Vaccine for preventing and treating sheep echinococcosis infection and preparation method thereof

Abstract

The invention relates to a vaccine for preventing and treating sheep echinococcosis infection and a preparation method thereof.The preparation method comprises the steps that echinococcosis Eg95 full-length gene antigen epitopes are screened through bioinformatics software, screened fragments with high antigenicity are optimized and then subjected to double-copy connection through a flexible linker, His tag sequences are added to the two ends of a tandem sequence, the sequence is as shown in SEQ ID NO.2, and the vaccine for preventing and treating sheep echinococcosis infection is obtained; and the pEE12.4-double Eg95 plasmid is connected with a CHO eukaryotic cell expression vector, so that the pEE12.4-double Eg95 plasmid is obtained. The plasmid is integrated into a CHO eukaryotic cell expression system, the expression quantity of the purified Eg95 protein reaches 10g / L, and a stable and high-yield veterinary vaccine is provided for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a vaccine for preventing and treating sheep echinococcosis infection and its application. Background Art

[0002] Echinococcosis is a parasitic disease caused by the larvae of the tapeworm Echinococcus. It is a zoonotic disease. It has a regional distribution worldwide, primarily in countries where animal husbandry is a major industry. Echinococcosis poses a serious threat to public health and hinders the development of animal husbandry. The World Health Organization (WHO) has designated it as one of the diseases to be controlled or eliminated by 2050.

[0003] The prevention and treatment of echinococcosis mainly relies on immune prevention. Currently, scholars have done a lot of work on the research of echinococcosis vaccines. For example, CN111675758A provides a genetically engineered subunit vaccine for sheep echinococcosis infection. However, the Fc fragment itself may induce an immune response to IgG in the host, leading to accelerated antibody clearance or autoimmune risks. When repeatedly vaccinated, the vaccine effect may be reduced due to the production of anti-Fc antibodies (similar to the pre-existing immunity problem of adenovirus vector vaccines). In addition, compared with a single Eg95 protein, the fusion protein not only requires more complex process development and quality control, increasing production costs, but also the glycosylation modification of Fc is sensitive to storage conditions (such as temperature and pH), thereby increasing the cold chain transportation and storage costs of the vaccine. CN108066755A provides a method for preparing a genetically engineered subunit vaccine by expressing the recombinant protein Eg95 using an E. coli expression system. However, the protein expressed by the E. coli expression system lacks or has abnormal glycosylation, is prone to forming insoluble inclusion bodies, is contaminated by endotoxins, and has low expression levels. CN112250748 achieved secretory expression of Eg95 protein in a Pichia pastoris constitutive expression system by constructing a p-GAPZαA-Eg95 recombinant plasmid. However, the protein concentration expressed by this method was still relatively low (0.05 g / L). In addition, the existing technology for expressing Eg95 in CHO cells has problems such as low expression level (usually <1 g / L) and high glycosylation heterogeneity. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present application provides a vaccine for preventing and treating ovine echinococcosis infection and a preparation method thereof, which can simplify the downstream purification process, reduce production costs, and achieve an Eg95 protein expression level of 10 g / L after purification, providing a stable and high-yield veterinary vaccine for large-scale production.

[0005] A recombinant protein comprises the amino acid sequence shown in SEQ ID NO.1.

[0006] The gene encoding the recombinant protein includes the nucleotide molecule shown in SEQ ID NO.2.

[0007] The recombinant vector containing the coding gene is a CHO eukaryotic cell expression vector.

[0008] The recombinant vector includes one of the pEE series, pcDNA series, and pCHO series vectors.

[0009] The recombinant vector includes one of the pEE series vectors.

[0010] A vaccine for preventing and treating ovine echinococcosis infection comprises the recombinant protein, an adjuvant, a stabilizer and a buffer.

[0011] A method for preparing a vaccine for preventing and treating ovine echinococcosis infection, characterized in that it comprises the following steps:

[0012] Cloning the coding gene of the recombinant protein into a eukaryotic expression vector to obtain a recombinant vector;

[0013] The recombinant vector is used to transfect host cells, and host cells that stably and efficiently express the recombinant protein in suspension are screened out, and then cultured at a lower temperature, and then the recombinant protein is separated and purified from the cell supernatant.

