An EgMDH DNA vaccine against Echinococcus granulosus and its preparation method and application

Abstract

The present invention discloses an EgMDH DNA vaccine against Echinococcus granulosus, a preparation method and an application, and belongs to the field of immunology. The DNA vaccine comprises a recombinant plasmid PVAX1‑EgMDH and an adjuvant. The DNA vaccine is prepared by mixing the recombinant plasmid PVAX1‑EgMDH with the adjuvant at a mass volume ratio of 1 μg:(1‑3) μL. Experiments have shown that the DNA vaccine can promote the production of humoral and cellular immune responses in the animal body, and tends to be Th1 cell-mediated cellular immune responses in the early stage of immunization, and tends to be Th2 cell-mediated humoral immune responses 20 days after attacking the worm; the cyst reduction rate can reach 80%, and it has a certain protective effect on the spleen. The PVAX1‑EgMDH DNA vaccine disclosed in the present invention has the potential to become a candidate vaccine for echinococcosis.

Description

Technical Field

[0001] The invention relates to the field of immunology, and in particular to an EgMDH DNA vaccine against Echinococcus granulosus, a preparation method and application thereof. Background Art

[0002] Cystic echinococcosis (CE) is a neglected zoonotic infectious disease with global public health significance, which can cause lasting impact on humans and increase economic burden. Although a praziquantel-based deworming program has been implemented for terminal host dogs, there are many factors that lead to ineffective deworming, and the incidence of CE in humans in some areas still exists. At present, the research on the terminal host vaccine of Echinococcus granulosus has shown satisfactory results, but it cannot be commercialized, mainly because the existing immunization scheme requires three immunizations and individual differences in experiments. The increase in the number of immunizations leads to an increase in workload and cost, and individual differences make it impossible to determine the safe value of vaccine antigen concentration. The research on the intermediate host vaccine of Echinococcus granulosus has made a key breakthrough, namely the Eg95 recombinant protein vaccine, which can produce more than 90% protection after vaccination of the intermediate host, but it only has a one-year protection period and only works during the migration of the six hook larvae. Therefore, the research on the vaccine of Echinococcus granulosus still needs to continue, and the emergence of a new DNA vaccine is needed to solve the problems existing in the existing technology.

[0003] In the preparation of vaccines, the role of adjuvants is very important. Adjuvants can non-specifically enhance the body's specific immune response to antigens and play an auxiliary role. Commonly used DNA vaccine adjuvants include bacterial toxin adjuvants (such as cholera toxin, Escherichia coli heat-labile enterotoxin and its derivatives), saponin adjuvants (QuilA), lipopolysaccharide derivative adjuvants (LPS), cell wall acyl dipeptide derivative adjuvants (Freund's complete adjuvant), hormone adjuvants (vitamin D3), lipopeptide adjuvants, etc. Saponin QuilA and its monomer components are extracted from the bark of the South American soap tree. Studies have shown that saponins can form complexes with cholesterol on the cell membrane and are potent cell membrane adjuvants. They can induce a variety of cytokines such as MHC-I restricted CTLs and have an enhancing effect on cytokines such as IL-10, γ-IFN, IL-4, IL-2, and IL-8. The role of cytokines in the body's immune regulation is a double-edged sword. It can promote the anti-infection effect of the immune system, but it can also cause immune pathological damage and participate in immune escape. Therefore, the selection of adjuvants is also a crucial step in the preparation of DNA vaccines. Summary of the invention

[0004] The purpose of the present invention is to provide an EgMDH DNA vaccine against Echinococcus granulosus, a preparation method and an application thereof, so as to solve the problems existing in the above-mentioned prior art. The PVAX1-EgMDH vaccine constructed by the present invention can reduce the cysts of Echinococcus granulosus by 80% and has a certain protective effect on the spleen.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides an EgMDH DNA vaccine for resisting Echinococcus granulosus. The EgMDH DNA vaccine comprises a recombinant plasmid PVAX1-EgMDH. The recombinant plasmid PVAX1-EgMDH is constructed by expressing an exogenous gene EgMDH by using an expression vector PVAX1. The nucleotide sequence of the exogenous gene EgMDH is shown in the accession number L08894.1 on Genbank.

