CN114921397A - Bacillus subtilis for expressing SAT2 type foot-and-mouth disease virus structural protein VP3 and application thereof - Google Patents
Bacillus subtilis for expressing SAT2 type foot-and-mouth disease virus structural protein VP3 and application thereof Download PDFInfo
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- CN114921397A CN114921397A CN202210619983.1A CN202210619983A CN114921397A CN 114921397 A CN114921397 A CN 114921397A CN 202210619983 A CN202210619983 A CN 202210619983A CN 114921397 A CN114921397 A CN 114921397A
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- bacillus subtilis
- pht43
- mouth disease
- disease virus
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Abstract
The invention belongs to the field of biological medicines, and relates to bacillus subtilis for expressing SAT2 type foot-and-mouth disease virus structural protein VP3 and application thereof. The invention transforms recombinant plasmid expressing SAT2 type foot-and-mouth disease virus structural protein VP3 into bacillus subtilis WB800N to obtain recombinant bacillus subtilis expressing SAT2 type foot-and-mouth disease virus structural protein VP 3; the recombinant bacillus subtilis has good immunoreactivity while efficiently expressing SAT2 type foot-and-mouth disease virus structural protein VP3, and can induce an organism to generate humoral immune response and cellular immune response. Can also be used asThe mucosal vaccine generates higher level of antigen-specific IgG antibody and mucosal sIgA antibody after being immunized orally, and raises the CD4 of peripheral blood + And CD8 + The proportion of T lymphocytes stimulates the proliferation of spleen lymphocytes and promotes the expression of cytokines IFN-gamma, IL-2 and IL-4.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to bacillus subtilis for expressing SAT2 type foot-and-mouth disease virus structural protein VP3 and application thereof.
Technical Field
Foot-and-mouth disease (FMD) is an acute, hot and highly contact infectious disease caused by FMDV (Foot-and-mouth disease virus), mainly infects artiodactyl animals such as pigs, cows and sheep, and causes huge economic loss to the livestock breeding industry. FMDV has 7 serotypes, i.e., type O, type a, type 1 Asia (Asia 1), type 1 south africa (Southern African terrorisites 1, SAT1), type 2 south africa (SAT2), type 3 south africa (SAT 3), and type C, and the prevalence of types O and a is predominant in our country. South African foot-and-mouth disease is regional and is mainly concentrated in the south of the Saharan desert, but recent reports show that the SAT2 type foot-and-mouth disease crosses Africa, spreads and erupts in the middle east, has higher risk of being transmitted to southeast Asia and China, and poses potential threat to the breeding industry of China. Therefore, the vaccine development aiming at SAT2 type FMDV is urgently needed, and technical reserve is reserved for the foot-and-mouth disease prevention and control in China.
At present, the most economical and effective measure for preventing and controlling the foot-and-mouth disease is an immune vaccine, and the foot-and-mouth disease inactivated vaccine is a leading product for preventing and controlling the foot-and-mouth disease in China, and makes a great contribution to the prevention and control of the foot-and-mouth disease in China, but has some defects, for example, the traditional manufacture may have incomplete virus inactivation, so that the virus-dispersing risk and the potential safety hazard exist, and the inactivated vaccine generally cannot induce organisms to generate effective mucosal immune response. Therefore, the development of safer, more efficient and more green foot-and-mouth disease vaccines is of great significance.
The mucous membrane is the first defense line for protecting the body from pathogen invasion, and FMDV mainly invades the body through the mucous membrane of the respiratory tract and the digestive tract, so that the induction of the body to generate mucosal immune response is particularly important for the prevention and treatment of foot-and-mouth disease. Compared with the traditional vaccine, the mucosal vaccine has the following advantages: the inoculation modes (eye drop, drinking, gastric lavage, sublingual administration, nasal drip and spraying) of the mucous membrane immunity are diversified and simpler, the immunization can be completed without professional technicians, and the pain and the stress to animals in the immunization process can be reduced; most mucosal immunization modes are suitable for large-scale vaccination, and a needle and a syringe are not needed during administration, so that the risk of spreading blood-borne diseases is avoided. Therefore, the foot-and-mouth disease mucosal vaccine is a novel vaccine with great potential.
Bacillus subtilis, as a probiotic, is a better candidate for mucosal vaccine delivery vehicles. Although the use of bacillus subtilis for antigen protein delivery has been studied in a large amount at present, no specific effective oral mucosal vaccine is used for foot-and-mouth disease prevention and control.
Disclosure of Invention
In order to solve the technical problems, firstly, in the research process of the invention, the inventor finds that when the recombinant bacillus subtilis WB800N/pHT43-VP0 is prepared by using the SAT2 type foot-and-mouth disease virus structural protein VP0, although the recombinant bacillus subtilis WB800N/pHT43-VP0 can efficiently express the SAT2 type foot-and-mouth disease virus structural protein VP0 and has good immunogenicity, the induced organism has low humoral immune response and cellular immune response level, and the organism cannot be induced to generate mucosal immune response and cannot be used as a mucosal vaccine; moreover, a pMA5 vector is used for constructing a recombinant plasmid for expressing the SAT2 type foot-and-mouth disease virus structural protein VP3, although the recombinant plasmid can be successfully electrically transformed into host bacteria B.S168, the target protein VP3 cannot be expressed; when a pWB980 vector is used for constructing a recombinant plasmid for expressing a structural protein VP3 of the SAT2 type foot-and-mouth disease virus, the VP3 gene cannot be successfully connected to the vector; the invention unexpectedly discovers that only the constructed recombinant bacillus subtilis WB800N/pHT43-VP3 expressing the SAT2 type foot and mouth disease virus structural protein VP3 can induce an organism to generate humoral immune response and cellular immune response, can induce the organism to generate high-level mucosal immune response, and is used for preparing the SAT2 type foot and mouth disease mucosal vaccine. The method specifically comprises the following steps:
in a first aspect, the invention provides recombinant bacillus subtilis WB800N/pHT43-VP3 for expressing SAT2 type foot-and-mouth disease virus structural protein VP3, wherein the recombinant bacillus subtilis WB800N/pHT43-VP3 is obtained by introducing a gene for encoding SAT2 type foot-and-mouth disease virus structural protein VP3 or a recombinant vector/plasmid for expressing SAT2 type foot-and-mouth disease virus structural protein VP3 into the bacillus subtilis WB 800N.
