CN113278575A

CN113278575A - Attenuated mutant strain of mycoplasma pneumoniae and application thereof

Info

Publication number: CN113278575A
Application number: CN202110788783.4A
Authority: CN
Inventors: 李水红; 邓仲良; 蒋竹林; 周润婕
Original assignee: Nanhua University
Current assignee: Nanhua University; University of South China
Priority date: 2021-07-13
Filing date: 2021-07-13
Publication date: 2021-08-20
Anticipated expiration: 2041-07-13
Also published as: CN113278575B

Abstract

The invention discloses a mycoplasma pneumoniae attenuated mutant strain and application thereof, and relates to the technical field of microorganisms. The bacterium of the present invention is Mycoplasma pneumoniaeMycoplasima pneumonia) The M129 gene mutant strain is classified and named as Mycoplasma pneumoniae Mut129 strain, and the preservation number is CCTCC NO: PB 2021038. The invention also provides a method for preparing viable bacteria vaccine by using the mutant strain to prevent mycoplasma pneumoniae infection. The mutant strain has better immunogenicity, the prepared live vaccine has better immune protection effect, and the experimental animals have no serious adverse reaction.

Description

Attenuated mutant strain of mycoplasma pneumoniae and application thereof

Technical Field

The invention relates to a mycoplasma pneumoniae attenuated mutant strain and application thereof as a live vaccine.

Background

Mycoplasma pneumoniae (Mycoplasma pneumoniae) belongs to the class of the mollicutes, the order Mycoplasmales, the family Mycoplasmataceae, a prokaryotic cell type microorganism lacking a cell wall and capable of passing through a 0.45 μm sterile filter under pressure. In 1962, Chanock and Hayflick isolated and cultured the pathogen from the sputum of primary atypical pneumonia patients using artificial medium and named "Mycoplasma pneumoniae". Mycoplasma pneumoniae is a common pathogen of community-acquired pneumonia, mainly causes upper and lower respiratory tract infection (such as pharyngitis, bronchitis, interstitial pneumonia and the like) of children or teenagers, can develop serious lung injury (such as lung abscess, necrotizing pneumonia, acute respiratory distress syndrome and the like) which can endanger life in some cases, and can be accompanied with pulmonary vascular embolism, pleural effusion, multi-organ dysfunction, serious long-term sequelae (such as obliterative bronchiolitis, atelectasis, bronchiectasis and the like) and extrapulmonary complications (such as rash, pleuritis, meningoencephalitis, myocarditis, nephritis and the like). In addition, mycoplasma pneumoniae infection is also closely associated with the pathogenesis of bronchial asthma and atherosclerosis.

The first medicine for clinically treating mycoplasma pneumoniae infection of children or teenagers is macrolide antibiotics, but 69-95% of clinical isolates of mycoplasma pneumoniae in China have drug resistance to macrolide antibiotics, and death cases of refractory mycoplasma pneumoniae (RMPP) caused by the drug-resistant strains are increased year by year, so that the clinical treatment medicine is limited, and the morbidity of severe pneumonia and extrapulmonary complications is increased. The vaccination is the most powerful weapon for controlling infectious diseases, but the application of the potential vaccine of mycoplasma pneumoniae at home and abroad has the defects of low immune protection efficiency, short immune duration, serious adverse reaction and the like, so that no vaccine is successfully put on the market at present.

Disclosure of Invention

The purpose of the invention is as follows:

the invention aims to provide a mycoplasma pneumoniae attenuated mutant strain which can provide excellent immune stimulation capability and immune protection efficacy by inducing the organism to generate cellular immunity or humoral immunity.

The technical scheme is as follows:

the purpose of the invention is realized by adopting the following technical scheme.

The attenuated mutant strain of mycoplasma pneumoniae is named as mycoplasma pneumoniae Mut129 (Mycoplasma pneumonia) in classification, is preserved in Wuhan, Wuhan university and China center for type culture Collection, has the preservation number of CCTCC PB2021038 and the preservation time of 2021.6.22.

The specific sources of the strain are as follows: the mutant strain with attenuated virulence after inoculation of Mycoplasma pneumoniae M129 strain (ATCC 29342) deposited by the institute of pathogenic biology of southern China into PPLO liquid medium and subculture at least 80 times (about 5-7 days, subculture after turning yellow from red in the color of the medium) was subjected to genome re-sequencing and single nucleotide mutation analysis, and was named Mycoplasma pneumoniae M129 (Mycoplasma pneumoniae CCTCC PB 2021038).

