K4 subcluster mycobacterium bacteriophage, separation method and application of K4 subcluster mycobacterium bacteriophage

Abstract

The invention discloses a K4 subcluster mycobacterium bacteriophage, a separation method and application thereof, Msm is used as a host to separate and purify a new bacteriophage K4SD6 from soil, the genome size of the new bacteriophage K4SD6 is 59.028 kb, the bacteriophage shows good lysis activity on mycobacterium tuberculosis H37Ra, and a mouse infection model verifies that the new bacteriophage K4SD6 has a good anti-tumor activity on mycobacterium tuberculosis H37Ra. The K4SD6 bacteriophage has an extremely strong capability of removing H37Ra in a mouse body, and the K4SD6 bacteriophage can be used for efficiently removing the H37Ra infected in the mouse body.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of mycobacterium tuberculosis lysis, and particularly relates to a K4 sub-cluster mycobacteriophage, a separation method and application thereof. BACKGROUND

[0002] Phages are viruses that specifically infect bacteria and have high bactericidal activity. As the most abundant microorganism on earth, it has been successfully applied to the clinical treatment of pathogenic bacterial infections and has shown good efficacy. In particular, with the emergence of antibiotic-resistant bacteria in recent years, phage therapy is currently considered a new solution for the treatment of drug-resistant pathogenic bacterial infections and has broad application prospects. For example, in 2018, the Shanghai Public Health Center applied phage therapy to successfully cure a urinary tract infection caused by multi-drug resistant Klebsiella pneumoniae. In 2021, the Huang Wei team carried out a clinical trial of phage therapy for the treatment of multi-drug resistant Acinetobacter baumannii infection, successfully eliminating drug-resistant bacteria from the patient's lung infection. In the same year, Dedrick et al. used three engineered mycobacteriophages to make a cocktail preparation, successfully curing a cystic fibrosis patient with disseminated drug-resistant Mycobacterium abscessus infection. In 2022, Nick et al. also cured a cystic fibrosis patient caused by drug-resistant Mycobacterium abscessus through two engineered phages.

[0003] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis infection, which has long been a threat to human health. In particular, with the spread of drug-resistant tuberculosis around the world, the prevention and control of tuberculosis is facing severe challenges, and there is an urgent need to develop new strategies against tuberculosis. Mycobacteriophages are a class of viruses that specifically infect Mycobacterium and can effectively treat drug-resistant Mycobacterium abscessus-induced lung infections and show good clinical efficacy. Moreover, phages have made good progress in the treatment of tuberculosis. Studies have found that some A, G, K, and L sub-cluster phages can effectively infect Mycobacterium tuberculosis. In addition, some phages have been proven to have good bactericidal activity against Mycobacterium tuberculosis infection in animals. Therefore, the isolation and identification of phages that can infect Mycobacterium tuberculosis will be expected to provide tool resources for the treatment of tuberculosis.

[0004] The prior art has proved that some mycobacteriophages can effectively infect Mycobacterium tuberculosis, and there are a few cases that prove the bactericidal effect of the phage on Mycobacterium tuberculosis in animal infection models. For example, A cluster phages DS6A and D29 are found to be able to significantly reduce the survival of Mycobacterium tuberculosis in guinea pigs. Although phages have achieved good effects in many pathogenic bacterial infections, there are few treatments for tuberculosis, mainly due to the following reasons: first, there are few phages that can infect Mycobacterium tuberculosis, and foreign countries almost have a monopoly. Second, most of the Mycobacterium tuberculosis infecting phages are lysogenic, and the lysis ability is poor. Finally, the bactericidal effect of these phages in animals needs to be verified, which greatly restricts the clinical application of Mycobacterium tuberculosis phage. SUMMARY

[0005] The purpose of the present application is to provide a K4 sub-cluster mycobacteriophage, a separation method and its application, using Mycobacterium smegmatis Msm as a host to isolate and purify a new phage K4SD6 from soil, and the genome size of the phage is 59.028kb. The phage shows good lysis activity to Mycobacterium tuberculosis H37Ra, and the K4SD6 phage can efficiently eliminate the H37Ra infection in mice through the mouse infection model verification.

