滤出管流出至第二储罐内，直至第一储罐内剩余预设体积量的噬菌体原始液。
S22：在第三储罐内加入指定体积量的LB液体培养基，将第三储罐内的LB液体培养基从进样管进入膜包装置，从回流管流出的液体回流到第三储罐中，第二滤出液从滤出管流出至第二储罐内，直至第三储罐内剩余第二预设体积量的噬菌体第二浓缩液。
S23：合并第一储罐内剩余的噬菌体原始液和第三储罐内剩余的噬菌体第二浓缩液，得到噬菌体浓缩液。
S24：使用LB液体培养基梯度稀释噬菌体浓缩液，通过双层琼脂平板法对噬菌体浓缩液的噬菌体浓度进行计数。
如图4所示，图4为图1中步骤S30的流程示意图。该步骤S30包括以下步骤：
S31：提供一离心管，其中，离心管内的底部为第一浓度的CsCl溶液，离心管内的中部为第二浓度的CsCl溶液，离心管内的上部为第三浓度的CsCl溶液，第一浓度大于第二浓度，第二浓度大于第三浓度。
S32：将一部分噬菌体浓缩液加入离心管，向离心管的底部依次注入第三浓度的CsCl溶液、第二浓度的CsCl溶液、第一浓度的CsCl溶液。
S33：对离心管内的各个分层进行离心处理，抽吸出噬菌体密度梯度液。
如图5所示，图5为图1中步骤S40的流程示意图。该步骤S40包括以下步骤：
S41：以噬菌体保存缓冲液为透析液，采用20KD膜对噬菌体密度梯度液进行多次透析，得到纯化后的噬菌体制剂。
S42：通过双层琼脂平板法对噬菌体制剂的噬菌体浓度进行计数。
本申请提供一种噬菌体制剂的制备方法，如图6所示，图6为本申请噬菌体制剂的制备方法另一实施例的流程示意图。
S50：复苏宿主菌。
S60：按预设比例将噬菌体加入包含宿主菌的培养基中，复苏噬菌体。
S70：扩大培养噬菌体，得到噬菌体混合培养液。
S10：通过切向流系统，将噬菌体混合培养液渗滤，得到噬菌体原始液。
S20：通过切向流系统，将噬菌体原始液浓缩，直到浓度为噬菌体原始液的浓度的7～10倍，得到噬菌体浓缩液。
S30：采用氯化铯溶液对噬菌体浓缩液进行氯化铯密度梯度离心，得到噬菌体密度梯度液。
S40：采用20KD膜对噬菌体密度梯度液进行透析，得到噬菌体制剂。
在一实施例中，上述预设比例为：噬菌体数量与宿主菌数量之间的比值为：1:10。
本申请提供一种噬菌体制剂的制备方法，如图7所示，图7为本申请噬菌体制剂的制备方法又一实施例的流程示意图。该方法包括以下步骤：
S10：通过切向流系统，将噬菌体混合培养液渗滤，得到噬菌体原始液。
S20：通过切向流系统，将噬菌体原始液浓缩，直到浓度为噬菌体原始液的浓度的7～10倍，得到噬菌体浓缩液。
S30：采用氯化铯溶液对噬菌体浓缩液进行氯化铯密度梯度离心，得到噬菌体密度梯度液。
S40：采用20KD膜对噬菌体密度梯度液进行透析，得到噬菌体制剂。
S80：使用内毒素检测鲎试剂盒检测噬菌体制剂的内毒素浓度。
本申请提供一种药物组合物，药物组合物包括：噬菌体制剂、以及药学上可接受的载体、赋形剂或稀释剂中的至少一种。
其中，噬菌体制剂为上述实施例的制备方法制备得到的噬菌体制剂。
本申请提供的方法上述实施例的制备方法制备得到的肺炎克雷伯菌噬菌体制剂、鲍曼不动杆菌噬菌体制剂、铜绿假单胞菌噬菌体制剂中的应用。
为了可以更好地理解本申请，记载以下实施例。这些实施例仅用于说明的目的，并且不应被解释为以任何方式限制本申请的范围。
实施例1
以铜绿假单胞菌噬菌体PA39为例，介绍本申请的噬菌体制剂的制备方法。
步骤1：复苏宿主菌。
具体如下：取出-80℃保存铜绿假单胞菌PAO1菌株，LB无抗平板划线，8小时后，挑单菌落到10mL LB培养基，隔夜培养(摇床37℃，摇床转速150rpm))。
步骤2：复苏噬菌体。
