Novel microecological preparation composition for preventing and treating feline plague and application of novel microecological preparation composition

Abstract

The invention discloses a novel microecological preparation composition for preventing and treating feline plague and application of the novel microecological preparation composition, and belongs to the technical field of microecological preparations. The recombinant lactobacillus plantarum NC8-VP2 constructed by a genetic engineering means can efficiently express the feline plague VP2 protein, and finally, a protective antigen expressed by the recombinant lactobacillus plantarum efficiently stimulates an organism to generate a specific antibody, so that the feline plague is prevented, and the use of chemical drugs is reduced. Recombinant escherichia coli BL21-pET32a-MLT constructed by the research can secrete and express melittin (MLT), large-scale production of melittin is realized, melittin and recombinant lactobacillus plantarum NC8-VP2 are further taken as active ingredients, a novel microecological preparation composition for preventing and controlling feline plague is developed and prepared, the morbidity of feline plague can be reduced, clinical symptoms of sick animals can be improved, and the novel microecological preparation composition has a good application prospect. Therefore, the compound has a relatively good application prospect in the aspect of preventing and treating feline plague.

Description

Technical Field

[0001] The present invention belongs to the technical field of microecological agents, and particularly relates to a novel microecological agent composition for preventing and treating feline panleukopenia and its application. Background Art

[0002] Feline panleukopenia (FP), also known as feline panleukopenia, is an acute, highly contagious, and highly lethal infectious disease caused by feline parvovirus (FPV). Cats of all ages can be infected, with an infection rate of up to 70% and a fatality rate of 50%-60%. FP has been defined as one of the most serious infectious diseases in cats in many countries in Europe and the Americas. Under natural conditions, FPV can also infect rare wild animals such as foxes, leopards, lions, tigers, and raccoons, posing a great threat to wildlife protection. Its prevention and control are the difficulties and key points of current research.

[0003] At present, the prevention and treatment of FP mainly involve vaccination. Timely vaccination with effective live attenuated vaccines or inactivated vaccines can provide certain immunity for animals. However, the protective antigen protein of the FPV inactivated vaccine is prone to denaturation during the preparation process, resulting in unsatisfactory immune effects; although the live attenuated vaccine can effectively induce specific immune responses in the body, there is a potential risk of reversion of virulence; antibody therapy mainly relies on a large amount of injection of antiviral monoclonal antibodies, etc., but most of them are murine-derived, which are prone to induce allergic reactions in the body, and due to factors such as maternal antibody interference, the vaccine effect is not obvious, and the incidence rate is still very high. Therefore, it is necessary to develop safe and effective new immune products for the prevention and treatment of FP.

[0004] Lactobacillus plantarum is a Gram-positive bacterium that can uptake sugar substances to produce lactic acid. Because of its probiotic characteristics such as regulating the intestine and enhancing immunity, it has been certified as a safe probiotic by FAO and WHO and is widely used in fields such as food and medicine. In addition, Lactobacillus has the advantages of a large genome, good biological safety, and high resistance to gastric acid and bile. After oral administration, it can adhere to the intestinal mucosa and can continuously stimulate the systemic humoral, mucosal, and cellular immune responses of the host. It is a good live vector for highly expressing foreign proteins. Recombinant Lactobacillus can induce mucosal immunity in the intestine, which is beneficial to the prevention and treatment of animal diseases.

[0005] Melittin (MLT) is the main component of bee venom and the main active substance of honeybee venom, accounting for about 40%-50% of the dry weight of bee venom. Melittin is an α-helical linear short peptide composed of 26 amino acids, which plays a major pharmacological role in bee venom and has anti-inflammatory, antibacterial, anti-radiation, antiviral, anti-tumor and other effects. At present, the common ways to obtain melittin are limited by factors such as low yield, complex extraction process, low purity, and high cost of chemical synthesis methods, which restrict the in-depth research and industrial production of melittin.

[0006] In the prior art, there has been no report on developing a new microecological preparation composition for preventing and controlling feline panleukopenia by using recombinant Lactobacillus plantarum expressing feline panleukopenia virus VP2 protein and melittin as active ingredients. Summary of the Invention

[0007] Aiming at the above-mentioned prior art, the purpose of the present invention is to provide a new microecological preparation composition for preventing and controlling feline panleukopenia and its application. The present invention uses genetic engineering means to insert the VP2 gene of feline parvovirus into an expression vector and introduce it into Lactobacillus plantarum NC8 to construct a genetically engineered recombinant Lactobacillus plantarum strain NC8 / Δalr-pSIP409-pgsA’(ata)-VP2-DCpep (abbreviated as NC8-VP2). Further, the recombinant Lactobacillus strain NC8-VP2 and melittin (MLT) are used in combination to develop a new immune microecological preparation composition for preventing and controlling feline panleukopenia, which can reduce the incidence of feline panleukopenia, improve the clinical symptoms of diseased animals, promote the health of the body, and promote the rapid development of the pet breeding industry.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] In the first aspect of the present invention, a microecological preparation composition is provided, and the microecological preparation composition includes recombinant Lactobacillus plantarum expressing feline panleukopenia virus VP2 protein and melittin.

[0010] In the microecological preparation composition, the viable count of recombinant Lactobacillus plantarum ≥ 1.0×10 10 CFU / g, and the content of melittin ≥ 50mg / g.

[0011] The microecological preparation composition may further include pharmaceutically acceptable excipient components.

[0012] The excipient components are one or more of fillers, sweeteners, binders, lubricants, disintegrants, preservatives and antioxidants.

[0013] The dosage form of the microecological preparation composition is a capsule dosage form, tablet, powder, pill, suppository or granule.

[0014] Preferably, the dosage form of the microecological preparation composition is a capsule dosage form, and the weight of the capsule is (0.4 - 0.5) g per capsule.

[0015] In the second aspect of the present invention, a preparation method of the above microecological preparation composition is provided, including the following steps:

[0016] (1) Ferment the recombinant Lactobacillus plantarum expressing feline panleukopenia virus VP2 protein under anaerobic conditions at 28 - 30 °C for 12 - 14 h, centrifuge to collect the bacterial cell precipitate, and then perform freeze-drying to prepare the freeze-dried powder of recombinant Lactobacillus plantarum.

[0017] (2) Compound the freeze-dried powder of recombinant Lactobacillus plantarum and melittin to obtain the microecological preparation composition.

