A Trichinella spiralis vaccine kit based on Prime-boost sequential immunization program and its application

Abstract

The present invention discloses a Trichinella vaccine kit based on Prime-boost sequential immunization program and its application, belonging to the field of genetic engineering technology. The present invention is to improve the protective effect of trichinosis. The present invention provides a Trichinella vaccine kit, the kit is divided into two parts, the first part is a recombinant protein vaccine of Trichinella with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, and the second part is a recombinant adenovirus expressing an amino acid sequence as shown in SEQ ID NO.1. The Prime-boost immunization strategy not only effectively avoids the shortcomings of a single type of Trichinella vaccine, but also significantly improves the efficacy and persistence of the immune response, providing a more efficient and comprehensive immune protection for the body.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a Trichinella spiralis vaccine kit based on a Prime-boost sequential immunization program and an application thereof. Background Art

[0002] Trichinellosis is a disease caused by the nematode Trichinella spiralis ( Trichinella spiralis The zoonotic parasitic disease caused by the infection of Trichinella spiralis is a huge threat to the livestock breeding industry, resulting in stunted animal growth, reduced meat quality, and even animal death, causing direct economic losses. At the same time, humans who eat meat infected with Trichinella spiralis may also become infected and have serious health problems, further affecting food safety and public health. Therefore, trichinellosis not only affects the sustainable development of animal husbandry, but also poses a potential risk to human health. Effective prevention and control measures need to be taken to ensure the safety of animal husbandry production and human health.

[0003] Although trichinellosis has a serious impact on animal economy and animal husbandry, there is currently no effective vaccine for trichinellosis. Traditional vaccine development methods, such as attenuated live vaccines and natural antigen vaccines, are difficult to prepare such antigens through standardized mass production methods of artificially infected animals due to the complex antigen composition of the Trichinella spiralis and the fact that Trichinella spiralis cannot complete its entire life cycle through in vitro culture. Therefore, they cannot meet the needs of antigens for immune diagnosis and immune prevention. With the rapid development of genetic engineering technology, new vaccines such as recombinant protein vaccines, peptide vaccines, and DNA vaccines have been continuously explored and applied in the field of trichinellosis prevention and control. Although they have certain protective power, they have not been able to achieve complete protection against Trichinella spiralis infection. Summary of the invention

[0004] The purpose of the present invention is to improve the protective effect of trichinellosis.

[0005] The present invention provides a Trichinella vaccine kit, which is divided into two parts. The first part is a Trichinella recombinant protein vaccine with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, and the second part is a recombinant adenovirus expressing the amino acid sequence as shown in SEQ ID NO.1.

[0006] It is further defined that the content of the recombinant protein of Trichinella spiralis is 60 μg; the content of the recombinant adenovirus expressing the amino acid sequence as shown in SEQ ID NO.1 is 10 8 PFU; the immunization method of the recombinant protein vaccine of Trichinella spiralis is intramuscular injection; the immunization method of the recombinant adenovirus expressing the amino acid sequence shown in SEQ ID NO.1 is nasal injection.

[0007] It is further defined that the Trichinella recombinant protein vaccine is a Trichinella recombinant protein with amino acids as shown in SEQ ID NO.2 and a CPG1018 adjuvant in a ratio of 1:1 / v:v.

[0008] The present invention provides an application of the above-mentioned kit in preparing a medicine for preventing or treating trichinellosis.

[0009] The invention provides a Trichinella spiralis recombinant protein, the amino acid sequence of the recombinant protein is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0010] The present invention provides the nucleic acid molecule of the above-mentioned vaccine.

[0011] The present invention provides a recombinant vector of the above nucleic acid molecule.

[0012] The present invention provides a recombinant microbial cell containing the above-mentioned nucleic acid molecule.

[0013] The present invention provides the use of the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the preparation of a vaccine or medicine for preventing or treating trichinellosis.

[0014] It is further defined that the vaccine is in the form of a nasal spray, an oral formulation, a suppository or a parenteral formulation.

