A recombinant Bacillus subtilis and its construction method and application

Abstract

The present invention discloses a recombinant Bacillus subtilis and a construction method and application thereof, belonging to the field of biological gene engineering technology. The recombinant Bacillus subtilis is constructed by introducing an expression vector containing a Bacillus subtilis spore capsid protein gene and a porcine reproductive and respiratory syndrome virus antigen gene; the capsid protein gene is CotB or CotC; the antigen gene is a fusion gene ORF56 of ORF5 and ORF6. The present invention also discloses the application of the recombinant Bacillus subtilis in the preparation of a vaccine for the prevention and treatment of porcine reproductive and respiratory syndrome. The recombinant Bacillus subtilis prepared by the present invention has anti-PRRSV virus activity, can produce humoral immunity and cellular immune response in mice, not only provides a new idea for the research and development of vaccines, but also provides a strong technical support for the healthy breeding of pigs.

Description

Technical Field

[0001] The present invention relates to the technical field of biological gene engineering, and in particular to a recombinant Bacillus subtilis and a construction method and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as Blue-ear Disease, is a typical immunosuppressive infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). It is one of the major diseases that seriously endanger the pig industry. PRRSV infection can cause sow abortion, return of estrus, stillbirth or weak piglets, piglet death and respiratory symptoms in pigs of all ages, causing immunosuppression and secondary / mixed infection, making the symptoms of respiratory diseases in pigs more severe and the mortality rate higher. In particular, the continuous emergence of highly pathogenic strains, NADC30-Like strains and even recombinant strains has made the prevention and control of the disease face huge challenges.

[0003] Currently, PRRSV has commercial inactivated vaccines, attenuated live vaccines, and gene chimeric live vaccines. Although these vaccines play an important role in the prevention and control of PRRS disease, they have disadvantages such as low immune efficacy of inactivated vaccines and the easy emergence of recombinant viruses in live vaccines. Therefore, it is necessary to develop a safe and efficient new PRRS vaccine. Summary of the invention

[0004] The purpose of the present invention is to provide a recombinant Bacillus subtilis and a construction method and application thereof to solve the problems existing in the above-mentioned prior art. The recombinant Bacillus subtilis can induce mucosal immunity and body immune response in mice, has the ability to resist PRRSV infection, can be used as a vaccine for preventing and treating PRRSV, and has practical application value.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a recombinant Bacillus subtilis, which is constructed by introducing an expression vector containing a Bacillus subtilis spore capsid protein gene and a porcine reproductive and respiratory syndrome virus antigen gene;

[0007] The capsid protein gene is CotB or CotC; the antigen gene is ORF56, a fusion gene of ORF5 and ORF6;

[0008] The accession number of CotB or CotC in NCBI is AL009126.3; the nucleotide sequence of the fusion gene ORF56 is shown in SEQ ID NO:1.

[0009] Furthermore, the fusion gene ORF56 is connected to the end of the CotB or CotC sequence.

[0010] The present invention also provides a method for constructing the recombinant Bacillus subtilis, comprising the following steps:

[0011] Recombinant plasmids of fusion gene ORF56, CotB or CotC were constructed respectively;

[0012] The recombinant plasmid of the fusion gene ORF56 and the recombinant plasmid of CotB or CotC were double-digested;

[0013] The double-enzyme digestion products are subjected to ligation reaction;

[0014] The ligation product is transformed into Bacillus subtilis.

[0015] Furthermore, the fusion gene ORF56 recombinant plasmid is pMD19-T-ORF56 recombinant plasmid; the CotB or CotC recombinant plasmid is pDG-CotB or pDG-CotC recombinant plasmid.

[0016] Furthermore, the ligation product is a pDG-CotB-ORF56 or pDG-CotC-ORF56 recombinant vector.

[0017] The present invention also provides a medicine for preventing and treating porcine reproductive and respiratory syndrome, comprising the recombinant Bacillus subtilis.

[0018] Furthermore, the medicament comprises an oral vaccine.

[0019] The present invention also provides the use of the recombinant Bacillus subtilis in preparing a vaccine for preventing and treating porcine reproductive and respiratory syndrome.

[0020] The present invention discloses the following technical effects:

[0021] The present invention successfully prepared a recombinant Bacillus subtilis with key antigenic proteins of PRRSV displayed on the surface of Bacillus subtilis spores. The recombinant bacteria has anti-PRRSV virus activity, which not only provides new ideas for the development of vaccines, but also provides strong technical support for the healthy breeding of pigs. The present invention induces mucosal immunity and body immune response in mice by intragastric administration of recombinant bacteria, and evaluates the ability of recombinant Bacillus subtilis oral vaccine to fight PRRSV infection. The results show that intragastric administration of recombinant Bacillus subtilis produces specific humoral immunity and cellular immune response in mice, revealing that it can be used as a vaccine for the prevention and treatment of PRRSV and has practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Figure 1 shows the restriction enzyme digestion of the recombinant plasmid pDG; M: DNA molecular mass standard marker; 1: recombinant plasmid pDG364-CotC-ORF56; 2: recombinant plasmid pDG364-CotC-ORF56 digested with BamHI and EcoRI (1300bp); 3: recombinant plasmid pDG364-CotC-ORF56 digested with BamHI and HindIII (220bp); 4: recombinant plasmid pDG364-CotC-ORF56 F56 was double-digested with HindⅢ and EcoRI (1110 bp); 5: recombinant plasmid pDG364-CotB-ORF56; 6: recombinant plasmid pDG364-CotB-ORF56 was double-digested with BamHI and EcoRI (2200 bp); 7: recombinant plasmid pDG364-CotB-ORF56 was double-digested with BamHI and HindⅢ (1100 bp); 8: recombinant plasmid pDG364-CotB-ORF56 was double-digested with HindⅢ and EcoRI (1110 bp);