[0018] In step (1), the recombinant Lactobacillus plantarum expressing feline panleukopenia virus VP2 protein is prepared by the following method:

[0019] After optimizing the VP2 gene of feline parvovirus according to the codons of Lactobacillus plantarum and adding the DC cell targeting peptide sequence, insert it into the cloning vector pUC to obtain the target fragment VP2-DCpep. Connect the target fragment VP2-DCpep to the antibiotic-free label expression vector pSIP409-pgsA’(ata) to obtain the recombinant plasmid pSIP409-pgsA’(ata)-VP2-DCpep. Transform the recombinant plasmid into Lactobacillus plantarum NC8 / Δalr to obtain the recombinant Lactobacillus plantarum NC8 / Δalr-pSIP409-pgsA’(ata)-VP2-DCpep (abbreviated as NC8-VP2) expressing feline panleukopenia virus VP2 protein.

[0020] The target fragment VP2-DCpep (1821 bp) sequentially includes: the optimized VP2 gene (1758 bp) + His.Tag gene sequence (18 bp) + DCpep sequence (36 bp) + terminator TAA (3 bp) + restriction enzyme site AAGCTT (6 bp).

[0021] The nucleotide sequence of the optimized VP2 gene (1758 bp) is as shown in SEQ ID NO.1.

[0022] The amino acid sequence of the protein encoded by the optimized VP2 gene is as shown in SEQ ID NO.2.

[0023] The His.Tag gene sequence is as shown in SEQ ID NO.3.

[0024] The DC cell targeting peptide (DCpep) sequence is as shown in SEQ ID NO.4.

[0025] In step (2), the amino acid sequence of melittin is as shown in SEQ ID NO.5.

[0026] In step (2), the melittin can be obtained by purchase, direct chemical synthesis, induction expression in a prokaryotic expression system or induction expression in a eukaryotic expression system.

[0027] Preferably, in step (2), the melittin is prepared by the following method:

[0028] Construct the recombinant Escherichia coli BL21-pET32a-MLT expressing melittin, homogenize the cells obtained by centrifuging the fermentation product of the recombinant Escherichia coli BL21-pET32a-MLT, and obtain it through affinity chromatography, followed by freeze-drying, desalting column desalting, enterokinase de-tagging, and ion exchange chromatography.

[0029] The construction method of the recombinant Escherichia coli BL21-pET32a-MLT is as follows:

[0030] Optimize and synthesize the MLT gene encoding the melittin sequence according to the codon preference of Escherichia coli, ligate it with the cloning vector pUC19, perform PCR amplification to obtain the MLT target gene with correct sequence, and obtain it by ligating the MLT target gene with the expression vector pET32a and then transforming it into Escherichia coli.

[0031] In the third aspect of the present invention, there is provided the use of the above-mentioned microecological preparation composition in the preparation of a drug for preventing and treating diseases caused by feline parvovirus.

[0032] The beneficial effects of the present invention:

[0033] (1) The recombinant Lactobacillus plantarum NC8-VP2 constructed by the present invention through genetic engineering means can efficiently express the feline panleukopenia VP2 protein. The inserted DC cell-targeting peptide sequence (DCpep) during the construction process can target the feline panleukopenia VP2 protein antigen expressed by the recombinant Lactobacillus plantarum NC8-VP2 to DC cells, enhance the immune response, improve the targeting delivery efficiency of the therapeutic VP2 protein, and ultimately enable the protective antigen expressed by the recombinant Lactobacillus plantarum to efficiently stimulate the body to produce specific antibodies, thereby preventing the occurrence of FP and reducing the use of chemical drugs.

[0034] (2) The recombinant Escherichia coli BL21-pET32a-MLT constructed by the present invention using genetic engineering means can secrete and express melittin (MLT). The recombinant Escherichia coli BL21-pET32a-MLT realizes the large-scale production of melittin, laying a foundation for better exerting the immunomodulatory effect of melittin.

[0035] (3) The present invention further uses melittin and recombinant Lactobacillus plantarum NC8-VP2 as active ingredients to develop and prepare a novel microecological preparation composition for preventing and controlling feline panleukopenia. This novel microecological preparation composition can reduce the incidence of feline panleukopenia, improve the clinical symptoms of diseased animals, promote the health of the body, has good application prospects in the prevention and treatment of feline panleukopenia, and promotes the rapid development of the pet breeding industry. Description of the Drawings

[0036] Figure 1 It is the PCR identification result of plasmid pSIP409-pgsA’(ata)-VP2-DCpep.

[0037] Figure 2 It is the expression diagram of FPV VP2 protein detected by Western Blot.

[0038] Figure 3 It is the map of recombinant plasmid pET32a-MLT.

[0039] Figure 4 It is the SDS-PAGE electrophoresis detection diagram of the fusion protein fermented and expressed by Escherichia coli BL21-pET32a-MLT; where M: Marker, sample well 1: BL21-pET32a empty vector induced for 4 h, sample well 2: BL21-pET32a-MLT induced for 0 h, sample wells 3-5: BL21-pET32a-MLT induced at 16 °C for 18 h.

[0040] Figure 5 It is the Western Blot detection diagram of the fusion protein fermented and expressed by Escherichia coli BL21-pET32a-MLT; where, M: Marker, sample well 1: pET32a empty vector induced for 4 h, sample well 2: the bacteria body induced and expressed by BL21-pET32a-MLT, sample well 3: the supernatant of the broken bacteria body induced and expressed by BL21-pET32a-MLT, sample well 4: the precipitate of the broken bacteria body induced and expressed by BL21-pET32a-MLT.

[0041] Figure 6 It is the SDS-PAGE electrophoresis diagram of the pET32a-MLT fusion protein after purification by nickel column; where, M: Marker, sample wells 1-14: elution tubes 1-14.

[0042] Figure 7SDS-PAGE electrophoresis detection chart of the pET32a-MLT fusion protein after cleavage by enterokinase; where M: Marker, sample well 1: pET32a-MLT fusion protein, sample well 2: pET32a-MLT fusion protein after cleavage by enterokinase, sample well 3: precipitate after centrifugation of the pET32a-MLT fusion protein after cleavage, sample well 4: flow-through, sample well 5: washing solution, sample well 6: eluate.

[0043] Figure 8 Western Blot electrophoresis detection chart of the purified pET32a-MLT fusion protein after cleavage by enterokinase; where M: Marker, sample well 1: pET32a-MLT fusion protein, sample well 2: pET32a-MLT fusion protein after cleavage by enterokinase, sample well 3: precipitate after centrifugation of the pET32a-MLT fusion protein after cleavage, sample well 4: flow-through, sample well 5: washing solution, sample well 6: eluate.

[0044] Figure 9 NO content in the supernatant of RAW264.7 cells after treatment in each group.

[0045] Figure 10 Fecal score of experimental cats after challenge.

[0046] Figure 11 White blood cell count of experimental cats after challenge.

[0047] Figure 12 SAA detection of experimental cats after challenge.

[0048] Figure 13 Intestinal condition of experimental cats after challenge; where group 1: NC8-VP2 + MLT protein composition group, group 2: NC8-VP2 group, group 3: MLT group, group 4: CK group.

