CN110724647B - Pseudomonas aeruginosa with immunoprotection effect of endotoxin protein on 7 serotype strains and application thereof - Google Patents
Pseudomonas aeruginosa with immunoprotection effect of endotoxin protein on 7 serotype strains and application thereof Download PDFInfo
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Abstract
The invention discloses pseudomonas aeruginosa capable of protecting 7 serotype strains from endotoxin protein produced by the pseudomonas aeruginosa and application of the pseudomonas aeruginosa, and belongs to the technical field of immunization. The strain is Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 with the preservation number of CGMCC NO.11268. The endotoxin protein produced by the strain has immune protection effect on 7 serotype pseudomonas aeruginosa strains. The invention also provides application of the strain and endotoxin protein produced by the strain in preparation of a medicament for preventing or treating pseudomonas aeruginosa infection, in particular application in preparation of pseudomonas aeruginosa vaccines. The invention also provides a preparation method of the endotoxin protein and a pseudomonas aeruginosa vaccine containing the endotoxin protein. The pseudomonas aeruginosa PA-017 and the endotoxin protein produced by the pseudomonas aeruginosa PA-017 can be applied to the field of immunity for preventing and treating pseudomonas aeruginosa infection.
Description
Technical Field
The invention relates to pseudomonas aeruginosa capable of producing endotoxin protein with immune protection effect on 7 serotype strains and application thereof, belonging to the technical field of immunity.
Background
Pseudomonas Aeruginosa (also known as Pseudomonas Aeruginosa) is a gram-negative bacterium belonging to the family of Pseudomonas of the gamma-proteobacteria. Pseudomonas aeruginosa is an ubiquitous microorganism capable of living in an inanimate or animate environment, and in humans it is an opportunistic pathogen that is only of value in healthy individuals to cause disease, infecting patients when the immune system is compromised, for example, patients with chronic diseases such as cystic fibrosis, cancer, post-burn acute hypoimmunity, trauma or critical illness. The use of invasive devices that disrupt physical defenses, such as urinary and vascular catheters, also increases the risk of infection with pseudomonas aeruginosa. Pseudomonas aeruginosa has become one of the important pathogenic bacteria of nosocomial infection and is also a common pathogenic bacteria of war wound infection. The pseudomonas aeruginosa can also cause septic death of livestock and the like, and causes great harm and economic loss to human health and the breeding industry.
Endotoxin protein (endotoxin protein) is a main immunogenic substance of pseudomonas aeruginosa, can stimulate an organism to generate humoral antibodies, and has the effects of stimulating the activity of reticuloendothelial system cells of the organism and enhancing the phagocytic function of macrophages. However, the current research shows that the endotoxin protein produced by the found strain has good immune protection effect on the same serotype strain, but has weak protection effect on other serotype pseudomonas aeruginosa, and because the serotypes of the pseudomonas aeruginosa are numerous and reach more than ten, the preparation of vaccines by using the endotoxin proteins of more than ten serotype strains is not only tedious and has large workload, but also has only low content of the endotoxin protein of each serotype, because the high content of the total protein can cause strong side effects of the organism, and the lowest dose for exciting the immune response of the organism is difficult to achieve. Therefore, it is of great interest to screen Pseudomonas aeruginosa for endotoxin proteins that are capable of immunoprotection against a variety of serotype strains.
Disclosure of Invention
In order to solve the problem that the endotoxin protein produced by the strain has good immune protection effect on the same serotype strain but has weak protection effect on other serotype Pseudomonas Aeruginosa, the invention provides a Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 strain with the endotoxin protein produced by the strain having immune protection effect on 7 serotype strains, and the technical scheme is as follows:
the invention aims to provide a Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 with the preservation number as follows: CGMCC NO.11268. The pseudomonas aeruginosa PA-017 strain has been preserved in 2015, 24 months and 08 months in the common microorganism center of the China microorganism strain preservation management committee of the institute of microbiology of the national academy of sciences No. 3 of the national institute of sciences, no.1 of the Xilu, beijing, the facing-yang district.
The endotoxin protein produced by Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 has immunoprotection effect on the virus attack of 7 serotypes of Pseudomonas Aeruginosa strains, and the 7 serotypes are II, IV, V, VIII, IX, X and XI respectively.
The pseudomonas aeruginosa PA-017 strain screen is selected from sputum of inpatients of a second hospital affiliated to Harbin medical university.