[0007] Preferably, the vaccine further comprises an adjuvant.

[0008] Preferably, the EgMDH DNA vaccine is prepared by mixing the recombinant plasmid PVAX1-EgMDH with the adjuvant at a mass volume ratio of 1 μg:(1-3) μL.

[0009] Preferably, the adjuvant comprises QuilA.

[0010] The present invention also provides a method for preparing an EgMDH DNA vaccine for preventing Echinococcus granulosus, comprising the following steps:

[0011] Cloning the nucleotide sequence of the EgMDH gene;

[0012] Connecting the nucleotide sequence of the EgMDH gene to the expression vector PVAX1 to construct a recombinant plasmid PVAX1-EgMDH;

[0013] The recombinant plasmid PVAX1-EgMDH is mixed with an adjuvant to obtain an EgMDH DNA vaccine for preventing Echinococcus granulosus.

[0014] Preferably, the nucleotide sequence of the EgMDH gene is as shown in Genbank accession number L08894.1.

[0015] Preferably, the recombinant plasmid PVAX1-EgMDH is mixed with the adjuvant at a mass volume ratio of 1 μg:(1-3) μL.

[0016] Preferably, the adjuvant comprises QuilA.

[0017] The invention also provides a drug for resisting Echinococcus granulosus, comprising the EgMDH DNA vaccine.

[0018] The present invention discloses the following technical effects:

[0019] The present invention expresses the exogenous gene EgMDH through the expression vector PVAX1 to construct a PVAX1-EgMDH DNA vaccine. Experimental verification shows that the PVAX1-EgMDH DNA vaccine can reduce the cysts of Echinococcus granulosus by 80% and has a certain protective effect on the spleen. The PVAX1-EgMDH DNA vaccine has the potential to become a candidate vaccine for echinococcosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 is the result of PCR amplification of EgMDH gene; M: DNA molecular mass standard; 1: EgMDH PCR product;

[0022] Figure 2 The results of enzyme digestion identification of the recombinant plasmids; A: pMD19-T-EgMDH, M is DNA marker, 1 is pMD19-T-EgMDH plasmid double enzyme digestion, 2 is pMD19-T-EgMDH plasmid control; B: PVAX1-EgMDH, M is DNA marker, 1 is PVAX1-EgMDH plasmid double enzyme digestion, 2 is PVAX1-EgMDH, and 3 is PVAX1 plasmid blank control;

[0023] Figure 3 This is the morphological structure of the protoscolex of Echinococcus granulosus under a 20× microscope;

[0024] Figure 4 The total IgG gradient of the experimental mouse serum; * indicates P < 0.05, significant expression; ** indicates P < 0.01, extremely significant expression;

[0025] Figure 5 The changes in serum antibody levels of experimental mice in the PVAX1-EgMDH DNA vaccine group; * indicates P < 0.05, significant expression; ** indicates P < 0.01, extremely significant expression;

[0026] Figure 6The results of IL-1 detection in mice of PVAX1-EgMDH DNA vaccine group; * indicates P < 0.05, significant expression; ** indicates P < 0.01, extremely significant difference;

[0027] Figure 7 The results of IL-4 detection in mice of PVAX1-EgMDH DNA vaccine group; * indicates P < 0.05, significant expression; ** indicates P < 0.01, extremely significant difference;

[0028] Figure 8 The results of IL-5 detection in mice of PVAX1-EgMDH DNA vaccine group; * indicates P < 0.05, significant expression; ** indicates P < 0.01, extremely significant difference;

[0029] Fig. 9 The results of IL-10 detection in mice of PVAX1-EgMDH DNA vaccine group; * indicates P < 0.05, significant expression; ** indicates P < 0.01, extremely significant difference;

[0030] Fig.10 The results of IFN-γ detection in mice of PVAX1-EgMDH DNA vaccine group; * indicates P < 0.05, significant expression; ** indicates P < 0.01, extremely significant difference;

[0031] Fig.11 These are pictures of liver cysts in some experimental mice; the black arrows point to the liver cysts;