[0006] The present application is realized by the following technical solutions: A separation method of a K4 sub-cluster mycobacteriophage, comprising the following steps: S1: obtaining a phage suspension by enriching phages in soil with host bacteria, and culturing and treating; S2: culturing and purifying the phage suspension with host bacteria to obtain a phage solution; S3: extracting the purified phage for detection.

[0007] Preferably, in steps S1 and S2, the host bacteria is Mycobacterium smegmatis strain.

[0008] Preferably, in step S1, the host strain is added to the soil sample, and centrifuged and cultured at 160rpm and 37℃ for 24h.

[0009] Preferably, in step S1, the host bacteria after culture is centrifuged at 9000rpm, and the supernatant is obtained, and the supernatant is filtered through a filter membrane to obtain a phage suspension.

[0010] Preferably, in step S2, the phage suspension and the host bacteria are mixed in a proper ratio, and then added to 5mL LB upper culture medium and mixed, and then poured into LB lower culture medium, and cultured for 24h.

[0011] Preferably, in step S2, the phage suspension is soaked in a biological buffer for 24 hours to obtain a phage solution.

[0012] Preferably, in step S3, the phage suspension is diluted, and the diluted phage suspension is mixed with 1 mL of host bacteria and cultured by a double-layer plate method to obtain a single phage plaque.

[0013] Preferably, in step S3, the single phage plaque is repeatedly picked and purified to obtain phage plaques with consistent morphology and transparency, the phage plaques are enriched, and the filtered enrichment liquid is stored at 4 DEG C for extraction of a genome and detection of a sequence.

[0014] An application of a K4 sub-cluster mycobacteriophage, which is applied to lyse a live attenuated strain of Mycobacterium tuberculosis.

[0015] Compared with the prior art, the present application has the following advantages and beneficial effects: In the present application, a new phage K4SD6 is isolated and purified from soil by using Msm as a host, the genome size of the phage is 59.028 kb, the phage exhibits good lytic activity on Mycobacterium tuberculosis H37Ra, and the K4SD6 phage is verified to have a strong clearing capacity on H37Ra in a mouse infection model. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings in the embodiments will be briefly introduced as follows, and it should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and other related drawings can also be obtained by those skilled in the art without creative labor on the basis of the drawings.

[0017] Figure 1 A phage plaque morphology diagram of the K4SD6 phage on a plate containing Msm bacteria in the present application.

[0018] Figure 2 A lytic capacity titer experiment analysis diagram of the K4SD6 phage on Msm and H37Ra in the present application.

[0019] Figure 3 A columnar diagram of the K4SD6 phage on H37Ra clearing results in a mouse in the present application. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application, and obviously, the described embodiments are some embodiments of the present application, but not all the embodiments.

[0021] Embodiment 1 A K4 sub-cluster mycobacteriophage, as shown in Figure 1 and Figure 2 Phage K4SD6 belonging to K4 sub-cluster was isolated from soil, preferably soil from Shandong, as shown in Figure 1 It can be seen that slightly turbid plaques are formed on the plate containing Msm bacterial lawn. Figure 2 In the embodiment, K4SD6 phage of different dilution gradients are analyzed by proliferation on plates containing Msm and Mra bacterial lawns, Msm represents Mycobacterium smegmatis, and Mra represents Mycobacterium tuberculosis H37Ra, and the results show that the lysis ability of K4SD6 phage to Mycobacterium tuberculosis H37Ra is very obvious; and in a mouse infection model, K4SD6 phage shows very strong clearing ability to H37Ra infected in mice; in addition, the genome size is 59.028 kb, which can be used as a chassis phage.

[0022] Embodiment 2 A method for isolating a K4 sub-cluster mycobacteriophage, comprising the following steps: S1: obtaining a phage suspension by enriching phages in soil through host bacteria, and culturing and treating; S2: adding host bacteria to the phage suspension for culture and purification to obtain a phage solution; S3: extracting the purified phage for detection sequence.

