Vaccine for preventing and treating staphylococcus aureus mastitis and application thereof

Abstract

The invention is applicable to the technical field of genetic engineering, and provides a vaccine for preventing and treating staphylococcus aureus mastitis and application of the vaccine. A recombinant bacillus subtilis construction method comprises the following steps: taking a DNA sequence as shown in SEQ ID NO.3 as a template, and performing PCR amplification to obtain a PCR product sequence; connecting the PCR product to a pHT43 vector, and converting the pHT43 vector into a competent cell DH5alpha, so as to obtain a recombinant plasmid pHT43-cCsa; the method comprises the following steps: transforming a recombinant plasmid pHT43-cCsa into a competent cell of bacillus subtilis WB800N, taking pHT43-F and pHT43-R as primers, and screening a positive transformant through a PCR (Polymerase Chain Reaction) technology; the recombinant bacillus subtilis for expressing the staphylococcus aureus Csa protein is constructed by utilizing a homologous recombination technology, so that pathological injury caused by staphylococcus aureus infection can be reduced, and staphylococcus aureus mastitis of dairy cattle can be prevented.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a vaccine for the prevention and treatment of Staphylococcus aureus mastitis and its application. Background Technology

[0002] Bovine mastitis is a major challenge facing the dairy farming and dairy industry, and is one of the most common and economically significant diseases in dairy cows. Currently, the high incidence of bovine mastitis and the difficulty in prevention and treatment make it a primary factor limiting the development of the dairy industry. Staphylococcus aureus is the main pathogen causing bovine mastitis. At present, antibiotics remain the main method for the clinical prevention and treatment of Staphylococcus aureus infection; however, the overuse and irrational use of antibiotics have increased the antibiotic resistance of Staphylococcus aureus.

[0003] The first step in Staphylococcus aureus infection relies on adhesion to host cells, mediated by Staphylococcus aureus surface proteins. Secondly, surface proteins also play a crucial role in Staphylococcus aureus biofilm formation, bacterial invasion and colonization, and evasion of host immune defenses. Therefore, surface proteins are essential for Staphylococcus aureus infection and are a hot research topic in vaccine development. Csa protein, belonging to the conserved Staphylococcus antigen family, plays an important role in the pathogenesis of Staphylococcus aureus. Studies have shown that Csa protein can induce a specific immune response in animal models of Staphylococcus aureus infection, demonstrating that Csa protein could be a potential new vaccine target for preventing Staphylococcus aureus infection.

[0004] Bacillus subtilis ( Bacillus subtilis , B. subtilis Bacillus subtilis is a recognized safe Gram-positive probiotic. This bacterium can produce metabolically dormant endospores under harsh conditions, thus endowing it with extremely high resistance to harsh environments such as high temperature, acidity, alkalinity, and radiation. As a probiotic, Bacillus subtilis not only improves feed conversion rate to promote animal growth performance, but also regulates intestinal flora and enhances host immunity. Furthermore... B. subtilis It has gradually been recognized as an ideal host for secreting heterologous proteins and a Bacillus subtilis expression system has been successfully developed.

[0005] Specifically, the challenges in vaccine development mainly lie in three aspects: First, the existing technological system has not yet identified specific Staphylococcus aureus biomarkers that can be used for vaccine development, resulting in a lack of precise anchors for research directions; second, research has found that the toxins produced by Staphylococcus aureus can interfere with the host's recognition process of pathogens. While the revelation of this mechanism provides a new perspective on understanding the interaction between the bacterium and the host, it also further highlights the complexity of its pathogenic mechanism; third, after infection with Staphylococcus aureus, the host often fails to form an effective memory immune response. This phenomenon indicates that the interaction between the pathogen and the host is far beyond current understanding, requiring further in-depth research to elucidate the relevant mechanisms to support vaccine development. Therefore, how to accurately screen for Staphylococcus aureus antigens that can induce protective immunity has become a core breakthrough direction in the current vaccine development field. In summary, developing a vaccine with a clear protective effect against Staphylococcus aureus mastitis has become an urgent task driven by clinical needs. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing recombinant Bacillus subtilis, thereby addressing the problems mentioned in the background section.

[0007] The present invention is implemented as follows: a method for constructing recombinant Bacillus subtilis includes the following steps: Step 1: Using the DNA sequence described in SEQ ID NO.3 as a template, and cCsa-U and cCsa-L 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.4. The PCR product was ligated into the pHT43 vector and transformed into competent DH5α cells to obtain the recombinant plasmid pHT43-cCsa. Step 2: Prepare competent cells using Bacillus subtilis WB800N; Step 3: Transform the recombinant plasmid pHT43-cCsa into Bacillus subtilis WB800N competent cells. Using pHT43-F and pHT43-R as primers, positive transformants are screened by PCR technology, which are the recombinant Bacillus subtilis.

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

[0009] Another objective of this invention is to provide a vaccine for the prevention and treatment of Staphylococcus aureus mastitis, the vaccine comprising the aforementioned recombinant Bacillus subtilis.

