CN114404583A - Preparation method of paramotoxin adjuvant vaccine - Google Patents
Preparation method of paramotoxin adjuvant vaccine Download PDFInfo
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- CN114404583A CN114404583A CN202210088216.2A CN202210088216A CN114404583A CN 114404583 A CN114404583 A CN 114404583A CN 202210088216 A CN202210088216 A CN 202210088216A CN 114404583 A CN114404583 A CN 114404583A
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Abstract
The invention discloses a preparation method of a haemophilus parasuis adjuvant vaccine, which is characterized in that an immune stimulating complex (ISCOM-OMP5) of an OMP5 antigen of haemophilus parasuis is prepared by a dialysis method, and the ISCOM-OMP5 is used as an adjuvant of the haemophilus parasuis vaccine to carry out immunological evaluation through a mouse model. The result shows that the ISCOM-OMP5 prepared by observation under an electron microscope has a typical honeycomb structure of ISCOM, the diameter is 30nm-40nm, and the encapsulation rate of OMP5 protein reaches 85.8%; the high-level specific H.parasus antibody can be detected in the serum of the mouse 1 week after the mouse is immunized twice, the mouse in-vitro lymphocyte proliferation and cytokine detection results show that the ISCOM-OMP5 has a remarkable enhancement effect (P <0.01) on the humoral immunity of the mouse body, and also has a remarkable promotion effect (P <0.001) on the expression of IL-10 and IFN-gamma. The ISCOM-OMP5 prepared by the invention can effectively induce mice to generate immune response aiming at H.parasuis, and can obviously enhance the immune effect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of a parasuis bacillus adjuvant vaccine.
Background
Haemophilus parasuis (h. parasuis) is currently the most serious bacterial disease that endangers large-scale swine farms. The main infected objects of the strain are piglets and growing pigs at the age of 2-28 weeks, wherein the harm and the influence on weaned nursery piglets at the age of 5-8 weeks are most prominent, the nursery pig infection is mainly clinically manifested by symptoms such as serositis, arthritis and the like, the infection rate is 30-60%, and the death rate is 50-70%. The serotype of haemophilus parasuis is complex and various, and at least 15 serotypes are contained according to the difference of outer membrane protein antigens of homotypic bacteria, and more than 30 percent of serotypes can not be identified. The currently dominant serotypes in China are mainly 4, 5, 12 and 13 types, and the serotypes in different regions have obvious difference. In clinic, drug resistance is easy to generate due to the use of antibiotics, so that the prevention and treatment difficulty is greatly improved, and the adoption of vaccine immunization is the most economical and effective way for preventing and treating the disease.
Since the vaccine adjuvant IS applied to veterinary vaccines for the first time in the 20 th century, because the traditional oil-containing adjuvant IS easy to cause immune side reactions such as granuloma, ulcer, fever and the like, with the development of scientific technology, the types of adjuvants discovered by people are more and more, and an immunostimulating complex (IS-COMs) adjuvant IS researched and developed by Morein et al (1984, Nature) of Sweden scholars in about 1984, mainly comprises lipid, cholesterol, antigen, glucoside Quil-A and the like, IS used as an adjuvant of an antigen delivery system, can capture protein antigen through surface hydrophobic effect, and IS taken up by APC through endocytosis. The Immune Stimulating Complex (ISCOM) vaccine antigen presentation system is very complete, and can effectively realize efficient antigen delivery and improve the immune enhancement system when in use. The ISCOM can obviously improve the proliferation and differentiation capacity of T cells, thereby stimulating the organism to generate immune response and enabling animals to generate antibody response to various antigens. ISCOM has the antigen demand little, lasting effect is long-lived, the appearance time is early, antibody level is high, and do not receive existing antibody or characteristics such as maternal antibody influence. The application of the hydrophobic membrane protein is not limited, and the production process is simple, so the application range is wider. Numerous experiments have shown that the adjuvant has high safety and immunogenicity in vaccine applications. At present, the immune stimulating complex is widely applied to veterinary vaccines, is suitable for various animals such as guinea pigs, cows, dogs, rabbits, sheep and the like, can mediate wide and comprehensive immune response, and has stronger immunogenicity and safety.