[0024] Figure 2 This is a picture of amylase activity identification; A: original strain; B: recombinant strain B56; C: recombinant strain C56;

[0025] Figure 3 The figure shows the results of PCR identification of recombinant Bacillus subtilis; M: DNA molecular mass standard marker; Y: original strain; K: blank control; Cb: recombinant strain B56; Cc: recombinant strain C56;

[0026] Figure 4 The Western-Blot analysis results of recombinant Bacillus subtilis B56 and C56; M: protein quality standard; Y: original strain;

[0027] Figure 5 Immunofluorescence microscopy images; A, B, C: bright field images; D, E, F: fluorescence images; A, D: original strain; B, E: recombinant strain B56; C, F: recombinant strain C56;

[0028] Figure 6 This is a statistical chart of the test results of mouse serum IgG antibody levels;

[0029] Figure 7 This is a statistical chart of the test results of mouse sIgA antibody levels;

[0030] Figure 8 Results of flow cytometry analysis of mice; A: CD4 + / CD3 + T cell; B: CD8 + / CD3 + Tcell;

[0031] Fig. 9 Effects of recombinant bacteria on the expression of cytokines in mice; A: IL-2; B: IL-4; C: IL-6; D: IL-10; E: TNF-α; F: IFN-γ;

[0032] In the above figures, ns: p>0.05; *: p≤0.05; **: p≤0.01; ***: p≤0.001. DETAILED DESCRIPTION

[0033] Example 1 Construction of recombinant Bacillus subtilis

[0034] 1 Experimental Materials

[0035] Porcine reproductive and respiratory syndrome virus R98 live vaccine is a product of Qilu Animal Health Products Co., Ltd.; Bacillus subtilis 12 was screened from pig environmental samples and preserved by the Key Laboratory of Animal Husbandry and Veterinary Medicine, Shandong Academy of Agricultural Sciences; pDG364 vector is preserved by the Key Laboratory of Animal Husbandry and Veterinary Medicine, Shandong Academy of Agricultural Sciences.

[0036] 2. Target gene amplification

[0037] 2.1 Primer design

[0038] Table 1 Primer list

[0039]

[0040] Note: The underlined sites are restriction enzyme cutting sites.

[0041] Based on the complete gene sequences of PRRSV and B. subtilis provided in GeneBank, 5 pairs of specific primers were designed using the software Primer5, which were used to amplify the spore capsid protein genes CotB and CotC of Bacillus subtilis No. 12, the GP5 (encoding gene ORF5) and M (encoding gene ORF6) proteins of PRRSV, and the amylase (amyE) gene.

[0042] 2.2 Genome extraction

[0043] Follow the instructions of FastPure Viral DNA / RNA Mini Kit (Nanjing Novozyme) to extract viral RNA. The instructions of IIIRT SuperMix for qPCR (+gDNAwiper) (Nanjing Novozyme) were used to reverse transcribe PRRSV viral RNA and convert it into cDNA for stable storage. The bacterial genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract the genome of Bacillus subtilis.

[0044] 2.3 Target fragment amplification

[0045] (1) Amplification of ORF5 and ORF6 antigen fragments: Using the extracted porcine reproductive and respiratory syndrome virus cDNA as a template and ORF5-F / ORF5-R and ORF6-F / ORF6-R as primers, the ORF5 and ORF6 fragments were amplified. The PCR reaction system and reaction procedure are shown in Tables 2 and 3.

[0046] Table 2 Amplification of ORF5 target gene

[0047]

[0048] Table 3 Amplification of ORF6 target gene

[0049]

[0050] (2) Amplification of ORF56 target gene: The ORF5 and ORF6 target genes were connected and the GP5-Linker-M fusion protein was amplified. The target gene obtained by the above PCR reaction was used as a template, and ORF5-F / ORF6-R were used as upstream and downstream primers for touchdown PCR reaction to obtain the long fragment ORF56 target gene. The reaction system and reaction procedure used in the experiment are shown in Table 4.

[0051] Table 4 ORF56 target gene amplification procedure

[0052]

[0053] (3) Amplification of CotB gene: Genomic DNA extracted from Bacillus subtilis was used as a template, and the CotB-F / R primer pair was selected to amplify the target gene fragment CotB. The PCR reaction system and reaction procedure are shown in Table 5.

[0054] Table 5 CotB target gene amplification

[0055]

[0056] (4) Amplification of CotC gene: Genomic DNA extracted from Bacillus subtilis was used as a template, and the CotC-F / R primer pair was selected to amplify the target gene fragment CotC. The PCR reaction system and reaction procedure are shown in Table 6.

[0057] Table 6 CotC target gene amplification

[0058]

[0059] After the PCR reaction is completed, agarose gel electrophoresis is performed to observe whether the size and position of the target gene fragment are consistent with expectations. If the electrophoresis results are correct, the PCR purification and recovery kit is used to purify and recover the target fragment according to the steps, and the recovered product is stored in a -20°C freezer.

