Genetic engineering bacillus subtilis and application thereof in prevention of mastitis

Abstract

The invention is applicable to the technical field of genetic engineering, and provides genetic engineering bacillus subtilis and application thereof in prevention of mastitis, a construction method comprises the following steps: taking a DNA sequence as shown in SEQ ID NO.1 as a template, and carrying out PCR (Polymerase Chain Reaction) amplification to obtain a PCR product sequence as shown in SEQ ID NO.3; the PCR product is connected to a pHT43 vector and is converted into a competent cell DH5-alpha, and a recombinant plasmid pHT43-Sp A is obtained; the method comprises the following steps: transforming a recombinant plasmid pHT43-Sp A into a competent cell of bacillus subtilis WB800N, and screening a positive transformant by virtue of a PCR (Polymerase Chain Reaction) technology; by means of the homologous recombination technology, bacillus subtilis is selected as a host strain, the genetic engineering bacillus subtilis strain capable of expressing staphylococcus aureus A (Sp A) protein is successfully constructed, and the pathological damage degree caused by staphylococcus aureus infection can be relieved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a genetically engineered Bacillus subtilis and its application in the prevention of mastitis. Background Technology

[0002] Mastitis in dairy cows is the leading cause of reduced milk production. Its high incidence and difficulty in prevention and treatment make it a primary factor limiting the development of the dairy industry. Staphylococcus aureus is the main pathogen causing mastitis in dairy cows. Currently, antibiotics remain the primary method for the clinical prevention and treatment of Staphylococcus aureus infections. However, the overuse and irrational use of antibiotics have increased the antibiotic resistance of Staphylococcus aureus, leading to substandard milk, antibiotic-resistant bacteria, and drug residues, which pose serious public health problems.

[0003] The first step in Staphylococcus aureus infection relies on adhesion to host cells, which is mediated by Staphylococcus aureus surface proteins. Furthermore, surface proteins are also essential for Staphylococcus aureus biofilm formation, bacterial invasion and colonization, and evasion of host immune defenses. Therefore, surface proteins are crucial for Staphylococcus aureus infection. Consequently, Staphylococcus aureus surface proteins are a research hotspot in vaccine development. Staphylococcus aureus protein A (SpA) is a highly conserved secreted protein. Studies have shown that it contains five immunoglobulin-binding domains (IgBDs): E, D, A, B, and C. Each module can bind to the Fcγ domain of human IgG1, IgG2, and IgG4, exhibiting good immunogenicity. These findings demonstrate that SpA protein could be a potential new vaccine target for preventing Staphylococcus aureus infection.

[0004] Bacillus subtilis ( Bacillus subtilis, B. subtilis Bacillus subtilis is a Gram-positive probiotic that can produce metabolically dormant endospores under harsh conditions, exhibiting strong resistance and surviving under conditions of high temperature, dryness, and acid / alkali attack. As a probiotic, Bacillus subtilis can regulate the intestinal flora of animals, improve their growth performance, and enhance host immunity, making it a widely used novel probiotic. Furthermore, B. subtilis Bacillus subtilis has gradually been recognized as an ideal host for secreting heterologous proteins, and a Bacillus subtilis expression system has been successfully developed. However, there are currently no reports on recombinant Bacillus subtilis live vector vaccines expressing Staphylococcus aureus SpA protein.

[0005] To avoid the above technical problems, it is necessary to provide a recombinant Bacillus subtilis to overcome the shortcomings of existing technologies in preventing Staphylococcus aureus mastitis. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing genetically engineered Bacillus subtilis, aiming to solve the problems mentioned in the background art.

[0007] The present invention is implemented as follows: a method for constructing genetically engineered Bacillus subtilis includes the following steps: Step 1: Using the DNA sequence described in SEQ ID NO.1 as a template, and using Sp AU and Sp AL as primers, homologous arms were introduced at the 5' ends of the upstream and downstream primers for PCR amplification. The PCR product sequence obtained is shown in SEQ ID NO.3. The PCR product was ligated into the pHT43 vector and transformed into competent cells DH5-α to obtain the recombinant plasmid pHT43-Sp A. Step 2: Prepare competent cells using Bacillus subtilis WB800N; Step 3: The recombinant plasmid pHT43-Sp A was transformed into Bacillus subtilis WB800N competent cells, and positive transformants were screened by PCR using SpA-F and Sp AR as primers. B. subtilis (pHT43-Sp A), namely genetically engineered Bacillus subtilis.