Preferably, the preparation method of the recombinant bacillus subtilis WB800N/pHT43-VP3 comprises the following steps: the full-gene synthesis of SAT2 type foot-and-mouth disease virus structural protein VP3 gene; connecting a SAT2 type foot-and-mouth disease virus structural protein VP3 gene to a pHT43 plasmid to construct a recombinant plasmid pHT43-VP 3; the recombinant plasmid pHT43-VP3 is transformed into bacillus subtilis WB800N competent cells to obtain recombinant bacillus subtilis WB800N/pHT43-VP 3.
Preferably, the primer for amplifying the SAT2 type foot-and-mouth disease virus structural protein VP3 gene is as follows:
pHT43-VP 3-F: atcagccgtaggatccTGCTCTAGAATGGGCATCATCCCGGTCGCTG, wherein the lower case part is a homology arm gene sequence;
pHT43-VP 3-R: tcattaggcgggctgcTCAATGGTGGTGATGGTGATGGGTGATGCTGTCGCACA, wherein the lower case portion is the homology arm gene sequence; the italic portion was the 6 × His tag gene sequence.
Preferably, the gene sequence of the SAT2 type foot-and-mouth disease virus structural protein VP3 is shown as SEQ ID NO. 1.
Preferably, the conversion is an electrical conversion.
Preferably, the electricity is converted into:
(1) adding recombinant plasmid pHT43-VP3 into competent cells of bacillus subtilis WB800N, adding an electric rotor after ice bath, setting an electric rotor at 2.5kv, 25 muF, 2000 omega, duration of 4.5ms, and electrically shocking for 1 time;
(2) the electric rotating cup is taken out, the recovery culture medium RM is added, the recovery is carried out for 3 hours at 37 ℃ and 200rpm, 200 mu L of bacterial liquid is taken and coated on 2 XYT solid plate culture medium with Cm resistance (10 mu g/mL), and the culture is carried out overnight at 37 ℃.
Preferably, the preparation method of the bacillus subtilis WB800N competent cell comprises the following steps:
(1) selecting a single colony of bacillus subtilis WB800N to inoculate in an LB culture medium, and culturing at 37 ℃ and 200rpm for 18 h;
(2) transferring the overnight culture in the step (1) into GM solution to make OD 600 Culturing at 37 deg.C and 200rpm to OD about 0.2 600 About 1.0;
(3) taking all the bacterial liquid obtained in the step (2) for ice bath, centrifuging for 8min at 4 ℃ and 5000rpm, removing the upper-layer waste liquid, and collecting thalli in the lower-layer precipitate;
(4) precooling an electrotransfer buffer solution ETM at 4 ℃, adding 40mL of the electrotransfer buffer solution ETM each time to wash the thalli in the step (3), centrifuging for 8min at 4 ℃ and 5000rpm, discarding the upper-layer waste liquid, retaining the lower-layer precipitate, and repeating for 3 times;
(5) and (5) resuspending the washed thallus in the step (4) in 500. mu.L of the ETM described in the step (4), and freezing at-80 ℃ for later use.
In a second aspect, the invention provides an application of the recombinant bacillus subtilis WB800N/pHT43-VP3 in preparation of SAT2 type foot-and-mouth disease virus vaccine.
In a third aspect, the invention provides an application of the recombinant bacillus subtilis WB800N/pHT43-VP3 in preparation of a medicine for preventing or treating SAT2 type foot-and-mouth disease virus infection.
Preferably, the recombinant bacillus subtilis WB800N/pHT43-VP3 is added with a pharmaceutically acceptable carrier to be prepared into any one of oral liquid, injection, spray, drops, patch, powder, tablet, granule, capsule, emulsion, suspension or ointment.
The invention has the beneficial effects that: the invention transforms recombinant plasmid expressing SAT2 type foot-and-mouth disease virus structural protein VP3 into bacillus subtilis WB800N to obtain recombinant bacillus subtilis capable of expressing SAT2 type foot-and-mouth disease virus structural protein VP 3; the recombinant bacillus subtilis has good immunoreaction while efficiently expressing SAT2 type foot-and-mouth disease virus structural protein VP3, and can induce an organism to generate humoral immune response and cellular immune response. Can also be used as mucosal vaccine, after oral immunization, it can produce high-level antigen-specific IgG antibody and mucosal sIgA antibody, and increase peripheral blood CD4 + And CD8 + The proportion of T lymphocytes stimulates the proliferation of spleen lymphocytes and promotes the expression of cytokines IFN-gamma, IL-2 and IL-4.
Drawings
FIG. 1 is a schematic diagram showing the construction of a recombinant Bacillus subtilis expression vector pHT43-VP3 of SAT2 type FMDV structural protein VP3 according to the present invention;
FIG. 2 shows the result of amplification of VP3 gene, a type SAT2 FMDV structural protein;
FIG. 3 shows the double digestion of pHT43 plasmid;
FIG. 4 is a diagram showing the results of PCR identification of the recombinant plasmid pHT43-VP 3;
FIG. 5 MegAlign software alignment analysis of recombinant plasmid pHT43-VP 3;
FIG. 6 is a diagram showing the PCR identification result of recombinant Bacillus subtilis WB800N/pHT43-VP 3;
FIG. 7 is a diagram showing the result of Western blot detection using a His-tagged monoclonal antibody as a primary antibody;
FIG. 8 is a Western blot assay using SAT2 type FMDV VP3 rabbit multiple antiserum Pep48 as a primary antibody;
FIG. 9 ELISA detection of IgG in serum;
FIG. 10 ELISA test results for sIgA in mucosal samples in lung wash;
FIG. 11 ELISA test results for sIgA in mucosal samples in intestinal lavage fluid;
FIG. 12 peripheral blood CD4 + And CD8 + Detecting the result of the T lymphocyte subpopulation;
FIG. 13 results of splenic lymphocyte proliferation assay;
FIG. 14 measurement results of cytokine levels.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the following examples further illustrate the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The reagent materials and the like used in the following examples are commercially available unless otherwise specified.