[0023] 10g of soil in Shandong area is taken, the surface layer of about 1cm soil sample is thrown away, then 10mL of Msm strain is added, and cultured at 160rpm and 37℃ constant temperature for 24h; then centrifuged at 9000rpm for 20min, 1mL of supernatant is taken and filtered with 0.22μm filter membrane to obtain a phage suspension; 0.5mL of phage suspension is mixed with 1mL of Msm strain, the mixture is added to 5mL of LB upper culture medium, quickly mixed and poured on LB lower culture medium, and cultured at 37℃ for 24h; plaques with different morphologies and transparency degrees are picked and placed in 0.1mL of MP buffer, filtered and added with 1mL of Msm strain for double-layer agar plate culture, and cultured at 37℃ for 24h; finally, 5mL of MP buffer is added for soaking for 12h, and filtered to obtain a phage solution.

[0024] During extraction and purification, the phage suspension was diluted, the diluted phage suspension was mixed with 1 mL of Msm strain and cultured by double-layer agar plate method to obtain single phage plaque; the plaque picking and purification were repeated for 3 times to obtain phage plaques with similar morphology and transparency for enrichment, and the enrichment liquid was filtered and stored at 4°C. The K4SD6 phage genome after extraction and purification was sent to a detection department for genome sequencing.

[0025] Example 3 A use of a high-efficiency mycobacterium tuberculosis phage for lysing H37Ra.

[0026] As shown in Figure 3 , the phage K4SD6 was verified for its ability to clear H37Ra in mice by a mouse infection experiment: first, the H37Ra strain was grown to OD600=0.8, and the bacteria were collected; then the bacteria were resuspended after being washed once with PBS to make a bacterial solution, and the concentration of the bacterial solution was

(1x10 7 CFU) / (40 μL)

[0027] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification or equivalent change made on the basis of the technical essence of the present application to the above embodiment falls within the protection scope of the present application.

Claims

1. A method for isolating a K4 sub-cluster mycobacteriophage, characterized in that, It comprises the following steps: S1: obtaining bacteriophage suspension by enriching bacteriophage in soil with host bacteria, and culturing and treating; S2: adding host bacteria to the bacteriophage suspension to culture and purify to obtain bacteriophage liquid; S3: extracting the purified bacteriophage for detection sequence.

2. The method of claim 1, wherein the K4 sub-cluster mycobacteriophage is isolated from a sample of a mycobacterium. In steps S1 and S2, the host bacteria is Mycobacterium smegmatis strain.

3. The method of claim 1, wherein the K4 sub-cluster mycobacteriophage is isolated from a sample of a mycobacterium. In step S1, the host strain is added to the soil sample, centrifuged at 160 rpm and 37℃, and cultured for 24h.

4. The method of claim 3, wherein the K4 sub-cluster mycobacteriophage is isolated from a sample of a patient with a mycobacterial infection. 5 In step S1, the host bacteria after culture is centrifuged at 9000 rpm and the supernatant is taken, and the supernatant is filtered through a filter membrane to obtain bacteriophage suspension.

5. The method of claim 1, wherein the K4 sub-cluster mycobacteriophage is isolated from a sample of a patient with a mycobacterial infection. 5 In step S2, the bacteriophage suspension and host bacteria are mixed in appropriate proportions, then added to 5mL LB upper culture medium, mixed and poured into LB lower culture medium, and cultured for 24h.

6. The method of claim 5, wherein the K4 sub-cluster mycobacteriophage is isolated from a sample of a patient with a mycobacterial infection. In step S2, the bacteriophage suspension is soaked in biological buffer for 24h to obtain bacteriophage liquid.

7. The method for isolating K4 subclusteric mycobacterial phage as described in claim 1, characterized in that, In step S3, the bacteriophage suspension is diluted, and the diluted bacteriophage suspension is mixed with 1mL host bacteria and cultured by double-layer plate method to obtain single bacteriophage plaque.

8. The method for isolating K4 subclusteric mycobacterial phage as described in claim 7, characterized in that, In step S3, the single bacteriophage plaque is repeatedly picked and purified multiple times to obtain bacteriophage plaques with consistent morphology and transparency, the bacteriophage plaques are enriched, the filtered enrichment liquid is stored at 4℃, and used for extraction of genome and detection sequence.

9. Use of a K4 sub-cluster mycobacteriophage, characterized in that, It is applied to lyse Mycobacterium tuberculosis attenuated strain.

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