[0014] Furthermore, the host cell is a CHO cell.

[0015] Furthermore, the concentration of the recombinant protein obtained by separation and recovery is ≥10 g / L, and the purity of the target protein is >95%.

[0016] The use of the recombinant protein or the immune composition in preparing a genetically engineered subunit vaccine against sheep echinococcosis infection.

[0017] The beneficial effects produced by the present invention are:

[0018] The present invention utilizes two copies of the truncated Eg95 gene in series to significantly improve transcriptional efficiency and protein expression levels. At the same time, through the efficient protein synthesis capacity of CHO eukaryotic cells, it ensures the correct folding and post-translational modification of the protein, thereby improving the immunogenicity of the vaccine. In addition, the cell line stably expressing the Eg95 protein can be grown in a glutamine-free culture medium, reducing the accumulation of harmful substances such as ammonia, thereby extending cell survival time and increasing protein production; reducing culture medium costs, reducing metabolic stress during the culture process, and being more suitable for industrial production. The His tag is introduced into the Eg95 gene sequence to simplify the nickel column affinity chromatography purification process, improve recovery and purity, and reduce production costs; at the same time, combined with the efficient expression of CHO cells, the purified Eg95 protein yield is as high as 10g / L, far exceeding traditional methods and meeting the large-scale demand for veterinary vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1These are the results of pEE12.4-double Eg95 digestion identification (1, 2, 3: pEE12.4-double Eg95 digestion samples). Figure 2 The expression of dual Eg95 proteins in cell lines at low temperature was shown in Figure 1. A is the SDS-PAGE result, and B is the Western Blot result.

[0020] Figure 3 The expression of SEQ ID NO.3 protein in cell lines after cooling down, A is the SDS-PAGE result, and B is the Western Blot result.

[0021] Figure 4 The expression of SEQ ID NO.4 protein in cell lines after cooling down, A is the SDS-PAGE result, and B is the Western Blot result.

[0022] Figure 5 The SDS-PAGE detection results of the purified Eg95 protein expressed by the double Eg95 recombinant plasmid (1, 2, 3: 10-fold diluted samples of purified protein).

[0023] Figure 6 The body temperature results of healthy susceptible lambs aged 2 to 4 months after vaccination (A, body temperature change curve before and after vaccination; B, difference between body temperature after vaccination and body temperature before vaccination).

[0024] Figure 7 These are the body temperature results of primigravid ewes after vaccination (A, body temperature change curve before and after vaccination; B, difference between body temperature after vaccination and body temperature before vaccination).

[0025] Figure 8 ELISA antibody results of healthy susceptible lambs aged 2 to 4 months after vaccination. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0027] Example 1 Construction of recombinant eukaryotic expression vector pEE12.4-double Eg95

[0028] The full-length gene of Echinococcus cysticercosis Eg95 (Sequence ID: AF199354.2) was screened for antigenic epitopes using bioinformatics software. The highly antigenic fragment (bases 70 to 414) was optimized and then ligated in duplicate using a flexible linker. His-tag sequences were designed at both ends of the tandem sequence, resulting in the nucleotide sequence shown in SEQ ID NO. 2, representing the double Eg95 recombinant sequence. This sequence was subsequently synthesized by GenScript Biotech to generate the plasmid pUC57-double Eg95.

[0029] 1.1 Digest the pEE12.4 vector and the pUC57-double Eg95 plasmid with Hind III and EcoRI, respectively, at 37°C for 3 hours. Enzyme digestion system: 5 μg of pUC57-double Eg95 / pEE12.4; 5 μl each of Hind III and EcoRI; 4 μl of 10× Buffer; and add HO to 40 μl.

[0030] 1.2 The above enzyme digestion products were recovered by agarose gel, and then the target fragments were cut out under ultraviolet light for gel recovery.

[0031] 1.3 Ligate the purified product at 22°C for 4 hours. The total volume is 20 μl, including 13 μl of the double Eg95 fragment, 4 μl of purified pEE12.4, 1 μl of T4 ligase, and 2 μl of 10× Buffer.