[0060] 3 Construction of recombinant vector plasmid

[0061] 3.1T carrier connection

[0062] The purified and recovered PCR products were prepared into a ligation system according to the instructions of the pMD19-T kit, and ligated overnight at 16°C to construct plasmids pMD19-T-ORF56, pMD19-T-CotB, and pMD19-T-CotC. An appropriate amount of ligation products was preliminarily detected and analyzed by agarose gel electrophoresis, and the products that were successfully verified were used in subsequent transformation experiments. The ligation system is shown in Table 7.

[0063] Table 7T vector plasmid connection system

[0064]

[0065] 3.2 Transformation experiment

[0066] The transformation experiment was carried out according to the instructions using Escherichia coli competent cells Trans 5α. After overnight culture on ampicillin (concentration of 50 μg / mL) resistant LB plates, single clone colonies were selected and inoculated in LB liquid medium containing the same resistance for shaking culture. The plasmids of the above bacterial solution were extracted according to the steps in the instructions of the Tiangen Plasmid Extraction Kit, the plasmid concentration was determined, and then double enzyme digestion identification was performed. The plasmids containing the CotB and CotC target gene fragments were double digested with BamHⅠ and HindⅢ, and the plasmids containing the ORF56 target gene fragments were double digested with HindⅢ and EcoRⅠ. The double enzyme digestion system is shown in Table 8. Take 5 μL of the double enzyme digestion product for detection by agarose gel electrophoresis. The successfully verified samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The plasmids with correct sequencing results were named pMD19-T-ORF56, pMD19-T-CotB, and pMD19-T-CotC.

[0067] Table 8T vector double restriction enzyme digestion system

[0068]

[0069] 3.3 Recombinant plasmid construction

[0070] Plasmids pMD19-T-CotB, pMD19-T-CotC and plasmid pDG364 were double-digested with BamHⅠ and HindⅢ (double-digestion system is the same as Table 8), and the double-digestion products were verified by agarose gel electrophoresis. After successful verification, a special DNA gel recovery kit was used to recover the linear double-digested plasmid pDG, double-digestion products CotB, CotC target genes. The pDG plasmid was connected to the target genes CotB and CotC. The connection reaction system and reaction conditions are shown in Table 9. After the connection product was transformed into Escherichia coli competent cells Trans 5α, a single colony was selected from the resistance plate for expansion culture, and then the plasmid was extracted for double-digestion and PCR amplification verification. After preliminary verification, the correct recombinant plasmids were named pDG-CotB and pDG-CotC.

[0071] Table 9pDG364 plasmid ligation system

[0072]

[0073] The plasmid pMD19-T-ORF56 and the recombinant plasmids pDG-CotB and pDG-CotC were double-digested with HindⅢ and EcoRⅠ (the double-digestion system is the same as Table 8), and then the gel recovery kit was used to recover and purify the target gene fragment ORF56 and the linear plasmids pDG-CotB and pDG-CotC after double-digestion. The purified and recovered target gene fragment ORF56 was connected to the linear recombinant plasmids pDG-CotB and pDG-CotC, respectively. The connection reaction system and conditions are shown in Table 10. Subsequently, transformation experiments, plasmid extraction, PCR amplification, double-digestion electrophoresis analysis were carried out. After preliminary verification, the recombinant plasmids were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The recombinant plasmids with correct sequences were named pDG-CotB-ORF56 and pDG-CotC-ORF56.

[0074] Table 10pDG recombinant vector connection system

[0075]

[0076]

[0077] 4 Construction of recombinant Bacillus subtilis

[0078] 4.1 Preparation of competent Bacillus subtilis

[0079] The two-step sampling method was used to prepare Bacillus subtilis competent cells. The formula and dosage of the competent culture medium are shown in Table 11. The specific steps are as follows:

[0080] Cultivate Bacillus subtilis No. 12 on a shaking table overnight, transfer 200 μL of bacterial solution to 10 mL of SPⅠ medium, and culture at 37℃ and 200 rpm for 3-4 hours. Take part of the bacterial solution every 0.5 hours to measure the OD value; when the OD value is about 1.1, take 1 mL of bacterial solution and inoculate it into 10 mL of SPⅡ medium, culture on a shaking table for 1.5 hours, add 100 μL of 100× EGTA solution for treatment, and after shaking culture for 10 minutes, it will be transformed into competent bacteria that can be used for transformation experiments; add 2 mL of 60% glycerol, and dispense 500 μL each into 1.5 mL centrifuge tubes, and store in a -80℃ refrigerator. All the above reagents must be prepared before use.

[0081] Table 11 Preparation of culture medium for competent Bacillus subtilis

[0082]

[0083] 4.2 Transformation of recombinant plasmid

[0084] The recombinant vector plasmids pDG-CotB-ORF56 and pDG-CotC-ORF56 (single enzyme digestion system, see Table 8) were digested with XbaⅠ restriction endonuclease to obtain recombinant linear plasmids. 10 μL of single enzyme digestion products pDG-CotB-ORF56 and pDG-CotC-ORF56 were transferred into 500 μL of Bacillus subtilis competent cells and gently mixed evenly. The transformation experiment was carried out, and chloramphenicol resistance plates (working concentration of 5 μg / mL) were used for culture, and the temperature was kept at 37°C overnight. Single colony transformants were selected, and PCR amplification and amylase activity analysis were performed to verify the characteristics of the recombinant bacteria.