The pseudomonas aeruginosa PA-017 strain has the following properties:
1. colony morphology: culturing the bacterial colony on a blood agar plate culture medium at 37 ℃ for 18-24 h, and observing by naked eyes, wherein the bacterial colony is smooth and slightly raised, neat in edge, gray, moist in surface, opaque and about 1.5-2.5 mm in diameter; the microscopic examination is gram negative short rod shape.
2. Culture characteristics: culturing for 18-24 h at 37 ℃ on a blood agar plate culture medium, wherein a hemolysis ring appears; culturing at 37 ℃ in an endotoxin protein production culture medium for 18-24 h to produce green pigment, wherein the culture medium is uniformly turbid and is yellow-green.
3. Physiological and biochemical characteristics: the oxidase test is positive, the MR test and the VP test are negative, the mannitol test is negative, glucose can be decomposed to produce acid without producing gas, gelatin can be liquefied, citrate can be utilized, and hydrogen sulfide is not produced.
4. Immunological properties: the endotoxin protein produced by the pseudomonas aeruginosa PA-017 strain has good immune protection effect on serotype II, IV, V, VIII, IX, X and XI pseudomonas aeruginosa strains, and the protection rate is more than 80%.
The invention also aims to provide application of the Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017, in particular to application of the Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 in preparing a medicament for preventing or treating Pseudomonas Aeruginosa infection; application of Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 in preparing Pseudomonas Aeruginosa vaccines.
Another objective of the invention is to provide an endotoxin protein produced by Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017.
The invention also aims to provide application of endotoxin protein produced by Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017, in particular to application of endotoxin protein produced by Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 in preparing a medicament for preventing or treating Pseudomonas Aeruginosa infection; application of endotoxin protein produced by Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 in preparing Pseudomonas Aeruginosa vaccines.
Another objective of the invention is to provide a preparation method of endotoxin protein produced by Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017:
(1) Inoculating Pseudomonas aeruginosa PA-017 strain to agar slant culture medium, culturing at 37 deg.C for 18 hr, washing, inoculating to endotoxin protein production culture medium, with the inoculation amount of 3.0 × 10 8 Performing shake culture at 37 deg.C and 175 rpm for 22 hr in CFU/mL to obtain culture;
(2) Adjusting pH of the culture obtained in step (1) to 8.5, autolyzing at 37 deg.C in toluene heavy layer for 48 hr, adjusting pH of the autolysate to 7.2 after complete autolysis under dark field microscope, vacuum filtering to obtain brown transparent liquid, and adjusting ZnCl content to 50% 2 Precipitating the protein, centrifuging at 4 deg.C, removing supernatant, precipitating with 20% Na 2 HPO 4 ·7H 2 Dissolving the O solution, standing at 37 ℃ for 2h, and storing in a refrigerator at 4 ℃ overnight;
(3) Centrifuging at 4 ℃, collecting supernatant, correcting the pH to 7.0-7.2, dialyzing for 48 hours by using running water of a dialysis bag with the molecular weight cutoff of 8000-14000, and blowing and concentrating by using an electric fan until the volume is 50-80 mL;
(4) Correcting the pH value of the concentrated solution to 7.0-7.2, and according to the volume of the concentrated solution: cold acetone volume =1:2, precipitating the concentrated solution by using cold acetone,centrifuging at 4 deg.C, removing supernatant, precipitating with ddH 2 Dissolving O, concentrating, and vacuum freeze drying to obtain endotoxin protein.
Another objective of the invention is to provide a Pseudomonas Aeruginosa vaccine containing endotoxin protein produced by Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017.
The invention has the beneficial effects that:
the invention provides a Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 strain, endotoxin protein produced by the strain has good immune protection effect on serotype II, IV, V, VIII, IX, X and XI Pseudomonas Aeruginosa strains, and the protection rate is more than 80%.
The endotoxin protein produced by using the pseudomonas aeruginosa PA-017 strain provided by the invention can be applied to preparing medicines for preventing and treating pseudomonas aeruginosa infection.
Drawings
FIG. 1 is a colony morphology of PA-017 strain (blood agar plate medium).
FIG. 2 is a gram-stained microscopic image of PA-017 strain.
FIG. 3 is a graph showing the production of green pigment by PA-017 strain in the production medium of endotoxin protein.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified. The media involved in the following examples are composed as follows:
1. blood agar plate medium:
per 1000mL of medium contained: 10.0g of pancreatic casein digest, 3.0g of pancreatic heart digest, 1.0g of corn starch, 14.0g of agar, 5.0g of gastric and meat enzyme digest, 5.0g of yeast extract powder, 5.0g of sodium chloride, 1000mL of distilled water and 60mL of aseptic defibrinated goat blood, and the pH value is corrected to 7.2-7.4.