[0032] Fig.12 Pathological sections of some livers of experimental mice; AC: 200x; ①: cysts; ②: germinal layer; ③: stratum corneum; ④: fibroblasts and fibrocytes; ⑤: ballooning degeneration of hepatocytes; ⑥: infiltration of lymphocytes, eosinophils and multinuclear macrophages;

[0033] Fig.13 This is a picture of part of the spleen of an experimental mouse. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Example 1 Construction of recombinant plasmid pMD19-T-EgMDH

[0040] 1. Main reagents and consumables, see Table 1 and Table 2:

[0041] Table 1 Main experimental instruments

[0042] Instrument Name Manufacturer Low temperature high speed centrifuge Eppendorf, Germany High Pressure Steam Sterilizer Sanoyo, MLS-3780 Ice Maker Ice Maker Japan Sanyo Company Products Water Purifier Millipore China Ltd. NanOD600rop2000c Ultra-micro Spectrophotometer Thereto Fisher Scientific, USA Constant temperature incubator Shanghai Yiheng Scientific Instrument Co., Ltd. Constant temperature water bath Jintan Medical Instrument Factory

[0043] Table 2 Main reagents and consumables

[0044] Reagent name Manufacturer PVAX1 eukaryotic vector Invitrogen E.coliDH5α competent cells Tiangen Biochemical Technology (Beijing) Co., Ltd. DNA Marker Tiangen Biochemical Technology (Beijing) Co., Ltd. 2×PCR Mix Takera Japan Agarose Gel DNA Recovery Kit Tiangen Biochemical Technology (Beijing) Co., Ltd. Plasmid Extraction Kit Tiangen Biochemical Technology (Beijing) Co., Ltd. Restriction endonucleases EcoRI, HindⅠⅠⅠ, XhoⅠ Takera Japan T4 DNA Ligase Takera Japan E. coli competent cells BL21(DE3) Tiangen Biochemical Technology (Beijing) Co., Ltd. Endotoxin-free plasmid kit Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0045] 2. Primer design of EgMDH gene

[0046] Specific PCR primers and qRT-PCR primers were designed according to EgMDH (sequence accession number: L08894.1) in Genbank, and the EcoRI, HindⅢ and XhoⅠ restriction sites and protective bases were underlined. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sequence accession number XM_024494574) gene of Echinococcus granulosus was designed as the internal reference primer of qRT-PCR. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., see Table 3.

[0047] Table 3

[0048]

[0049] 3. Total RNA Extraction

[0050] Take out the Echinococcus granulosus adults and protoscolex frozen in TRIZOL Reagent in the laboratory refrigerator at -80℃, and follow the instructions of the RNA extraction kit.

[0051] 4. Reverse transcription of total RNA and cloning of EgMDH gene

[0052] 4.1 Reverse transcription of total RNA from Echinococcus granulosus protoscolex

[0053] The extracted RNA of Echinococcus granulosus adults and protoscolex was used as the first chain of reverse transcription cDNA according to the instructions of the reverse transcription kit. The reaction conditions were 42°C water bath for 2 min, 50°C water bath for 30 s, and 85°C water bath for 30 s. After the reaction, the product was stored at -20°C. The reverse transcription system is shown in Table 4.

[0054] Table 4 Reverse transcription system of total RNA from Echinococcus granulosus protoscolex

[0055]

[0056] 4.2 Amplification of EgMDH gene

[0057] The reverse transcribed Echinococcus granulosus cDNA was used as a template and PCR amplification was performed using the EgMDH primer pair in Table 3. The PCR system and program are shown in Tables 5 and 6.

[0058] Table 5 Amplification reaction system

[0059]

[0060] Table 6 Amplification reaction program

[0061] program condition Number of cycles Pre-denaturation 95℃5min 1 transsexual 95℃30s 35 annealing 60℃30s 35 extend 72℃1min 35 Further extension 72℃5min 1

[0062] 4.3 Gel recovery of PCR products

[0063] The PCR product obtained by the above amplification was subjected to agarose gel electrophoresis at 120V and 400mA for 30min using 1.5% agarose gel. After the electrophoresis was completed, the target band was recovered using an agarose gel DNA recovery kit.