Disclosure of Invention
The invention aims to provide a preparation method of a haemophilus parasuis adjuvant vaccine to improve the immune effect of the haemophilus parasuis vaccine.
In order to achieve the purpose, the technical method adopted by the invention is as follows:
a preparation method of a parasuis adjuvant vaccine comprises the following steps:
(1) HPS OMP5 PCR primer design and Synthesis
The whole gene of HPS OMP5 was amplified using the biological software Premier Primer5.0 design 1 pair of specific primers P1/P2(P1: CAACCGTAATAGCGTGACCT; P2: CACTTCTTTACTACCCTGAACCT) based on the HPSOMP5 gene sequence (CP 001321). Sent to Shanghai Biotechnology Co., Ltd for synthesis.
(2) Cloning of HPS OMP5 gene and construction of expression vector
The experiment uses a specific primer P1/P2, clones a vector according to a molecular cloning mode, then amplifies a Haemophilus parasuis OMP5 gene from a cloning plasmid pMD18T-OMP5 by a PCR method, performs double enzyme digestion by BamH I and Xho I, subclones the gene into a prokaryotic expression vector pET-28a (+), constructs a recombinant expression vector, and performs PCR identification and enzyme digestion identification.
(3) Induced expression of recombinant pET-28a-OMP5 fusion protein and purification and detection of protein
Inoculating the positive recombinant expression plasmid pET-28a-OMP5 to LB plate culture medium with corresponding resistance, culturing overnight at 37 ℃, selecting a single colony from the LB culture medium to inoculate 5ml of LB liquid culture medium with corresponding resistance, 200rpm, 37 ℃ overnight culture, inoculating the overnight culture solution into 200ml LB liquid culture medium added with corresponding resistance according to the inoculum size of 150-, culturing at 37 deg.C and 200rpm until OD600 of the bacterial liquid is 0.6-0.8, adding IPTG (final concentration of 0.4mM) to start induction expression, culturing at 200rpm and 37 deg.C until the OD600 of the bacterial liquid is 1.48-1.52, collecting bacteria, centrifuging the bacterial liquid (6000r/min, 15min), removing supernatant, resuspending the bacteria with 10ml of inclusion body buffer solution, centrifuging (6000r/min, 10min), removing supernatant, and purifying the bacteria for inclusion body protein.
(4) Preparation of ultrasonic crushing antigen of haemophilus parasuis
And (3) resuspending the thalli prepared in the step (3) by using 10ml of inclusion body buffer solution, and ultrasonically crushing the thalli at the temperature of 4 ℃, wherein the main modes are as follows: and (4) performing ultrasonic treatment for 3s, stopping for 6s, performing amplitude 38% and performing ultrasonic treatment for 15min, wherein the steps are performed in an ice bath condition until the bacteria liquid is transparent. After ultrasonic treatment, the mixture is centrifuged at 10000r/min at 4 ℃ for 20min, 20 mul of supernatant is taken, the sample is electrophoresed, the precipitate is resuspended and centrifuged by 10ml of inclusion body buffer solution (12000r/min, 4 ℃, 10min), and the supernatant is removed. The lysed pellet was resuspended in 10ml of inclusion body wash, centrifuged (12000r/min, 4 ℃, 10min), the pellet was collected, resuspended in inclusion body wash and centrifuged. This was repeated 3 times to wash out the water-soluble protein from the inclusion bodies. And finally washing the collected precipitate, re-suspending with 10ml of inclusion body dissolving solution, uniformly blowing and vibrating on a vortex instrument for 1-3min to fully dissolve the inclusion body protein. Then, the mixture was centrifuged at 12000r/min for 10min, and 20. mu.l of the supernatant was subjected to electrophoresis. 10ml of the supernatant was placed in a dialysis bag and sealed, and placed in a 5L beaker containing 400ml of peripheral dialysate, and left to equilibrate well at 4 ℃. Then, clean water was gradually and slowly added dropwise to the peripheral dialysate by a peristaltic pump until the peripheral dialysate was diluted 10 times (time was 8 hours at minimum depending on flow rate). And putting the sample after the first dialysis into a second dialysate for dialysis until the concentration of urea in the protein solution is lower than 0.04M (dialysis time is 3-4 h). Sampling, measuring protein concentration and electrophoresis. And (4) subpackaging and storing the protein sample with successful renaturation in a refrigerator at the temperature of-20 ℃ for later use.