2. Nutrient agar slant culture medium:
per 1000mL of medium contained: 10.0g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride and 15.0g of agar, and the final pH value is 7.3 +/-0.2.
3. General broth culture:
per 1000mL of medium contained: 10.0g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride, and the final pH value is 7.4 +/-0.2.
4. Endotoxin protein production medium: 20.0g of sodium glutamate, 7.5g of glucose 4 ·7H 2 O 0.2g，Ca(NO 3 ) 2 0.01g，FeSO 4 ·7H 2 O 0.00005g，KH 2 PO 4 0.252g，Na 2 HPO 4 ·12H 2 O5.63 g, dissolved in 1000mL of distilled water, pH adjusted to 7.2-7.4, and autoclaved at 115 ℃ for 25 min.
Example 1: isolation and identification of Pseudomonas aeruginosa PA-017 strain
1. Isolation of Pseudomonas aeruginosa PA-017 Strain:
(1) The strain isolation sample was sputum of an inpatient collected from the secondary hospital affiliated to the Harbin medical university.
(2) Adding appropriate amount of sterile double distilled water into the sputum sample, mixing, sucking 30uL with sterile sample gun, uniformly spreading, inoculating into blood agar plate culture medium, and culturing at 37 deg.C for 24 hr. Typical single colonies with large lysosomes on the plate were picked with an inoculating loop, streaked on a blood agar plate medium, and incubated at 37 ℃ for 22h. Purifying the blood agar plate culture medium for 2 generations to obtain the pseudomonas aeruginosa strain, naming the strain as PA-017, preserving in nutrient agar slant culture medium and identifying.
2. Identification of Pseudomonas aeruginosa PA-017 strain:
(1) And (3) colony morphology characteristics:
PA-017 strain is cultured on blood agar plate culture medium at 37 deg.c for 18-24 hr, and its thallus morphology is shown in figure 1, and its colony is smooth, slightly raised, neat in edge, gray, moist in surface, opaque, about 1.5-2.5 mm in diameter and hemolytic.
The result of microscopic examination of the strain PA-017 is shown in FIG. 2, and the microscopic examination is gram-negative short rod-shaped.
The PA-017 strain is inoculated into an endotoxin protein production culture medium and fermented for 18-24 h at 37 ℃ to produce green pigment, and the culture medium is uniformly turbid and is in yellow green, as shown in figure 3.
(2) Biochemical identification: the PA-017 strain has positive oxidase test, negative MR test and VP test, negative mannitol test, capability of decomposing glucose, producing acid and no gas, capability of liquefying gelatin and utilizing citrate, and no hydrogen sulfide, so the PA-017 strain is identified and proved to be pseudomonas aeruginosa as shown in Table 1.
TABLE 1 major biochemical characteristics of the PA-017 strain
Note: "+": positive; "-": and (4) negative.
Example 2: test for preparing endotoxin protein by PA-017 strain fermentation
1. Preparing a production strain:
and (3) aseptically opening a PA-017 strain freeze-drying tube, inoculating into a common broth culture medium, and standing and culturing for 22h at 37 ℃. Sucking 30uL with a sterile sample-adding gun, uniformly spreading and inoculating into a blood agar plate culture medium, and culturing at 37 ℃ for 24h. Typical single colonies with large lysosomes on the plate were picked with an inoculating loop, streaked on a blood agar plate, and incubated at 37 ℃ for 22h. Typical single colonies with large hemolysis circle on the plate are selected to be inoculated on nutrient agar slant culture medium and cultured for 24h at 37 ℃. Microscopic examination to determine pseudomonas aeruginosa.
2. Preparing endotoxin protein:
(1) Taking PA-017 strain, inoculating on common agar slant culture medium, culturing at 37 deg.C for 18h, washing with endotoxin protein production culture medium, inoculating into triangular flask containing 800-1000 mL endotoxin protein production culture medium, with inoculation amount of 3.0 × 10 8 CFU/mL (bacterioturbidimeter TA-2XJ, beijing Tianan Co., ltd.), and shake-culturing at 37 ℃ for 22 hours by a rotary shaker (175 rpm).