[0085] 5. Identification, screening and analysis of recombinant Bacillus subtilis

[0086] 5.1 Identification of amylase activity and PCR identification

[0087] Single colonies were selected from the resistant plates for expansion culture. 2.5 μL of bacterial solution was gently dripped onto the starch hydrolysis solid medium. Bacillus subtilis No. 12 was used as a control. After overnight culture at 37°C, the amylase activity was analyzed.

[0088] The genome of the recombinant Bacillus subtilis was extracted, and PCR amplification was performed on the original bacteria and the recombinant bacteria using specific primers amyE-F / R, amyE-F / CotB(C)-R, ORF56-F / amyE-R, and ORF56-F / R, respectively. The PCR reaction system and conditions were based on the amplification of the target gene in 2.3. The correct recombinant Bacillus subtilis strains verified by electrophoresis were named B56 and C56, respectively.

[0089] 5.2 Preparation of Bacillus subtilis spores

[0090] The recombinant bacteria B56, C56 and the original strain cultured overnight were added to DSM spore medium at a ratio of 1:100 and cultured at 37°C with constant temperature shaking. The bacterial concentration was detected every 0.5 h. When the bacterial OD value was about 0.1, the constant temperature shaking culture was continued for 48 h. The spore production in the culture medium was observed under a microscope using a spore staining kit. When the spore rate reached more than 90%, the bacterial solution was centrifuged at 4000 rpm for 30 min at 4°C, and the collected spores were temporarily stored at -20°C.

[0091] 5.3 Western-Blot analysis of recombinant spore capsid protein

[0092] The collected spores were treated with 50 μg / mL lysozyme Tris-HCl buffer at 37°C for 1 h, centrifuged to obtain the precipitate, washed with sterile deionized water, and then resuspended in PBS. Then, an appropriate amount of loading buffer was added, and the mixture was boiled in boiling water for 10 min. Then, Western-blot analysis of the expression of the recombinant protein was performed. The blocking solution was 5% skim milk powder, the primary antibody was a rabbit PRRSV GP5 protein polyclonal antibody (1:3000 dilution), and the secondary antibody was HRP-labeled goat anti-rabbit IgG (1:2000 dilution). The GE Image Quant LAS 500 integrated imager was used for imaging.

[0093] 5.4 Immunofluorescence microscopy of recombinant spores

[0094] The collected spores were diluted and evenly spread on polylysine anti-smear slides, air-dried and washed with PBS, fixed with paraformaldehyde, blocked with 5% BSA for 1 hour, incubated with PRRSV GP5 polyclonal antibody (1:300 dilution) at 4°C overnight, and then incubated with CoraLite 594-labeled goat anti-rabbit IgG (1:500 dilution) at room temperature in the dark for 1 hour. Finally, the slides were evenly covered with anti-fluorescence attenuation quencher, covered with coverslips, and observed and imaged under a fluorescence microscope.

[0095] 6 Results

[0096] 6.1 Amplification of target gene

[0097] Using the extracted Bacillus subtilis DNA as a template, the Bacillus subtilis spore capsid protein CotB gene (1100bp) and CotC gene (215bp) were amplified by PCR; the PRRSV ORF5 gene (642bp) and ORF6 gene (547bp) were amplified by RT-PCR technology, and the fusion fragment ORF56 was amplified using the respectively amplified ORF5 and ORF6 gene fragments as templates. The accession numbers of CotB gene and CotC gene in NCBI are both AL009126.3, the sequence of CotB amplified fragment is located at 3715057bp-3716144bp of the accession number sequence, and the sequence of CotC gene amplified fragment is located at 1904995bp-1905192bp of the accession number sequence; the accession numbers of ORF5 gene and ORF6 gene in NCBI are both DQ355796.1, the sequence of ORF5 gene fragment is located at 1096bp-1692bp of the accession number of NCBI, and the sequence of ORF6 gene fragment is located at 1683bp-2204bp of the accession number of NCBI; the nucleotide sequence of fusion fragment ORF56 is shown in SEQ ID NO: 1:

[0098]

[0099] The results of gel electrophoresis analysis of all PCR products proved that all target fragments were correctly amplified and could be used for subsequent experimental research.

[0100] 6.2 Plasmid digestion identification results

[0101] The recombinant plasmid pDG364-CotB-ORF56 had obvious bands at 1100bp and 7400bp after double digestion with BamHI and HindⅢ, obvious bands at 1120bp and 7400bp after double digestion with HindⅢ and EcoRI, and obvious bands at 2200bp and 6300bp after double digestion with BamHI and EcoRI. The recombinant plasmid pDG364-CotC-ORF56 had obvious bands at 1100bp and 7400bp after double digestion with HindⅢ and EcoRI. After double digestion, there were obvious bands at 220bp and 7400bp, after double digestion with BamHⅠ and EcoRⅠ, there were obvious bands at 1300bp and 6300bp, and after double digestion with HindⅢ and EcoRⅠ, there were obvious bands at 1120bp and 6500bp. The size of the double digestion product bands was consistent with the theory. All the above experimental results proved that the recombinant plasmids pDG364-CotB-ORF56 and pDG364-CotC-ORF56 were successfully constructed. Figure 1 ).