[0049] Figure 14 Intestinal pathological sections of experimental cats after challenge; where group 1: NC8-VP2 + MLT protein composition group, group 2: NC8-VP2 group, group 3: MLT group; group 4: CK group. Detailed implementation mode

[0050] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0051] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The experimental methods without specified detailed conditions are carried out according to conventional test methods or the operation manuals recommended by the suppliers. Among them:

[0052] NC8 / Δalr is a Lactobacillus plantarum with alanine racemase gene deficiency, which is recorded in Patent CN 112300976B; the expression vector pSIP409-pgsA’(ata) is constructed by removing EGFP from the "plasmid pSIP409-pgsA’-EGFP(ata)" recorded in Patent CN 112300976B.

[0053] Example 1: Construction of recombinant Lactobacillus plantarum strain NC8 / Δalr-pSIP409-pgsA’(ata)-VP2-DCpep (NC8-VP2)

[0054] (1) Construction of pSIP409-pgsA’(ata)-VP2-DCpep plasmid vector

[0055] The VP2 gene sequence was selected, optimized and synthesized according to the codon preference of Lactobacillus plantarum. After placing the DC cell-targeting peptide (DCpep) sequence at the 3' end of the optimized VP2 gene, it was inserted into the cloning vector pUC to obtain pUC-VP2-DCpep. Subsequently, PCR was carried out with primers VP2-Xba I-F / VP2-HindⅢ-DCpep-R (shown in SEQ ID NO.6-SEQ ID NO.7). The PCR reaction system is shown in Table 1, and the PCR reaction conditions are: pre-denaturation at 98°C for 20 s, denaturation at 98°C for 10 s, annealing at 50°C for 5 s, extension at 72°C for 30 s, 30 cycles, and finally reaction at 72°C for 10 min. After electrophoresis of the PCR product, it was recovered using a gel recovery kit. The recovered product was double-digested with Xba I / HindⅢ, placed in a water bath at 37°C for overnight digestion, the digestion system is shown in Table 2, and the digested product was recovered using a DNA purification kit to obtain the target fragment VP2-DCpep.

[0056] Table 1 PCR reaction system of plasmid pUC-VP2-DCpep

[0057]

[0058] Table 2 Enzyme digestion system of plasmid pUC-VP2-DCpep

[0059]

[0060] Subsequently, the antibiotic-free label expression vector pSIP409-pgsA’(ata) and the target fragment VP2-DCpep were ligated using T4 ligase and placed in a metal bath at 16°C for overnight ligation.

[0061] Table 3 DNA ligation reaction system

[0062]

[0063] The above ligation product was transformed into electrocompetent Escherichia coli χ6212. After transformation, 100 μL of the bacterial solution was taken and spread on an LB agar (peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, pH 7.0 ± 0.2, sterilized at 121°C for 20 min) plate; the next day, colonies were picked for plasmid extraction and sequencing verification, and the obtained positive plasmid was named pSIP409-pgsA’(ata)-VP2-DCpep.

[0064] (II) Construction of recombinant Lactobacillus plantarum NC8 / Δalr-pSIP409-pgsA’(ata)-VP2-DCpep

[0065] 1 Preparation of electrocompetent Lactobacillus plantarum

[0066] (1) Inoculate Lactobacillus plantarum NC8 / Δalr stored at -80°C into 5 mL of MRS liquid medium containing D-alanine (0.2 mg / mL) and culture overnight at 30°C under anaerobic conditions;

[0067] MRS liquid medium: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, Tween-80 1 mL / L, pH value 6.0 ± 0.05, sterilized at 118°C for 20 min.

[0068] (2) Inoculate an appropriate amount of the bacterial solution on MRS solid medium containing D-alanine (0.2 mg / mL) and culture at 30°C under anaerobic conditions until well-grown single colonies appear, about 36 - 48 h;

[0069] MRS solid medium: based on the formula of MRS liquid medium, add another 15 g / L of agar and sterilize at 118°C for 20 min.

[0070] (3) Inoculate a well-grown single colony from step (2) into 5 mL of MRS liquid medium containing D-alanine (0.2 mg / mL) (containing 2% Gly) and culture at 30°C under anaerobic conditions until the OD 600nm value reaches 0.6;

[0071] (4) Take 20 mL of MRS liquid medium containing D-alanine (0.2 mg / mL) (containing 2% Gly) and inoculate it with 30 μL of the bacterial liquid in the above step (3). Continue culturing under anaerobic conditions at 30 °C until the OD 600nm value is 0.4;

[0072] (5) Ice-bath the bacterial liquid with an OD 600nm value of 0.4 for 20 min, centrifuge at 5000 r / min for 10 min at 4 °C, and collect the bacterial cell precipitate;

[0073] (6) Resuspend the bacterial cell precipitate with 2 mL of ice-cold washing buffer (Na3PO4: 0.19 g, anhydrous MgCl2: 0.09 g, ddH2O: 80 mL. Adjust the pH to 7.4 with dilute HCl, make up the volume to 100 mL, aliquot in small amounts, autoclave at 121 °C for 20 min, and store at -20 °C for later use), centrifuge at 5000 r / min for 10 min at 4 °C, and repeat the washing twice. Collect the bacterial cell precipitate;

[0074] (7) Resuspend the bacterial cell precipitate with 400 μL of ice-cold electroporation buffer (sucrose: 34.21 g, anhydrous MgCl2: 0.029 g, ddH2O: 80 mL. Adjust the pH to 7.4 with dilute HCl, make up the volume to 100 mL, aliquot in small amounts, autoclave at 121 °C for 20 min, and store at -20 °C for later use), ice-bath for 10 min, and then aliquot 100 μL per tube for later use.

[0075] 2 Electroporation of Lactobacillus plantarum NC8 / Δalr

[0076] (1) Take 5 μL of the recombinant plasmid pSIP409-pgsA’(ata)-VP2-DCpep and add it to two tubes of 100 μL of ice-bathed Lactobacillus plantarum competent cells. Gently mix, transfer it into a pre-cooled electroporation cuvette with a 0.2 cm gap, let it stand and ice-bath for 5 min, and then place it in an electroporator (2.5 Kv, 6 ms) for electroporation;

[0077] (2) After the electroporation is completed, immediately let it stand and ice-bath for 5 min. Take the liquid in the electroporation cuvette and add it to 800 mL of MRS culture medium preheated to 30 °C, and culture it under anaerobic conditions at 30 °C for 3 h;

[0078] (3) Uniformly coat 100 μL of the bacterial liquid obtained in step (2) on the MRS solid medium, and culture it under anaerobic conditions at 30 °C for 36 - 48 h until well-grown single colonies appear.