The invention provides an application of the attenuated mutant strain of mycoplasma pneumoniae in constructing live vaccine of mycoplasma pneumoniae, and the preferable technical scheme is as follows: the mycoplasma pneumoniae attenuated mutant strain pure culture is combined with cyclic diguanylic acid as an adjuvant, and immunization is carried out in a nasal drip mode. The pure culture has a bacterial density of about 3.5X 108CFU/mL, a total volume of the inoculation solution of about 30. mu.L, a total amount of inoculated viable bacteria of 107CFU/mL, and a dose of cyclodiguanylic acid of about 5 ng.

Advantageous effects

The attenuated strains of the present invention have three advantageous features. First, after the attenuated strain immunization, no antigen can be detected in lung tissue, no inflammatory cell infiltration occurs, and no obvious adverse reaction occurs. Secondly, after attenuated strain immunization, the body can be induced to generate stable and durable specific IgG type antibodies (mainly IgG1 and IgG2a subclasses, and the total titer can be as high as 1: 6400) and IgA type antibodies. Thirdly, after attenuated strain immunization, the strain is detoxified again by using wild mycoplasma pneumoniae, so that the proliferation of the strain in lung tissues can be completely prevented, and the lung tissues have no obvious pathological changes. Therefore, the live vaccine prepared by the attenuated mutant strain of the mycoplasma pneumoniae has good biological safety and can provide good immune stimulation capability and virus attack protection efficacy, and the three advantageous characteristics are beneficial to the wide application of the vaccine.

Description of the drawings:

FIG. 1: colony morphology of Mycoplasma pneumoniae Mut129 strain on solid agar plate medium. (A) The method comprises the following steps Direct observation with a microscope (10 × 10); (B) the method comprises the following steps Microscopic observation (10X 10) after Dienes staining.

FIG. 2: electrophoresis gel image of p1 gene PCR amplified fragment. 1-2: mut129 strain; 3: marker; 4-6: m129 standard strain.

FIG. 3: after the mycoplasma pneumoniae Mut129 strain and the M129 strain are respectively dripped into a nose to attack a mouse, the number (A) of viable bacteria and (B) of Mp antigen in lung tissue homogenate are measured.

FIG. 4: after the mycoplasma pneumoniae Mut129 strain is dripped into a nose attacking mouse, pathological changes are observed after lung tissue sections are subjected to HE staining, wherein A is 100 Xvisual field, and B is 400 Xvisual field.

FIG. 5: indirect ELISA method for determining IgG antibody level in serum of 14d, 28d, 42d immunized mice, and enzyme-catalyzed color depth of solution (absorbance value at 450nm, A)₄₅₀) Representing the relative high or low levels of the antibody tested.

FIG. 6: after a mouse is immunized by the mycoplasma pneumoniae Mut129 strain, M129 strain is infected by virus, and the expression level of antibodies in mouse serum is increased, wherein A is IgA, B is IgG, C is IgG1, and D is IgG2 a.

FIG. 7: after a mouse is immunized by the mycoplasma pneumoniae Mut129 strain, M129 strain is infected by counteracting toxic substances, viable mycoplasma pneumoniae bacteria in homogenate of a mouse lung tissue are counted, A is PPLO solid plate counting method detection, and B is qPCR detection result.

FIG. 8: after a mouse is immunized by the mycoplasma pneumoniae Mut129 strain, the mouse is infected by M129 strain through virus challenge, and the pathological change of the lung tissue of the mouse is shown, wherein A is 100 Xvisual field, and B is 400 Xvisual field.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The present invention has various known alternatives and modifications in the technical field, and those alternatives and modifications are within the scope of the present invention without departing from the spirit of the present invention.

Preparing PPLO liquid/solid culture medium: 21g of PPLO dry powder, 4g of yeast powder and 0.04g of phenol red are weighed and fully dissolved in 700mL of double distilled water, the pH value of the culture medium is adjusted to 7.9 +/-0.02 by using 3mol/L sodium hydroxide solution, and the volume is fixed to 780mL by using the double distilled water. Sterilizing with high pressure steam at 121 deg.C for 15min, cooling to 60 deg.C, adding 160 million units of penicillin solution, 10mL of 50% sterile glucose solution and 200mL fetal calf serum, mixing, and storing in refrigerator at 4 deg.C. PPLO solid medium: 1.5% (w/v) agar was added and mixed well before the above autoclaving step, and other steps such as PPLO broth preparation were performed in the same manner.