具体如下：取0.5mL菌液加入20mL LB培养基，培养2小时(摇床37℃，摇床转速150转/分钟(Revolutions Per minute，rpm)后，从噬菌体库中取样噬菌体PA39，加入噬菌体与宿主菌数量的比值为1：10，继续培养(摇床37℃，摇床转速150rpm)。待宿主菌裂解完全，约4小时后，高速离心混合培养液(8000rpm，10min)，取上清液通过0.22μm滤膜过滤，过滤液即时复苏后的噬菌体。使用LB液体培养基梯度对原始液进行倍比稀释至10 -8，通过双层琼脂平板法对噬菌体浓度进行计数。详细操作如下：向灭菌八连管中(最大250μL体积)加入90μL LB液体培养基，共8个稀释度，分别编号10 -1、10 -2、10 -3、10 -4、10 -5、10 -6、10 -7和10 -8。取10μL噬菌体原液，注入10 -1的管中，使用100μL移液枪反复吹打3～5次，从10 -1管中吸10μL加入10 -2管中，混匀，余类推，稀释至10-8管。准备好LB琼脂平板，熔化LB软琼脂(琼脂浓度0.75％)培养基，并在50度下保温；将铜绿假单胞杆菌PAO1的过夜培养液摇匀，取菌液0.5mL加入含有15mL的LB软琼脂培养基的试管中，轻晃混匀。将该培养基迅速倒入底层琼脂平板上，放在台面上摇匀，使上层培养基铺满平板；凝固后，取稀释好的不同浓度的噬菌体样品10μL，点于该平板上，待液体晾干后，置37℃过夜培养。观察平板中的噬菌斑，将每一稀释度的噬菌斑形成单位记录于实验报告表格内，并选取10～50个PFU数的平板计算每毫升未稀释的原液的噬菌体浓度；噬菌体浓度＝噬菌斑形成数(PFU)×稀释倍数×100，其中，噬菌体浓度为4×10 9PFU/mL。
步骤3：复苏宿主菌。
具体如下：重新挑取单菌落到100mL LB培养基，隔夜培养(摇床37℃，摇床转速150rpm)。
步骤4：2L体系扩培噬菌体。
具体如下：取50mL菌液加入2L LB培养基，培养(摇床37℃，摇床转速150rpm))2小时后，加入复苏后的噬菌体PA39，噬菌体与宿主菌数量的比值为1：10(不同噬菌体该比值可不同)，继续培养(摇床37℃，摇床转速150rpm)，得到噬菌体混合培养液。
步骤5：通过切向流系统渗滤噬菌体混合培养液。
具体如下：待宿主菌裂解完全，约4小时后，离心噬菌体混合培养液(其中，离心转速8000rpm，10min)，取上清液通过切向流系统过滤 (切向流系统的过滤膜包孔径为0.22μm)。先将进样管通过蠕动泵，随后将进样管和回流管置于储罐内的上清液中，将滤出管单独放入无菌的1L空瓶内；储罐内的上清液从进样管进入切向流系统，滤出液从切向流系统中的侧后方的滤出管流出，即为噬菌体原始液；而从切向流系统的回流管流出的液体回流到储罐内，直到储罐内的所有上清液均从滤出管滤出，过滤结束。
使用LB液体培养基梯度对噬菌体原始液进行倍比稀释至10 -8，通过双层琼脂平板法对噬菌体原始液的噬菌体浓度进行计数，其中，噬菌体浓度为4×10 9PFU/mL。
步骤6：通过切向流系统浓缩噬菌体原始液。
具体如下：将噬菌体原始液通过切向流系统(超滤膜包的孔径为100KDa)进行浓缩。先将进样管通过蠕动泵，随后将进样管和回流管置于储罐内的噬菌体原始液中，将滤出管单独放入无菌的1L空瓶内；滤出液从滤出管流出；回流管中流出的液体回流到储罐中；该系统不断的滤出液体，直到储罐内的噬菌体原始液仅剩约50mL时，暂停该切向流系统的工作。储罐内剩余的噬菌体原始液即为第一浓缩液。然后加入400mL LB液体培养基到另一储罐中，以它作为样品，再次通过切向流系统，获得约50mL的第二浓缩液。将第一浓缩液和第二浓缩液结合，最终获得约100mL噬菌体浓缩液。