The existing nucleotide sequences involved in the present invention can be obtained by searching published academic publications in the field and GenBank, and those skilled in the art can complete the molecular biology and cell biology tests involved in the present invention based on the reference of textbooks or experimental manuals in the field.
Example 1 preparation of recombinant Bacillus subtilis WB800N/pHT43-VP3
Construction of SAT2 type FMDV structural protein VP3 recombinant Bacillus subtilis expression plasmid
The construction schematic diagram of the recombinant bacillus subtilis expression vector pHT43-VP3 is shown in figure 1; the method comprises the following specific steps:
(1) designing and synthesizing a primer: according to the FMDV-SAT2-VII-VP3 (SEQ ID NO: JX014256) gene published by GenBank as a reference sequence, recombinant plasmids GS1803U-pUC57 are synthesized by Nanjing Kingsry Bio-Inc., and are transformed into Top10 to be stored as templates GS1803U-pUC57-Top10, and primers are designed and synthesized by Xian engine Biotech, Inc.:
pHT43-VP 3-F: atcagccgtaggatccTGCTCTAGAATGGGCATCATCCCGGTCGCTG, wherein the lower case part is a homology arm gene sequence;
pHT43-VP 3-R: tcattaggcgggctgcTCAATGGTGGTGATGGTGATGGGTGATGCTGTCGCACA, wherein the lower case portion is the homology arm gene sequence; the italic portion was the 6 × His tag gene sequence.
Amplifying VP3 gene by PCR: taking GS1803U-pUC57 recombinant plasmid as a template, and carrying out PCR amplification system in a volume of 50 μ L: namely, 25. mu.L of Ex-Taq Mix, 2. mu.L of each of the upstream and downstream primers, 2. mu.L of the template, ddH 2 O19. mu.L. PCR amplification procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 deg.C for 30s, annealing at 58 deg.C for 45s, and extension at 72 deg.C for 1min, and circulating for 30 times; extension at 72 ℃ for 10 min. Detecting the amplification result of the PCR product by 1% agarose gel electrophoresis, recovering the target fragment by using a DNA gel recovery kit, and storing the sample at-20 ℃. The amplification result is shown in FIG. 2, wherein M is the DNA molecular mass standard; 1 is negative control; 2 is a VP3 gene PCR amplification product; the results show that the PCR amplification is carried out by using the primers by using the recombinant plasmid GS1803U-PUC57 as a template, and a specific band appears at the VP3-His-684bp, which is consistent with the expected size of the SAT2 type FMDV structural gene VP 3.
(2) Construction and identification of recombinant plasmid pHT43-VP 3: carrying out double enzyme digestion on pHT43 plasmid by using Xba I and Sma I restriction endonucleases according to the enzyme digestion site of pHT43 plasmid, carrying out 1% agarose gel electrophoresis on the enzyme digestion product, recovering a linearized vector by using a gel recovery kit, carrying out recombination reaction and recombination product transformation, transforming the recombination product into E.coli DH5 alpha competent cells, coating the competent cells on an LB plate containing ampicillin resistance, culturing at 37 ℃ for 12-16h, selecting a single colony for culturing for 8h, centrifuging at 12000rpm to collect thalli, extracting plasmid, taking the extracted recombinant plasmid as a template of PCR reaction, and carrying out PCR amplification identification by using the primer in the step (1), wherein the PCR reaction system and the reaction conditions are as in the step (1) and the nucleic acid electrophoresis observation result is an amplification product; the positive plasmids were sent to the bio-corporation for sequencing and the results were analyzed by the MegAlign software alignment.
The pHT43 plasmid was double digested with Xba I and SmaI restriction endonucleases, the result is shown in FIG. 3, where M is the molecular mass standard of DNA; 1 is pHT43 plasmid double enzyme digestion product; the result shows that the pHT43 plasmid is subjected to double enzyme digestion to obtain a linearized vector with the size of about 8057bp, which is consistent with the expected result.
The amplification result is shown in FIG. 4, wherein M is the DNA molecular mass standard; 1.3 is a negative control; 2 is PCR amplification product of VP3 gene; 4-6 are PCR products of recombinant plasmid pHT43-VP 3. The result shows that the target fragment with expected size is amplified and the negative control hole has no band, and the extracted plasmid is considered as the positive recombinant plasmid.
The result of comparison and analysis of MegAlign software is shown in FIG. 5, after the recombinant plasmid is sent to the company for sequencing, the MegAlign software is used for analysis, and the homology with the target gene reaches 100 percent, which shows that the recombinant plasmid pHT43-VP3 is successfully constructed, namely the SAT2 type FMDV structural protein VP3 recombinant Bacillus subtilis expression vector.