(2) The cultures were pH adjusted to 8.5 and autolysed in a toluene layer at 37 ℃ for 48h. After completion of autolysis by dark field microscopy, the autolysate was adjusted to pH 7.2, suction filtered through a G5 sand-core funnel to give a brown transparent liquid, protein was precipitated with 50% ZnCl2 (3.2mL 50% ZnCl2/100mL of the filtrate), centrifuged at 4000rpm for 20min at 4 ℃ and the supernatant was discarded. The precipitate was dissolved in an appropriate amount of 20% Na2HPO4.7H2O solution, allowed to stand at 37 ℃ for 2 hours, and stored in a refrigerator at 4 ℃ overnight.
(3) Centrifuging at the temperature of 4 ℃ and the rpm of 4000 for 20min, collecting supernatant, correcting the pH to 7.0-7.2, putting into a dialysis bag (molecular weight cut-off: 8000-14000) for running water dialysis for 48h, and blowing and concentrating by an electric fan to the volume of 50-80 mL.
(4) Adjusting the pH of the concentrated solution to 7.0-7.2, precipitating with cold acetone (the volume of the concentrated solution: the volume of the cold acetone =1: 2), centrifuging at 4 ℃ and 4000rpm for 20min, discarding the supernatant, dissolving the precipitate with a small amount of ddH2O, blowing with an electric fan for concentration, and performing vacuum freeze drying to obtain the endotoxin protein.
Example 3: endotoxin protein immunoprotection assay
1. Preparing a virus attacking strain:
pseudomonas aeruginosa PA-393 strain (serotype I), PA-251 strain (serotype II), PA-526 strain (serotype III), PA-182 strain (serotype IV), PA-109 strain (serotype V), PA-189 strain (serotype VI), PA-894 strain (serotype VII), PA-1537 strain (serotype VIII), PA-025 strain (serotype IX), PA-261 strain (serotype X) and PA-1206 strain (serotype XI) are provided for the professional laboratory of Pseudomonas aeruginosa in the China center for medical bacterial conservation management.
The freeze-drying tube of each strain is aseptically opened, inoculated into a common broth culture medium, and subjected to static culture at 37 ℃ for 22h. Sucking 30uL with a sterile sample-adding gun, uniformly spreading and inoculating into a blood agar plate culture medium, and culturing at 37 ℃ for 24h. Typical single colonies with large lysosomes on the plate were picked with an inoculating loop, streaked on a blood agar plate, and incubated at 37 ℃ for 22h. Typical single colony with large hemolytic ring on the plate is selected to be inoculated on nutrient agar slant culture medium and cultured for 24h at 37 ℃. Microscopic examination to determine pseudomonas aeruginosa.
2. Determination of minimum lethal dose of challenge strain:
aseptic operation, culturing the strain at 37 deg.C for 12-16 hThe nutrient agar culture is washed with sodium chloride injection to obtain thallus, diluted, mixed, diluted with bacteria turbidimeter to obtain 4.0 × 10 thallus 8 /mL、3.6×10 8 /mL、3.2×10 8 /mL、2.8×10 8 /mL、2.4×10 8 /mL、2.0×10 8 /mL、1.6×10 8 /mL、1.2×10 8 /mL、8.0×10 7 /mL、4.0×10 7 /mL、2.0×10 7 PermL of the same concentration of bacterial suspension, 5 mice with the weight of 14-16 g were intraperitoneally injected with each dilution of bacterial suspension, each mouse was injected with 0.5mL, and 3 days of observation were observed, so that the minimum dose of all the mice died within 3 days after infection was 1 Minimum Lethal Dose (MLD). When the above-mentioned challenge dose did not die completely or all the dose groups died completely, the minimum challenge dose for killing all the mice was searched upward or downward again and refined. MLD was measured for the above 11 serotype strains as shown in Table 2.
TABLE 2MLD assay results for 11 serotypes of Pseudomonas aeruginosa
3. And (3) immunity test:
(1) Clean-grade mice weighing 14-16 g were selected and divided into 11 groups of 32 mice each. Immunizing the 11 groups of mice with endotoxin protein for 3 times, and performing first immunization by injecting 40 mu g of endotoxin protein into each mouse subcutaneously; two immunizations were performed at 3-day intervals, and each mouse was injected subcutaneously with 80 μ g; three immunizations were performed 3 days apart and each mouse was injected subcutaneously with 120 μ g.
(2) On day 7 after the end of the third immunization, the 11 immunization groups were each injected with 1MLD of fresh bacterial suspension into the abdominal cavity of each mouse, and the 1MLD of virulent bacteria was contained in 0.5 mL.