[0008] Another objective of this invention is to provide a genetically engineered Bacillus subtilis strain, which is constructed using the above-described method.

[0009] Another objective of this invention is to provide the application of genetically engineered Bacillus subtilis in the preparation of a vaccine to prevent mastitis, wherein the mastitis is caused by Staphylococcus aureus.

[0010] Another objective of this invention is to provide a vaccine for the prevention and treatment of Staphylococcus aureus mastitis, the vaccine comprising the aforementioned genetically engineered Bacillus subtilis.

[0011] This invention provides a genetically engineered Bacillus subtilis strain that, using homologous recombination technology, selects Bacillus subtilis as the host strain and successfully constructs a genetically engineered Bacillus subtilis strain that can express Staphylococcus aureus A (SpA) protein. Experimental results show that this genetically engineered Bacillus subtilis can induce a specific immune response against SpA protein in mice, significantly reducing the severity of pathological damage caused by Staphylococcus aureus infection, and ultimately achieving effective prevention of Staphylococcus aureus mastitis, providing a new technical approach for the prevention and control of this disease. Attached Figure Description

[0012] Figure 1 A flowchart illustrating the construction process of genetically engineered Bacillus subtilis is provided for an embodiment of the present invention. Figure 2 This invention provides an analysis of SpA protein antigenic epitopes in an embodiment of the invention. Figure 3 This is a nucleic acid gel electrophoresis image of the SpA gene PCR amplification product provided in an embodiment of the present invention; Figure 4 The PCR results of positive clone plasmids obtained by screening plasmids pHT43-Sp A transformed into Escherichia coli competent cells DH5α provided in the embodiments of the present invention are shown in the figure. 1: Escherichia coli DH5α competent cells before transformation; 2: nucleic acid marker; 3-11: Escherichia coli DH5α competent cells after transformation. Figure 5 Nucleic acid gel electrophoresis image of plasmid pHT43-Sp A provided in the embodiments of the present invention, verified by BamHI and SmaI enzyme digestion; Figure 6 Positive recombinant Bacillus subtilis obtained by screening after transforming plasmid pHT43-Sp A into Bacillus subtilis WB800N, as provided in this embodiment of the invention. B. subtilis (pHT43 Sp A) PCR results, where 1: Bacillus subtilis WB800N competent cells before transformation; 2: nucleic acid marker; 3~11: Bacillus subtilis WB800N competent cells after transformation; Figure 7 Provided for embodiments of the present invention B. subtilis (pHT43-Sp A) Western Blot validation of Sp A protein expression results, where 1: protein marker; 2: empty vector supernatant of induced group; 3: empty vector precipitate of induced group; 4: recombinant Bacillus subtilis supernatant of induced group; 5: recombinant Bacillus subtilis precipitate of induced group; 6: empty vector supernatant of non-induced group; 7: empty vector precipitate of non-induced group; 8: recombinant Bacillus subtilis supernatant of non-induced group; 9: recombinant Bacillus subtilis precipitate of non-induced group; Figure 8 Provided for embodiments of the present invention B. subtilis Results of genetic stability testing for (pHT43-Sp A); Figure 9 The number of Staphylococcus aureus colonies in breast tissue provided in this embodiment of the invention; Figure 10 This is a bar chart showing the number of Staphylococcus aureus colonies in breast tissue provided in an embodiment of the present invention; Figure 11 Small intestine and lung tissue provided in the embodiments of the present invention B. subtilis (pHT43-Sp A) colony count; Figure 12 HE staining results of breast tissue provided in the embodiments of the present invention; Figure 13 The serum IL-1β content provided in the embodiments of the present invention; Figure 14 The serum TNF-α content provided in the embodiments of the present invention; Figure 15 The IL-1β content in the mammary gland provided in the embodiments of the present invention; Figure 16 The TNF-α content in the breast provided in the embodiments of the present invention; Figure 17 The detection results of specific IgG in serum provided in the embodiments of the present invention; Figure 18 The results of the detection of specific sIg A in the small bowel lavage fluid provided in this embodiment of the invention; Figure 19 The detection results of specific sIg A in the lung lavage fluid provided in the embodiments of the present invention; In the attached figure, the number of asterisks corresponds to the significance level (p-value) thresholds, i.e., no asterisk (ns): p > 0.05, no statistical significance; *: p ≤ 0.05, significant; **: p ≤ 0.01, highly significant; ***: p ≤ 0.001, extremely significant; ****: p ≤ 0.0001, extremely significant. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0014] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0015] Example 1: A genetically engineered Bacillus subtilis, which uses the nucleic acid sequence shown in SEQ ID NO.1, or a nucleic acid sequence with greater than 95% homology to the nucleic acid sequence shown in SEQ ID NO.1, to encode a protein with the same function, the amino acid sequence of which is shown in SEQ ID NO.2; like Figure 2 As shown, antigenic epitope analysis was performed on the protein; The above-mentioned method for constructing genetically engineered Bacillus subtilis is as follows: Figure 1 As shown, the specific steps include: 1. Construction of Bacillus subtilis recombinant shuttle expression vector: (1) The SpA protein gene sequence was amplified using ApexHFHS DNA Polymerase FS and SpA AU, SpA-L primers (as shown in SEQ ID NO. 4-5) from Aikerui Biotechnology Co., Ltd. (Hunan, China). Figure 3 (As shown), the plasmid pHT43 was then digested with Takara BamHI and Sma I (China, Dalian) to obtain the linearized vector plasmid; (2) The amplified SpA protein gene fragment and the linearized vector were ligated using the ClonExpress UltraOne Step Cloning Kit V2 from Novizan Biosciences (Nanjing, China). The ligation product was transformed into Escherichia coli DH5-α (purchased from Tulugang Biotechnology Co., Ltd., Shanghai, China) and plated on 100 μg / mL ampicillin-resistant LB plates for overnight culture. Positive transformants were screened by PCR the next day (e.g., Figure 4 (As shown), plasmids were then extracted from the positive transformants and verified by sequencing and enzyme digestion (e.g. Figure 5 As shown), the Bacillus subtilis shuttle vector pHT43-Sp A was obtained; 2. Preparation of Bacillus subtilis WB800N competent cells: (1) Bacillus subtilis WB800N (purchased from Changsha Aibiwei Biotechnology Co., Ltd.) was revived and cultured overnight on 25 μg / mL kanamycin-resistant LB plates. The next day, single colonies were selected and inoculated into 25 μg / mL kanamycin-resistant liquid LB for expansion culture. (2) The next day, the culture was transferred to 50 mL of electroporation transformation medium A at a ratio of 1:100 and cultured until the OD reached 600 nm to between 0.85 and 0.95. (3) Transfer the above bacterial solution to a sterile and cooled centrifuge tube and incubate on ice for 15 min; (4) Place at 4℃, centrifuge at 5000 rpm for 10 min, and collect the bacterial precipitate; (5) Resuspend the bacterial pellet in pre-cooled electroporation transformation medium B, centrifuge again following the above steps, and wash repeatedly 5 times; (6) Wash with 1 mL of electroporation medium B for the last time and dispense into chilled centrifuge tubes at a rate of 60 μL / tube for later use; 3. Constructing genetically engineered Bacillus subtilis B. subtilis (pHT43-Sp A): The shuttle vector pHT43-SpA was electroporated into Bacillus subtilis WB800N competent cells using electroporation at 2.5 kV, 5.5 ms, and 200 Ω. Immediately after electroporation, resuscitation medium was added, and the cells were incubated at 37°C for 3 hours. The cells were then plated onto 25 μg / mL chloramphenicol-resistant LB agar plates and incubated overnight. Transformants were identified and screened using PCR with primers Sp AF / R (as shown in SEQ ID NO. 6-7). Figure 6 (As shown), finally, for the positive transformants B. subtilis (pHT43-Sp A) induces protein expression; Simultaneously, the pHT43 plasmid without the SpA gene was transformed into [a specific gene] using the same method. B. subtilis In the middle, we obtained B. subtilis (pHT43); Performance testing: Genetically engineered Bacillus subtilis B. subtilis (pHT43-Sp A) Immunization: Seven-week-old Balb / c mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd., Liaoning, China) were randomly divided into a negative control group (Con) and a positive control group (Con). S.aureus +PBS), empty vector group ( S . aureus + B. subtilis (Pht43)) S.aureus + B. subtilis (pHT43-Sp A)G immunization group (by gavage). S.aureus + B. subtilis (pHT43-Sp A)D immunization group (nasal drops), immunized every 14 days, each immunization was performed by continuous gavage and nasal drops 1×10 10 CFU / 0.2mL, for 5 consecutive days. The negative control group received no treatment, while the positive control group received 0.2mL of sterile PBS by gavage. Serum was collected via orbital sampling on days 0, 14, 28, and 35 of immunization. On day 34, 1×10 7 Mice were infected with CFU of Staphylococcus aureus (USA300, purchased from the American Culture Collection Center, ATCC), and mammary glands, small intestines, and serum were collected on day 35. Test results: like Figure 7 As shown, detection was performed using Western blotting. B. subtilis The expression of (pHT43-Sp A) protein was investigated, and the results showed that a specific protein band of approximately 45.5 kDa, located between 55 kDa and 42 kDa, appeared in lane 4.5, consistent with the size of Staphylococcus aureus protein A. like Figure 8 As shown, after 35 generations of continuous culture in antibiotic-free LB medium, the results indicated... B. subtilisThe (pHT43-Sp A) plasmid can be stably inherited; like Figure 9 , Figure 10 As shown, mammary gland tissue from mice after immunization and challenge was ground and plated. The results indicated that... B. subtilis (pHT43-Sp A) significantly inhibited the colonization ability of Staphylococcus aureus in mouse mammary glands; like Figure 11 As shown, the small intestine of mice immunized by gavage and the lungs of mice immunized by intranasal drops were ground and plated. The results showed that... B. subtilis (pHT43-Sp A) was successfully colonized in the intestines of mice by gavage and in the lungs of mice by nasal drops; like Figure 12 As shown, mammary gland tissue from mice after immune challenge was stained with hematoxylin and eosin (HE) and observed under a microscope. The results showed that... B. subtilis (pHT43-Sp A) effectively alleviates pathological damage to mammary tissue in mice caused by Staphylococcus aureus infection; like Figures 13 to 16 As shown, the serum and mammary tissue of mice after immunization with the virus were detected using an ELISA kit. The results indicated that... B. subtilis (pHT43-Sp A) significantly reduced the levels of TNF-α and IL-1β in the serum and mammary glands of mice infected with Staphylococcus aureus; like Figures 17 to 19 As shown, the serum, small intestinal lavage fluid, and lung lavage fluid of mice after immunization and challenge were detected using a specific IgG and sIgA ELISA kit. The results showed that... B. subtilis (pHT43-Sp A) successfully induced specific antibodies in mice after gavage and nasal immunization.