2. Preparation of recombinant Bacillus subtilis WB800N/pHT43-VP3
2.1 preparation of Bacillus subtilis WB800N competent cells:
(1) picking a single colony of bacillus subtilis WB800N on a fresh plate, inoculating the single colony bacillus subtilis WB800N in 5mL of LB culture medium, and culturing at 37 ℃ and 200rpm for 18 h;
(2) 2.6mL of overnight culture were transferred to 50mL of GM medium to OD 600 Culturing at 37 deg.C and 200rpm to OD of about 0.2 600 About 1.0;
(3) performing ice-bath on all bacteria liquid for 10min, centrifuging at 4 ℃ and 5000rpm for 8min, removing the upper-layer waste liquid, and collecting thalli in the lower-layer precipitate;
(4) precooling an electrotransformation buffer solution ETM in a refrigerator at 4 ℃, adding 40mL of washing thalli each time, centrifuging at 4 ℃ and 5000rpm for 8min, discarding upper-layer waste liquid, reserving lower-layer precipitate, and repeatedly rinsing for 3 times;
(5) the washed cells were resuspended in 500. mu.L of ETM obtained in step (4), and the cells were aliquoted into sterile 1.5mL tubes and frozen at-80 ℃ for future use at 60. mu.L/tube.
2.2 transformation of competent cells of Bacillus subtilis WB800N with recombinant plasmids
(1) Adding 5 μ L of recombinant plasmid into 60 μ L of WB800N competent cells, adding 2mm electric rotor after ice bath for 5min, setting the electric rotor at 2.5kv, 25 μ F, 2000 Ω, duration 4.5ms, and shocking for 1 time;
(2) the electric rotating cup is taken out, 1mL of recovery culture medium RM is added, recovery is carried out at 37 ℃ and 200rpm for 3h, 200 mu L of bacterial liquid is taken and coated on a Cm resistant (10 mu g/mL) 2 XYT solid plate culture medium, overnight culture is carried out at 37 ℃, and positive transformants are screened.
2.3 identification of recombinant Bacillus subtilis expressing SAT2 type FMDV structural protein VP3
(1) Single colonies of Bacillus subtilis on the transformed plates were picked, inoculated into 5mL of Cm-resistant (10. mu.g/mL) 2 XYT liquid medium, and cultured overnight at 37 ℃ and 220 rpm.
(2) The glycerol strain is preserved at-40 ℃, the recombinant expression plasmid is extracted by adopting a kit, and the glycerol strain is preserved at-20 ℃ for later use.
(3) The extracted recombinant expression plasmid was used as a template, PCR identification was performed using the primers described in (1) above in step 1, and the positive transformant was named WB800N/pHT43VP 3.
The amplification result is shown in FIG. 6, wherein M is DNA molecular mass standard DL 2000; 1 is negative control; 2 is a VP3 gene PCR amplification product; 3-7 is PCR amplification product of recombinant bacteria WB800N/pHT43-VP 3. The result shows that the recombinant bacillus subtilis WB800N/pHT43-VP3 constructed by the method has a VP3-His band which is about 684bp long and is consistent with the size of an expected target fragment, and a negative control hole has no band, so that the recombinant bacillus subtilis WB800N/pHT43-VP3 capable of expressing SAT2 type FMDV structural protein VP3 is correctly constructed.
2.4 expression and identification of SAT2 type FMDV structural protein VP3 in recombinant Bacillus subtilis expression plasmid
(1) Inducible expression of the SAT2 type FMDV structural protein VP 3: single colonies identified as positive were picked aseptically, inoculated in 5mL of Cm-resistant 2 XYT liquid medium, and cultured overnight at 37 ℃ and 220 rpm. Inoculating fresh bacteria liquid to 100mL of Cm resistant 2 XYT liquid culture medium according to the proportion of 2%, when culturing until OD600 is about 0.6, adding 100 mu L of IPTG with the concentration of 1mol/L, continuing to induce for 14h at 37 ℃, centrifuging at 6000rpm for 10min to collect bacteria, discarding the upper layer waste liquid, and resuspending the lower layer precipitate with PBS solution.
(2) Western blot identification of the SAT2 type FMDV structural protein VP 3: preparing a sample: taking 80 μ L of each of the induced and non-induced WB800N/pHT43-VP3 heavy suspension, adding 20 μ L of 5 Xprotein loading buffer, mixing well, performing metal bath at 100 ℃ for 10min, standing and cooling at room temperature, and centrifuging at room temperature for 2 min; (ii) electrophoresis: SDS-PAGE gels (12% gel) were fixed on a protein electrophoresis apparatus and sufficient 1 XSDS-PAGE running buffer was added to load 10. mu.L per lane. Performing electrophoresis for 20min under the condition of 80V voltage, and adjusting the voltage to be 120V till the electrophoresis is finished; transferring: the transfer printing liquid is placed in a refrigerator for precooling at 4 ℃ in advance, and the surface of the gel is cleaned by deionized water. A PVDF membrane slightly larger than the gel area is cut and soaked in 100% methanol for 1-2 min. Stacking the membrane and SDS-PAGE gel, putting the membrane and the SDS-PAGE gel into an electrophoresis tank, adding sufficient transfer solution, and rotating the membrane for 1.5 hours under the current condition of 400 mA; sealing: after the transfer printing is finished, taking out the PVDF membrane, and sealing 5% skimmed milk powder at room temperature for 2 hours; incubation primary antibody: adding 8mL diluted His-tagged murine monoclonal antibody (1:2000 dilution) or SAT2 type FMDV VP3 rabbit multiple antiserum Pep48(1:500 dilution), and incubating overnight at 4 ℃; sixthly, incubation of the secondary antibody: washing with 1 XPBST for 7-10 times, adding HRP-labeled goat anti-mouse IgG antibody (diluted 1: 5000) diluted with 8mL or HRP-labeled goat anti-rabbit IgG antibody (diluted 1: 5000), and incubating at room temperature for 1 h; exposure: washing with 1 XPBST for 5 times, dropping proper amount of ECL developing liquid onto PVDF film, scanning and exposing, and storing the picture.
The result of Western blot detection with His-tagged monoclonal antibody as the primary antibody is shown in FIG. 7, where M is the protein molecular standard; 1 is VP3 protein; 2 is WB800N/pHT43 induced whole thallus; 3 is recombinant bacteria WB800N/pHT43-VP3 induced whole bacteria; 4 is the supernatant of the recombinant bacteria WB800N/pHT43-VP3 after induction; 5, precipitation is carried out after induction of recombinant bacteria WB800N/p HT43-VP 3; the results show that the proteins in the supernatant and the precipitate after the ultrasonic treatment have obvious bands near 25kDa, and the molecular weight of the bands is consistent with that of the target protein VP 3-His.