[0064] 4.4 Ligation, transformation and sequencing of gel-recovered products

[0065] The recovered target band was connected to the pMD19-T vector at 4°C overnight. The connection system (10μL) was as follows: 4.5μL of the target band recovered by gel, 5μL of solutionⅠ, and 0.5μL of pMD19-T vector. The connection product was transformed into the cloning bacteria DH5α competent cells. After culturing at 37°C overnight for 12h, a single colony was picked and cultured in a centrifuge tube at 180rpm for 4h. The bacterial liquid PCR identification was performed. The bacterial liquid PCR system and procedure were the same as those in Tables 5 and 6. For the strains identified as positive, 50% glycerol was used to preserve the bacteria. Then the culture was expanded and the plasmid was extracted using a plasmid mini-extraction kit.

[0066] The extracted plasmid pMD19-T-EgMDH was double-digested with restriction endonucleases EcoRI and XhoI for identification. The double-digestion system is shown in Table 7, and the reaction conditions are 37°C water bath for 1 hour. After the double-digestion is completed, 1.5% agarose gel electrophoresis is used for 30 minutes at 120V and 400mA for verification.

[0067] The recombinant plasmid with the correct double enzyme digestion was sent to Shanghai Bioengineering Co., Ltd. for sequencing. The recombinant plasmid that was verified to be correct by double enzyme digestion and sequencing was named pMD19-T-EgMDH.

[0068] Table 7 Plasmid double restriction enzyme digestion system

[0069]

[0070]

[0071] 4.3 Construction of PVAX1-EgMDH eukaryotic vector

[0072] The correctly sequenced pMD19-T-EgMDH recombinant plasmid was inoculated into 5 mL of LB liquid culture medium containing ampicillin and cultured at 37°C and 180 rpm for 8 h. After 8 h, the pMD19-T-EgMDH recombinant plasmid was extracted using a plasmid extraction kit.

[0073] After plasmid extraction, restriction endonucleases EcoRI and XhoI were used to perform double enzyme digestion on the PVAX1 eukaryotic vector plasmid and pMD19-T-EgMDH plasmid, respectively. The double enzyme digestion system was the same as in Table 7. After double enzyme digestion, 1.5% agarose gel was used for 30 min electrophoresis at 120 V and 400 mA, and the expected target gene fragment was recovered using a gel recovery kit.

[0074] The PVAX1 eukaryotic vector plasmid and the EgMDH fragment were digested with restriction endonucleases EcoRI and XhoI and connected overnight at 4°C using T4 DNA ligase. The next day, the ligation product was transformed into cloning bacteria DH5α competent cells. After culturing at 37°C overnight for 12 hours, a single colony was picked and cultured in a centrifuge tube at 180 rpm for 4 hours. The bacterial liquid PCR identification was performed. The bacterial liquid PCR system and procedure were the same as 4.2, except that the EgMDH annealing temperature was different. For strains identified as positive, 50% glycerol was used to preserve the bacteria. Then the culture was expanded and the plasmid was extracted using a plasmid mini-extraction kit.

[0075] The two extracted plasmids were double-digested with restriction endonucleases EcoRI and XhoI, respectively, and the double-digestion system was the same as in Table 7. The reaction conditions were 37°C water bath for 1 hour. After the double-digestion was completed, 1.5% agarose gel was used for 30 minutes of electrophoresis verification at 120V and 400mA.

[0076] The recombinant plasmid with the correct double enzyme digestion was sent to Shanghai Bioengineering Co., Ltd. for sequencing. The recombinant plasmid that was positive after double enzyme digestion verification and correct sequencing was named PVAX1-EgMDH.

[0077] 5. Results and Analysis

[0078] 5.1 EgMDH gene amplification

[0079] The EgMDH gene was amplified by PCR using specific primers. After 1.5% agarose gel electrophoresis, a specific band of the EgMDH gene at 975 bp was found (see Figure 1 ), which is consistent with the expected result size.