(5) Preparation and characterization of immunostimulatory Complex (ISCOMs) matrices
Referring to the Wayanna method, in obtaining an immunostimulant, phosphatidylcholine and cholesterol are first added to 20% mega-10, so that they are completely dissolved, dispensed and stored in a low temperature environment to prevent denaturation. 0.1g of QuilA was then dissolved in 5mL of PBS buffer pH7.2 to a final concentration of 20 mg/mL. Taking 10mg/mL OMP 51 mL, adding non-ionic active agent mega-10 to make the final concentration 2%, treating at 37 deg.C for 12-18 h, and cracking. This was then added to 0.1mL of lipid stock and 5mL of QuilA, shaken and then cleaved at RT for 240 min. Sonicate in ice bath for 1.5h at room temperature, then place in a 4 ℃ environment for 3 days (PBS pH7.2-7.4), place in a 12000 MW dialysis bag for the first two days and a 30000 MW dialysis bag for the last day), and filter with a 0.22. mu.L filter. Taking 20 mu L, adopting a BCA protein concentration determination kit to determine the protein content in ISCOM-OMP5, performing the measuring steps according to the instruction, taking a sample, carrying out phosphotungstic acid negative staining, sending the sample to a Wuhan virus institute electron microscope chamber, and observing the sample under a transmission electron microscope. Filtering, sterilizing and storing at 4 ℃.
The immune stimulating complex (ISCOM-OMP5) containing the Haemophilus parasuis OMP5 antigen is prepared by a dialysis method, and the ISCOM-OMP5 is used as the antigen of the Haemophilus parasuis vaccine to carry out immunological evaluation through a mouse model. The ISCOM-OMP5 vaccine was used to immunize mice, and after two immunizations, the ISCOM-OMP5 vaccine effect was evaluated by measuring the levels of H.parauis antibodies, cytokines such as IL-2, IL-4, IL-10, TNF- α, and IFN- γ, and lymphocyte proliferation in mice. The results show that the ISCOM-OMP5 prepared by observation under an electron microscope has a typical honeycomb structure of ISCOM, the diameter is 30nm-40nm, and the encapsulation rate of OMP5 protein reaches 85.8%. The high-level specific H.parasus antibody can be detected in the serum of the mouse 1 week after the mouse is immunized twice, the mouse in-vitro lymphocyte proliferation and cytokine detection results show that the ISCOM-OMP5 has a remarkable enhancement effect (P <0.01) on the humoral immunity of the mouse body, and also has a remarkable promotion effect (P <0.001) on the expression of IL-10 and IFN-gamma. The results show that the ISCOM-OMP5 can effectively induce mice to generate immune response aiming at H.
Drawings
FIG. 1 is the result of SDS-PAGE analysis of ISCOM-HPS OMP 5; wherein M is Marker; 1 HPS OMP 5.
FIG. 2 is an electron micrograph (Bar 200nm) of ISCOM-HPSOMP 5.
FIG. 3 is mouse HPS antibody levels; *: p is less than 0.05; **: p is less than 0.01; ***: p is less than 0.001.
FIG. 4 shows the gene expression level of mouse peripheral blood cytokines; *: p is less than 0.05; **: p is less than 0.01; ***: p is less than 0.001.
FIG. 5 shows the results of mouse lymphocyte proliferation; *: p is less than 0.05; **: p is less than 0.01; ***: p is less than 0.001.
Detailed Description
The invention relates to the following materials:
test strains: the haemophilus parasuis serotype 5 strain is saved in the laboratory of the swine epidemic disease prevention and control engineering research center of Fujian province.