[0079] 3 Extraction and identification of recombinant Lactobacillus plantarum plasmid

[0080] Inoculate a single colony of Lactobacillus plantarum in good condition into MRS culture medium and culture it overnight under anaerobic conditions at 30 °C. Extract the plasmid using a small-scale plasmid extraction kit (for Gram-positive bacteria). Perform double digestion and PCR identification on the extracted plasmids. The enzyme digestion system and PCR reaction system are shown in Table 4 and Table 5. PCR reaction conditions: pre-denaturation at 95 °C for 5 min, then denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 135 s, 30 cycles, and finally reaction at 72 °C for 10 min, store at 4 °C. After the reaction, perform 1.0% agarose gel electrophoresis and load 5 μL of the PCR product.

[0081] Table 4 Enzyme digestion system of plasmid pSIP409-pgsA’(ata)-VP2-DCpep

[0082]

[0083]

[0084] Table 5 PCR reaction system of plasmid pSIP409-pgsA’(ata)-VP2-DCpep

[0085]

[0086] Use HindⅢ and Xba I to perform double digestion identification on the extracted plasmid, and use the vector primers (SEQ ID NO.8 - SEQ ID NO.9) for PCR identification of the extracted plasmid. The results are shown in Figure 1 . Bands corresponding to the target were observed around 2000 bp in both double digestion and PCR results. Sequencing of the PCR product revealed that the size of the recombinant Lactobacillus plantarum plasmid fragment was 1920 bp, and the specific sequence was as shown in SEQ ID NO.10.

[0087] 4 Detection of the expression of the target protein by recombinant Lactobacillus plantarum (NC8-VP2)

[0088] Thaw the cryopreservation solution stored in a -80 °C freezer on ice, streak and inoculate it on MRS solid medium for 1 passage, then pick a single colony and inoculate it into MRS liquid medium for overnight culture. Then transfer the bacterial solution to MRS liquid medium with an inoculation amount of 2%, and wait until the cell density reaches OD 600nmWhen it reached 0.3 - 0.4, SppIP with a final concentration of 100 ng / mL was added, and anaerobic induction culture was carried out overnight at 30°C. The extraction method of the recombinant protein was as follows: 200 mL of the cultured bacterial liquid was collected by centrifugation. After the bacterial cells were washed twice with sterile ice-cold PBS, the precipitate was resuspended with PBS containing 0.1% PMSF. After adding 100 μL of lysozyme (10 mg / mL), ultrasonic disruption was carried out under ice-bath conditions (ultrasonic power 300 W, ultrasonic for 2 s, intermittent for 5 s, 99 times) to obtain the protein sample. Western Blot analysis was performed on the extracted protein sample. The primary antibody used for detection was Flag antibody (mouse monoclonal antibody, 1:1000, purchased from Beyotime Biotechnology), and the secondary antibody used was goat anti-mouse IgG (H+L) labeled with horseradish peroxidase (Goat Anti-Mouse (H+L):HRP, 1:1000, purchased from Beyotime Biotechnology). The detection results were as Figure 2 shown. A protein band with a size of approximately 90 kDa was exposed in the recombinant Lactobacillus plantarum, which was consistent with the expected result, proving the successful expression of FPV VP2 protein in the recombinant Lactobacillus plantarum (NC8-VP2).

[0089] Example 2: Study on the fermentation process of recombinant Lactobacillus plantarum NC8-VP2

[0090] 1 Experimental materials

[0091] Strain: Recombinant Lactobacillus plantarum NC8-VP2 prepared in Example 1.

[0092] MRS liquid medium: Glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, Tween-80 1 mL / L, pH value 6.0 ± 0.05, sterilized at 118°C for 20 min.

[0093] Inducer: SppIP.

[0094] 2 Experimental methods

[0095] 1 mL of the bacterial liquid was aspirated from the glycerol tube preservation solution of recombinant Lactobacillus plantarum NC8-VP2 and inoculated into 100 mL of MRS liquid medium, and static culture was carried out overnight at 30°C to prepare the first-stage seed liquid. The first-stage seed liquid was inoculated into 3 L of MRS liquid medium at an inoculation amount of 3%, and cultured overnight at 30°C to obtain the second-stage seed liquid. 60 L of the 3 L second-stage seed liquid was inoculated into a 100 L seed tank (liquid loading amount: 60 L of MRS liquid medium), and activated at 30°C for 10 - 12 h under anaerobic conditions to obtain the seed tank fermentation liquid. 60 L of the activated seed tank fermentation liquid was transferred to a 500 L fermentation tank (liquid loading amount: 400 L of MRS liquid medium), and cultured at 30°C for 1 h under anaerobic conditions. The cell density was detected to reach OD600nm When it reaches 0.3 - 0.4, SppIP with a final concentration of 100 ng / mL is added for induced culture. The fermentation ends when the pH of the fermentation broth drops below 4.0 (or the pH does not continuously decrease within 2 h). The total fermentation time is generally controlled at 12 - 14 h. Samples are taken every 2 h during the fermentation process to detect and record the OD 600nm , pH value and viable cell count of the fermentation broth.

[0096] 3 Experimental Results

[0097] The viable cell count during the fermentation of recombinant Lactobacillus plantarum NC8-VP2 was counted. As can be seen from Table 6, the highest viable cell count in the stationary phase of recombinant Lactobacillus plantarum NC8-VP2 was 5.1×10 9 CFU / mL.

[0098] Table 6 Statistical Results of Viable Cell Count during the Fermentation of Recombinant Lactobacillus plantarum NC8-VP2

[0099]

[0100] Example 3: Construction of Recombinant Escherichia coli BL21-pET32a-MLT

[0101] According to the gene sequence encoding melittin (MLT), it was analyzed using the E.coli Codon Usage Analyzer website and optimized and synthesized according to the codon preference of Escherichia coli. Using this as a template for PCR amplification, the PCR reaction system is shown in Table 7, and the reaction conditions are: pre-denaturation at 98℃ for 5 min, denaturation at 98℃ for 10 s, annealing at 55℃ for 5 s, extension at 72℃ for 5 s, for 30 cycles.

[0102] Table 7 PCR Reaction System

[0103]

[0104] The primer sequences of MLT-F (EcoRⅠ) and MLT-R (Hind III) are shown in SEQ ID NO.11 - SEQ ID NO.12. After the PCR product was preliminarily confirmed to be correct by agarose gel electrophoresis, it was purified using a DNA purification and recovery kit. The purified PCR product and the pET32a vector were respectively digested with EcoR I and Hind III at 37℃ for 2 h, and the digestion system is shown in Table 8.