[0080] 5.2 Construction of recombinant plasmid and double enzyme digestion identification

[0081] After pMD19-T-EgMDH was digested with restriction endonucleases EcoRI and XhoI, the digestion results were detected by electrophoresis. The results showed that pMD19-T-EgMDH was successfully cut by restriction endonucleases EcoRI and XhoI, and the target fragment was consistent with the expected size, indicating that the pMD19-T-EgMDH plasmid was successfully constructed. The double enzyme digestion identification results are as follows: Figure 2 As shown in A.

[0082] After the PVAX1-EgMDH DNA plasmid was extracted, the PVAX1-EgMDH DNA plasmid was double-digested with restriction endonucleases EcoRI and XhoI. Figure 2 As shown in B.

[0083] Example 2 Preparation of EgMDH DNA vaccine and study of immune effects

[0084] 1. Experimental Materials

[0085] (1) Experimental animals and protoscolex of Echinococcus granulosus

[0086] The animals used in the present invention are Kunming mice purchased from the Experimental Center of Xinjiang Medical University. The protoscolex used in the experiment was collected from the fresh livers of sheep with echinococcosis purchased from designated slaughterhouses in Urumqi, Xinjiang and Shihezi City.

[0087] (2) Main reagents and consumables

[0088] Adjuvant was purchased from Invivogen, USA; rabbit anti-mouse IgG-HRP and TMB single-component colorimetric solution were purchased from Beijing Solebow Technology Co., Ltd.; mouse interleukin 1 ELISA kit, mouse interleukin 4 ELISA kit, mouse interleukin 5 ELISA kit, mouse interleukin 10 ELISA kit, and mouse interleukin IFN-γ ELISA kit were purchased from Shenzhen Jingmei Bioengineering Co., Ltd.

[0089] 2. Vaccine preparation

[0090] Take out the PVAX1-EgMDH prepared in Example 1 from a -20°C refrigerator. Wait for it to melt at room temperature. After melting, flick to mix, then take out 1 μL of each vaccine and use a nucleic acid protein analyzer to determine its concentration and purity (to detect whether degradation and contamination occur). Vaccine preparation: The concentration of Quil A adjuvant is 1 μg / μL. PVAX1-EgMDH DNA vaccine group: 50 μg PVAX1-EgMDH DNA plasmid + 50 μL Quil A adjuvant are mixed for each mouse to be immunized; Quil A group: 50 μg QuilA + 50 μL PBS are mixed for each mouse to be immunized; PBS group: 100 μL PBS for each mouse;

[0091] Each time the vaccine was prepared, it was sealed with a sealing film after preparation and shaken overnight at 4°C to mix thoroughly.

[0092] 3. Animal immunization

[0093] There were a total of 24 experimental mice in this experiment. After they were purchased, they were observed for one week to see if they had any stress reactions. After one week, it was determined that each experimental mouse was healthy and randomly divided into three groups, namely the PBS group, the QuilA group, and the PVAX1-EgMDH DNA vaccine group. The above-prepared vaccine was immunized to the experimental mice by intramuscular injection. A total of 3 immunizations were performed, once every 15 days, for a total of 3 immunizations. The parasite attack began 30 days after the three immunizations. Blood was collected from the mouse orbital vein before each immunization, centrifuged at 3000rpm for 5 minutes, and the serum was collected and stored at -80℃ for later use.

[0094] 4. Animals attack insects

[0095] (1) Collection and activity detection of Echinococcus granulosus protoscolex

[0096] The cyst-containing sheep livers were collected from the slaughterhouse, kept fresh at low temperature and brought back to the laboratory. The surface of the liver was rinsed with sterile water and then wiped with a 75% alcohol cotton ball. The liver cyst fluid was collected aseptically and the protoscolex in the cyst fluid was collected by centrifugation at 3000 rpm. The activity was observed under an optical microscope using 0.4% trypan blue staining.

[0097] (2) Insect attack experiment

[0098] 30 days after the completion of the three vaccinations, the mice were challenged with parasites by intraperitoneal injection using a syringe. The collected protoscolex were turned upside down, mixed thoroughly, and 50 μL was aspirated onto a glass slide. The protoscolex activity was counted and observed under a 40× microscope. Each experimental mouse was challenged with about 2,000 parasites.