[0032] 1.4 Add the ligation product to the DH5α competent medium and mix well. Incubate on ice for 30 minutes, then at 42°C for 90 seconds. Then quickly transfer to an ice bath and incubate for 3 minutes. Add 300 μl of LB medium and incubate on a shaker at 37°C for 1 hour. Then spread the plate onto a plate containing ampicillin resistance and invert it into a 37°C incubator for overnight incubation.

[0033] 1.5 Extraction and identification

[0034] Pick a single colony for amplification, and then extract according to the instructions of the plasmid extraction kit.

[0035] The enzyme digestion reaction system for the recombinant plasmid was as follows: pEE12.4-double Eg95 2 μl; Hind III and EcoRI 1 μl each; 10× Buffer 1 μl; H2O 5 μl; digestion at 37°C for 1 hour. 5 μl of the digestion product was then analyzed by agarose gel electrophoresis. Figure 1 The results of pEE12.4-double Eg95 digestion are shown (1, 2, 3: pEE12.4-double Eg95 digestion samples). The positively identified plasmids were sent to Qingke for sequencing, and the correct plasmids were selected for amplification to obtain the eukaryotic expression plasmid pEE12.4-double Eg95.

[0036] The double Eg95 recombinant sequence shown in SEQ ID NO.2 was replaced with other optimized fragments shown in SEQ ID NO.3 and SEQ ID NO.4, and other conditions remained unchanged. A comparative experiment was performed to obtain two recombinant plasmids synthesized according to SEQ ID NO.3 and SEQ ID NO.4.

[0037] Example 2 Construction and screening of recombinant CHO cells

[0038] 2.1 Plasmid linearization and recovery

[0039] Plasmid pEE12.4-double Eg95 was linearized using Pvu I enzyme and incubated at 37°C for 3 hours. The digestion system consisted of: 100 ng of plasmid; 10 μl of Pvu I; 20 μl of 10× Buffer; and HO added to 200 μl. The digestion product was purified using a gel extraction kit. Finally, the DNA was dissolved in sterile water and stored at -20°C until ready for use.

[0040] 2.2 Cell transfection and cell line screening

[0041] The CHO cells were centrifuged at 1000 rpm for 5 minutes to collect the precipitate, and the cells were gently resuspended and washed with CD-CHO-AGT medium without glutamine to remove residual glutamine. The medium was removed by centrifugation at 1000 rpm for 5 minutes, and the cells were gently resuspended with electroporation buffer to adjust the density to 3 × 10 7 cells / ml. Take two sterile 1.5ml EP tubes, add 0.5ml of cell suspension resuspended in electroporation buffer to each tube, then add 5μg of eukaryotic expression plasmid pEE12.4-double Eg95 to each tube, mix gently, and incubate at room temperature for 15 minutes, gently flicking the tube every 5 minutes to mix. After incubation, place the tube in an ice bath for 5 minutes. Add 0.5ml of the cell and plasmid mixture to each electroporation cup, place the tube in the electroporator, and start the electroporation program. After the electroporation is complete, transfer the cells to a conical flask containing 20ml of CD-AGT medium and culture in a 37°C shaker.

[0042] The plasmid pEE12.4-double Eg95 was replaced with two recombinant plasmids constructed according to SEQ ID NO.3 and SEQ ID NO.4, and other experimental conditions remained unchanged for a comparative experiment.

[0043] After 48 hours of culture, the cell culture medium was replaced with CD-CHO-AGT medium containing 50 μM MSX and the cells were cultured in a shaking incubator at 37°C. From the 7th day, the cell density and activity were observed. The cell density increased from 1.2×10 6cells / ml began to decrease, and around the 10th to 15th day, the cell density began to increase. When the cell density increased to 1.0×10 6 cells / ml, the cells were passaged to a density of approximately 1.0×10 5 cells / ml and continue culturing.

[0044] The cells were switched to CHO-2 suspension culture for expanded production, and after adaptation, they were frozen to eventually obtain a high-expression dual Eg95 cell line.