[0016] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing genetically engineered Bacillus subtilis, characterized in that, Includes the following steps: Step 1: Using the DNA sequence described in SEQ ID NO.1 as a template, and using Sp AU and Sp AL as primers, homologous arms were introduced at the 5' ends of the upstream and downstream primers for PCR amplification. The obtained PCR product sequence is shown in SEQ ID NO.

3. The PCR product was ligated into the pHT43 vector and transformed into competent DH5α cells to obtain the recombinant plasmid pHT43-Sp A; Step 2: Prepare competent cells using Bacillus subtilis WB800N; Step 3: The recombinant plasmid pHT43-Sp A was transformed into Bacillus subtilis WB800N competent cells, and positive transformants were screened by PCR using Sp AF and Sp AR as primers. B. subtilis (pHT43-Sp A), namely genetically engineered Bacillus subtilis.

2. The method for constructing genetically engineered Bacillus subtilis according to claim 1, characterized in that, The sequence of primer SpA-U is shown in SEQ ID NO.4, and the sequence of primer SpAL is shown in SEQ ID NO.

5.

3. The method for constructing genetically engineered Bacillus subtilis according to claim 1, characterized in that, The sequences of primers SpA-F are shown in SEQ ID NO.6, and the sequences of primers SpAR are shown in SEQ ID NO.

7.

4. A genetically engineered Bacillus subtilis strain, characterized in that, It is constructed using the construction method described in any one of claims 1-3.

5. The application of the genetically engineered Bacillus subtilis as described in claim 4 in the preparation of a mastitis prevention vaccine, characterized in that, The mastitis was caused by Staphylococcus aureus infection.

6. A vaccine for the prevention and treatment of Staphylococcus aureus mastitis, characterized in that, The vaccine includes the genetically engineered Bacillus subtilis as described in claim 4.

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