Experimental animals: the clean grade ICR laboratory mouse has a male and female half, and a body weight of 22-25g (provided by Fujian Wu's laboratory animals Co., Ltd., certification number: SCXK (Min) 2016-.
The main reagents are as follows: TSA and TSB are purchased from Kyork, Guangdong, Microbiol technologies, Inc.; NAD was purchased from shanghai krammar; cholesterol was purchased from Sigma; phosphatidylcholine, N-decanoyl-N-methylglucamine (Mega-10) from Amersham; QuilA was purchased from Accurate; the BCA protein concentration determination kit is purchased from Biyuntian biotechnology limited company; the HPS antibody detection kit is purchased from Peking Firey Biotech. Blood RNA, DNA reverse transcription and fluorescence quantitative detection kit purchased from TAKARA biological company.
Example 1
A preparation method of a parasuis adjuvant vaccine comprises the following steps:
(1) HPS OMP5 PCR primer design and Synthesis
The whole gene of HPS OMP5 was amplified using the biological software Premier Primer5.0 design 1 pair of specific primers P1/P2(P1: CAACCGTAATAGCGTGACCT; P2: CACTTCTTTACTACCCTGAACCT) based on the HPSOMP5 gene sequence (CP 001321). Sent to Shanghai Biotechnology Co., Ltd for synthesis.
(2) Cloning of HPS OMP5 gene and construction of expression vector
The experiment uses a specific primer P1/P2, clones a vector according to a molecular cloning mode, then amplifies a Haemophilus parasuis OMP5 gene from a cloning plasmid pMD18T-OMP5 by a PCR method, performs double enzyme digestion by BamH I and Xho I, subclones the gene into a prokaryotic expression vector pET-28a (+), constructs a recombinant expression vector, and performs PCR identification and enzyme digestion identification.
(3) Induced expression of recombinant pET-28a-OMP5 fusion protein and purification and detection of protein
Inoculating positive recombinant expression plasmid pET-28a-OMP5 to LB plate culture medium with corresponding resistance, culturing overnight at 37 ℃, selecting a single colony from the LB culture medium, inoculating to 5ml of LB liquid culture medium with corresponding resistance, culturing overnight at 200rpm and 37 ℃, inoculating 200 mul of bacterial liquid cultured overnight according to the inoculation amount of 150-.
(4) Preparation of ultrasonic crushing antigen of haemophilus parasuis
And (3) resuspending the thalli prepared in the step (3) by using 10ml of inclusion body buffer solution, and ultrasonically crushing the thalli at the temperature of 4 ℃, wherein the main modes are as follows: and (4) performing ultrasonic treatment for 3s, stopping for 6s, performing amplitude 38% and performing ultrasonic treatment for 15min, wherein the steps are performed in an ice bath condition until the bacteria liquid is transparent. After ultrasonic treatment, the mixture is centrifuged at 10000r/min at 4 ℃ for 20min, 20 mul of supernatant is taken, the sample is electrophoresed, the precipitate is resuspended and centrifuged by 10ml of inclusion body buffer solution (12000r/min, 4 ℃, 10min), and the supernatant is removed. The lysed pellet was resuspended in 10ml of inclusion body wash, centrifuged (12000r/min, 4 ℃, 10min), the pellet was collected, resuspended in inclusion body wash and centrifuged. This was repeated 3 times to wash out the water-soluble protein from the inclusion bodies. And finally washing the collected precipitate, re-suspending with 10ml of inclusion body dissolving solution, uniformly blowing and vibrating on a vortex instrument for 1-3min to fully dissolve the inclusion body protein. Then, the mixture was centrifuged at 12000r/min for 10min, and 20. mu.l of the supernatant was subjected to electrophoresis. 10ml of the supernatant was placed in a dialysis bag and sealed, and placed in a 5L beaker containing 400ml of peripheral dialysate, and left to equilibrate well at 4 ℃. Then, clean water was gradually and slowly added dropwise to the peripheral dialysate by a peristaltic pump until the peripheral dialysate was diluted 10 times (time was 8 hours at minimum depending on flow rate). And putting the sample after the first dialysis into a second dialysate for dialysis until the concentration of urea in the protein solution is lower than 0.04M (dialysis time is 3-4 h). Sampling, measuring protein concentration and electrophoresis. And (4) subpackaging and storing the protein sample with successful renaturation in a refrigerator at the temperature of-20 ℃ for later use.