Example 1: obtaining attenuated mutant strain of mycoplasma pneumoniae and detecting gene mutation

The M.pneumoniae Mp 129 standard strain (ATCC 29342) deposited at the institute of pathogenic biology, institute of Hengyang medical school, university of south China was inoculated into PPLO liquid medium and placed at 37 deg.C (5% CO)₂,95％N₂) Culturing in an incubator, and after the color of the culture medium changes from red to yellow (about 7 days), subculturing the culture medium into a fresh PPLO culture medium, and so on, and subculturing the obtained strain for about 80 times. The strain appeared as an oil-fried egg-like colony on PPLO solid agar plates (as shown in FIG. 1). A synthetic Mycoplasma pneumoniae p1 gene specific primer (upstream primer: GCCACCCTCGGGGGCAGTCAG; downstream primer: GAGTCGGGATTCCCCGCGGAGG) is designed for PCR amplification, and the Mycoplasma pneumoniae is identified by detecting the size (209bp) of a target gene fragment in an amplification product (shown in figure 2). By genome re-sequencing and M129 reference genome alignment analysis, it was found that: mycoplasma pneumoniae has mutations in several genes that encode adhesin-related proteins, resulting in changes in the amino acids that they encode (see tables 1 and 2). The gene of the P1 adhesion protein and the family protein thereof has synonymous mutation, heterosis mutation, frame shift mutation and non-frame shift mutation. The MgpC, P65 adhesion related protein and terminal organelle protein (topJ, hmw1-3, P200) gene have synonym mutation and heteronym mutation. We named this mutant strain Mycoplasma pneumoniae Mut 129. The strain specification preservation is carried out on the mycoplasma pneumoniae Mut129 strain, and the preservation information is as follows:

and (3) classification and naming: mycoplasma pneumoniae Mut129 strain;

latin article name: mycoplasma pneumonia;

the preservation unit: china center for type culture Collection;

address: wuhan, Wuhan university;

the preservation date is as follows: 6 months and 15 days 2021;

the preservation number is: CCTCC NO: PB 2021038.

TABLE 1 comparison of Mycoplasma pneumoniae Mut129 Strain with the wild type Strain (M129 Strain), the mutation of the gene coding for the adhesion-related protein (part)

TABLE 2 mutation status of genes encoding adhesion-related proteins (section)

Example 2: virulence assessment of Mycoplasma pneumoniae Mut129 Strain

Experimental animals: 24 SPF-grade BALB/c mice of 4-6 weeks old are randomly divided into 8 mice respectively in a normal group, a Mut129 challenge group and an M129 challenge group, and are raised for 1 week before the experiment to adapt to the environment.

Test materials: mycoplasma pneumoniae Mut129 strain was inoculated into PPLO liquid medium and incubated at 37 deg.C (5% CO)₂,95％N₂) After 1-3 days of culture recovery, subculturing for 1-2 generations, centrifuging fresh pure culture at 10000 Xg and 4 ℃ for 20min, collecting thallus precipitate, and counting viable bacteria after resuspension with PBS for later use.

And (3) testing toxicity: isoflurane anesthetizing the mice, after the mice are changed from chest to abdominal breathing, respectively sucking 30 μ L of 3.5 × 10⁸The bacterial solution of CFU/ml Mycoplasma pneumoniae Mut129 or M129 is slowly dropped into the nasal cavity of the experimental mice, and 30. mu.L PBS (pH 7.4) is dropped into the normal mice according to the same method. After feeding for 3 days, taking 50 mu L of lung homogenate, uniformly coating the lung homogenate on a PPLO solid agar plate for culturing, and observing the colony formation quantity condition of an 'oil-fried egg sample' under a microscope; meanwhile, the content of mycoplasma pneumoniae antigens in the lung homogenate of the mice is measured by using an Mp antigen ELISA kit (Shanghai Hengyuan). The whole lung tissue is taken, immersed in 12% acidic formalin (containing 5% glacial acetic acid) for fixation, embedded in paraffin, cut into 5 mu m pieces, stained with Hematoxylin and Eosin (HE), and the pathological changes of the lung tissue are observed.

