Mycobacterium abscessus and marine mycobacterium vaccine and preparation method thereof

Abstract

The invention relates to a mycobacterium abscessus and marine mycobacterium vaccine and a preparation method thereof, the mycobacterium abscessus and marine mycobacterium vaccine comprises a recombinant plasmid based on an antigen protein gene, the recombinant plasmid is used for stably expressing cell wall antigen protein, and the recombinant plasmid is used for stably expressing cell wall antigen protein. The recombinant plasmid sequentially contains a resistance screening gene Kan, an escherichia coli replicon oriE, a promoter hsp60, an antigen protein gene MMAR1296 and a gene tag Flag-tag in the clockwise direction, wherein the DNA (deoxyribonucleic acid) sequence of the promoter hsp60 is as shown in SEQ ID No.1; the recombinant plasmid sequentially contains a resistance screening gene Kan, an escherichia coli replicon oriE, a promoter hsp60, an antigen protein gene MMAR1296 and a gene tag Flag-tag in the clockwise direction; the sequence of the antigen protein gene MMAR1296 is as shown in SEQ ID No. 2; and the DNA (Deoxyribose Nucleic Acid) sequence of the gene tag Flag-tag is as shown in SEQ ID No. 3. According to the invention, cell wall antigen protein MMAR1296 from marine mycobacterium is displayed on the surface of a mycobacterium smegmatis Msmeg vaccine carrier without any toxicity, a recombinant strain and a vaccine thereof are constructed, and subcellular component separation and western-blot experiments prove that the antigen protein MMAR1296 is successfully displayed on the surface of mycobacterium smegmatis MC2155; balb / c mouse in-vivo experiments prove that the immune effect of the recombinant mycobacterium smegmatis vaccine with the MMAR1296 antigen protein displayed on the surface is far superior to that of a bacillus calmette guerin vaccine BCG.

Description

Technical Field

[0001] The present invention relates to a technology in the field of biomedicine, specifically a vaccine of Mycobacterium abscessus and Mycobacterium marineum and a preparation method thereof. Background Art

[0002] Mycobacteria are a diverse group of bacteria characterized by different abilities to cause disease. Mycobacteria are classified into nontuberculous mycobacteria (NTM) and those that cause tuberculosis. NTM are ubiquitous, heterogeneous, and cause lung infections, disseminated infections, cervical lymphadenitis, and bone and soft tissue infections. NTM infections are increasing worldwide and have surpassed the incidence of tuberculosis in many countries. Age, bronchiectasis, exposure to aerosols, indiscriminate use of antibiotics and immunosuppressants, use of showers, and human-pathogen interactions, coupled with improved diagnostic capabilities are responsible for the increased incidence of NTM-induced diseases. The cost of treating NTM is high. Long-term treatment, widespread antibiotic resistance, recurrence of infection, and lack of a suitable vaccine have become a concern. Summary of the invention

[0003] In view of the shortcomings of the prior art in that the vaccine cannot provide protection against non-tuberculous mycobacterium infection and cannot more effectively stimulate the body to produce anti-mycobacterial inflammatory factors, the present invention proposes a vaccine for Mycobacterium abscessus and marine Mycobacterium and a preparation method thereof. The cell wall antigen protein MMAR_1296 derived from marine Mycobacterium is displayed on the surface of a non-toxic Mycobacterium smegmatis Msmeg vaccine carrier to construct a recombinant strain and a vaccine thereof. Subcellular component separation and western-blot experiments prove that the antigen protein MMAR_1296 is successfully displayed on the surface of the Mycobacterium smegmatis Msmeg vaccine carrier. 2 155 surface; in vivo experiments in Balb / c mice proved that the immune effect of the recombinant Mycobacterium smegmatis vaccine displaying MMAR_1296 antigen protein on the surface is far superior to that of the BCG vaccine.

[0004] The present invention is achieved through the following technical solutions:

[0005] The invention relates to a recombinant Mycobacterium smegmatis, which comprises a recombinant plasmid based on an antigen protein gene and is used for stably expressing a cell wall antigen protein.

[0006] The recombinant plasmid contains, in clockwise direction, a resistance screening gene Kan, an Escherichia coli replicon oriE, a promoter hsp60, an antigen protein gene MMAR_1296, and a gene tag Flag-tag, wherein: the DNA sequence of the promoter hsp60 is shown in SEQ ID No.1; the sequence of the antigen protein gene MMAR_1296 is shown in SEQ ID No.2; and the DNA sequence of the gene tag Flag-tag is shown in SEQ ID No.3.