[0045] 2.3 Identification of Eg95 Protein by Western Blot and SDS-PAGE

[0046] The supernatant of Eg95 protein expressed in cell line at 33℃ was collected by centrifugation, and the expressed product was identified by Western Blot using His mouse antibody as primary antibody and HRP-labeled goat anti-mouse IgG (H+L) as secondary antibody. The supernatant of the expressed product was also detected by SDS-PAGE.

[0047] The Eg95 protein expressed by the double Eg95 recombinant plasmid was as follows Figure 2 As shown, a relevant target band was observed around 35 kd in size.

[0048] The Eg95 protein was expressed by the recombinant plasmid synthesized with SEQ ID NO.3. Figure 3 As shown, SDS-PAGE ( Figure 3 The results of A) in the figure showed that no relevant target band was observed around 45kd. Western Blot ( Figure 3 The results in B) show that the target band was observed around 45 kd. Figure 3 Analysis of results A and B in Figure 1: After the optimized sequence was electroporated into CHO cells, expression was observed upon cooling, but the expression level was so low that the target band could not be seen with the naked eye on SDS-PAGE.

[0049] like Figure 4 As shown, the Eg95 protein expressed by the recombinant plasmid synthesized with SEQ ID NO.4 sequence did not show the target band near 20 kd in SDS-PAGE and Western Blot results, indicating that the optimized protein was not expressed after being electroporated into CHO cells and then cooled.

[0050] Example 3 Recombinant protein purification

[0051] 3.1 Eg95 protein purification

[0052] The Eg95 protein expression supernatant was collected and filtered through a 0.22 μm filter. The supernatant was loaded onto a Ni-TED gravity column, and the flow-through was collected and washed with 50 mmol / L imidazole and then eluted with 500 mmol / L imidazole. The eluate was placed in a 40 kDa dialysis bag and dialyzed at 4°C for 48 hours. The total protein content of the dialyzed protein was determined using a BCA protein concentration assay kit. The purified protein was diluted 10-fold and subjected to SDS-PAGE. The gel was scanned with an imager, and purity was analyzed by peak area using Image J software.

[0053] The Eg95 protein expressed by the double Eg95 recombinant plasmid was as follows Figure 5 As shown (1, 2, 3: 10-fold diluted samples of purified protein), the concentration of the purified recombinant protein was 10.007 mg / ml, and the purity of the target protein was >95%.

[0054] The recombinant plasmid constructed according to SEQ ID NO.3 and SEQ ID NO.4 expressed too low or no expression after electroporation.

[0055] Example 4 Vaccine Preparation

[0056] 4.1 Preparation of aqueous phase

[0057] Dilute the purified protein Eg95 with physiological saline to the vaccine concentration (protein antigen content is 100 μg / ml), mix well, and preheat to 32°C.

[0058] 4.2 Emulsification

[0059] Preheat ISA201 VG adjuvant to 32°C. Slowly add the aqueous phase to the ISA201 VG adjuvant at a ratio of 1:1 (mass ratio) and emulsify at 100-120 r / min for 60 minutes.

[0060] 4.3 Packaging

[0061] Pack in quantitative quantities, seal with stoppers, affix labels, and store at 2-8°C.

[0062] Example 5 Safety Test of Eg95 Genetically Engineered Subunit Vaccine in Sheep

[0063] 5.1 Safety study of Eg95 genetically engineered subunit vaccine in healthy susceptible lambs aged 2 to 4 months

[0064] Five healthy susceptible lambs aged 2 to 4 months with negative serum antibody test results for echinococcosis were selected and 2.0 ml of vaccine was subcutaneously injected into the neck of each lamb. A corresponding blank control group was also set up. 28 days after the first vaccination, a second vaccination was performed with the same dose. Under the same feeding conditions, the rectal temperature of the test lambs and the control lambs was measured regularly, the clinical manifestations of the lambs were observed, and the reaction at the injection site was checked. Table 1 shows the observation results of clinical manifestations and injection site after vaccination of lambs aged 2 to 4 months with Eg95 genetically engineered subunit vaccine. Figure 4 The results show the body temperature of healthy, susceptible lambs aged 2 to 4 months after vaccination (A, temperature curve before and after vaccination; B, temperature difference between before and after vaccination). The results show that all healthy, susceptible lambs aged 2 to 4 months remained in good spirits and behavior throughout the observation period, with no swelling at the vaccination site. The body temperature of the vaccinated lambs increased slightly 2 hours after vaccination, but never exceeded 1°C. These results indicate that the Eg95 genetically engineered subunit vaccine is safe for healthy, susceptible lambs aged 2 to 4 months.