The result of Western blot detection using SAT2 type FMDV VP3 rabbit multiple antiserum Pep48 as primary antibody is shown in FIG. 8, wherein M is protein molecule standard; 1 is WB800N/pHT43 induced whole thallus; 2 is the whole thallus after induction of the recombinant bacteria WB800N/p HT43-VP 3; 3 is the supernatant of the recombinant bacteria WB800N/pHT43-VP3 after induction; 4, precipitation is carried out after induction of recombinant bacteria WB800N/pHT43-VP 3; 5 is VP3 protein; the results show that the proteins in the supernatant and the precipitate after the ultrasonic treatment have obvious bands near 25kDa, and the molecular weight of the bands is consistent with that of the target protein VP 3-His.
The test results show that the recombinant bacillus subtilis can express the SAT2 FMDV structural protein VP3 and has good immunoreaction.
Example 2 preparation of recombinant Bacillus subtilis WB800N/pHT43-VP3 mucosal vaccine and immunogenicity evaluation of the vaccine
1. Preparation of recombinant bacillus subtilis WB800N/pHT43-VP3 mucosal vaccine
(1) Seed bacteria recovery: recombinant Bacillus subtilis WB800N/pHT43-VP3 and recombinant Bacillus subtilis WB800N/pH T43 were inoculated to 5mL of Cm-resistant (10. mu.g/mL) 2 XYT liquid medium, respectively, and cultured overnight at 37 ℃ and 220 rpm.
(2) Seed bacteria amplification culture: fresh bacteria was inoculated at a rate of 2% to 50mL of Cm-resistant 2 XYT liquid medium and cultured overnight at 37 ℃ and 220 rpm.
(3) Induced expression of the protein of interest: inoculating fresh bacteria liquid at a ratio of 2% to 2L Cm resistant 2 XYT liquid culture medium, and culturing to OD 600 At about 0.6, 2mL of IPTG with a concentration of 1mol/L was added and induction was continued at 37 ℃ for 14 hours.
(4) Preparing a mucosal vaccine: the thalli is collected by centrifugation at 6000rpm for 10min, the upper waste liquid is discarded, and the lower sediment is resuspended by PBS solution. Washing the collected thallus with sterile PBS for 3 times, resuspending the thallus in PBS, counting bacteria by 10 × dilution method (three replicates per group, averaging), and adjusting the concentration of the bacteria solution to 5 × 10 10 CFU/mL, stored at 4 ℃ until use.
2. Evaluation of immunogenicity of mucosal vaccines
(1) Grouping, immunizing and collecting samples of mice: randomly dividing 75 female BALB/c mice with the age of 6-8 weeks into 3 groups, 25 mice in each group, respectively immunizing WB800N/pHT43-VP3, WB800N/pHT43 negative control group and PBS blank control group, respectively immunizing recombinant Bacillus subtilis WB800N/pHT43-VP3, idle bacteria WB800N/pHT43 and 1 XPBS buffer, and carrying out oral immunization for three days continuously for each time, namely, the 1d-3d, 11d-13d, 1 XPS,21d-23d, each group was drenched with a gavage needle, and the immunization dose was 0.2mL (1X 10) 10 CFU). 3 mice with different time periods (10d, 20d, 30d, 37d and 44d) are respectively extracted from each group of the 3 groups to carry out orbital venous blood collection and collect serum, and meanwhile, intestinal lotion and lung lotion are collected and are frozen and stored at the temperature of minus 20 ℃ for standby.
(2) ELISA detection of IgG in immune mouse serum
The indirect ELISA method is used for measuring the IgG antibody level in the serum of the immunized mouse, and comprises the following specific steps:
antigen coating: using SAT2 type FMDV VP3 protein as coating antigen, diluting the antigen with coating buffer, adding into ELISA microplate, 200 ng/well, 100. mu.L/well, standing at 4 deg.C for 12 h.
Sealing: the ELISA reaction plate coated with the antigen is taken out, discarded, washed for 5 times and patted dry. Add 1% BSA, 220. mu.L/well, block for 2h at 37 ℃, wash 5 times with 1 XPBST and pat dry, store at-40 ℃ until needed.
Adding the serum to be detected: the collected mouse serum samples were diluted with serum diluent and added to ELISA reaction plates, and the sera of WB800N/pHT43 control group and PBS group were used as negative controls, 100. mu.L/well, and 2 wells were repeated for each sample. And (3) standing and incubating for 1h at 37 ℃, discarding the solution, washing the plate for 5 times and patting dry.
Adding a second antibody: HRP-labeled goat anti-mouse IgG was diluted with 1 XPBST (1:50000) and added to each well at 100. mu.L/well, incubated at 37 ℃ for 1 hour, discarded, washed 5 times and patted dry.
Color development: adding TMB substrate color development solution, standing at 50 μ L/hole at 37 deg.C in dark for 15 min.
And sixthly, terminating: stop solution was added at 50. mu.L/well. OD determination on enzyme-linked immunosorbent assay (ELIASA) within 5min 450 And (4) storing the result.
(3) Detection of mucosal immune sIgA level of immune mice
Collecting intestinal lotion: before sampling, the mice are fasted and water is forbidden for 6h, 3 mice with different time periods (0d, 10d, 20d, 30d, 37d and 44d) are selected in each group, cervical vertebra dislocation is killed, the mice are fixed on a foam plate, 75% alcohol cotton balls are used for body surface disinfection, small intestines (the length is about 6cm) are cut after the abdominal cavity is opened, the mice are placed into 200 mu L precooled PBS (1.5mM EDTA, 0.2mM PMSF), surgical cutting is carried out, then a vortex instrument is used for shaking and mixing evenly, centrifugation is carried out for 5min at 4 ℃ and 12000rpm, supernatant is gently sucked into a centrifuge tube with the volume of 1.5mL, and the whole process is operated on ice and stored for standby at-70 ℃.