[0007] The recombinant plasmid is transferred into Mycobacterium smegmatis MC by electroporation 2 Obtained from 155.

[0008] The antigen protein gene is specifically MMAR_1296, and its nucleotide sequence is shown in SEQ ID No.2.

[0009] The recombinant plasmid is prepared by the following method: constructing plasmid PMV261-MMAR_1296: digesting the starting plasmid PMV261 with restriction endonuclease EcoRV, and recovering a functional fragment A of about 5 kb; using the genome of marine mycobacterium as a template, amplifying a 199 bp fragment MMAR_1296 with primers MMAR_1296-F and MMAR_1296-R respectively, and connecting MMAR_1296 with homologous recombinase functional fragment A to obtain a plasmid PMV261-MMAR_1296 of about 4.7 kb.

[0010] The sequences of the primer MMAR_1296-F and the primer MMAR_1296-R are shown in SEQ ID No.4 and SEQ ID No.5 respectively.

[0011] The present invention relates to an application based on the above-mentioned recombinant Mycobacterium smegmatis, which is used for preparing a vaccine.

[0012] The preparation specifically comprises: using Mycobacterium smegmatis MC as an empty vector 2 155. The commercially available Bacillus Calmette-Guérin (BCG) vaccine made from a suspension of attenuated bovine tuberculosis bacteria and the recombinant Mycobacterium smegmatis prepared above are cultured to obtain a bacterial solution and emulsified for injection. Technical Effects

[0013] The present invention constructs a recombinant Mycobacterium smegmatis by fusion expression of the uncharacterized marine Mycobacterium membrane protein MMAR_1296 by using an episomal plasmid PMV261 containing a strong hsp60 promoter. Compared with the prior art, the recombinant Mycobacterium smegmatis prepared by the present invention can more effectively provide protection against infection with Mycobacterium abscessus and Mycobacterium marineum. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the episomal plasmid PMV261 containing the hsp60 strong promoter for fusion expression of MMAR_1296;

[0015] Figure 2 Schematic diagram of the process for constructing the fusion expression plasmid of MMAR_1296;

[0016] Figure 3 The colony of recombinant Mycobacterium smegmatis expressing MMAR_1296 and the schematic diagram of scanning electron microscopy;

[0017] Figure 4 Schematic diagram of Western blotting to verify the recombinant Mycobacterium smegmatis expressing MMAR_1296;

[0018] Figure 5 Schematic diagram of the immunization process of recombinantly expressing MMAR_1296 Mycobacterium smegmatis;

[0019] Figure 6 This is a schematic diagram of the lung bacterial load on the seventh day after mice immunized with recombinant Mycobacterium smegmatis expressing MMAR_1296 were infected with Mycobacterium abscessus;

[0020] Figure 7 This is a schematic diagram of the lung bacterial load on the tenth day after mice immunized with recombinant Mycobacterium smegmatis expressing MMAR_1296 were infected with Mycobacterium abscessus;

[0021] Figure 8 Schematic diagram of Elisa detection of IL-10 and IFN-γ in the blood of mice immunized with recombinant Mycobacterium smegmatis expressing MMAR_1296 and infected with Mycobacterium abscessus;

[0022] Fig. 9 Schematic diagram of Elisa detection of IL-10 and IFN-γ in the blood of mice immunized with recombinant Mycobacterium smegmatis expressing MMAR_1296 and infected with Mycobacterium marinum. DETAILED DESCRIPTION

[0023] The molecular biology experimental techniques used in the following examples include PCR amplification, plasmid extraction, plasmid transformation, DNA fragment ligation, enzyme digestion, gel electrophoresis, etc. Unless otherwise specified, they are usually operated according to conventional methods. For details, see "Molecular Cloning Experiment Guide" (3rd edition) (Sambrook J, Rμssell DW, Janssen K, Argentine J. Translated by Huang Peitang et al., 2002, Beijing: Science Press), or according to the conditions recommended by the manufacturer. TransTaq-T DNA Polymerase, dNTP and related reagents used in the PCR reaction in the following examples were purchased from Beijing Jinsha Biotechnology; antibiotic kanamycin was purchased from Meilun Biotechnology Co., Ltd.; Escherichia coli competent DH5a was purchased from Shanghai Qingke Biotechnology Co., Ltd.; DNA ligation reactions were all carried out using Takara Biotechnology's T4DNA ligation kit; plasmid DNA extraction kit, DNA recovery kit, and PCR purification kit were purchased from Magen (Meiji) Biotechnology Co., Ltd. The PCR primers and DNA fragments used in the following examples were synthesized by Shanghai Qingke Biotechnology Co., Ltd. Example 1