(5) Preparation and characterization of immunostimulatory Complex (ISCOMs) matrices
Referring to the Wayanna method, in obtaining an immunostimulant, phosphatidylcholine and cholesterol are first added to 20% mega-10, so that they are completely dissolved, dispensed and stored in a low temperature environment to prevent denaturation. 0.1g of QuilA was then dissolved in 5mL of PBS buffer pH7.2 to a final concentration of 20 mg/mL. Taking 10mg/mL OMP 51 mL, adding non-ionic active agent mega-10 to make the final concentration 2%, treating at 37 deg.C for 12-18 h, and cracking. This was then added to 0.1mL of lipid stock and 5mL of QuilA, shaken and then cleaved at RT for 240 min. Sonicate in ice bath for 1.5h at room temperature, then place in a 4 ℃ environment for 3 days (PBS pH7.2-7.4), place in a 12000 MW dialysis bag for the first two days and a 30000 MW dialysis bag for the last day), and filter with a 0.22. mu.L filter. Taking 20 mu L, adopting a BCA protein concentration determination kit to determine the protein content in ISCOM-OMP5, performing the measuring steps according to the instruction, taking a sample, carrying out phosphotungstic acid negative staining, sending the sample to a Wuhan virus institute electron microscope chamber, and observing the sample under a transmission electron microscope. Filtering, sterilizing and storing at 4 ℃.
Example 2
Evaluation of adjuvant Activity of immunostimulatory complexes (ISCOMs) to generate specific immune responses in mice
60 clean ICR mice are randomly selected, 3 experimental groups are set, and 20 mice in each group are half female and half male. ISCOM-OMP5 vaccine group, inactivated vaccine control group, negative control group, and first immunization were injected subcutaneously at a concentration of 0.2 ml each, and second immunization was performed in the same manner after 14 days.
Blood collection of mice: blood was collected from the orbital wells of mice on days 7 and 14 after the secondary immunization, respectively, and a portion of the blood was centrifuged at 4000r/min for 20min to collect serum, which was stored at 2-8 ℃ for detection of IgG antibodies; the other part of the blood was treated with anticoagulant and stored at-70 deg.C for testing mouse peripheral blood lymphocyte proliferation and cytokine in serum.
Detection of mouse haemophilus parasuis IgG antibody: the relevant detection steps are carried out according to the instructions of the kit.
Detection of mouse cytokines: primers and primer sequences of an internal reference gene beta-Actin are designed according to a mouse cytokine gene sequence published by Genbank and are synthesized and provided by Shanghai Biotechnology GmbH. The extraction is carried out according to the instruction, and the amplification process is as follows: the pre-denaturation and denaturation was carried out at 95 ℃ for 30 seconds and the annealing was carried out at 60 ℃ for 30 seconds for a total of 40 cycles (see Table 1).