使用LB液体培养基梯度对噬菌体浓缩液进行倍比稀释至10 -8，通过双层琼脂平板法对噬菌体浓缩液的噬菌体浓度进行计数，计数方法同上，其中，噬菌体浓度为3×10 10PFU/mL。
步骤7：将噬菌体浓缩液通过氯化铯密度梯度离心进行纯化。
具体如下：将20mL噬菌体浓缩液加入离心管(最大体积40mL)，离心管内的底部为第一浓度(1.7g/L)的CsCl溶液，离心管内的中部为第二浓度(1.5g/L)的CsCl溶液，离心管内的上部为第三浓度(1.3g/L)CsCl溶液。构建浓度梯度时，先加20mL噬菌体浓缩液，随后在巴斯德吸管中吸入4mL第三浓度(1.3g/L)的CsCl溶液，将其头部触碰到离心管底部，缓慢注入，可见明显的页面分层；随后在离心管底部注入4mL第二浓度(1.5g/L)的CsCl溶液，最后离心管底部注入4mL第二浓度(1.7g/L)的CsCl溶液。
步骤8：离心处理。
具体如下：使用HiMAC超速离心机离心2小时(4℃，24000rpm)。离心完成后，在不同浓度的CsCl溶液之间可见一层白褐色分层(如见多个分层，需将各个分层分别取样)，用注射器针头在噬菌体平面以下2mm左右的位置进针抽吸出所有白褐色分层，一个离心管可抽吸出约3mL的噬菌体密度梯度液。
步骤9：以噬菌体保存缓冲液为透析液，采用20KDa透析膜对噬菌 体密度梯度液进行多次透析，得到纯化后的噬菌体制剂。
具体如下：以噬菌体保存缓冲液(SM buffer)为透析液，采用20KDa透析膜对噬菌体密度梯度液进行多次透析，去除噬菌体样品中的氯化铯，每次透析使用5L透析液，每隔4小时更换一次透析液，至少透析3次，最终得到约15mL纯化后的噬菌体制剂。
使用双层琼脂平板法检测噬菌体制剂的噬菌体浓度，其中，噬菌体浓度为2×10 11PFU/mL。
步骤10：使用内毒素检测鲎试剂盒检测噬菌体制剂的内毒素浓度。
具体如下：使用内毒素检测鲎试剂盒(试管定量显色基质法，厦门鲎试剂生物科技有限公司，EC80545)检测噬菌体制剂的内毒素浓度。该检测试剂盒是利用人工合成的显色基质配套的偶氮化试剂使鲎试剂产生的玫瑰红显色反应定量检测内毒素。该试剂盒检测范围为0.1-1EU/mL。严格按照操作手册进行实验，测得上述步骤5制得的噬菌体原始液的内毒素浓度约为10000EU/mL，测得上述步骤9制得的噬菌体制剂的内毒素含量为500EU/mL。将上述步骤9制得的噬菌体制剂进行倍比稀释至噬菌体浓度为10 9PFU/mL后，测得每毫升稀释后的噬菌体制剂的内毒素含量为2.5EU。
实施例2
采用本申请噬菌体制剂的制备方法制备铜绿假单胞菌噬菌体PA39、鲍曼不动杆菌噬菌体AB6、鲍曼不动杆菌噬菌体Ab_SZ3以及肺炎克雷伯菌噬菌体KPh，其中，制作步骤可参考上述噬菌体制剂的制备方法的实施例。
检测上述实施例2中各噬菌体原始液的内毒素浓度、各噬菌体制剂的噬菌体浓度、各噬菌体制剂的内毒素浓度、以及将噬菌体制剂进行倍比稀释至噬菌体浓度为10 9PFU/mL后，每毫升稀释后的噬菌体制剂的内毒素含量(以下简称为：10 9PFU单位剂量的内毒素含量)。检测结果如下表1所示。
表1各噬菌体制剂的噬菌体浓度、内毒素浓度以及噬菌体原始液的内毒素浓度
Figure PCTCN2021097556-appb-000001
Figure PCTCN2021097556-appb-000002
美国食品药品管理局((Food and Drug Administration，FDA)规定规定内毒素入血的标准为小于5EU/Kg×h，以成人(50Kg)单次给药为例，即在1小时内可接受的内毒素含量最大为250EU，而在临床上应用时，一般推荐噬菌体的单次使用剂量为10 9PFU。如表1所示，将噬菌体制剂进行倍比稀释至噬菌体浓度为10 9PFU/mL后，每毫升稀释后的噬菌体制剂的内毒素含量均低于250EU，可直接应用于临床治疗，适用于各种应用场景包括雾化、静脉注射、肌肉注射、外敷、冲洗等。