[0105] Table 8 Double Digestion System

[0106]

[0107] The digested PCR products and the pET32a vector were respectively subjected to gel recovery using a DNA purification and recovery kit, and then ligated overnight at 16°C using T4 ligase. The ligation reaction system is shown in Table 9.

[0108] Table 9 Ligation System

[0109]

[0110] The obtained ligation products were subjected to the following operations: Take 10 μL of the ligation product and add it to 100 μL of Escherichia coli E. coli BL21(DE3) competent cells, then incubate on ice for 30 min, heat shock at 42°C for 90 s, then incubate on ice for 5 min. Add 900 μL of antibiotic-free LB culture medium at 37°C to the tube, shake on a shaker at 37°C and 200 rpm for 1 h, then spread it on an LB agar plate containing ampicillin resistance, incubate at 37°C, pick a single colony for activation after colonies grow, and perform colony PCR identification. After PCR amplification, electrophoresis was observed. The colonies with positive PCR results were sent to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing. After sequencing confirmation, a successfully constructed recombinant Escherichia coli was finally obtained. The recombinant Escherichia coli was named Escherichia coli BL21-pET32a-MLT, and its plasmid map is shown in Figure 3 。

[0111] Example 4: Recombinant Escherichia coli BL21-pET32a-MLT was induced to express melittin (MLT)

[0112] 1 Fermentation and expression of the strain

[0113] The Escherichia coli BL21-pET32a-MLT prepared in Example 3 was plated and incubated overnight, and a single colony was picked into LB medium and cultured overnight at 37°C and 220 rpm; the culture was transferred to LB medium at a ratio of 1:100 and cultured at 37°C and 220 rpm until the OD 600nm reached 0.6, and 2 mL of pre-induction bacteria were collected for use as a negative control; 1 mol / L IPTG was added to the LB medium to a final concentration of 1 mmol / L, and induced to culture at 16°C for 18 h.

[0114] 2 Bacterial cell disruption and sample treatment and detection

[0115] Collect the induced bacteria, centrifuge at 6000 rpm for 10 min, discard the supernatant, and keep the pellet; resuspend the collected bacteria with 25 mmol / L Tris·HCl buffer, sonicate for 2 s and pause for 4 s until the cell suspension becomes clear, centrifuge at 4 °C and 12,000 rpm for 5 min; take 20 μL of the protein supernatant into a centrifuge tube, add 5 μL of 5×SDS loading buffer and mix well. After resuspending the cell pellet with 200 μL of PBS buffer, similarly take 20 μL of the suspension into a centrifuge tube, add 5 μL of 5×SDS loading buffer and mix well.

[0116] The bacteria before induction (negative control) were treated in the same way.

[0117] Boil all the samples to be tested in a water bath for 10 min, centrifuge at 12,000 rpm for 1 min, and then perform SDS-PAGE electrophoresis detection. One gel is used for Coomassie Brilliant Blue staining, and one gel is used for Western Blot analysis. As Figure 4 shown, Coomassie Brilliant Blue staining shows that this strain has obvious expression at 25 kDa, which is consistent with the expected size.

[0118] 3 Western Blot detection

[0119] For the gel after the above SDS-PAGE, strip the gel, cut the gel, cut the membrane, and activate the membrane (methanol for 10 s, electrolyte for 15 min) in the electrotransfer solution. "Sandwich": blackboard - sponge - three layers of filter paper - gel - membrane - three layers of filter paper - sponge - whiteboard, clamp it and put it into the electrotransfer tank; connect the power supply, set the constant current and time (300 mA, 1 h); after the membrane transfer is completed, take out the PVDF membrane and block it in 5% skim milk TBST at room temperature for 2 h; dilute the primary antibody His-tag antibody (mouse monoclonal antibody, 1:1000, purchased from Beyotime Biotechnology) with TBST containing 5% skim milk, immerse the PVDF membrane in the primary antibody incubation solution, and incubate overnight at 4 °C; wash the PVDF membrane 3 times with TBST, 10 min each time; the secondary antibody is horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (Goat Anti-Mouse (H+L):HRP, purchased from Beyotime Biotechnology), dilute the secondary antibody with TBST containing 5% skim milk (1:1000), and incubate at room temperature for 1 h; wash the PVDF membrane thoroughly with TBST 3 times, 10 min each time; mix the ECL luminescent solution A and B at 1:1; after taking out the PVDF membrane from the TBST washing solution, blot it dry with filter paper; place the PVDF on the imager, evenly add the ECL working solution, remove the bubbles, and start exposure. As Figure 5 can be seen, there is a luminescent band at the corresponding band of SDS-PAGE, and the obtained expression band is the target protein, that is, melittin (pET32a-MLT fusion protein).

[0120] Purification, Acquisition and Identification of Melittin

[0121] (1) Treatment of Ni Column

[0122] Mix 5 mL of Ni-Agarose Resin filler and add it to the chromatography column. Let it stand at room temperature for 10 min. After the gel and the solution are separated, open the outlet at the bottom and let the ethanol flow out slowly by gravity. Add 5 column volumes of deionized water to the loaded column to wash the ethanol clean, and then equilibrate the column with 10 column volumes of Binding Buffer. After equilibration, the sample can be loaded.

[0123] (2) Purification of Melittin (pET32a-MLT Fusion Protein)

[0124] Dilute the supernatant solution of the obtained melittin (pET32a-MLT fusion protein) with Binding Buffer at a ratio of 1:1, load it onto the column at a flow rate of 10 column volumes / hour, and collect the flow-through fraction. Wash the column with 15 column volumes of Binding Buffer to wash away the miscellaneous proteins. Elute with an appropriate amount of Elution Buffer and collect the elution peak. After elution, wash the column with 10 column volumes of deionized water, then equilibrate with 3 column volumes of 20% ethanol, seal the column and store it at 2 - 8 °C. Then, perform SDS-PAGE detection on the eluted sample solution, and the detection results are as Figure 6 shown.

[0125] (3) Desalting Dialysis

[0126] Cut the dialysis bag into small segments of appropriate length (about 10 cm), place it in the treatment solution (1 mmol / L EDTA, pH 8.0) and boil it for 10 min. Take it out and wash the dialysis bag thoroughly with distilled water. Add the purified melittin (pET32a-MLT fusion protein) into the dialysis bag, clamp both ends with clips, and place it in the dialysis solution (25 mmol / L Tris·HCl) for overnight dialysis at 4 °C, with stirring several times during the process. After dialysis, suck out the liquid in the dialysis bag to obtain the purified fusion protein solution (pET32a-MLT fusion protein). After cleaning the dialysis bag, store it in 50% ethanol.