(3) Simultaneously, 165 clean-grade mice which are fed in the same batch and have the same weight as the immune group are used as controls, the mice are divided into 11 groups, each group is 15, each group is divided into 3 groups of 2MLD, 1MLD and 1/2MLD, and each group is provided with 5 mice; fresh suspensions of the 11 serotypes (contained in 0.5 mL) were intraperitoneally injected with 2MLD, 1MLD and 1/2MLD, respectively, and observed for 3 days, and the results were determined: and (3) the control group mice infected with 2MLD and 1MLD should die completely, the 1/2MLD infected individuals should die partially, and the protection rate of the mice in the immune group is calculated.
TABLE 3 endotoxin protein test results on the immunity of 11 serotype P.aeruginosa strains
The result shows that the endotoxin protein prepared by the pseudomonas aeruginosa PA-017 strain has good immune protection effect on serotype II, IV, V, VIII, IX, X and XI pseudomonas aeruginosa strains, and the protection rate is over 80 percent, as shown in the table 3. Can show endotoxin protein produced by the PA-017 strain and can be applied to preparing pseudomonas aeruginosa vaccines.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (2)
1. Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 with the preservation number as follows: CGMCC NO.11268.
2. A method for producing endotoxin protein produced by Pseudomonas Aeruginosa (Pseudomonas Aeruginosa) PA-017 as claimed in claim 1, wherein the method comprises:
(1) Taking pseudomonas aeruginosa PA-017 strain, inoculating on agar slant culture medium, culturing at 37 deg.C for 18h, washing the strain, inoculating to endotoxin protein production culture medium, wherein the inoculation amount is 3.0 × 10 8 Performing shake culture at 37 deg.C and 175 rpm for 22 hr in CFU/mL to obtain culture;
(2) Adjusting the pH of the culture obtained in the step (1) to 8.5, carrying out autolysis on a toluene heavy layer at 37 ℃ for 48h, and carrying out autolysis by dark field microscopyAfter completion, the pH of the autolysate is corrected to 7.2, the clear brown liquid is filtered off with suction and 50% ZnCl are used 2 Precipitating protein, centrifuging at 4 deg.C, removing supernatant, and precipitating with 20% Na 2 HPO 4 ·7H 2 Dissolving the O solution, standing at 37 ℃ for 2h, and storing in a refrigerator at 4 ℃ overnight;
(3) Centrifuging at 4 ℃, collecting supernatant, correcting the pH to 7.0-7.2, dialyzing for 48 hours by using running water of a dialysis bag with the molecular weight cutoff of 8000-14000, and blowing and concentrating by using an electric fan until the volume is 50-80 mL;
(4) Correcting the pH value of the concentrated solution to 7.0-7.2, and according to the volume of the concentrated solution: cold acetone volume =1:2 precipitating the concentrated solution with cold acetone, centrifuging at 4 deg.C, removing supernatant, and precipitating with ddH 2 Dissolving O, concentrating, and vacuum freeze drying to obtain endotoxin protein.
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|---|---|---|---|---|
| US4157389A (en) * | 1976-02-05 | 1979-06-05 | The Kitasato Institute | Mixed vaccine against infections by pseudomonas aeruginosa |
| WO1994028928A1 (en) * | 1993-06-07 | 1994-12-22 | Cheil Foods & Chemicals Inc. | Novel attenuated pseudomonas aeruginosa strains |
| CN101406697A (en) * | 2008-11-21 | 2009-04-15 | 黑龙江省科学院微生物研究所 | Bacillus pyocyaneus vaccine and preparation method thereof |
| CN105749266A (en) * | 2016-04-26 | 2016-07-13 | 齐鲁动物保健品有限公司 | Mink hemorrhagic pneumonia and botulism combined inactivate vaccine and preparing method thereof |
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2018
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4157389A (en) * | 1976-02-05 | 1979-06-05 | The Kitasato Institute | Mixed vaccine against infections by pseudomonas aeruginosa |
| WO1994028928A1 (en) * | 1993-06-07 | 1994-12-22 | Cheil Foods & Chemicals Inc. | Novel attenuated pseudomonas aeruginosa strains |
| CN101406697A (en) * | 2008-11-21 | 2009-04-15 | 黑龙江省科学院微生物研究所 | Bacillus pyocyaneus vaccine and preparation method thereof |
| CN105749266A (en) * | 2016-04-26 | 2016-07-13 | 齐鲁动物保健品有限公司 | Mink hemorrhagic pneumonia and botulism combined inactivate vaccine and preparing method thereof |
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| 铜绿假单胞菌内毒素蛋白的提取及其临床应用;张桂勤等;《临床检验杂志》;19931231;第11卷(第2期);第一段、3 结果部分 * |
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