[0015] Beneficial effects: By using recombinant adenovirus vaccines for primary immunization (prime) and recombinant protein vaccines for boost immunization (boost), the immunogenicity of the vaccine can be effectively improved, thereby stimulating the body to produce a stronger immune response. This strategy combines the high immunogenicity of recombinant adenovirus vaccines and the high safety of recombinant protein vaccines, synergistically enhancing the immune response and providing solid immune protection for the body. In addition, the prime-boost immunization scheme can also stimulate the body to form long-term immune memory, which means that once the body encounters an insect infection again, it can quickly initiate a strong immune response and quickly eliminate the pathogen, thereby effectively reducing the risk of disease.

[0016] The Prime-boost immunization strategy shows unique advantages in the development of Trichinella vaccine, aiming to overcome the limitations of a single vaccine type. This strategy first uses recombinant adenovirus vaccine for primary immunization (prime), which can efficiently and short-term activate humoral immunity and cellular immunity, including stimulating B lymphocytes to produce plasma cells to secrete antibodies such as IgG to neutralize the virus, and activating cytotoxic T lymphocytes to recognize and kill virus-infected target cells, thereby clearing the virus. However, homologous immune Trichinella recombinant adenovirus vaccine may cause pre-existing immune problems, reduce immunogenicity, and cause adverse reactions such as fever. The Trichinella recombinant protein vaccine as a booster shot (boost) can make up for the shortcomings of the recombinant adenovirus vaccine. It has strong immunogenicity and can significantly improve the level of neutralizing antibodies. It is also highly safe, has few adverse reactions, has good stability, and is not demanding on storage and transportation conditions. Therefore, the Prime-boost immunization strategy not only effectively avoids the shortcomings of a single type of Trichinella vaccine, but also significantly improves the efficacy and durability of the immune response, providing the body with more efficient and comprehensive immune protection.

[0017] Adenovirus has the problem of pre-existing immunity. If two injections of adenovirus vaccine are given, the immune effect will be affected. Recombinant protein vaccines require adjuvants and 2-3 injections. Considering that the trichinellosis vaccine is mainly used in pigs (pigs are the main source of infection), and the release of pigs is time-limited (about half a year). Adenovirus vaccines do not require adjuvants, and single-dose vaccination is highly effective. Therefore, adenovirus vaccines are used for the first immunization to activate the immune system, and only one recombinant protein vaccine is used for booster immunization, avoiding the problem of long immunization cycles. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the recombinant adenovirus plasmid rAd5TsCLP and the identification result. M is DL15000 DNA Marker. Lane 1 is the recombinant adenovirus plasmid rAd5TsCLP. Lane 2 is the PacI restriction linearization identification result.

[0019] Figure 2 The results of IFA analysis of Ts-CLP protein expression in HEK293A cells infected with recombinant adenovirus rAd5TsCLP;

[0020] Figure 3 This is the prediction result of Trichinella spiralis protein signal peptide;

[0021] Figure 4 This is a flowchart of prokaryotic expression and immunization of recombinant proteins;

[0022] Figure 5 This is the result of SDS-PAGE and WB verification of Ts-CLP protein expression of Trichinella spiralis recombinant protein vaccine;

[0023] Figure 6 The following are the Prime-boost animal immunization program diagram and specific immunization dosage result diagram; A is the animal immunization program diagram; B is the immunization dosage result diagram;

[0024] Figure 7 The results of the protection analysis of mice immunized with rAd5TsCLP after being challenged with 250 Trichinella spiralis muscle larvae. A is the average number of larvae per gram of muscle (LPG), and B is the average number of AD3 adults per gram of muscle. The data are mean ± SEM.

[0025] Figure 8 This is the result of HE staining of diaphragm tissue;

[0026] Fig. 9 Figure 1 is the result of the detection of IgG, IgG1, IgG2a, IgA, IgM, and NAb specific antibody levels in mouse serum (*, P<0.05; **, P<0.01; ***, P<0.001); A is the result of the detection of IgG specific antibody levels in mouse serum; B is the result of the detection of IgG1 specific antibody levels in mouse serum; C is the result of the detection of IgG2a specific antibody levels in mouse serum; D is the result of the detection of IgA specific antibody levels in mouse serum; E is the result of the detection of IgM specific antibody levels in mouse serum; F is the result of the detection of neutralizing antibody NAb levels in mouse serum;