[0024] Step 1) Construction of recombinant plasmid: Figure 1 The free plasmid PMV261-MMAR_1296 shown in the figure contains the resistance screening gene Kan, the Escherichia coli replicon oriE, the promoter hsp60, the antigen protein gene MMAR_1296, and the gene tag Flag-tag in a clockwise direction, specifically: Figure 5 As shown, after the starting plasmid PMV261 was digested with the restriction endonuclease EcoRV, a functional fragment A of about 5 kb was recovered; using the genome of marine mycobacterium as a template, a 199 bp fragment MMAR_1296 was amplified using primers MMAR_1296-F and primers MMAR_1296-R, respectively, and MMAR_1296 was connected with the homologous recombinase functional fragment A to obtain a plasmid PMV261-MMAR_1296 of about 4.7 kb.

[0025] Step 2) Construction of recombinant Mycobacterium smegmatis: The recombinant plasmid constructed in step 1 is transformed into Mycobacterium smegmatis MC 2 155, and the expression of antigen protein gene MMA-1296 in the strain was verified by western-blot, and it was expressed in Mycobacterium smegmatis MC 2 The surface of 155 strains, including:

[0026] 2.1. Transformation: Plasmids PMV261-MMAR_1296 and PMV261 were transformed into Mycobacterium smegmatis MC by electroporation. 2 155 in.

[0027] 2.2. Western-blot verification of the surface display of the antigen protein gene MMAR_1296: A subcellular component separation experiment was performed on the PMV261-MMAR_1296 strain, and western-blot was performed after obtaining the cell components to verify the surface display effect of MMAR_1296.

[0028] i) Subcellular component separation: For the component separation experiment of strain MV261-MMAR_1296, see Mycobacteria Protocols (Fourth Edition) (Tanya Parish and Anuradha Kumar, 2021, Springer Nature).

[0029] ii) Western-blot expression and identification analysis: The cell components of PMV261-MMAR_1296 strain were verified by Western-blot experiment. The verification results are as follows Figure 3 shown.

[0030] Step 3) Immunogenicity test of Mycobacterium abscessus and Mycobacterium marinum vaccines: The immunogenicity of the vaccines was evaluated by animal experiments. Forty-four 6-week-old Balb / c female mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. were randomly divided into 5 groups: MC 2 155 strain group, BCG strain, MC 2 155_MMAR-1296 strain group, adjuvant group, and PBS group (negative control), and the number of animals in each group are shown in Table 1.

[0031] Table 1 Mouse grouping and immunization details

[0032] Step 4) vaccine preparation, specifically comprising:

[0033] 4.1. Vaccine preparation: Bacterial vaccine MC 2 The 155 strain, BCG, and MC2155_MMAR-1296 strain were cultured in a 50 mL conical flask containing 30 mL 7H9 medium (containing 10% OADC) in a shaking incubator at 37 °C until the OD600 was 0.6-0.8;

[0034] 4.2. Pipette 100 μL of the above three types of bacteria into a 15 mL centrifuge tube, add to 9.9 mL 7H9 medium (containing 10% OADC), centrifuge at 4000 r / min at room temperature, add 10 mL PBS to resuspend, and centrifuge at 4000 r / min; repeat washing with PBS three times; finally, resuspend each group of bacterial liquid in 0.5 mL PBS, and add an equal volume of adjuvant to emulsify for injection.

[0035] Step 5) Vaccine testing: Figure 5 As shown, subcutaneous injections were used for 3 times of immunization, with an interval of 2 weeks between each immunization, and immune protection was evaluated two weeks after the final immunization. Bacterial load evaluation and blood cytokine expression level detection were performed after eye blood sampling and tissue sampling at the specified sampling time points. Specifically, the following were performed:

[0036] CFU evaluation: After the mice were anesthetized, blood was collected from their eyeballs. After the blood was collected, the mice were euthanized and then soaked in 75% alcohol. The lungs or foot pads were taken from the clean bench and placed in a grinding tube containing PBS. Steel beads were added for grinding. After grinding at the highest speed for 5 minutes, the samples were graded and spread on 7H10 plates for colony counting. The results are as follows Figure 6 and Figure 7 shown.

[0037] Elisa evaluation: After centrifugation of blood to obtain serum, dilute it proportionally and refer to the instructions of the Elisa kit for operation. In this study, the Elisa kit of Shanghai Qincheng Biological was used for detection. The results are as follows Figure 8 and Fig. 9 shown.