实施例3
本申请研究了实施例1制得的铜绿假单胞菌噬菌体PA39制剂在静 脉注射时对小鼠机体的影响。
本实施例将小鼠分为四组，每组6例，分别是溶媒对照组、低剂量组(2×10 8PFU/Kg/)、中剂量组(2×10 10PFU/Kg)、高剂量组(2×10 11PFU/Kg),将不同浓度铜绿假单胞菌噬菌体PA39制剂经尾静脉注入小鼠体内，分两次给药，首剂后3天再给第二剂。
结果表明经实施例1制得的铜绿假单胞菌噬菌体PA39制剂在静脉注射到小鼠体内后不会对其机体产生明显毒副作用。详细结果如下：
如图8a～8i所示，在首剂后1小时、6小时、24小时、72小时检测小鼠血液中炎症指标。在中剂量组、高剂量组中，1小时后小鼠部分炎性指标升高，在24小时后，炎性指标基本回落到正常水平，在低剂量组中未见明显炎症指标变化。
在第二剂后24小时，采集小鼠血液，分析血常规及各项生化指标。如图9a～9e所示，显示各组各项指标均在正常范围内波动。
同时在第二剂后，解剖小鼠。低、中、高剂量组皮肤、被毛、嘴唇及口腔、心、肝、脾、肺、胸腺、肾、肾上腺、胰腺、胃肠道、睾丸和附睾、子宫和卵巢等未见肉眼形态学改变。在高剂量组中，有3例的脾脏出现相关的组织病理学改变，表现为白髓淋巴细胞增多(如图10所示)，该结果提示铜绿假单胞菌噬菌体PA39制剂可引起机体免疫反应；理论上如果后期继续观察该组，随着噬菌体的清除，脾脏可恢复正常，有待进一步实验验证。其他受检心脏、肝脏、肺、脑、十二指肠、及给药部位等组织未见明显组织病理学变化。
区别于现有技术的情况，本申请能够快速、低成本、高效地去除噬菌体制剂中的内毒素，其中，切向流系统具有高通量、可反复使用的优点，采用切向流系统过滤结合切向流系统浓缩，可以实现快速、高效、低成本的噬菌体混合培养液的渗滤和噬菌体原始液的浓缩；此外，通过氯化铯密度梯度离心结合透析去除氯化铯，可高效、快速地获得高纯度的噬菌体制剂。
以上所述仅为本申请的实施方式，并非因此限制本申请的专利范围，凡是利用本申请说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本申请的专利保护范围内。

Claims (10)

  1. 一种噬菌体制剂的制备方法，其特征在于，所述方法包括：
    通过切向流系统，将噬菌体混合培养液渗滤，得到噬菌体原始液；
    通过所述切向流系统，将所述噬菌体原始液浓缩，直到浓度为所述噬菌体原始液的浓度的7～10倍，得到噬菌体浓缩液；
    采用氯化铯溶液对所述噬菌体浓缩液进行氯化铯密度梯度离心，得到噬菌体密度梯度液；
    采用20KD膜对所述噬菌体密度梯度液进行透析，得到所述噬菌体制剂。
  2. 根据权利要求1所述的制备方法，其特征在于，所述切向流系统包括：第一储罐、进样管、回流管、滤出管、蠕动泵、膜包装置、第二储罐，所述蠕动泵包括滚轮，所述进样管夹在所述滚轮之间以蠕动所述进样管中的液体，其中，所述进样管的一端和所述回流管的一端置于所述第一储罐中，所述滤出管单独放入所述第二储罐内；
    所述通过切向流系统，将噬菌体混合培养液渗滤，得到噬菌体原始液的步骤，包括：
    待所述噬菌体混合培养液中的宿主菌裂解完全，高速离心所述噬菌体混合培养液，得到上清液；
    通过所述切向流系统过滤所述上清液，其中，将所述第一储罐内的所述上清液从所述进样管进入所述膜包装置，从所述回流管流出的液体回流到所述第一储罐中，滤出液从所述滤出管流出至所述第二储罐内，得到所述噬菌体原始液；