[0127] (4) Enzymatic Cleavage Treatment and Detection of Purified Melittin (pET32a-MLT Fusion Protein)

[0128] Using the BCA concentration assay method, the concentration of the pET32a-MLT fusion protein was determined to be 995.496 μg / mL. Calculated based on 0.1 - 0.2 U of enterokinase per 1 mg of protein, digestion was carried out overnight at 25 °C and 50 rpm on a shaker; after digestion, it was purified by ion exchange chromatography, and the flow-through and elution fractions were collected for Tricine-SDS-PAGE( Figure 7 ) and Western Blot detection( Figure 8 ). According to the Tricine-SDS-PAGE and Western Blot detections, after digestion of the pET32a-MLT fusion protein with enterokinase, the vector tag could be obtained and the target band appeared below 10 kDa. The melittin (MLT) cut off was mainly in the flow-through fraction.

[0129] Example 5: Cytotoxicity test of NC8-VP2 protein and MLT (CCK8 method)

[0130] RAW264.7 murine monocyte-macrophage leukemia cells were seeded in a 96-well cell culture plate at a density of 5×10 3 cells / well, and 100 μL of 1% FBS-DMEM medium was added. The cells were incubated in a 37 °C, 5% CO2 incubator for 12 - 24 h until they adhered to the plate.

[0131] The drug groups were as follows: ① Blank control group: containing only 1% FBS-DMEM medium; ② LPS gradient group: LPS concentrations were 12.5, 25, 50, 100, 200 μmol / L respectively; ③ Melittin standard group: melittin standard (Shanghai Yuanye Bio-Technology, product number T71338) concentrations were 5.6, 2.8, 1.4, 0.7, 0.35 μmol / L respectively; ④ MLT group: melittin purified by enterokinase digestion had concentrations of 11.2, 5.6, 2.8, 1.4, 0.7, 0.35 μmol / L respectively; ⑤ NC8-VP2 recombinant protein group: NC8-VP2 recombinant protein concentrations were 8.8, 4.4, 2.2, 1.1, 0.55, 0.28 μmol / L respectively. All treatments were diluted with 1% FBS-DMEM.

[0132] Drug treatment and cell viability detection: The original cell medium was discarded, and the cells were washed once with PBS. Then, 100 μL of the corresponding concentration of the drug solution was added according to the drug groups, and each group had 6 replicates. Another 100 μL of 1% FBS-DMEM medium was added to make the final volume 200 μL, and the cells were cultured for another 24 h. Then the supernatant was discarded, and 100 μL of DMEM solution containing 10% CCK-8 was added to each well. After 4 h of color development, OD 450nm The absorbance value was read.

[0133] Result analysis (see Table 10): When the concentrations of melittin standard and MLT were ≥ 2.8 μmol / L, the survival rate of RAW264.7 cells was significantly lower than that of the blank control group (P < 0.05), showing concentration-dependent toxicity; while within the range of 0.28 - 8.8 μmol / L of NC8-VP2 protein, there was no significant difference in cell survival rate compared with the blank control group, indicating no cytotoxicity.

[0134] Table 10 Results of cell safety test

[0135]

[0136]

[0137] (Note: The superscript * indicates a significant difference between groups (P < 0.05).)

[0138] Example 6: Detection of anti-inflammatory activities of NC8-VP2 protein and MLT

[0139] Seed RAW264.7 cells into a 96-well cell culture plate at a density of 1×10 5 cells / well, add 100 μL of 1% FBS-DMEM medium, and incubate in a 37 °C, 5% CO2 incubator for 12 - 24 h until the cells adhere to the plate.

[0140] The drug groups are as follows:

[0141] ① Blank control group: 100 μL of 1% FBS-DMEM + 100 μL of PBS as the medium (without LPS and protein);

[0142] ② Inflammatory model group: 100 μL of 1% FBS-DMEM + 100 μL of PBS as the medium, and add LPS with a final concentration of 5 μg / mL to the medium;

[0143] ③ Test group 1 (MLT group): Dissolve the melittin prepared in Example 4 with PBS to prepare an MLT solution with a mass concentration of 20 μg / mL; use 100 μL of 1% FBS-DMEM + 100 μL of the MLT solution as the medium, and add LPS with a final concentration of 5 μg / mL to the medium.

[0144] ④ Test group 2 (NC8-VP2 protein group): Dissolve NC8-VP2 protein with PBS to prepare an NC8-VP2 protein solution with a mass concentration of 20 μg / mL; use 100 μL of 1% FBS-DMEM + 100 μL of the NC8-VP2 protein solution as the medium, and add LPS with a final concentration of 5 μg / mL to the medium.

[0145] ⑤Experimental group 3 (NC8-VP2-MLT mixed protein group): Mix NC8-VP2 protein and MLT at a mass ratio of 1:200. Dissolve the mixed protein in PBS to prepare an NC8-VP2-MLT mixed protein solution with a mass concentration of 20 μg / mL. Use 100 μL of 1% FBS-DMEM + 100 μL of the NC8-VP2-MLT mixed protein solution as the culture medium, and add LPS with a final concentration of 5 μg / mL to the culture medium.

[0146] It was found that compared with the LPS-induced inflammation model group, MLT could significantly reduce the secretion of NO, and the NC8-VP2 protein had no obvious effect on reducing the secretion of NO. When NC8-VP2 and MLT were mixed at a ratio of 1:200, under the condition of the same protein addition concentration, the mixed protein had a more significant effect on reducing the NO concentration, indicating that the combination of NC8-VP2 and MLT had a synergistic anti-inflammatory effect ( Figure 9 ).

[0147] Example 7: Preparation of a novel microecological preparation

[0148] (I) Preparation of freeze-dried powder of recombinant Lactobacillus plantarum NC8-VP2

[0149] Centrifuge the bacterial liquid of recombinant Lactobacillus plantarum NC8-VP2 fermented for 12 h according to the fermentation process described in Example 2, collect the bacterial cell precipitate, and mix the bacterial cell precipitate with the freeze-drying protectant at a mass ratio of 1:1. The freeze-drying protectant is composed of the following raw materials in parts by mass: 22.5 parts of skim milk powder, 9 parts of sucrose, 1 part of glycerol, and 67.5 parts of water.

[0150] Subsequently, freeze-dry for 48 h using conventional vacuum freeze-drying technology to obtain the freeze-dried powder of recombinant Lactobacillus plantarum NC8-VP2, and the viable count of the powder is not less than 4.0×10 11 CFU / g.

[0151] (II) Preparation of melittin freeze-dried powder

[0152] 1 Preparation of seed liquid

[0153] Seed medium: LB medium (10.0 g / L of peptone, 5.0 g / L of yeast extract, 10.0 g / L of sodium chloride, pH 7.0 ± 0.2), autoclave at 121 °C for 30 min, and add ampicillin with a final concentration of 100 μg / mL after cooling to room temperature;

[0154] Fermentation medium: 0.2% glucose, 1% peptone, 0.5% sodium chloride, 0.5% yeast extract, adjust the pH to 7.0, and autoclave at 118 °C for 20 min.