[0027] Fig.10 are the levels of cytokines in mouse serum (*, P<0.05; **, P<0.01; ***, P<0.001); A is the result graph of IFN-γ level in mouse serum; B is the result graph of TNF-α level in mouse serum; C is the result graph of IL-4 level in mouse serum; D is the result graph of IL-10 level in mouse serum;

[0028] Fig.11 The numbers of CD3+CD4+, CD3+CD8+, and CD3+CD4+CD8+ T lymphocytes (*, P<0.05; **, P<0.01; ***, P<0.001). DETAILED DESCRIPTION

[0029] Example 1 Preparation of recombinant adenovirus vaccine

[0030] 1. Construction of rAd5TsCLP recombinant adenovirus

[0031] The Ts-CLP target gene was obtained. The CDS sequence of Ts-CLP was obtained from PubMed (SEQ ID NO.3). The optimized codon nucleotide sequence suitable for expression in mammalian cells was designed, with a total length of 1218 bp. The Ts-CLP fragment was amplified and identified. The recombinant adenovirus expressing the Ts-CLP gene was constructed using the AdEasy system of adenovirus type 5 with E1 and E3 deleted. The pShuttle-CMV-Ts-CLP shuttle plasmid was constructed, transformed into DH5α competent cells for amplification, linearized by PmeI, and transformed into BJ5183 bacteria already carrying the pAdEasy-1 plasmid for homologous recombination, and finally screened by kanamycin.

[0032] SEQ ID NO.3:

[0033]

[0034] A small amount of amplified bacteria was used to extract plasmids, which were then digested with PacI for identification. After PacI digestion, the recombinant adenovirus plasmid obtained a large fragment of about 30Kb and a small fragment of 3Kb or 4.5Kb. The correctly identified recombinant adenovirus plasmid rAd5TsCLP was transformed into XL10-Gold super competent bacteria for amplification. The linearized recombinant adenovirus plasmid digested with PacI was used for adenovirus packaging after DNA purification ( Figure 1 ).

[0035] 2. Packaging of rAd5TsCLP recombinant adenovirus

[0036] Inoculate 7-8×10 5 cells / mL of HEK293A cells were placed in a 60-mm cell culture dish and cultured for 24 hours. The recombinant adenovirus plasmid after PacI digestion was transfected into HEK293A cells cultured overnight by Lipofectamine 3000. After 6 hours of transfection, fresh cell culture medium was replaced and cultured for 7 to 10 days. The cells were obviously floating, and the adenovirus was collected to obtain the recombinant adenovirus vaccine rAd5TsCLP.

[0037] 3. Collection and amplification of rAd5TsCLP recombinant adenovirus

[0038] Aspirate the adenovirus packaging cell culture medium, add 0.5mL PBS to scrape the cells and transfer them to a centrifuge tube. Repeatedly freeze and thaw at -80℃ and 37℃ for four times to lyse the cells, centrifuge and take the supernatant to obtain the P0 generation adenovirus, and store it for later use. The P0 generation virus was used to infect HEK293A cells for amplification, and the P1 generation virus was obtained after a small amount of amplification and repeated freezing and thawing. The virus was amplified in large quantities for three consecutive generations to the P4 generation, purified and concentrated. After determining the titer, the recombinant adenovirus rAd5TsCLP was packaged and stored. The results showed that IFA confirmed that rAd5TsCLP expressed Ts-CLP protein in HEK293A cells ( Figure 2 ).

[0039] SEQ ID NO.1:

[0040] MSFMHCIFVVLFFAVGEAQILGETTHYGRNDPVMLRNAHEALFSSDLKQESGVFHKLLELEESSTMGILTTMKVVMQDTDCPVSFALLSYYDVLVNCQGEGRRKHCTMEYTHRNPSKATVSKCFEEVEEPLIIPQRVKMIGGRAVYIDSNADVEEQMQMLGETTHYGRNDPVMLPKAREALFSSDSKEQSGVLHKLVELEESS TMGILTTMKVVIQDTECRVSSAYSSYYDVLHYCHGKGPRKHCTLEYRHRTPSTATVSECFEEVEEPLIVPQRVQRVNGRTIYLDSSDDVEEQVVSQRSQMLG GTTKYTDSNVHIKEEVKQAIFESDKKKSSGTYLLLDKIVEGFNMGISSRFQVLVKETECGIKEKAFNSYEDVYKNCSGSGDSKVCSVEYKYFDPTKSTVEC.