[0102] 6.3 Recombinant Bacillus subtilis amylase activity and PCR identification

[0103] Bacillus subtilis contains amylase gene. After the recombinant plasmid pDG364-CotB-ORF56 and pDG364-CotC-ORF56 are introduced into Bacillus subtilis, the homologous fragments of amylase are fused and crossed. The constructed and expressed CotB-ORF56 and CotC-ORF56 target genes are introduced into the amylase gene fragments, resulting in the inactivation of amylase in the recombinant bacteria. Wild-type Bacillus subtilis and recombinant Bacillus subtilis are added dropwise on the soluble starch medium plate. After overnight culture, iodine solution is sprayed. The wild-type Bacillus subtilis colony has complete amylase activity to decompose starch to form a transparent enzymatic hydrolysis circle, while the recombinant Bacillus subtilis is the opposite ( Figure 2 ).

[0104] To further prove that the insertion of CotB-ORF56 and CotC-ORF56 genes in recombinant Bacillus subtilis leads to the inactivation of amylase, this study used wild-type Bacillus subtilis as a control and used four pairs of primers, amyE-F / R, amyE-F / CotB-R, amyE-F / CotC-R, and ORF56-F / R, to perform PCR amplification to identify whether the recombinant Bacillus subtilis was successfully constructed. The results showed that under the three pairs of primers amyE-F / CotB-R, amyE-F / CotC-R, and ORF56-F / R, the wild-type Bacillus could not amplify bands, while the recombinant Bacillus had obvious bands at the theoretical position. Under the amyE-F / R primer amplification, the wild-type Bacillus amplified a DNA fragment of about 760bp, and the recombinant Bacillus subtilis amplified a DNA fragment of about 3700bp and 3000bp, respectively ( Figure 3 ). Both amylase activity and PCR amplification proved that the recombinant Bacillus subtilis was successfully constructed. The recombinant strain containing the CotB-ORF56 target fragment was named B56, and the recombinant strain containing the CotC-ORF56 target fragment was named C56.

[0105] 6.4 Western-Blot Identification and Immunofluorescence Microscopy Analysis of Recombinant Spores

[0106] In order to identify whether the fusion genes CotB-ORF56 and CotC-ORF56 were successfully expressed on the spore surface, Western-Blot method was used for detection. Figure 4 The results showed that no specific bands were observed in the spore capsid protein component of the original strain, but a clear target band of about 48 kDa appeared in the spore capsid protein component of the recombinant strain C56; a target band of about 68 kDa also appeared in the spore capsid protein component of the recombinant strain B56. The sizes of these protein target bands were completely consistent with the experimental expectations, thus confirming the successful expression of the fusion gene on the spore surface. At the same time, the results of the immunofluorescence test showed that under the fluorescence microscope, the original strain had no fluorescence, while the recombinant strains B56 and C56 had obvious red fluorescence on the spore surface ( Figure 5 ). Western-Blot analysis and immunofluorescence microscopy observations confirmed that the GP5-M fusion protein was successfully displayed on the surface of Bacillus subtilis spores. The fragment was effectively expressed under the induction of CotB and CotC coat protein promoters and could be accurately recognized and bound by PRRSV-specific antibodies.

[0107] 7 Results Analysis

[0108] The GP5 protein encoded by ORF5 of PRRSV virus is the receptor binding protein of the virus, and the M protein encoded by ORF6 is closely related to the assembly and budding of the virus, and are two key immunogenic proteins of the virus; the spores produced by Bacillus subtilis in harsh environments can survive for a long time, and the coat protein gene of the spore is connected to the target protein to display the key protein of PRRSV on the surface of the spore. In this experiment, the GP5 and M proteins of PRRSV virus were connected by Linker protein, and the foreign protein gene was connected to the coat protein gene CotB of Bacillus subtilis spore and the CotC terminus to construct the fusion protein fragments CotB-ORF56 and CotC-ORF56, and the fusion fragments were introduced into Bacillus subtilis through the expression vector, and the target gene was introduced into the chromosome through homologous recombination, so that the GP5 and M proteins of PRRSV virus were expressed with the production of spores, and recombinant Bacillus subtilis B56 and C56 were successfully constructed. The exogenous target protein gene ORF56 is integrated into the Bacillus subtilis sequence and expressed along with the production of spores, thereby avoiding the loss of the target gene fragment, and can be replicated along with the replication of the host chromosome, and protein preparation and purification can be carried out along with the amplification and purification of the spore coat protein.

[0109] The amylase activity of the successfully constructed recombinant bacteria B56 and C56 was identified, and it was found that the amylase activity of the recombinant bacteria was lost, and starch was no longer decomposed. The colonies were sprayed with iodine solution and turned blue; the original Bacillus subtilis could decompose starch, and a transparent enzymatic circle appeared around the colonies sprayed with iodine solution. The PCR amplification results proved that the exogenous protein gene was successfully inserted into the Bacillus chromosome sequence; the recombinant bacterial spores were analyzed by immunofluorescence microscopy, and specific fluorescent signals were found on the surface of the recombinant spores, proving that the exogenous protein was successfully fused with the spore coat protein; the purified spore capsid protein was analyzed by total protein Western-Blot, and specific bands appeared at 68KDa for the recombinant bacteria B56 and 48KDa for the recombinant bacteria C56, proving that the exogenous protein expressed on the surface of the recombinant bacteria is immunogenic and can be used in subsequent mouse animal experiments to test its immune effect.