Collecting lung washing liquid: the mice which have collected the intestinal lavage fluid continue to open the chest cavity, the lungs are cut, the mice are washed by precooled PBS to remove redundant blood, the mice are repeatedly washed by precooled PBS (1.5mM EDTA, 0.2mM PMSF) with 200 mu L, the lung lavage fluid is collected, the mice are centrifuged at 12000rpm for 5min at 4 ℃, and the supernatant is gently sucked into a centrifuge tube with 1.5mL and stored at 70 ℃ for standby.
Detecting mucous membrane sIgA in lung lotion and intestinal lotion of a mouse by adopting an indirect ELISA method, which comprises the following steps:
antigen coating: using SAT2 type FMDV VP3 protein as coating antigen, diluting the antigen with coating buffer, adding into ELISA microplate, 200 ng/well, 100. mu.L/well, standing at 4 deg.C for 12 h.
Sealing: the antigen-coated ELISA reaction plate was removed, discarded, washed 5 times, and patted dry. Add 1% BSA, 220. mu.L/well, block for 2h at 37 ℃, wash 5 times with 1 XPBST and pat dry, store at-40 ℃ until needed.
Adding a sample to be detected: the mouse intestinal wash was filtered through a 0.45 μm filter, and the intestinal wash and the lung wash were diluted with a serum diluent and added to the reaction plate, while the WB800N/pHT43 control group and PBS group were used as negative controls, 100 μ L/well, and 2 wells were repeated for each sample. And (3) standing and incubating for 1h at 37 ℃, discarding the solution, washing the plate for 5 times and patting dry.
Adding a second antibody: goat anti-mouse IgA antibody to HRP was diluted at a ratio of 1:10000 in 1 XPBST at 100. mu.L/well, incubated at 37 ℃ for 1h, discarded, washed 5 times and patted dry.
Color development: adding TMB substrate developing solution, 50 mu L/hole, keeping away from light at 37 ℃, and standing and incubating for 15 min.
And sixthly, terminating: stop solution was added at 50. mu.L/well. Determination of OD on enzyme-linked immunosorbent assay (ELIASA) within 5min 450 And (4) storing the result.
(4) Peripheral blood CD4 + 、CD8 + Flow cytometry detection of T lymphocyte subsets
The anticoagulation of the eyeball venous plexus of an immunized 33d mouse is collected through a capillary, 100 mu L of the collected anticoagulation is respectively sucked into a marked clean 1.5mL centrifuge tube, 12 mu L of antibodies (2 mu L of FITC-CD4, 5 mu L of APC- CD 3 and 5 mu L of PE-CD8) are added into each tube, and the voltage of a blank set of flow regulating cytometry is set. After the corresponding antibody is added, the tube wall of the centrifuge tube is flicked, turned upside down and mixed evenly, and incubated for 30min on ice in the dark. 1.3mL of erythrocyte lysate was added and lysed at room temperature for 10 min. Centrifuging at 3500rpm for 10min, discarding supernatant, flicking the tube wall of the centrifuge tube, washing with PBS for 1 time, centrifuging at 3500rpm for 5min, discarding supernatant, adding 150 μ L PBS to resuspend cells, and detecting on machine.
(5) Spleen lymphocyte proliferation response assay
The proliferation condition of spleen lymphocytes of mice immunized with 33d is detected by a CCK-8 method, and the method comprises the following steps:
after the eyeball of a mouse immunized with 33d is picked and killed, the mouse is soaked in 75% ethanol for less than 5min, and the body surface is disinfected.
And carefully opening the abdominal cavity of the mouse by using sterile forceps and scissors in the biological safety cabinet, and performing sterile separation to obtain the spleen. The mouse spleen was placed in a bacterial culture dish containing 5mL of tissue diluent, and an appropriate amount of tissue diluent was aspirated with a 2mL sterile syringe and blown up to a pale pink color. The spleen was then placed in a cell mesh (400 mesh) and ground to leave white connective tissue.
③ 5mL of the spleen cell suspension was gently added to the top of 7mL of the lymphocyte separation medium, and the balance was taken.
Fourthly, horizontally centrifuging the mixture for 30min at the temperature of 20 ℃ at 2000g (acel 0, brake 0), and obtaining the whole process for about 50 min.
Fifthly, sucking the leucocyte layer to a sterile 15mL centrifuge tube, adding sterile PBS containing 1% 1640 complete culture medium to supplement to 14 mL. Centrifugation was carried out horizontally at 2000g (acell 9, brake 9) for 20min at 4 ℃.
Sixthly, the supernatant is discarded, 1mL of erythrocyte lysate is added, the erythrocyte lysate is lysed for 1-2min at room temperature, the lysis is stopped by PBS, the volume is supplemented to 14mL, and the centrifugation is carried out horizontally for 20min at 2000g (acell 9 and break 9) at 4 ℃. .
Seventhly, resuspending the lymphocytes by using 500. mu.L of 1640 medium, and counting.
Each holeInoculation of 2.5X 10 5 Individual viable cells, 150 μ L/well, blank control: 1640 medium (no cells).
Ninthly, negative control group: 1640 complete medium, experimental group: antigen stimulus (5 μ g/mL), positive control group: con A (5. mu.g/mL), 6 replicate wells, 50. mu.L/well were set for each group.
⑩37℃、5％CO 2 Culturing for 72h under the condition, adding CCK-8 solution, 10 μ L/well, culturing for 1-4h, reading OD 450 The value is obtained. Stimulation Index (SI) ═ experimental OD value-blank OD value)/(negative control OD value-blank OD value).