[0041] Example 2 Preparation of recombinant protein vaccine

[0042] 1. Preparation of expression bacteria

[0043] SignalP 6.0 software was used to predict and analyze the signal peptide of the sequence. The advantage of using a prokaryotic expression system to remove the signal peptide (amino acid sequence such as SEQ ID NO.2) is that it can avoid protein insolubility, inclusion body formation and potential processing problems caused by the signal peptide, thereby improving the soluble expression and purification efficiency of the protein ( Figure 3 ).

[0044] SEQ ID NO.2:

[0045] QILGETTHYGRNDPVMLRNAHEALFSSDLKQESGVFHKLLELEESSTMGILTTMKVVMQDTDCPVSFALLSYYDVLVNCQGEGRRKHCTMEYTHRNPSKATVSKCFEEVEEPLIIPQRVKMIGGRAVYIDSNADVEEQMQMLGETTHYGRNDPVMLPKAREALFSSDSKEQSGVLHKLVELEESSTMGILTTMK VVIQDTECRVSSAYSSYYDVLHYCHGKGPRKHCTLEYRHRTPSTATVSECFEEVEEPLIVPQRVQRVNGRTIYLDSSDDVEEQVVSQRSQMLGGTTKYTDSNVHIKEEVKQAIFESDKKKSSGTYLLLDKIVEGFNMGISSRFQVLVKETECGIKEKAFNSYEDVYKNCSGSGDSKVCSVEYKYFDPTKSTVEC;

[0046] The Trichinella spiralis protein gene without a signal peptide was cloned into the pET-28a(+) vector and transformed into BL21 competent cells. After overnight culture, a single clone was picked and placed in LB medium for expansion culture to prepare the expression bacteria pET28a-TsCLP and stored at -80°C.

[0047] 2. Induced expression of recombinant protein

[0048] Take out the expression bacteria pET28a-TsCLP stored in the -80℃ refrigerator, streak on the LB plate containing kanamycin with an inoculation loop, pick a single colony on the LB plate, inoculate it in 5 mL LB liquid medium, and expand the culture with 1 L liquid LB medium. When the OD600 of the bacterial solution is 0.4-0.6 detected by the microplate reader, add 1 mL of 1 mol / L IPTG, and then culture it on a horizontal shaker at 37℃ for 6 h. The induced bacterial solution was divided into 500 mL centrifuge bottles, centrifuged at 5000 g for 30 min, discarded the supernatant, took the bacterial pellet, and resuspended the bacterial pellet with PBS. Add the resuspended bacterial pellet to a 50 mL centrifuge tube and freeze-thaw three times. The resuspended bacteria were treated with an ultrasonic disruptor. The conditions for ultrasonic disruption were: ultrasonic power 200 W, duration 3 s, interval 3 s, total time 45 min, temperature 4℃. The bacterial suspension after ultrasonic disruption was centrifuged at 5000 g for 30 min, the supernatant was discarded, and the bacterial precipitate was collected.

[0049] 3. Gel excision and purification

[0050] The purified recombinant protein sample was mixed evenly with the protein loading buffer in proportion, and after boiling, it was used for SDS-PAGE electrophoresis. After the electrophoresis, the concentrated gel was removed, and the separation gel was placed in a 2.5% KCL solution, and then placed in a 4°C refrigerator. After 5 minutes, a white band appeared in the concentrated gel, and the white band was cut off with a sterile knife. The white band was cut into pieces and placed in a 2 mL centrifuge tube, and an appropriate amount of high-pressure sterilized PBS solution was added, and shaken in a 4°C refrigerator overnight. After centrifugation, the supernatant was extracted with a pipette, and then the target protein was concentrated to the desired concentration by centrifugation using a sterile ultrafiltration tube. The recombinant protein rTsCLP was obtained by removing endotoxins from the purified protein using a large-capacity endotoxin removal kit. The Ts-CLP protein was verified by SDS-PAGE and Western blot ( Figure 5 ).