[0110] Example 2 Study on the immune effect of recombinant Bacillus subtilis on mice

[0111] 1 Experimental methods

[0112] 1.1 Mouse grouping and spore preparation

[0113] 80 mice (5-week-old BalB-C female mice and feed were purchased from Pengyue Co., Ltd.) were randomly divided into 4 groups. The specific grouping is shown in Table 12. The mice were gavaged on the 0th, 14th, and 21st days after the start of the experiment. The PBS group was gavaged with 100 μL PBS each time, and the original bacteria group was gavaged with 100 μL Bacillus subtilis spore solution (spore concentration was 2.0×10 10CFU / mL), and the B56 and C56 experimental groups were gavaged with 100 μL of recombinant Bacillus subtilis spore solution each time (spore concentration was 2.0×10 10 CFU / mL). The mice were raised in an animal room with good ventilation and a stable indoor temperature of 18-24℃. The bedding was changed regularly. The mouse room was fully fumigated and disinfected before the mouse experiment. The mice were pre-raised for 7 days to make them better adapt to the experimental environment and eliminate environmental influences. Each group of mice had free access to food and water, and the feeding period was set at 35 days.

[0114] Table 12 Mouse experimental grouping

[0115]

[0116] 1.2 Sample collection

[0117] Three mice were randomly selected from each group on days 0, 7, 14, 21, 28, and 35 of the experiment. The mice were enucleated to collect blood samples and euthanized after the blood collection was completed. The small intestinal contents of these mice were collected under sterile conditions; the spleen of the mice was collected on day 35 for lymphocyte factor detection, CCK8 cell proliferation experiment, and flow cytometry; the cecal contents of the mice were collected for microbial community analysis.

[0118] 1.3 Determination of mouse weight growth

[0119] Bacillus subtilis is one of the probiotics that enhances the body's immune function. The weight gain of mice is the most intuitive indicator to measure the biological effect of recombinant Bacillus on the body. Starting from the first day of the formal experiment, the weight of each group of mice was weighed every week to calculate the average weekly weight gain of mice and the weight gain ratio of mice during the experimental period.

[0120] 1.4 ELISA detection of mouse antibody levels

[0121] Blood was collected from the mouse eyeballs and collected in a pre-sterilized 1.5 mL centrifuge tube. After standing for 2 hours, the blood was centrifuged at 3000 rpm for 20 minutes to collect serum and temporarily stored in a -80°C refrigerator for subsequent detection of mouse serum IgG antibody levels. The mice were killed after blood was collected, and after sterile dissection of the mice, 0.5 g of the mouse small intestinal contents were collected and placed in a 5 mL centrifuge tube with 3 mL of sterile pre-cooled PBS added. The supernatant was taken and temporarily stored in a -80°C refrigerator for detection of sIgA antibody levels in the mouse small intestinal contents.

[0122] The Haibolai PRRSV indirect ELISA antibody detection kit is used to detect serum antibody levels, and the Jianglai (sIgA) enzyme-linked immunosorbent assay kit is used to determine the sIgA antibody level in the small intestinal contents of mice.

[0123] 1.5 Preparation of mouse spleen lymphocytes

[0124] The spleens of 35-day-old mice were collected aseptically, washed once in the previously prepared PBS and 1640 basal medium, and a soybean-sized spleen was placed in a 1.5 mL sterile centrifuge tube and temporarily stored in a -80°C refrigerator for subsequent lymphocyte factor level detection experiments. Lymphocytes were extracted from the remaining spleen.

[0125] 1.6 Flow cytometry to detect the number of mouse lymphocyte subsets

[0126] The effect of oral immunization on the number of lymphocyte subsets in mice was further tested, and flow cytometry was used to detect the changes in the number of cells in each group of mice.

[0127] 1.7 CCK8 detection of mouse lymphocyte proliferation

[0128] In order to detect the effect of oral immunization on the specific cellular immune response and cell activity in mice, CCK8 kit (Abcam (Shanghai) Trading Co., Ltd.) was used to detect cell proliferation.

[0129] 1.8 Detection of lymphocyte factor levels in mouse spleen

[0130] Conventional fluorescence quantitative PCR was used to detect the expression of cytokines IL-2, IL-4, IL-6, IL-10, TNFα, and IFN-γ in mice to evaluate the immune response of mice.

[0131] Table 13 Mouse cytokine related primer sequences

[0132]

[0133]

[0134] 1.9 Data Statistics and Processing

[0135] Graphpad Prism5 was used for data processing. R language was used for Venn diagram analysis. Qiime software was used to calculate Alpha and Beta diversity. Gephi was used to draw the network diagram of related item analysis. The groups were compared by t-test, and the results were expressed as the mean.

[0136] 2 Results

[0137] 2.1 Effect of recombinant spores on mouse body weight

[0138] Before the experiment, there was no significant difference in the initial weight of the mice in each group, but on the 35th day, the weight gain of the experimental group mice was significantly higher than that of the control group mice (P≤0.05). The weight gain of B56 and C56 mice gavaged with recombinant spores was more obvious than that of the original strains, and the weight gain ratio of the C56 group mice was significantly different from that of the original bacteria group. The results of mouse weight gain showed that the recombinant bacteria improved the intestinal environment of mice to a certain extent, promoted the absorption of nutrients, and increased the weight gain ratio of experimental mice.