TABLE 1 fluorescent quantitative PCR primer sequences and annealing temperatures
Mouse lymphocyte proliferation assay: 100 mu.l of peripheral blood of the mouse is taken, 6mL of Ficoll-Paque separating medium is added, and the peripheral blood of the mouse is carefully paved on the Ficoll-Paque separating medium. Centrifuging at 18-20 deg.c and 400r/min for 30-40 min. The upper plasma and platelets were aspirated off with a sterile pipette, and the mononuclear cell layer was transferred to a sterile centrifuge tube. The volume of transferred mononuclear cells is estimated. Adding at least 3 times volume of PBS solution, using a pipette to gently resuspend the cells, performing centrifugation at the temperature of 18-20 ℃ and the temperature of 400-. 6-8mL PBS solution heavy suspension of mononuclear cells. Centrifuging at the temperature of 18-20 ℃ and the temperature of 400r/min for 10min, and removing supernatant to obtain lymphocyte suspension. The cell concentration was prepared by dilution to 7.5X 106/mL for use, the final concentration of LPS in the culture solution was 12.5. mu.g/mL, and the final concentration of ConA was 4. mu.g/mL, while a blank group and a control group were set. Incubating for 44h, after incubation, adding 20 μ L CCK8 to all wells, and further incubating at 37 deg.C and 5% CO2Culturing for 1h in an incubator, and measuring the OD450 nm value by using an enzyme-labeling instrument. Lymphocyte Stimulation Index (SI) ═ OD stimulated wells-OD blank wells)/(OD unstimulated wells-OD blank wells.
And (3) data analysis: statistical analysis of experimental data is carried out by using an SPSS 20.0 statistical software tool, single-factor analysis of variance and an LSD method for comparison among groups, and when P is less than 0.05, the difference is considered to have statistical value and can explain experimental conclusions; calculating the gene expression of the fluorescent quantitative PCR by using a 2-delta Ct method, selecting beta-Actin as an internal reference gene, and adopting the calculation formula as follows:
ΔΔCT＝(CTTarget-CTβ-Actin)experimental-(CTTarge-CTβ-Actin)control。 (2)
results of the experiment
(1) SDS-PAGE analysis
The SDS-PAGE result showed that the band size of the prepared ISCOM-HPSOMP5 was about 40.0kDa, which is the same as the band of HPSOMP5 (see FIG. 1).
(2) Preparation of ISCOM-OMP5
As a result of electron microscope observation, the prepared ISCOM-OMP5 has uniform size, diameter distribution of 30-40 nm, and honeycomb structure (see FIG. 2).
(3) Protein encapsulation efficiency
The prepared ISCOM-OMP5 was diluted 10-fold, the A600 value was determined to be 2.29, and the protein concentration after dilution was 0.29mg/mL, i.e., the protein encapsulation amount was 8.58mg according to the standard curve, thereby calculating the encapsulation efficiency of HPSOMP5 to be 85.8%.
(4) Mouse antibody detection results
The level of IgG antibody of the immunized mouse is detected, and 1 week after the second immunization, the level of H.parauis antibody of ISCOM-OMP5 vaccine group is obviously higher than that of the negative control group (P < 0.001); the ISCOM-OMP5 vaccine group had significantly or very significantly higher levels of h.parauis antibodies than the inactivated vaccine group (P <0.05) and the negative control group (P <0.001) 2 weeks after the second immunization, indicating that the ISCOM-OMP5 vaccine induced good immune responses in mice (see fig. 3).
(5) Results of cytokine detection
The CT value obtained by the fluorescent quantitative PCR of the mice 2 weeks after immunization is quantitatively analyzed, and the result shows that compared with a negative control group, the ISCOM-OMP5 vaccine group has obvious promotion effect on the expression of IL-10 and IFN-gamma (P is less than 0.001), and has no obvious effect on the expression of IL-2, IL-4 and TNF-alpha (P is more than 0.05); compared with the inactivated vaccine group, the ISCOM-OMP5 vaccine group has a remarkable promotion effect on the expression of IFN-gamma (P <0.001) (see FIG. 4).
(6) Results of lymphocyte proliferation assay
Lymphocytes are separated at 7d and 14d after the secondary immunization of the mice respectively, the proliferation capacity of the lymphocytes generated by the lymphocytes stimulated by ConA and LPS in vitro is obviously higher than that of a negative control group (P <0.05) but is not significantly different from that of the inactivated vaccine group (P >0.05) in 7d after the secondary immunization of the mice, ISCOM-OMP5 vaccine group and inactivated vaccine group, and the proliferation capacity of the lymphocytes stimulated by the ConA and the LPS in vitro is significantly higher than that of the negative control group (P <0.05) in 14d after the secondary immunization of the mice, and the proliferation capacity of the lymphocytes stimulated by the ConA and the LPS in vitro of the ISCOM-OMP5 vaccine group is significantly higher than that of the negative control group (P <0.05), wherein the proliferation capacity of the B cells stimulated by the LPS in vitro of the ISCOM-OMP5 vaccine group is far higher than that of the inactivated vaccine group (P <0.05) and the negative control group (P <0.001), which shows that the ISCOM-OMP5 vaccine has a significant enhancement effect on the humoral immunity of the mouse organism (5).