[0155] 2 Fermentation tank (20 L) fermentation and induction expression

[0156] (1) Preparation of seed culture: Inoculate from the glycerol-preserved bacteria (recombinant Escherichia coli BL21-pET32a-MLT) into a 5 mL test tube containing seed culture medium, and culture overnight with shaking at 37°C; Take the culture and inoculate it into an Erlenmeyer flask containing seed culture medium at a ratio of 1:100, and culture overnight with shaking at 37°C.

[0157] (2) Fermentation in fermenter: After sterilizing the fermentation medium prepared in the fermenter, add the seed culture into the fermenter at a ratio of 2% by volume. The fermentation temperature is 37°C, the aeration is 2 VVM, and the rotation speed is 200 rpm for constant-temperature fermentation. According to the dissolved oxygen situation, glucose feeding is carried out. When the fermentation OD 600nm ≈50, add IPTG for induction, and the final concentration of IPTG is 0.2 μmol / L. Induce for 12 h to terminate fermentation.

[0158] (3) Centrifugation and homogenization: Centrifuge the fermented bacterial liquid at 7000 rpm for 20 min, collect the bacterial cells, resuspend them with normal saline and centrifuge again. Resuspend the centrifuged bacterial cell precipitate with chromatography equilibration buffer, and homogenize in batches, 3 times each time, with a pressure of 10 8 Pa each time. Centrifuge the homogenate at 7500 rpm for 15 min, take the supernatant and filter it successively through a 0.22 μm PP filter membrane and a 0.4 m 2 PES membrane of hollow fiber column. After two filtrations, make the turbidity of the homogenate supernatant < 50 NTU.

[0159] (4) Affinity chromatography: Use an ÄKTA protein purifier and a Ni affinity chromatography column to purify the protein. Equilibrate with a buffer containing 0.02 M PB, 0.5 mol / L NaCl, and 30 mmol / L imidazole at a flow rate of 40 mL / min; The sample loading volume is determined according to the protein concentration, and the sample loading flow rate is 40 mL / min; Wash the impurities with the same buffer at a flow rate of 40 mL / min; Elute with a buffer containing 0.02 M PB, 0.5 M NaCl, and 200 mmol / L imidazole at a flow rate of 40 mL / min. Collect the eluate and ultrafiltrate and concentrate it to about 200 mL, and then ultrafilter and change it to a pH 7.4, 20 mmol / L PB buffer. Place the concentrated solution in a -80°C refrigerator and freeze it overnight, and then use a freeze dryer for freeze-drying to obtain a freeze-dried powder.

[0160] (5) Detection of freeze-dried powder: Detect the moisture content, protein content of the freeze-dried powder, and use SDS-PAGE gel electrophoresis to detect the purity of the target protein and whether degradation occurs. The content of MLT in the freeze-dried powder ≥ 125 mg / g.

[0161] (III) Preparation of the novel microecological preparation composition

[0162] The freeze-dried powder of recombinant Lactobacillus plantarum NC8-VP2 obtained in (1) and the freeze-dried powder of melittin obtained in (2) are compounded. After compounding, the viable count of recombinant Lactobacillus plantarum NC8-VP2 in the powder is 1.0×10 10 CFU / g, and the content of melittin is 50 mg / g.

[0163] The compounded powder is encapsulated with an edible glutinous rice capsule shell to prepare a composition capsule. Each capsule of the composition capsule contains recombinant Lactobacillus plantarum NC8-VP2 (content 5.0×10 9 CFU / capsule) and MLT (25 mg) expressed by recombinant Escherichia coli BL21-pET32a-MLT. The weight of the capsule is 0.5 g / capsule.

[0164] Control Example 1: Preparation of freeze-dried powder capsule of recombinant Lactobacillus plantarum NC8-VP2

[0165] The preparation of the freeze-dried powder capsule of recombinant Lactobacillus plantarum NC8-VP2 is different from the preparation of the novel microecological preparation (composition capsule) in Example 7 only in that the freeze-dried powder capsule of recombinant Lactobacillus plantarum NC8-VP2 only contains recombinant Lactobacillus plantarum NC8-VP2 (content 5.0×10 9 CFU / capsule).

[0166] Control Example 2: Preparation of melittin capsule

[0167] The preparation of the melittin capsule is different from the preparation of the novel microecological preparation (composition capsule) only in that the melittin capsule only contains MLT (25 mg) expressed by recombinant Escherichia coli BL21-pET32a-MLT.

[0168] Example 8: Evaluation of the prevention and treatment effect of the novel microecological preparation composition on feline panleukopenia

[0169] 1 Experimental protocol

[0170] According to the screening rules for feline panleukopenia-susceptible cats, 12 domestic cats about 10 weeks old are screened. Their backgrounds have not been injected with FPV-related vaccines and the anal swab FPV PCR test is negative.

[0171] All the experimental cats are divided into 4 groups, with 3 cats in each group. Among them, Experimental Group 1: Oral administration of the composition capsule prepared in Example 7 (NC8-VP2 + MLT protein composition group); Experimental Group 2: Oral administration of the freeze-dried powder capsule of recombinant Lactobacillus plantarum NC8-VP2 prepared in Control Example 1 (NC8-VP2 group); Experimental Group 3: Oral administration of the melittin capsule prepared in Control Example 2 (MLT group); Experimental Groups 1 to 3 are all 1 capsule / time / day and are continuously administered for 14 days. Not taking the capsule is used as the control group (CK group). On the 15th day, all the experimental cats are challenged (virus strain: FPV-S2, virus content 105.0 TCID 50 / mL, 1 mL per cat, intranasal inoculation for virus challenge). Subsequently, observe continuously for another 14 days, and record the infection rate of cats, positive rate of anal swab PCR, fecal score, white blood cell count, inflammatory factors, intestinal lesions, etc.

[0172] 2 Test results

[0173] (1) Infection rate and positive rate of anal swab PCR

[0174] The results after virus challenge showed that none of the test cats died, but all had varying degrees of infection symptoms. Especially in the CK group, the main manifestations were loss of appetite, listlessness, vomiting, diarrhea, etc. From the perspective of the positive rate of anal swab PCR (see Table 11), the positive rates in the CK group were all relatively high and all turned negative on the 14th day after virus challenge; in the NC8-VP2 + MLT protein composition group, 1 cat turned negative on the 1st day after virus challenge, and then the negative rate increased, and all turned negative on the 5th day after virus challenge; when NC8-VP2 and MLT were used alone, the negative conversion rates were all delayed compared with the composition group. The NC8-VP2 group all turned negative on the 8th day after virus challenge, while the MLT group all turned negative on the 11th day after virus challenge. This indicates that the NC8-VP2 + MLT protein composition is beneficial for the FPV-infected cats to excrete the virus and turn negative.