The experimental results are as follows: PPLO solid Flat after nose drop test of Mycoplasma pneumoniae Mut129 and M129 strainsThe plate count results were: the number of live Mut129 challenge groups is not detected, and the number of the live M129 challenge groups is (2.67 +/-0.65) multiplied by 10⁵CFU/mL (as shown in FIG. 3A). The result of measuring the content of the Mp antigen in the lung homogenate by the ELISA method is as follows: the Mp antigen content of the Mut129 challenge group is 0.20 +/-0.02 ng/mL, the Mp antigen content of the normal group is 0.19 +/-0.02 ng/mL, and the Mp antigen content of the M129 group is 0.53 +/-0.14 ng/mL. The Mp antigen content of Mut129 and the normal group is not statistically different, but is significantly reduced compared with the M129 group (P<0.05) (as shown in fig. 3B). From the pathological section of lung tissue, it can be seen that: compared with the normal mice, the mice in the experimental group have no obvious inflammatory cell infiltration around the bronchioles/bronchi of the lung tissues, no obvious change of the alveolar septal width and no obvious edema of the blood vessels and the bronchi, which indicates that no pathological damage and inflammatory reaction of the lung are caused (as shown in figure 4). In conclusion, the mycoplasma pneumoniae Mut129 strain is very weak in toxicity, and has almost no obvious pathogenic effect on experimental mice.

Example 3: evaluation of immunogenicity of Mycoplasma pneumoniae Mut129 Strain

Experimental animals: 24 SPF-grade BALB/c mice of 4-6 weeks old are randomly divided into 6 mice respectively in a normal group, an adjuvant control group, an immune group and a combined immune group, and are raised for 1 week before the experiment to adapt to the environment.

Test materials: the same as in example 2.

The immunization method comprises the following steps: mice were anesthetized with isoflurane and immunized with Mut129 strain after the mice were observed to switch from chest to abdominal breathing. The method comprises the following steps: the normal group was titrated with 30. mu.L PBS, the adjuvant control group was titrated with 30. mu.L 5ng c-di-GMP (solvent PBS), and the immune group was titrated with 30. mu.L 3.5X 10⁸mu.L of the CFU/ml Mut129 strain was applied nasally to the combined immunization groups with 5ng c-di-GMP + 3.5X 10⁸CFU/ml Mut129 was dripped into the nose and serum was collected at

day

14, 28, and 42 after immunization for future use.

Immunogenicity testing: take 3.5X 10⁸The preparation method comprises the steps of preparing an Mp antigen (5 s of ultrasonic time, 5s of interval time and 10min of whole course time) by ultrasonic treatment of a CFU/mL mycoplasma pneumoniae Mut129 strain under an ice bath condition, and detecting the levels of specific antibodies IgG in 14d, 28d and 42d mouse serum after immunization according to an indirect ELSIA method (a kit and an operation flow thereof). The microplate reader detects the absorbance value of each hole of the microplate at 450nm (A)₄₅₀) With A₄₅₀Semi-quantitatively represents antibody levels in mouse serum. The titer of specific IgG antibody in mouse serum was the highest dilution of positive results (A)₄₅₀>0.4，A₄₅₀(non-Normal/Normal group)>2.1 positive antibody titer can be determined).

The experimental results are as follows: the anti-mycoplasma pneumoniae specific IgG antibody of the adjuvant control group is not obviously increased, the antibody levels of the combined immune group on the 28 th day and the 42 th day are respectively 2.16 +/-0.07 and 2.85 +/-0.27, and the specific IgG antibody titer on the 42 th day can reach 1: 6400. The antibody level of the combined immune group is obviously higher than that of the immune group (as shown in figure 5).

Example 4: immunoprotection evaluation of Mycoplasma pneumoniae Mut129 strain

Experimental animals: 24 SPF-grade BALB/c mice of 4-6 weeks old are randomly divided into 8 mice respectively in a normal control group, a Mut129 strain immunization group and a non-immune M129 challenge group, and are raised for 1 week before the experiment to adapt to the environment.

Test materials: the same as in example 2.