(6) Cytokine level detection
And detecting cytokines INF-gamma, IL-2 and IL-4 in the serum of the mice at 37d after immunization by adopting a commercial IL-2, IL-4 and IFN-gamma kit of Shenzhen Xin Bosheng company according to the instruction, and evaluating the immune effect of the recombinant bacillus subtilis in the mice.
3. Results of the experiment
(1) ELISA detection result of IgG in serum
IgG antibody in serum is an important antibody generated by the secondary immune response of an animal body, so the level of the IgG antibody in the serum of an immunized mouse is detected by indirect ELISA. The results are shown in fig. 9, compared with the PBS blank control group, the WB800N/pHT43 negative control group mice had a small increase in IgG antibody level after oral immunization, and the recombinant bacillus subtilis WB800N/pHT43-VP3 immunized group mice continued to increase in IgG antibody level after boost immunization, reached a peak in antibody level one week after the triple immunization (i.e., 30d), and had a significant difference (P <0.001) compared with the negative control group, which indicates that the recombinant bacillus subtilis WB800N/pHT43-VP3 can induce a higher level of antigen-specific IgG antibody.
(2) ELISA detection result of sIgA in mucous membrane sample
sIgA is the most abundant immunoglobulin subtype in mucosa, is involved in mucosal local immunity, and is mainly secreted on the mucosal surface of the intestinal tract, particularly in the small intestine. The lung washing liquid and the intestinal washing liquid of the mice in different time periods are collected, the mucosa immune sIgA antibody level of the immunized mice is detected through indirect ELISA, the mucosa immune sIgA antibody level result in the lung washing liquid is shown in figure 10, and the mucosa immune sIgA antibody level result in the intestinal washing liquid is shown in figure 11. After the mice are subjected to gastric lavage and immunization of the unloaded bacteria WB800N/pHT43, the sIgA antibody level in lung washing liquid and intestinal washing liquid is slightly increased, but the sIgA antibody level is not significantly different from that in a PBS blank control group, and the situation that the live loaded bacteria of the bacillus subtilis have a certain enhancement effect on the mucosal immune response of the mice is shown. After the booster immunization, the sIgA antibody level in the lung wash and intestinal wash of the recombinant Bacillus subtilis WB800N/pHT43-VP3 immunized group mice increased with the number and time of immunization, and reached a peak in antibody level one week after the three immunization (i.e., 30 d). The higher level of sIgA antibody in intestinal washings compared to pulmonary washings further suggests that sIgA is mainly secreted on the mucosal surface of the intestinal tract. The results show that the recombinant bacillus subtilis WB800N/pHT43-VP3 can effectively induce the local mucosal immune response of mice after gastric lavage.
(3) Peripheral blood CD4 + And CD8 + T lymphocyte subpopulation detection result
Peripheral blood lymphocytes of each group of mice were isolated and CD4 was detected by flow cytometry + And CD8 + Percentage of T lymphocytes. 10000 lymphocytes are counted in each group, the average value of each group is taken, and the detection result is shown in figure 12. CD3 of recombinant bacillus subtilis WB800N/pHT43-VP3 immunization group + 、CD4 + And CD8 + The percentage of T lymphocytes is obviously higher than that of a WB800N/p HT43 negative control group; and the recombinant bacillus subtilis WB800N/pHT43-VP3 immunization group CD4 + /CD8 + The ratio is obviously higher than that of a WB800N/pHT43 negative control group. The results show that by maintaining the normal range of CD4 + And CD8 + T lymphocyte, recombinant bacillus subtilis WB800N/pHT43-VP3 can stimulate T lymphocyte typing change and improve humoral immunity and cellular immunity level of mice.
(4) Results of splenic lymphocyte proliferation assay
The lymphocytes can proliferate after being stimulated by antigens during in vitro culture, the number of the proliferating cells can reflect the cellular immune function of animal organisms, and the lymphocyte proliferation assay is also an important index for evaluating the cellular immune response level of the animal organisms. Therefore, at 33d after immunization, spleen lymphocytes were isolated from the collected spleens of mice, and the lymphocyte proliferation ability of the immunized mice was measured by the CCK-8 method, and the results are shown in FIG. 13. Under ConA stimulation, spleen lymphocyte Stimulation Indexes (SI) of mice in each group have no significant difference, but under the stimulation of corresponding antigens, the spleen lymphocyte stimulation indexes of the mice in the recombinant bacillus subtilis WB800N/pHT43-VP3 immunization group are significantly higher than those of the WB800N/pHT43 negative control group and the PBS blank control group, which shows that the recombinant bacillus subtilis WB800N/pH T43-VP3 can effectively stimulate spleen lymphocyte proliferation.
(5) Results of cytokine level detection
Cytokines are protein active molecules secreted by cells and capable of performing signal transduction, participate in immune response and immune regulation of organisms, and play an important regulation role in the process of resisting invasion of viruses and the like. The cytokine assay results of this assay are shown in FIG. 14. The levels of the cytokines IFN-gamma, IL-2 and IL-4 in the serum of the WB800N/pHT43 negative control group are slightly higher than that of the PBS blank control group, but no significant difference exists; the levels of IFN-gamma, IL-2 and IL-4 in the serum of the recombinant bacillus subtilis WB800N/pHT43-VP3 immune group are extremely higher than those of a WB800N/pHT43 negative control group (P is less than 0.001). IL-2, IFN-. gamma.is produced by T lymphocytes (Th1) and primarily mediates cellular immune responses, whereas IL-4 is produced by T lymphocytes (Th2) and primarily regulates humoral immune responses. The results show that the recombinant bacillus subtilis WB800N/pHT43-VP3 can simultaneously induce an organism to generate cellular immune response and humoral immune response after gastric lavage of an immune mouse.