[0038] Compared with the prior art, the present invention uses the free plasmid PMV261 containing the hsp60 strong promoter to fuse and express the marine mycobacterium membrane protein MMAR_1296 whose function has not been resolved to construct a recombinant Mycobacterium smegmatis. After the bacteria are prepared into a vaccine according to the above process, they can effectively provide protection against Mycobacterium abscessus and Mycobacterium marineum, and can effectively stimulate the significant production of anti-mycobacterial cytokines IL-10 and IFN-γ.

[0039] 1) The present invention is the first to express the hsp60 promoter gene, an antigen protein gene MMAR_1296 derived from marine mycobacterium and a gene tag Flag-tag by fusion. Figure 1 As shown, all three can be integrated into the mycobacterial genome for stable expression.

[0040] 2) The present invention uses Mycobacterium smegmatis as a carrier for the design of Mycobacterium abscessus and Mycobacterium marinum vaccines for the first time. It can not only induce long-term mucosal immune response and strongly stimulate macrophages to produce biological factors, but also quickly stimulate dendritic cell maturation and effectively present antigens to corresponding immune cells through the MHC presentation pathway. In addition, it grows rapidly, so it can be quickly cultured or even mass-produced; at the same time, it cannot enter the host epithelial cells, nor can it remain in the host's phagocytes, so its toxicity is relatively lower, so it is safer for the human body.

[0041] 4) The present invention is the first to display the antigen protein MMAR_1296 derived from marine mycobacterium on Mycobacterium smegmatis MC2 155 surface, respectively Figure 3 As shown;

[0042] 5) The abscess mycobacterium and marine mycobacterium vaccines prepared by the present invention are easy to culture, grow rapidly, and save time, manpower, material resources and financial resources. 2 The 155 vaccine can stably display the antigen protein MMAR_1296 on the surface of Mycobacterium smegmatis MC 2 155 surface, such as Figure 3 and Figure 4 Therefore, its immune effect in Balb / c mice is much better than that of BCG vaccine. Figures 6 to 9 shown.

[0043] The above-mentioned specific implementation can be partially adjusted in different ways by those skilled in the art without departing from the principle and purpose of the present invention. The protection scope of the present invention shall be based on the claims and shall not be limited by the above-mentioned specific implementation. Each implementation scheme within its scope shall be subject to the constraints of the present invention.

Claims

1. A recombinant Mycobacterium smegmatis, characterized in that: A recombinant plasmid containing an antigen protein gene is used to stably express the cell wall antigen protein; The recombinant plasmid contains, in clockwise direction, a resistance screening gene Kan, an Escherichia coli replicon oriE, a promoter hsp60, an antigen protein gene MMAR_1296, and a gene tag Flag-tag, wherein: the DNA sequence of the promoter hsp60 is shown in SEQ ID No.1; the sequence of the antigen protein gene MMAR_1296 is shown in SEQID No.2; and the DNA sequence of the gene tag Flag-tag is shown in SEQ ID No.

3.

2. The recombinant Mycobacterium smegmatis according to claim 1, characterized in that: The recombinant plasmid is transferred into Mycobacterium smegmatis MC by electroporation 2 Obtained from 155.

3. The recombinant Mycobacterium smegmatis according to claim 1, characterized in that: The antigen protein gene is specifically MMAR_1296, and its nucleotide sequence is shown in SEQ ID No.

2.

4. The recombinant Mycobacterium smegmatis according to claim 1, characterized in that: The recombinant plasmid is prepared by the following method: constructing plasmid PMV261-MMAR_1296: digesting the starting plasmid PMV261 with restriction endonuclease EcoRV, and recovering a functional fragment A of about 5 kb; using the genome of marine mycobacterium as a template, amplifying a 199 bp fragment MMAR_1296 with primers MMAR_1296-F and MMAR_1296-R respectively, and connecting MMAR_1296 with homologous recombinase functional fragment A to obtain a plasmid PMV261-MMAR_1296 of about 4.7 kb.

5. The recombinant Mycobacterium smegmatis according to claim 4, characterized in that: The sequences of the primer MMAR_1296-F and the primer MMAR_1296-R are shown in SEQ ID No.4 and SEQ ID No.5 respectively.

6. A use of the recombinant Mycobacterium smegmatis according to any one of claims 1 to 5, characterized in that: It is used to prepare vaccines.

7. The use according to claim 6, characterized in that: The preparation specifically comprises: using Mycobacterium smegmatis MC as an empty vector 2 155. The commercially available Bacillus Calmette-Guérin (BCG) vaccine made from a suspension of attenuated bovine tuberculosis bacteria and the recombinant Mycobacterium smegmatis prepared above are cultured to obtain a bacterial solution and emulsified for injection.

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