    使用LB液体培养基梯度稀释所述噬菌体原始液，通过双层琼脂平板法对所述噬菌体原始液的噬菌体浓度进行计数。
  3. 根据权利要求1所述的制备方法，其特征在于，所述切向流系统包括：第一储罐、进样管、回流管、滤出管、蠕动泵、膜包装置、第二储罐、第三储罐，所述蠕动泵包括滚轮，所述进样管夹在所述滚轮之间以蠕动所述进样管中的液体，其中，所述进样管的一端和所述回流管的一端置于所述第一储罐中，所述滤出管单独放入所述第二储罐内；
    所述通过切向流系统，将所述噬菌体原始液浓缩，直到浓度为所述噬菌体原始液浓度的7～10倍，得到噬菌体浓缩液的步骤，包括：
    将所述第一储罐内的所述噬菌体原始液从所述进样管进入所述膜包装置，从所述回流管流出的液体回流到所述第一储罐中，第一滤出液从所述滤出管流出至所述第二储罐内，直至所述第一储罐内剩余第一预设体积量的所述噬菌体原始液；
    在所述第三储罐内加入指定体积量的LB液体培养基，将所述第三储罐内的LB液体培养基从所述进样管进入所述膜包装置，从所述回流管流出的液体回流到所述第三储罐中，第二滤出液从所述滤出管流出至 所述第二储罐内，直至所述第三储罐内剩余第二预设体积量的噬菌体第二浓缩液；
    合并所述第一储罐内剩余的所述噬菌体原始液和所述第三储罐内剩余的所述噬菌体第二浓缩液，得到噬菌体浓缩液；
    使用LB液体培养基梯度稀释所述噬菌体浓缩液，通过双层琼脂平板法对所述噬菌体浓缩液的噬菌体浓度进行计数。
  4. 根据权利要求1所述的制备方法，其特征在于，所述采用氯化铯溶液对所述噬菌体浓缩液进行氯化铯密度梯度离心，得到噬菌体密度梯度液的步骤，包括：
    提供一离心管，其中，所述离心管内的底部为第一浓度的CsCl溶液，所述离心管内的中部为第二浓度的CsCl溶液，所述离心管内的上部为第三浓度的CsCl溶液，所述第一浓度大于所述第二浓度，所述第二浓度大于所述第三浓度，其中，将一部分所述噬菌体浓缩液加入所述离心管，向所述离心管的底部依次注入所述第三浓度的CsCl溶液、所述第二浓度的CsCl溶液、所述第一浓度的CsCl溶液；
    对所述离心管内的各个分层进行离心处理，抽吸出所述噬菌体密度梯度液。
  5. 根据权利要求1所述的制备方法，其特征在于，所述采用20KD膜对所述噬菌体密度梯度液进行透析，得到所述噬菌体制剂的步骤，包括：
    以噬菌体保存缓冲液为透析液，采用20KDa透析膜对所述噬菌体密度梯度液进行多次透析，得到纯化后的所述噬菌体制剂；
    通过双层琼脂平板法对所述噬菌体制剂的噬菌体浓度进行计数。
  6. 根据权利要求1所述的制备方法，其特征在于，所述方法包括：
    使用内毒素检测鲎试剂盒检测所述噬菌体制剂的内毒素浓度。
  7. 根据权利要求1所述的制备方法，其特征在于，所述通过切向流系统，将噬菌体混合培养液渗滤，得到噬菌体原始液的步骤之前，所述方法还包括：
    复苏宿主菌；
    按预设比例将噬菌体加入包含所述宿主菌的培养基中，复苏所述噬菌体；
    扩大培养所述噬菌体，得到所述噬菌体混合培养液。
  8. 根据权利要求7所述的制备方法，其特征在于，所述预设比例为：噬菌体数量与宿主菌数量之间的比值为：1:10。
  9. 一种药物组合物，其特征在于，所述药物组合物包括：
    如权利要求1～8任一项所述的方法制备得到的噬菌体制剂；
    药学上可接受的载体、赋形剂或稀释剂中的至少一种。
  10. 如权利要求1～8任一项所述的方法制备肺炎克雷伯菌噬菌体制 剂、鲍曼不动杆菌噬菌体制剂、铜绿假单胞菌噬菌体制剂中的应用。
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