The above description is only exemplary of the present invention and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> Bacillus subtilis for expressing SAT2 type foot-and-mouth disease virus structural protein VP3 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 666
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggcatcatcc cggtcgctgc cgctgacggg tatggtggtt tccagaacac cgacccgaaa 60
acggctgacc ccatttacgg gcatgtgtac aacccgtcca gaaacgactg ccacgggcga 120
tactccaatc ttatggatgt cgccgaggcg tgtccaacac tcctcaactt tgatggcaag 180
ccctacgtcg tgaccaagaa caacggtgac aaggtgatga cacgcttcga cgtcgccttt 240
acacacaagg tgcatgggaa cacgtttctg gcgggcttgg ccgactacta cacacagtat 300
tcaggcagcc taaactacca cttcatgtac actggaccca cacatcacaa ggcaaagttc 360
atggtggcat acgtgccccc tggtgttgca gttgaccagc tgcctagcac accggaggat 420
gctgcgcact gctaccatgc ggaatgggac accgggttga attcttcttt ctcgttcgca 480
gtgccttaca tctccgctgc ggacttttct tacacacaca cagacacacc ggccatggcc 540
accaccaatg gctgggtggt tgtactgcag gtcaccgaca cgcactctgc ggaagctgcc 600
gtggtggtgt cagtcagcgc gggaccagat ttggaattcc gattccctat cgaccctgtg 660
cgacag 666
Claims (10)
1. A recombinant bacillus subtilis WB800N/pHT43-VP3 for expressing SAT2 type foot-and-mouth disease virus structural protein VP3 is characterized in that the recombinant bacillus subtilis WB800N/pHT43-VP3 is obtained by introducing a gene for coding SAT2 type foot-and-mouth disease virus structural protein VP3 or a recombinant vector/plasmid for expressing SAT2 type foot-and-mouth disease virus structural protein VP3 into bacillus subtilis WB 800N.
2. The recombinant bacillus subtilis WB800N/pHT43-VP3 of claim 1, wherein the recombinant bacillus subtilis WB800N/pHT43-VP3 is prepared by the method comprising: the full-gene synthesis of a SAT2 type foot-and-mouth disease virus structural protein VP3 gene; connecting a SAT2 type foot-and-mouth disease virus structural protein VP3 gene to a pHT43 plasmid to construct a recombinant plasmid pHT43-VP 3; the recombinant plasmid pHT43-VP3 is transformed into competent cells of Bacillus subtilis WB800N to obtain recombinant Bacillus subtilis WB800N/pHT43-VP 3.
3. The recombinant Bacillus subtilis WB800N/pHT43-VP3 of claim 2, wherein the primers for amplifying the SAT2 type foot and mouth disease virus structural protein VP3 gene are:
pHT43-VP 3-F: atcagccgtaggatccTGCTCTAGAATGGGCATCATCCCGGTCGCTG, wherein the lower case part is a homology arm gene sequence;
pHT43-VP 3-R: tcattaggcgggctgcTCAATGGTGGTGATGGTGATGGGTGATGCTGTCGCACA, wherein the lower case portion is the homology arm gene sequence; the italic portion was the 6 × His tag gene sequence.
4. The recombinant Bacillus subtilis WB800N/pHT43-VP3 of claim 3, wherein the gene sequence of the structural protein VP3 of SAT2 type foot-and-mouth disease virus is represented by SEQ ID No. 1.
5. The recombinant bacillus subtilis WB800N/pHT43-VP3 of claim 2, wherein said conversion is electrotransformation.
6. The recombinant bacillus subtilis WB800N/pHT43-VP3 of claim 5, wherein said electrical conversion is:
(1) adding recombinant plasmid pHT43-VP3 into a bacillus subtilis WB800N competent cell, adding an electric rotor after ice bath, setting an electric rotor at 2.5kv, 25 muF and 2000 omega, setting the duration of 4.5ms, and electrically shocking for 1 time;
(2) the electric rotating cup is taken out, the recovery culture medium RM is added, the recovery is carried out for 3 hours at 37 ℃ and 200rpm, 200 mu L of bacterial liquid is taken and coated on 2 XYT solid plate culture medium with Cm resistance (10 mu g/mL), and the culture is carried out overnight at 37 ℃.
7. The recombinant Bacillus subtilis WB800N/pHT43-VP3 of claim 5, wherein the competent cells of Bacillus subtilis WB800N are prepared by:
(1) selecting a single colony of the bacillus subtilis WB800N to be inoculated in an LB culture medium, and culturing at 37 ℃ and 200rpm for 18 h;
(2) transferring the overnight culture in the step (1) into GM solution to make OD 600 Culturing at 37 deg.C and 200rpm to OD about 0.2 600 About 1.0;
(3) taking all the bacterial liquid obtained in the step (2) for ice bath, centrifuging for 8min at 4 ℃ and 5000rpm, discarding the upper-layer waste liquid, and collecting thalli in the lower-layer precipitate;
(4) precooling an electrotransfer buffer solution ETM at 4 ℃, adding 40mL of electrotransfer buffer solution ETM each time to wash the thalli in the step (3), centrifuging for 8min at 4 ℃ and 5000rpm, discarding an upper-layer waste liquid, reserving a lower-layer precipitate, and repeating for 3 times;
(5) and (3) resuspending the thallus washed in the step (4) in 500. mu.L of the ETM washed in the step (4), and freezing and storing at-80 ℃ for later use.
8. Use of the recombinant Bacillus subtilis WB800N/pHT43-VP3 of any one of claims 1-7 in the preparation of a SAT2 type foot and mouth disease virus vaccine.
9. The use of recombinant Bacillus subtilis WB800N/pHT43-VP3 as claimed in any one of claims 1 to 7 in the preparation of a medicament for preventing or treating SAT2 type foot and mouth disease virus infection.
10. The use of claim 9, wherein the recombinant bacillus subtilis WB800N/pHT43-VP3 is added with a pharmaceutically acceptable carrier to make any one of oral liquid, powder, tablet, granule, capsule, emulsion, suspension, injection, spray, drop, patch or ointment.
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