[0051] The recombinant protein vaccine rTsCLP: adjuvant and amino acid as shown in SEQ ID NO.2 were prepared in a ratio of 1:1 / v:v, and then immunized.

[0052] Example 3 Prime-boost immune vaccine kit

[0053] 1. Composition of the kit: The first part is the recombinant protein obtained in Example 2; the second part is the recombinant adenovirus obtained in Example 1.

[0054] 2. Specific plan for animal experiment

[0055] Female BALB / c mice aged 6-8 weeks were randomly divided into 5 groups, with 10 mice in each group. 8 PFU rAd5TsCLP, 60μg recombinant protein vaccine rTsCLP (rTsCLP: CPG1018 = 1:1 / v:v) was injected into the thighs in the second week, and the total immunization volume was 100μL (rAd5TsCLP:rTsCLP). At the same time, the recombinant protein vaccine (rTsCLP:rTsCLP) for two immunizations and the recombinant protein vaccine (Ad5Neg:rTsCLP) for the first immunization with an empty adenovirus vector were used as the vaccine group control. At each immunization time point, the same amount of PBS was used as a blank control, and the immunization was 10 8PFU of empty adenovirus Ad5Neg without the target gene was used as an empty vector control. Two weeks after the last immunization, each mouse was orally infected with 250 Trichinella muscle larvae. The mice were euthanized on the 3rd day and 5th week after the worm infection, and the number of intestinal adults and muscle larvae was determined. According to the number of collected muscle larvae and muscle weight, the number of muscle larvae (LPG) per gram of muscle was calculated, and the worm reduction rate was calculated by comparing the average LPG of each group of mice with that of the PBS control group. Serum, spleen and small intestine were collected at 1w, 3w, and 9w to detect the humoral immunity, cellular immunity and mucosal immunity levels of each group. At the same time, the diaphragm of the mice was stained with HE for parasite load assessment ( Figure 6 ).

[0056] Example 4 Evaluation of the immune effect of the Prime-boost immune strategy

[0057] 10 were immunized by intramuscular injection and nasal drops respectively. 8 PFU recombinant adenovirus rAd5TsCLP was inoculated into 6-8 week old female BALB / c mice (10 mice per group), and the mice inoculated with adenovirus Ad5Neg were used as vaccine control, and the mice inoculated with PBS were used as blank control.

[0058] Detection of the number of intestinal adults and muscle larvae in mice: The mice were euthanized on the 3rd day and 5th week after infection with Trichinella spiralis, and the number of intestinal adults and muscle larvae in mice was determined, and the worm reduction rate was calculated. The number of muscle larvae in mice was determined by pepsin digestion method, as follows: pepsin and hydrochloric acid were added to distilled water at 37°C to prepare digestion solution, and the final concentrations of pepsin and hydrochloric acid were 1%. Each mouse was digested with 500 mL of digestion solution, placed on a magnetic stirrer in a 37°C thermostat, and digested for 2 h. After the entire digestion process was completed, the residue in the digestion solution was filtered out with a 60-mesh sieve, and allowed to stand at room temperature for 1 h. The upper layer of liquid was gently extracted with a sterile syringe, 100 mL of liquid was retained, 400 mL of distilled water was added again, and the solution was repeatedly washed. Finally, the liquid was transferred to a 50 mL beaker, and the muscle larvae of Trichinella spiralis were counted under a microscope.

[0059] 1. Calculation of muscle larvae reduction rate

[0060] Add pepsin and hydrochloric acid at a final concentration of 1% to distilled water at 37°C to prepare 500 mL of digestive solution for digestion for 2 hours per mouse. After digestion, filter the residue with a 60-mesh sieve, let it stand for 1 hour, extract the upper liquid and keep 100 mL, wash it repeatedly and transfer it to a 50 mL beaker. Count the muscle larvae of Trichinella spiralis under a microscope, calculate the number of muscle larvae per gram of muscle of each mouse (LPG), and compare it with the PBS control group to calculate the worm reduction rate of each experimental group. The formula is as follows:

[0061] Muscle larvae reduction rate (%) = 1- ×100%

[0062] The results showed that 5 weeks after the infection, compared with the PBS group, the muscle larval loads of Ad5Neg (adenovirus empty vector), Ad5Neg:rTs-CLP (adenovirus empty vector primary immunization, recombinant protein secondary immunization), rAd5TsCLP:rTs-CLP (adenovirus primary immunization, recombinant protein secondary immunization), and rTs-CLP:rTs-CLP (recombinant protein secondary immunization) were reduced by 9.88%, 21.29%, 58.22%, and 53.39%, respectively, and the adult loads were reduced by 9.28%, 34.36%, 61.17%, and 54.3%, respectively. Figure 7 ).

[0063] Combined immunization with rAd5TsCLP: rTsCLP muscle larvae reduced by 58.22%, while single immunization with rAd5TsCLP reduced by 45.61% with intramuscular injection and 47.91% with nasal drops.

[0064] Combined immunization with rAd5TsCLP:rTsCLP reduced the number of adult worms by 61.17%, while immunization with rAd5TsCLP alone reduced the number by 36.49% after intramuscular injection and 42.57% after intranasal drops.

[0065] 2. HE staining of diaphragm tissue

[0066] The diaphragm tissue was dehydrated, transparent, waxed, embedded, and then sliced ​​and stained with hematoxylin-eosin (HE) to observe its tissue structure and pathological changes. The results showed that compared with the PBS and Ad5Neg groups, more intense inflammatory cell infiltration appeared around the rAd5TsCLP:rTs-CLP cysts, and more inflammatory cells entered the cysts and came into direct contact with the worms ( Figure 8 ).

[0067] 3. Specific antibody levels

[0068] Specific antibody ELISA test

[0069] Indirect enzyme-linked immunosorbent assay (ELISA) was used to detect specific antibodies (IgG, IgG1, IgG2a, IgM, and IgA) in the serum of each group of mice. 5 μg / mL of purified recombinant protein rTsCLP was coated on a 96-well plate at 4°C overnight. Subsequently, 100 μL / well blocking solution (5% skim milk powder) was added and blocked at 37°C for 1 hour. The collected serum was diluted 10 times in series, incubated at 37°C for 1 hour, and then goat anti-mouse IgG-HRP was added for incubation. Finally, TMB substrate was added for color development, the reaction was terminated with 2M H2SO4 sulfuric acid, and the OD450 value was measured with an enzyme reader.

[0070] 4. Neutralizing Antibody NAb Detection

[0071] The collected sera were inactivated at 56°C for 30 min and then serially diluted 10-fold in a 96-well plate (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280 in 100 μL DMEM) and mixed with an equal volume of 100 TCID 50 Recombinant adenovirus rAd5TsCLP was mixed and incubated at 37°C for 1 hour, and then 200 μL of serum-virus mixture was added to the monolayer of HEK293A cells. Ad5Neg and HEK293A were used as control groups. The cells were cultured at 37°C and 5% CO2 for 72 hours, and the expression of viral fluorescent protein was observed and the neutralizing antibody titer was calculated. If the number of green fluorescent foci in the sample dilution was reduced by more than 50% compared with the infected control group, the neutralizing antibody was judged to be positive.

[0072] The results showed that rAd5TsCLP:rTs-CLP could stimulate higher levels of IgG, IgG1, IgG2a, IgA, IgM and neutralizing antibody NAb. After the challenge, the levels of these antibodies detected continued to increase gradually throughout the process from the invasion of the parasite to the completion of its life cycle. This may mean that the body's immune system continues to strengthen its defense against the invasion of the parasite in an attempt to resist the possible harm caused by the parasite ( Fig. 9 ).

[0073] The values ​​of the average antibody titers of neutralizing antibodies NAb were as follows: the average antibody titer of the PBS group at the 9th week (35 days after challenge) was 40, the average antibody titer of the Ad5Neg group at the 9th week (35 days after challenge) was 40, the average antibody titer of the Ad5Neg:rTsCLP group at the 9th week (35 days after challenge) was 320, the average antibody titer of the rTsCLP:rTsCLP group at the 9th week (35 days after challenge) was 640, and the average antibody titer of the rAd5TsCLP:rTsCLP group at the 9th week (35 days after challenge) was 1024 (determined by 2-fold serial dilution retest). The average antibody titer of the rAd5TsCLP group at 7 weeks after a single intramuscular injection (35 days after challenge) was 512 (determined by 2-fold serial dilution retest), and the average antibody titer of the rAd5TsCLP group at 7 weeks after a single intranasal injection (35 days after challenge) was 320.