[0139] Table 14 Body weight changes of mice after feeding spores (g)

[0140]

[0141] 2.2 Indirect ELISA antibody level detection

[0142] 2.2.1 Serum IgG antibody level test results

[0143] IgG antibodies, as a key component of the immune system, play an important role in controlling viral infection. Their levels directly reflect the immune status of mice. Figure 6 ), obvious specific antibodies can be detected in the serum of B56 and C56 recombinant Bacillus subtilis groups. After the first oral gavage, there was no obvious fluctuation in the antibodies in the mice, and the difference in antibody levels on the 14th day was not significant. After the third oral gavage, the serum IgG antibody level in the mice reached the highest level on the 28th day. The antibody levels of the recombinant bacteria group mice and the original bacteria group mice were significantly different, which was significantly higher than the amount of antibodies produced by the original bacteria group mice. The antibody levels of the control group and the original bacteria group did not change significantly during the whole experiment, proving that only recombinant bacteria can induce specific immune responses in mice. On the 35th day, the antibody level in the mice tended to stabilize and decreased, but the antibody level of the recombinant bacteria C56 was significantly higher than that of the recombinant bacteria B56 (P<0.01).

[0144] 2.2.2 Results of sIgA antibody level detection in mouse small intestine contents

[0145] As a key effector of mucosal immunity, the formation process of secretory IgA includes several steps: first, in the intestinal mucosa, monomeric IgA binds to the J chain to form a dimer, and then the dimer binds to the component secreted by the epithelial cells (i.e., the secretory component, abbreviated as SC), and finally forms secretory IgA, which is an important indicator for detecting mucosal immune responses. During the experiment, the collected samples of the small intestinal contents of mice were specially tested for specific sIgA antibody levels. According to the final test results, it can be clearly known that mice treated with B56 and C56 recombinant Bacillus subtilis by gavage successfully induced specific sIgA antibodies. This finding directly verifies that recombinant Bacillus subtilis can induce mucosal immune responses in mice. On the 21st day, obvious specific antibody levels were detected in the B56 and C56 groups, and the antibody level peaked on the 28th day of the experiment. Starting from the 14th day of the experiment, the antibody levels of mice gavaged with recombinant bacteria were significantly different from those of mice in the original bacteria group. The secretion of sIgA antibodies increased significantly, and the specific antibodies in the mice in the gavage C56 recombinant Bacillus group were higher than those in the B56 recombinant bacteria group. In the control group and the original bacteria group, the specific sIgA antibody levels of mice were only slightly increased before and after the experiment, and there was no significant change ( Figure 7 ).

[0146] 2.3 Results of mouse flow cytometry analysis

[0147] In order to observe the effect of recombinant Bacillus B56 and C56 on the number of spleen lymphocyte subsets after intragastric immunization of mice, the number of spleen cell subsets of mice was detected by flow cytometry, and the CD8 + / CD3 + , CD4 + / CD3 + The experimental data revealed that ( Figure 8 ), compared with the control group, mice treated with recombinant Bacillus had a significantly higher CD8 + and CD4 + The percentages of cell subpopulations increased, and significant differences were found between the group gavage with Bacillus subtilis and the control group, proving that the recombinant bacterial spores have an effect on cellular immune response.

[0148] 2.4 Results of mouse lymphocyte proliferation experiment

[0149] On the 35th day of the experiment, the CCK8 proliferation experiment was used to measure the lymphocyte proliferation index of each group of mice. The results showed that the lymphocyte proliferation index of the experimental group of mice was higher than that of the control group. Bacillus subtilis can stimulate the proliferation of lymphocytes to a certain extent, and there was a significant difference in the proliferation index between the mice gavage with recombinant Bacillus subtilis and the mice gavage with original bacteria, indicating that gavage with recombinant Bacillus subtilis spores can effectively promote the growth and activation of mouse lymphocytes.

[0150] Table 15 CCK8 proliferation index of mouse lymphocytes on day 35

[0151]

[0152] 2.5 Results of detection of lymphocyte factor levels in mouse spleen

[0153] On the 35th day of the experiment, the mice were aseptically dissected and part of the spleen was taken to extract the total cellular RNA content. This was used as a template for reverse transcription into cDNA for fluorescent quantitative PCR experiments to detect the expression levels of different cytokines in mouse lymphocytes. The various cytokines secreted by mouse spleen cells play an important immunoregulatory role in mice and can affect the function of immune cells. Specifically, the secretion of IFN-γ, TNF-α, and IL-2 can promote the proliferation of T cells, thereby stimulating the body to produce a cellular immune response; while the secretion of IL-4, IL-6, and IL-10 helps B cells produce specific antibodies, thereby stimulating the body's humoral immune response. The cytokines work synergistically to maintain the immune balance and health of mice. Fig. 9 It can be seen that the mRNA content of cytokines in the spleen of the experimental group mice increased significantly, which was significantly different from that of the original bacteria group mice, proving that oral administration of Bacillus subtilis enhanced the immunity of mice and stimulated the expression of cytokines in mice. Among them, the change in the content of IFN-γ cytokines was not as obvious as the changes in the levels of other cytokines. This may be related to the fact that the recombinant spores mainly stimulated the mucosal immunity of mice and enhanced the humoral immune response of mice. There was a difference in the expression of cytokines between the mice in the oral administration of the original Bacillus subtilis group and the control group. As a probiotic, Bacillus subtilis regulates a variety of metabolic reactions in the body, enhances the immunity of mice, and may stimulate the production of lymphocyte factors.