[0010] Another objective of this invention is to provide the application of a vaccine for preventing Staphylococcus aureus mastitis in the preparation of products for preventing Staphylococcus aureus mastitis.

[0011] This invention utilizes homologous recombination technology to creatively construct a recombinant Bacillus subtilis expressing Staphylococcus aureus Csa protein using Bacillus subtilis as the host bacterium. This Bacillus subtilis can induce specific immunity against Csa protein in mice, reduce pathological damage caused by Staphylococcus aureus infection, and thus effectively prevent Staphylococcus aureus mastitis in dairy cows. Attached Figure Description

[0012] Figure 1 The recombinant Bacillus subtilis provided in the embodiments of the present invention B. subtilis Flowchart of the construction process of (pHT43-cCsa); Figure 2 The codon alignment results before and after optimization of the target gene sequence encoding Csa protein provided in the embodiments of the present invention; Figure 3 This invention provides a prediction of protein tertiary structure before and after Csa codon optimization in an embodiment of the invention. Figure 4 Csa protein antigenic epitope analysis provided in the embodiments of the present invention; Figure 5 This is a nucleic acid gel electrophoresis image of the cCsa gene PCR amplification product provided in an embodiment of the present invention; Figure 6 The PCR results of positive clone plasmids obtained by screening the constructing plasmid pHT43-cCsa after transformation into Escherichia coli competent cells DH5α provided in the embodiments of the present invention; Figure 7 Nucleic acid gel electrophoresis image of the constructed plasmid pHT43-cCsa provided in this embodiment of the invention, verified by BamHI and SmaI enzyme digestion; Figure 8 The positive recombinant Bacillus subtilis obtained by transforming the constructed plasmid pHT43-cCsa into Bacillus subtilis WB800N according to the embodiments of the present invention was screened. B. subtilis (pHT43-cCsa) PCR results; Figure 9 Provided for embodiments of the present invention B. subtilis (pHT43-cCsa) Western Blot verification of cCsa protein expression results. Lane 1 is the protein marker, and lanes 2-9 are respectively: induced supernatant, uninduced supernatant, induced empty supernatant, uninduced empty supernatant, induced precipitation, uninduced precipitation, induced empty precipitation, and uninduced empty precipitation. Figure 10 Provided for embodiments of the present invention B. subtilisThe genetic stability test results of (pHT43-cCsa) are shown in the following figures: a is the identification result of F1 plasmid, b is the identification result of F10 plasmid, c is the identification result of F20 plasmid, and d is the identification result of F30 plasmid. Figure 11 The number of Staphylococcus aureus colonies in breast tissue provided in this embodiment of the invention; Figure 12 The small intestinal tissue provided in the embodiments of the present invention B. subtilis (pHT43-cCsa) colony count; Figure 13 HE staining results of breast tissue provided in the embodiments of the present invention; Figure 14 The results of TNF-α and IL-1β content provided in the embodiments of the present invention are as follows: a is the serum TNF-α content, b is the serum IL-1β content, c is the breast TNF-α content, and d is the breast IL-1β content. Figure 15 The MPO content of mouse mammary tissue provided in the embodiments of the present invention; Figure 16 The values ​​of serum-specific IgG and small intestinal lavage fluid-specific sIgA in the immunized mouse serum provided in this embodiment of the invention are: a is the serum-specific IgG content, and b is the small intestinal lavage fluid-specific sIgA content. 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 recombinant 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; The nucleic acid sequence shown in SEQ ID NO.1 underwent codon optimization, such as... Figure 2As shown, the optimized cCsa protein DNA sequence is shown in SEQ ID NO.3; like Figure 3 and Figure 4 As shown, the optimized protein was subjected to three-dimensional structure and antigenic epitope analysis; The above-mentioned method for constructing recombinant Bacillus subtilis is as follows: Figure 1 As shown, the specific steps include: 1. Construction of Bacillus subtilis recombinant shuttle expression vector: (1) The optimized Csa protein gene sequence was amplified using ApexHFHS DNA Polymerase FS and cCsa-U and cCsa-L primers (as shown in SEQ ID NO. 5-6) from Aikerui Biotechnology Co., Ltd. (Hunan, China). Figure 5 (As shown), the plasmid pHT43 was then digested with Takara BamHI and SmaI (China, Dalian) to obtain the linearized vector plasmid; (2) The amplified cCsa protein gene fragment and the linearized vector were ligated using the ClonExpress UltraOne Step Cloning Kit V2 from Novizam Biotechnology Co., Ltd. (Nanjing, China). The ligation product was transformed into Escherichia coli DH5α (Tolo Harbor, Shanghai, China) and plated on 100 μg / mL ampicillin-resistant LB plates for overnight culture. The transformants were screened by PCR the next day (e.g., Figure 6 (As shown), plasmids were then extracted from positive transformants and verified by sequencing and enzyme digestion (e.g.) Figure 7 As shown), the Bacillus subtilis shuttle vector pHT43-cCsa 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. Restructuring B. subtilis (pHT43-cCsa): The shuttle vector pHT43-cCsa 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 pHT43-F / R (as shown in SEQ ID NO. 7-8). Figure 8 As shown), B. subtilis (pHT43-cCsa) was passaged under antibiotic-free conditions, and the plasmid was tested every 10 generations. Finally, positive transformants were analyzed. B. subtilis (pHT43-cCsa) induces protein expression; Simultaneously, the pHT43 plasmid without the cCsa gene was transformed into [a specific gene] using the same method. B. subtilis In the middle, we obtained B. subtilis (pHT43); Performance testing: Recombinant Bacillus subtilis B. subtilis (pHT43-cCsa) Immunity: Sixty 7-week-old Balb / c mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China) and randomly divided into a negative control group (Con group) and a positive control group (PBS+). S.aureus Group), empty vector control group ( S.aureus + B. subtilis (pHT43) group), immune group ( S.aureus + B. subtilis (pHT43-cCsa group), immunized every 14 days, each time by gavage 1×10 10 CFU / 0.2mL was administered by gavage for 5 consecutive days. The negative control group received no treatment, the positive control group received 0.2mL of sterile PBS by gavage, and the empty vector control group received 1×10⁻⁶ CFU / mL by gavage. 10 CFU / 0.2mL B. subtilis (pHT43); Serum and small bowel lavage fluid were collected on days 0, 14, and 28 of immunization. Except for the negative control group, 1×10⁻⁶ was administered on day 29. 7 Mice were infected with CFU of Staphylococcus aureus SA113 (ATCC35556, laboratory preserved), and mammary glands and small intestines were collected on day 30. Test results: like Figure 9As shown, recombination was detected by Western blotting. B. subtilis The expression status of (pHT43-cCsa) protein was analyzed, and the results showed that the recombinant bacteria successfully expressed the Staphylococcus aureus Csa protein. like Figure 10 As shown, the gene carrying status of the recombinant bacterial progeny was detected by PCR, and the results indicated that... B. subtilis (pHT43-cCsa) The target gene can still be detected in F30 (30th generation) under antibiotic-free conditions, and the plasmid can be stably inherited up to 30 generations; like Figure 11 As shown, breast tissue was ground, plated, and Staphylococcus aureus colonies were counted. The results indicated that the immune response was positive. B. subtilis (pHT43-cCsa) mice showed a significant decrease in CFU in mammary tissue. B. subtilis (pHT43-cCsa) significantly inhibited the colonization of Staphylococcus aureus in mouse mammary glands; like Figure 12 As shown, the small intestinal tissue was ground, plated, and statistically analyzed. B. subtilis (pHT43-cCsa) colony count, the results showed that, B. subtilis (pHT43-cCsa) was successfully colonized in the mouse intestine; like Figure 13 As shown, pathological changes were observed and tissue damage was scored by HE staining of breast tissue. The results showed that immunization of mice with recombinant bacteria alleviated breast structural damage and inflammatory infiltration. B. subtilis (pHT43-cCsa) effectively alleviated the pathological damage to mammary tissue caused by Staphylococcus aureus infection in mice; like Figure 14 As shown, by detecting serum and breast inflammatory factors TNF-α and IL-1β, it was found that mouse immunity... B. subtilis (pHT43-cCsa) significantly reduced the levels of TNF-α and IL-1β in the serum and mammary glands of mice infected with Staphylococcus aureus; like Figure 15 As shown, by detecting the MPO content in mouse mammary tissue, it was found that the mouse immune system... B. subtilis (pHT43-cCsa) significantly reduced the MPO level in the mammary glands of mice infected with Staphylococcus aureus; like Figure 16 As shown, serum-specific IgG and small intestinal wash fluid-specific sIgA were significantly elevated in immunized mice, indicating that... B. subtilis Immunizing mice with (pHT43-cCsa) successfully induced the production of specific antibodies in the mice.