[0074] 5. Serum cytokine detection

[0075] The Luminex detection platform was used to detect the secretion levels of IFN-γ, TNF-α, IL-4, and IL-10 cytokines in mouse serum.

[0076] The results showed that compared with other immune groups, the rAd5TsCLP:rTsCLP group had the highest concentrations of IFN-γ, TNF-α, IL-4, and IL-10 cytokines in the serum. Although Th2 response and mucosal immunity play an important role in the clearance of Trichinella spiralis, Th1 response also plays an equally important role ( Fig.10 ).

[0077] 6. T lymphocyte detection

[0078] After euthanasia of mice, the spleen of mice was removed and separated into single cell suspension and passed through a 100-mesh cell screen. After lysis with red blood cell lysis buffer, the cells were resuspended in PBS. In the dark, the cells were stained with anti-mouse CD3-APC, CD4-FITC, and CD8-PE surface marker antibodies at 4°C for 30 minutes. All samples were detected using a BD FACSCanton flow cytometer, and data were analyzed using FlowJo10 software.

[0079] The results showed that compared with the PBS group, the numbers of CD3+CD4+ T, CD3+CD8+ T, and CD3+CD4+CD8+ T lymphocytes in the other immune groups increased, among which the rAd5TsCLP:rTs-CLP group had the largest increase in the number of secreted T lymphocytes. Therefore, the prime-boost immune strategy can stimulate the proliferation of T lymphocytes, promote the secretion of cytokines, and then regulate and enhance the intensity and breadth of the immune response ( Fig.11 ).

[0080] The above results show that: (1) the present invention provides a method for preparing a recombinant adenovirus vaccine of Trichinella spiralis protein; (2) the present invention provides a method for preparing a recombinant protein vaccine of Trichinella spiralis protein; (3) the present invention combines the advantages of recombinant adenovirus vaccine and recombinant protein vaccine, and adopts prime-boost immunization strategy to immunize BALB / c mice; (4) the prime-boost immunization strategy adopted in the present invention has many advantages, which can effectively improve the systemic immunity level of the body, strongly stimulate the body to produce a strong mucosal immune response, promote Th1 / Th2 to maintain a dynamic equilibrium state, and enhance the proliferation capacity of T lymphocytes; (5) the prime-boost immunization strategy described in this application shows unique advantages in dealing with trichinellosis. It can effectively save the immunization dose, avoid the many inconveniences and potential risks that may be caused by large-scale vaccination, and significantly improve the immune effect, so that the body can more effectively resist the invasion of Trichinella spiralis, and provide a new idea and reliable way for the prevention and control of trichinellosis.

Claims

1. A Trichinella spiralis vaccine kit, characterized in that: The kit is divided into two parts. The first part is a recombinant protein vaccine of Trichinella spiralis with an amino acid sequence as shown in SEQ ID NO.2, and the second part is a recombinant adenovirus expressing an amino acid sequence as shown in SEQ ID NO.

1.

2. The kit according to claim 1, characterized in that The content of the recombinant protein of Trichinella spiralis is 60 μg; the content of the recombinant adenovirus expressing the amino acid sequence as shown in SEQ ID NO.1 is 10 8 PFU; the immunization method of the recombinant protein vaccine of Trichinella spiralis is intramuscular injection; the immunization method of the recombinant adenovirus expressing the amino acid sequence shown in SEQ ID NO.1 is nasal injection.

3. The kit according to claim 1, characterized in that The Trichinella spiralis recombinant protein vaccine is a Trichinella spiralis recombinant protein with amino acids as shown in SEQ ID NO.2 and a CPG1018 adjuvant in a volume ratio of 1:

1.

4. Use of the kit according to any one of claims 1 to 3 in the preparation of a medicament for preventing trichinellosis.

Images