The immunization and challenge method comprises the following steps: the nasal drop of the same amount of mixed solution (5ng c-di-GMP + 3.5X 10) was administered to the combined immunization group⁸CFU/ml) Mut129 at 42 days post immunization (same immunization as example 3), 30. mu.L of 3.5X 10⁸CFU/ml M129 challenge mice, normal groups were not treated with subsequent challenge.

Immunoprotection test: after 7 days of virus challenge, blood is collected from the eyeball vein, a serum sample is separated, and the expression level of the antibody is measured. The mice are killed by dislocation of the neck, lung tissues of the mice are taken in a sterile environment to prepare homogenate, and the number of viable bacteria in the lung tissues is measured by a PPLO solid plate counting method. Extracting total DNA of the lung tissue homogenate, and detecting the number of viable mycoplasma pneumoniae bacteria by using an RT-qPCR method. CCAACCAAACAACAACGTTCA as upstream primer; ACCTTGACTGGAGGCCGTTA as downstream primer, the size of the target fragment is 76bp, and the number of viable mycoplasma pneumoniae bacteria is calculated according to a qPCR reaction standard curve. Meanwhile, lung tissue sections were prepared and HE stained (same method as in example 2), and pathological changes of lung tissue were observed.

The experimental results are as follows: the results of the serum antibody levels of the mice after challenge show that: IgA (0.69 + -0.10), IgG (2.96 + -0.20), IgG1(0.36 + -0.01) and IgG2a (0.32 + -0.013) antibodies in the immune groupThe water mean is higher than IgA (0.07 + -0.01), IgG (0.34 + -0.07), IgG1(0.18 + -0.01) and IgG2a (0.018 + -0.01) antibodies (P)<0.05) (as shown in fig. 6). PPLO solid plate counting method results show that no mycoplasma pneumoniae viable bacteria are detected in lung tissues of immunized mice, and the number of viable bacteria of unimmunized virus-counteracting groups is 0.87 multiplied by 10⁵±0.17×10⁵CFU/mL (as shown in FIG. 7A). The qPCR result shows that the number of the live bacteria in the lung tissue of the immunized mice is 546.04 +/-96.71 CFU/mL, and the number of the live bacteria in the non-immunized counteracting group is 1.08 multiplied by 10⁵±0.21×10⁵CFU/mL (as shown in FIG. 7B). Lung histology results show: compared with the non-immunized group, the lung tissues and tissues of the mice in the immunized group have obviously reduced bronchiole/peribronchial inflammatory cell infiltration, the alveolar septal width is reduced, and the edema degree of blood vessels and bronchi is reduced (as shown in figure 8). The above results confirm that: after the Mut129 strain is immunized, a good immune protection effect can be formed for the mice.

Claims

1. Attenuated Mycoplasma pneumoniae mutant strain designated by classification as Mycoplasma pneumoniae (I), (II) or (III)Mycolplasma pneumonia) The culture medium is preserved in Wuhan, Wuhan university, China center for type culture Collection, with the preservation number of CCTCC PB2021038 and the preservation time of 2021.6.22.

2. Use of an attenuated mutant strain of mycoplasma pneumoniae as claimed in claim 1, for the preparation of a live vaccine against mycoplasma pneumoniae.

3. The use according to claim 2, wherein said attenuated mutant strain of Mycoplasma pneumoniae is an attenuated mutant strain obtained by passaging M129 Mycoplasma pneumoniae in vitro a plurality of times.

4. Use according to claim 2, characterized in that the live vaccine against mycoplasma pneumoniae is a pure culture in vitro of attenuated mutants of mycoplasma pneumoniae.

5. Use according to claim 2, wherein the pure culture of attenuated mutant strains of Mycoplasma pneumoniae is administered as a live vaccine by nasal drip.

6. Use according to claim 2, characterized in that the pure culture of the attenuated mutant strain of Mycoplasma pneumoniae is used as live vaccine, the number of live bacteria inoculated is about 1X 10⁷And (4) respectively.

7. A live vaccine of Mycoplasma pneumoniae comprising as an active ingredient the attenuated mutant strain of Mycoplasma pneumoniae of claim 1.

8. A live Mycoplasma pneumoniae vaccine according to claim 7, further comprising an adjuvant.

9. A live Mycoplasma pneumoniae vaccine according to claim 8, wherein the adjuvant is cyclic diguanylic acid.