[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 recombinant Bacillus subtilis, characterized in that, Includes the following steps: Step 1: Using the DNA sequence described in SEQ ID NO.3 as a template, and cCsa-U and cCsa-L 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.

4. The PCR product was ligated into the pHT43 vector and transformed into competent DH5α cells to obtain the recombinant plasmid pHT43-cCsa. Step 2: Prepare competent cells using Bacillus subtilis WB800N; Step 3: Transform the recombinant plasmid pHT43-cCsa into Bacillus subtilis WB800N competent cells. Using pHT43-F and pHT43-R as primers, positive transformants are screened by PCR technology, which are the recombinant Bacillus subtilis.

2. The method for constructing recombinant Bacillus subtilis according to claim 1, characterized in that, The sequence of primer cCsa-U is shown in SEQ ID NO.5, and the sequence of primer cCsa-L is shown in SEQ ID NO.

6.

3. The method for constructing recombinant Bacillus subtilis according to claim 1, characterized in that, The sequence of primer pHT43-F is shown in SEQ ID NO.7, and the sequence of primer pHT43-R is shown in SEQ ID NO.

8.

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

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

6. The use of the vaccine for preventing Staphylococcus aureus mastitis as described in claim 5 in the preparation of products for preventing Staphylococcus aureus mastitis.

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