In conclusion, the invention has the following beneficial effects:
(1) in the invention, 2 weeks after the secondary immunization, the level of the mouse haemophilus parasuis specific H.parasuis antibody for immunizing the ISCOM-OMP5 vaccine is obviously higher than that of an inactivated vaccine group without an adjuvant (P <0.01), which indicates that the ISCOM-OMP5 vaccine can play an effective immune enhancement role for experimental mice.
(2) The ISCOM-OMP5 vaccine group is remarkably higher in lymphocyte proliferation capability generated by in vitro stimulation of ConA and LPS than a negative control group (P <0.05) when the ISCOM-OMP5 vaccine group is directly acted on mouse lymphocytes in vitro, wherein the ISCOM-OMP5 antigen is combined with an immunostimulation compound to prepare the ISCOM-OMP5 vaccine, and the lymphocyte proliferation capability generated by in vitro stimulation of ConA and LPS is remarkably higher than that of the negative control group (P <0.05) after the nonimmunization, wherein the ISCOM-OMP5 vaccine group is remarkably improved in vitro stimulation and B cell proliferation capability brought by LPS and far exceeds that of an inactivated vaccine group (P <0.05) and the negative control group (P <0.001), and the ISCOM-OMP5 vaccine has a remarkable enhancing effect on humoral immunity of mouse organisms.
(3) After the mice are immunized twice, the ISCOM-OMP5 vaccine group has obvious promotion effect on the expression of IL-10 and IFN-gamma (P is less than 0.01), and the ISCOM-OMP5 vaccine prepared by the method has the effects of promoting T cell activation and promoting B cell transformation.
(4) The results of preliminary experiments on mice show that ISCOM-OMP5 vaccine prepared by ISCOM has better immune effect: on one hand, the antigenicity of the HPS thallus outer membrane protein OMP5 is proved to have better immunogenicity; on the other hand, experiments show that the ISCOM-OMP5 vaccine has obvious adjuvant effect and can obviously enhance the immune effect, and the immune mechanism of the ISCOM-OMP5 vaccine is probably related to the induction of higher humoral immunity.
Claims (6)
1. A preparation method of a parasuis adjuvant vaccine is characterized by comprising the following steps:
(1) designing and synthesizing HPS OMP5 PCR primer;
(2) cloning HPS OMP5 gene and constructing expression vector;
(3) induction expression of the recombinant pET-28a-OMP5 fusion protein and purification detection of the protein;
(4) preparing an ultrasonic crushing antigen of haemophilus parasuis;
(5) preparing and identifying an immune stimulation compound matrix;
the preparation and identification steps of the immune stimulation compound matrix are as follows: in obtaining the immunostimulant, phosphatidylcholine and cholesterol are first added to 20% mega-10 to be completely dissolved, then subpackaged and stored in a low-temperature environment, and 0.1g of QuilA is then dissolved in 5mL of PBS buffer solution with pH of 7.2 to obtain a solution with a final concentration of 20 mg/mL; taking 10mg/mL OMP 51 mL, adding a non-ionic active agent mega-10 to make the final concentration 2%, and treating at 37 ℃ for 12h-18h to crack; then adding 0.1mL of lipid stock solution and 5mL of QuilA, shaking uniformly, and then cracking for 240min at RT; ultrasonic treating in ice bath at room temperature for 1.5 hr, standing at 4 deg.C for 3 days, placing in 12000 molecular dialysis bag in the first two days, and filtering with 30000 molecular dialysis bag in 0.22 μ L filter in the last day; taking 20 mu L, adopting a BCA protein concentration determination kit to determine the protein content in ISCOM-OMP5, taking a sample, carrying out phosphotungstic acid negative staining on the sample, observing the sample under a transmission electron microscope, and storing the sample at 4 ℃ after filtering and sterilizing.