[0154] 3. Results summary and analysis

[0155] This study used the spore coat protein of Bacillus subtilis as a surface display carrier of PRRSV protein, aiming to construct an efficient recombinant spore vaccine to prevent and treat PRRSV infection. This study successfully prepared a recombinant spore vaccine with anti-PRRSV virus activity. This achievement not only provides new ideas for vaccine research and development, but also provides strong technical support for the healthy breeding of pigs. By inducing mucosal immunity and specific body immune response in mice through gavage, the ability of recombinant Bacillus spore oral vaccine to fight PRRSV infection was evaluated, and whether it could provide antibodies to block viral infection when PRRSV infects the body through the mucosa, preventing the virus from invading the body from the source. The results showed that after feeding mice with recombinant Bacillus subtilis, the weight gain of mice changed significantly. Bacillus subtilis as a probiotic supplement improved the intestinal health of mice, increased feed utilization, and increased feed-to-weight ratio; the detection of mouse serum IgG-specific antibodies and the detection of sIgA antibody levels in mouse small intestine contents proved that the recombinant spores stimulated the immune response of mice to a certain extent; the results of flow cytometry analysis showed that the distribution of cell subpopulations in the mice gavaged with Bacillus subtilis was significantly different from that in the control group. This difference showed that the introduction of Bacillus subtilis changed the proportion of cell subpopulations in mice, thereby triggering the cellular immune response of mice. In order to further verify this finding, a CCK8 cell proliferation experiment was conducted, and the experimental results showed that the recombinant spores can effectively promote the proliferation and activation of mouse spleen lymphocytes. The results of mouse lymphocyte factor mRNA content showed that the content of various cytokines in the mice gavage group with recombinant Bacillus was significantly higher than that in the original bacteria group, and the cytokine content in the mice gavage group with original Bacillus subtilis was also slightly increased compared with the control group. Gavage with recombinant spores produced humoral and cellular immune responses in mice, stimulating the mice to produce immune responses.

[0156] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A use of a recombinant Bacillus subtilis in the preparation of a vaccine for preventing and treating porcine reproductive and respiratory syndrome, characterized in that: The construction method of the recombinant Bacillus subtilis is as follows: Recombinant plasmids of the fusion gene ORF56 of GP5 and M protein of porcine reproductive and respiratory syndrome virus and the spore capsid protein gene CotB or CotC of Bacillus subtilis were constructed respectively; The recombinant plasmid of the fusion gene ORF56 and the recombinant plasmid of CotB or CotC were double-digested; The double-enzyme digestion products are subjected to ligation reaction; The ligation product is transformed into Bacillus subtilis; The recombinant plasmid of the fusion gene ORF56 is the pMD19-T-ORF56 recombinant plasmid; the recombinant plasmid of CotB or CotC is the pDG-CotB or pDG-CotC recombinant plasmid; the ligation product is the pDG-CotB-ORF56 or pDG-CotC-ORF56 recombinant vector; The fusion gene ORF56 is connected to the end of the CotB or CotC sequence; the accession number of the CotB or CotC in NCBI is AL009126.3, the amplified fragment sequence of the CotB is located at 3715057 bp-3716144 bp of the accession number sequence, and the gene amplified fragment sequence of the CotC is located at 1904995 bp-1905192 bp of the accession number sequence; The nucleotide sequence of the fusion gene ORF56 is shown in SEQ ID NO:

1.

2. A method for constructing a recombinant Bacillus subtilis, characterized in that: The following steps are involved: Recombinant plasmids of the fusion gene ORF56 of GP5 and M protein of porcine reproductive and respiratory syndrome virus and the spore capsid protein gene CotB or CotC of Bacillus subtilis were constructed respectively; The recombinant plasmid of the fusion gene ORF56 and the recombinant plasmid of CotB or CotC were double-digested; The double-enzyme digestion products are subjected to ligation reaction; The ligation product is transformed into Bacillus subtilis; The recombinant plasmid of the fusion gene ORF56 is the pMD19-T-ORF56 recombinant plasmid; the recombinant plasmid of CotB or CotC is the pDG-CotB or pDG-CotC recombinant plasmid; The fusion gene ORF56 is connected to the end of the CotB or CotC sequence; the connection product is a pDG-CotB-ORF56 or pDG-CotC-ORF56 recombinant vector; The accession number of the CotB or CotC in NCBI is AL009126.3, the amplified fragment sequence of the CotB is located at 3715057 bp-3716144 bp of the accession number sequence, and the gene amplified fragment sequence of the CotC is located at 1904995 bp-1905192 bp of the accession number sequence; The nucleotide sequence of the fusion gene ORF56 is shown in SEQ ID NO:

1.

3. A drug for preventing and treating porcine reproductive and respiratory syndrome, characterized in that: The invention comprises the recombinant Bacillus subtilis obtained by the construction method according to claim 2.

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