[0099] 5. Changes in antibody levels in experimental mice

[0100] Take out the experimental mouse serum from the -80℃ refrigerator and wait for it to melt naturally. If precipitation occurs, centrifuge again to obtain the supernatant, and dilute it to 1 / 3 of the original concentration with sample diluent, that is, take 50μL serum and add 100μL sample diluent and mix well. Use ELISA to detect the levels of IL-1, IL-4, IL-5, IL-10, and IFN-γ in mouse serum. For specific detection steps, refer to the instructions of the kit.

[0101] 6. Cytokine detection

[0102] The mouse interleukin ELISA kit was used to measure the experimental mouse cytokines, and the absorbance (OD value) was measured at a wavelength of 600 nm using an enzyme marker. The specific operation steps refer to the kit instructions.

[0103] 7. Observation of liver morphology

[0104] 90 days after the attack, that is, 150 days after the immunization, the experimental mice were killed and the livers were removed to count the liver cysts of the experimental mice. The cyst area of ​​the liver was fixed with 4% paraformaldehyde and prepared into paraffin sections. The pathological changes of the liver were observed after HE staining.

[0105] 8. Observation of spleen morphology

[0106] 90 days after infection, i.e. 150 days after immunization, the experimental mice were killed and the spleens were removed to observe the morphology of the spleens, measure the size of the spleens and weigh them.

[0107] 9. Data processing and analysis

[0108] The antibody levels and cytokine detection data were statistically analyzed using the one-way variance procedure in SPSS software. When P < 0.05, the differences between the groups were statistically significant, and the graphs were drawn using GraphPad prism software.

[0109] 10. Results and Analysis

[0110] 10.1 Detection of protoscolex activity of Echinococcus granulosus

[0111] The collected Echinococcus granulosus were observed to have complete morphology and structure under a 20× microscope (see Figure 3 ), all of them have peristalsis, indicating that the protoscolex of Echinococcus granulosus is quite active.

[0112] 10.2 Changes in Antibody Levels in Mouse Serum

[0113] The ELISA method was used to detect the changes in antibody levels in experimental mice. The results were as follows Figure 4 As shown, the secretion of total IgG antibodies in the PVAX1-EgMDH DNA vaccine group was higher than that in the QuilA group and the PBS group at 15d, 30d, 60d, and 150d after immunization. Compared with the PBS group, the secretion of total IgG antibodies in the PVAX1-EgMDH DNA vaccine group was significantly enhanced at 30d after immunization, i.e., the second immunization (P < 0.05). The secretion of total IgG antibodies was significantly enhanced at 150d after immunization, i.e., 90d after the attack of the insects (P < 0.05). Compared with the QuilA group, the secretion of total IgG antibodies in the PVAX1-EgMDH DNA vaccine group was significantly enhanced at 15d, 30d, and 60d after immunization (P < 0.05). There was no significant change at 150d (P > 0.05). The total IgG of the PVAX1-EgMDH DNA vaccine group at 0 day before immunization was compared with that at 15 days, 30 days, 60 days, and 150 days after immunization. Figure 5As shown. The total IgG in the PVAX1-EgMDH DNA vaccine group at 15d, 30d, 60d, and 150d showed an upward trend, and reached the highest at 150d (P < 0.05), but there was no significant change in the total IgG at 15d, 30d, and 60d (P > 0.05), and there was a slight decrease in the total IgG at 60d. In other words, the overall trend of total IgG antibodies in the PVAX1-EgMDH vaccine group was on the rise, but there was a slight decrease in total IgG at 60d.

[0114] 10.3 Changes of Cytokines in Serum of Experimental Mice

[0115] ELISA test results Figure 6-10 As shown, compared with PBS, the PVAX1-EgMDH DNA vaccine group had no significant effect on enhancing the secretion of IL-1 at 0d and 15d (P>0.05), but was significantly higher than the PBS group at 30d (P<0.05). The PVAX1-EgMDH DNA vaccine group had significantly higher secretion of IL-4 than the PBS group at 15d, 30d, 60d and 150d (P<0.05). The PVAX1-EgMDH DNA vaccine group had significantly higher secretion of IL-5 than the PBS group at 30d and 60d (P<0.05). The PVAX1-EgMDH DNA vaccine group had significantly higher secretion of IL-10 than the PBS group at 0d, 15d and 60d (P<0.05), and was significantly higher than the QA group at 0d, 15d, 30d, 60d and 150d (P<0.05). The secretion of IFN-γ in the PVAX1-EgMDH DNA vaccine group was significantly higher than that in the PBS group at 0d, 15d, and 30d, showing an enhancing effect (p<0.05).