2. The method for preparing a parasuis adjuvant vaccine according to claim 1, wherein the PCR primer design and synthesis steps are as follows: amplifying the whole gene of HPS OMP5 by using 1 pair of specific primers P1/P2 designed by biological software Premier Primer5.0 according to the HPSOMP5 gene sequence;
P1:CAACCGTAATAGCGTGACCT；
P2:CACTTCTTTACTACCCTGAACCT。
3. the method for preparing a parasuis adjuvant vaccine according to claim 2, wherein the cloning of the HPS OMP5 gene and the construction of the expression vector comprise the following steps: cloning the vector in a molecular cloning manner by using specific primers P1/P2, and then usingThe PCR method is used to amplify the OMP5 gene of haemophilus parasuis from the cloning plasmid pMD18T-OMP5BamHI andXhoafter double enzyme digestion, the gene is subcloned into a prokaryotic expression vector pET-28a (+), a recombinant expression vector is constructed, and PCR identification and enzyme digestion identification are carried out.
4. The method for preparing the adjuvant vaccine of Bacteroides parasuis according to claim 3, wherein the steps of inducing expression of the recombinant pET-28a-OMP5 fusion protein and purifying and detecting the protein are as follows: inoculating the positive recombinant expression plasmid pET-28a-OMP5 to an LB plate culture medium with corresponding resistance, culturing overnight at 37 ℃, selecting a single colony from the LB culture medium, inoculating the single colony to 5ml of LB liquid culture medium with corresponding resistance, culturing overnight at 200rpm and 37 ℃, inoculating 200 mul of the overnight-cultured bacterial liquid into 200ml of LB liquid culture medium with corresponding resistance according to the inoculation amount of 150-.
5. The method for preparing a parasuis adjuvant vaccine according to claim 4, wherein the ultrasonication antigen is prepared by the steps of: resuspending the thalli prepared in the step (3) by using 10ml of inclusion body buffer solution, and ultrasonically crushing the thalli at the temperature of 4 ℃ until bacterial liquid is transparent; centrifuging at 4 ℃ after ultrasonic treatment, taking 20 mu l of supernatant, carrying out sample electrophoresis, resuspending the precipitate by using 10ml of inclusion body buffer solution, and centrifuging to remove the supernatant; resuspending the cracked precipitate with 10ml of inclusion body washing solution, centrifuging, collecting the precipitate, then resuspending with the inclusion body washing solution, centrifuging, repeating the operation for 3 times in this way, fully washing off the water-soluble protein in the inclusion body, washing the collected precipitate for the last time, resuspending with 10ml of inclusion body dissolving solution, blowing, uniformly mixing, vibrating on a vortex instrument to fully dissolve the inclusion body protein, centrifuging, and taking 20 mu l of supernatant for electrophoresis; placing 10ml of supernatant in a dialysis bag, sealing, placing in a beaker filled with 400ml of peripheral dialysate, and placing at 4 ℃ for full balance; then gradually and slowly dripping clear water into the peripheral dialysate by using a peristaltic pump until the peripheral dialysate is diluted by 10 times, putting the sample after the first dialysis into the secondary dialysate for dialysis until the concentration of urea in the protein liquid is lower than 0.04M, sampling to measure the protein concentration, performing electrophoresis, and finally subpackaging and storing the successfully renatured protein sample in a refrigerator at the temperature of-20 ℃ for later use.
6. The method for preparing a parasuis adjuvant vaccine according to claim 5, wherein the ultrasonication step comprises: performing ultrasonic treatment for 3s, stopping for 6s, performing ultrasonic treatment for 15min at amplitude of 38%, wherein the steps are performed under the ice bath condition until the bacterial liquid is transparent; the time of the first dialysis is not less than 8h, and the time of the second dialysis is 3-4 h.
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