[0175] Table 11 Summary of FPV PCR results of anal swabs of test cats

[0176]

[0177]

[0178] Note: "+" represents positive for FPV PCR; "-" represents negative for FPV PCR.

[0179] (2) Diarrhea situation

[0180] Fecal score standard: normal feces = 0, loose feces but formed = 1, loose feces not formed = 2, watery feces or bloody feces = 3. The fecal scores are as Figure 10 shown. The fecal scores of the test cats in the CK group were all relatively high, and the diarrhea or vomiting situation worsened with the passage of time, and even watery bloody feces appeared in the later stage; while the fecal scores of the test cats in the NC8-VP2 + MLT protein composition group continued to decline and tended to be normal, and there was no vomiting or diarrhea situation; when NC8-VP2 and MLT were used alone, although the diarrhea or vomiting situation was alleviated to some extent, the fecal scores of the test cats were all higher than those in the composition group.

[0181] (3) White blood cell count

[0182] The number of white blood cells in the blood decreased significantly (lower than 4×10 9(cells / mL) are typical symptoms of feline panleukopenia. As Figure 11 shown, the white blood cell count of the test cats in the NC8-VP2+MLT protein composition group increased to normal after a brief decrease; while the white blood cell count of the test cats in the CK group continued to decline, and decreased to below 4×10 9 (cells / mL) on the 5th day after virus challenge, and then remained at a low level; when NC8-VP2 and MLT were used alone, the white blood cell count was lower than that of the NC8-VP2+MLT protein composition group and higher than that of the CK group.

[0183] (4) SAA detection

[0184] Serum amyloid A (SAA) is one of the most effective inflammatory markers in cats, and its level will increase significantly under the stimulation of the acute phase caused by virus infection. In this study, the enzyme-linked immunosorbent assay (ELISA) method was used to detect the SAA level in cat plasma. The detection results showed that after virus challenge, the SAA levels in all four test groups increased significantly, indicating that all the cats participating in the experiment had severe virus infections. In the composition group, the SAA level began to decline from the 5th day after virus challenge; while in the test groups using NC8-VP2 and MLT alone, the decline rate of the SAA level was lower than that of the composition group ( Figure 12 ). This result indicates that the composition can effectively relieve the inflammatory response in cats.

[0185] (5) Intestinal lesions

[0186] The autopsy results showed that the anuses of the test cats in the NC8-VP2+MLT protein composition group were clean and there was no phenomenon of anal soiling. In contrast, the anuses of the other test groups, especially the CK group, were red and swollen, and there was anal soiling to varying degrees; the lymph nodes of the test cats were slightly enlarged, but there was no congestion, and the intestinal pathology observations were all normal; when NC8-VP2 and MLT were used alone, the mesenteric lymph nodes of the test cats were abnormal, and the intestinal villi were partially dissolved and ruptured, without normal tissue morphology; the intestinal congestion of the test cats in the CK group was more severe, and the intestinal villi were severely damaged. (See Figure 13 、 Figure 14 )

[0187] The above results indicate that the NC8-VP2+MLT protein composition can effectively relieve the clinical symptoms such as increased mortality, decreased white blood cells, diarrhea or vomiting caused by FPV virus; compared with the use of NC8-VP2 and MLT alone, the NC8-VP2+MLT protein composition has a synergistic effect on the prevention and treatment of feline panleukopenia.

[0188] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A probiotic preparation composition, characterized in that, Comprising recombinant Lactobacillus plantarum expressing feline panleukopenia virus VP2 protein and melittin; In the microecological preparation composition, the viable count of recombinant Lactobacillus plantarum ≥ 1.0×10 10 CFU / g, and the content of melittin ≥ 50 mg / g.

2. The microecological preparation composition according to claim 1, wherein The microecological preparation composition further comprises pharmaceutically acceptable excipient components.

3. The microecological preparation composition according to claim 1 or 2, characterized in that, The dosage form of the microecological preparation composition is capsule dosage form, tablet, powder, pill, suppository or granule.

4. The microecological preparation composition according to claim 3, characterized in that The dosage form of the microecological preparation composition is capsule, and the capsule weight is (0.4 - 0.5) g / capsule.

5. The preparation method of the microecological preparation composition according to claim 1, characterized in that, Comprising the following steps: (1) Ferment recombinant Lactobacillus plantarum expressing feline panleukopenia virus VP2 protein under anaerobic conditions at 28 - 30 °C for 12 - 14 h, centrifuge to collect the cell precipitate and then perform freeze-drying to prepare the freeze-dried bacterial powder of recombinant Lactobacillus plantarum; (2) Compound the freeze-dried bacterial powder of recombinant Lactobacillus plantarum and melittin to obtain the microecological preparation composition.

6. The preparation method according to claim 5, characterized in that, In step (1), the recombinant Lactobacillus plantarum expressing feline panleukopenia virus VP2 protein is prepared by the following method: After optimizing the VP2 gene of feline parvovirus according to the codon preference of Lactobacillus plantarum and adding a DC cell targeting peptide sequence, insert it into a cloning vector to obtain the target fragment VP2-DCpep, ligate the target fragment VP2-DCpep to an expression vector to obtain a recombinant plasmid, and transform the recombinant plasmid into Lactobacillus plantarum to obtain recombinant Lactobacillus plantarum expressing feline panleukopenia virus VP2 protein.

7. The preparation method according to claim 6, characterized in that, The target fragment VP2-Dcpep sequentially comprises: the optimized VP2 gene sequence, His.Tag gene sequence, DCpep sequence, terminator and restriction enzyme site.

8. The preparation method according to claim 5, characterized in that, In step (2), the melittin is prepared by the following method: Construct recombinant Escherichia coli BL21-pET32a-MLT expressing melittin, homogenize the cells of the fermentation product of recombinant Escherichia coli BL21-pET32a-MLT by centrifugation, and obtain it through affinity chromatography, followed by freeze-drying, desalting column desalting, enterokinase de-tagging, and ion exchange chromatography.

9. The preparation method according to claim 8, wherein The construction method of the recombinant Escherichia coli BL21-pET32a-MLT is as follows: Optimize and synthesize the MLT gene encoding the melittin sequence according to the codon preference of Escherichia coli, ligate it to the cloning vector pUC19, perform PCR amplification to obtain the MLT target gene with correct sequence, and transform it into Escherichia coli after ligating the MLT target gene to the expression vector pET32a.

10. Use of the microecological preparation composition according to any one of claims 1 - 4 in the preparation of a drug for preventing and treating diseases caused by feline parvovirus.

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