[0116] The above results suggest that PVAX1-EgMDH DNA vaccine can promote humoral and cellular immune responses in animals.

[0117] 10.4 Counting of cysts in the liver of experimental mice and observation of pathological sections

[0118] 90 days after the challenge, i.e. 150 days after the immunization, the experimental mice were killed and their livers were removed. The number of cysts in the livers of the experimental mice was counted, as shown in Table 8 and Fig.11 It can be seen that the individual differences between the groups are very obvious. From the average value, the liver cysts of the experimental mice in the PVAX1-EgMDH DNA vaccine group were much lower than those in the QA group and the PBS group. Taking the PBS group as the control, it was found that the cyst reduction rate in the PVAX1-EgMDH DNA vaccine group could reach 80%, and the cyst reduction rate in the QA group was 19.05% (cyst reduction rate = (experimental group-PBS group) / PBS group × 100%).

[0119] like Fig.12As shown, pathological section observation revealed that inflammatory cell infiltration occurred at the junction of the cyst and liver tissue, and hepatocyte nuclear degeneration was common. The degree of pathological changes was similar among the groups, with no significant differences.

[0120] Table 8 Statistics of liver cysts in experimental mice

[0121]

[0122]

[0123] 10.5 Observation of spleen morphology

[0124] 28 days after the attack, the spleens of the mice in each group were taken out to observe the changes in spleen size and morphology. Fig.13 As shown in the table, it can be seen from the data that the average spleen weight of the experimental mice in the PVAX1-EgMDH DNA vaccine group is smaller than that in the QA group, but there is no significant difference in the data (P>0.05). The average spleen length is higher than that in the PBS group and lower than that in the QA group, but there is no significant difference in the data (P>0.05).

[0125] Table 9 Statistics of spleen size and weight of mice in different experimental groups

[0126]

[0127] In summary, the results of the present invention show that the cyst reduction rate of experimental mice in the PVAX1-EgMDH DNA vaccine group can reach 80%, and has a certain protective effect on the spleen. The PVAX1-EgMDH DNA vaccine has the potential to become a candidate vaccine for echinococcosis.

[0128] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An EgMDH DNA vaccine against Echinococcus granulosus, characterized in that: The EgMDH DNA vaccine comprises a recombinant plasmid PVAX1-EgMDH, wherein the recombinant plasmid PVAX1-EgMDH is constructed by using the expression vector PVAX1 to express the exogenous gene EgMDH, and the nucleotide sequence of the exogenous gene EgMDH is shown in the accession number L08894.1 on Genbank; The EgMDH DNA vaccine also includes an adjuvant; The EgMDH DNA vaccine is prepared by mixing the recombinant plasmid PVAX1-EgMDH with the adjuvant at a mass volume ratio of 1 μg:1 μL; The adjuvant includes QuilA; The encapsulation reduction rate of the EgMDH DNA vaccine reached 80%.

2. A method for preparing an EgMDH DNA vaccine for preventing Echinococcus granulosus, characterized in that: The following steps are involved: Cloning the nucleotide sequence of the EgMDH gene; Connecting the nucleotide sequence of the EgMDH gene to the expression vector PVAX1 to construct a recombinant plasmid PVAX1-EgMDH; The recombinant plasmid PVAX1-EgMDH is mixed with an adjuvant to obtain an EgMDH DNA vaccine for preventing Echinococcus granulosus; The nucleotide sequence of the EgMDH gene is shown in Genbank accession number L08894.1; The recombinant plasmid PVAX1-EgMDH and the adjuvant are mixed at a mass volume ratio of 1 μg:1 μL; The adjuvant includes QuilA.

3. A drug for resisting Echinococcus granulosus, characterized in that: Comprising the EgMDH DNA vaccine according to claim 1.

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