[0065] Table 1 Observation results of clinical manifestations and vaccination sites in 2-4 month old lambs after vaccination with Eg95 genetically engineered subunit vaccine

[0066]

[0067] 5.2 Safety Test of Eg95 Genetically Engineered Subunit Vaccine in Healthy Primigratory Ewes

[0068] Five healthy primigravid ewes with negative serum antibody test results for echinococcosis were selected and 2.0 ml of vaccine was subcutaneously inoculated into the neck of each sheep. A corresponding blank control group was also set up. 28 days after the first vaccination, a second immunization was performed with the same dose. After vaccination, the ewes were observed for 7 consecutive days and their temperatures were measured. The clinical manifestations and local reactions at the vaccination site of the ewes were observed, and the production status of the ewes was recorded. As shown in Table 2, Table 5, Figure 5 The body temperature results of primigravid ewes after vaccination (A, body temperature change curve before and after vaccination; B, body temperature difference between after vaccination and before vaccination) show: after vaccination of the ewes, all ewes were in normal spirits and movements, and there was no swelling or adverse reaction at the vaccination site; the body temperature of the ewes after vaccination did not exceed the basal body temperature by 1°C 2 hours after vaccination, and there was no significant difference in the body temperature between the vaccine group and the blank control group; no miscarriage, stillbirth or weak lambs occurred in either the vaccine group or the blank control group.

[0069] Table 2 Observation results of clinical manifestations and vaccination sites in primigravid ewes after vaccination with Eg95 genetically engineered subunit vaccine

[0070]

[0071] Table 3 Delivery results of primigravid ewes after vaccination with Eg95 genetically engineered subunit vaccine

[0072]

[0073] Example 6 Immunogenicity Test of Eg95 Genetically Engineered Subunit Vaccine

[0074] Five healthy susceptible lambs aged 2 to 4 months with negative serum antibody test for echinococcosis were taken, and 1.0 ml of vaccine was subcutaneously injected into the neck of each lamb. A corresponding blank control group was set up at the same time. 28 days after the first vaccination, a second immunization was carried out with the same dose. Blood was collected from the jugular vein before the first vaccination, 14 days, 28 days, 42 days (14 days after the second vaccination), and 56 days (28 days after the second vaccination), and the serum was separated and tested for ELISA antibodies. The results showed that 14 days after the first vaccination, if Figure 6 (ELISA antibody results in healthy, susceptible lambs aged 2 to 4 months after vaccination) All lambs in the immunized group were antibody-positive. These results demonstrate that the subunit vaccine exhibits strong immunogenicity in healthy, susceptible lambs aged 2 to 4 months, protecting them from Echinococcus infection, starting 14 days after vaccination.

[0075] The reason this data is called an inhibition rate is due to the assay principle of the kit: the target molecule (e.g., antibody) in the sample competes with the labeled antibody for binding to a limited amount of antigen. The more target molecules there are, the less the labeled antibody binds to the antigen, resulting in a weaker signal. A higher inhibition rate indicates a higher concentration of the target molecule in the sample. Compared to sandwich ELISA, the competitive assay is more sensitive.

[0076] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant protein, characterized in that It comprises the amino acid sequence shown in SEQ ID NO.

1.

2. The gene encoding the recombinant protein according to claim 1, characterized in that The coding gene includes the nucleotide molecule shown in SEQ ID NO.

2.

3. A recombinant vector comprising the encoding gene according to claim 2, characterized in that The recombinant vector is a CHO eukaryotic cell expression vector.

4. The recombinant vector according to claim 3, characterized in that Including one of the pEE series, pcDNA series, and pCHO series vectors.

5. The recombinant vector according to claim 3, characterized in that Includes one of the pEE series vectors.

6. A vaccine for preventing and treating ovine echinococcosis infection, characterized in that: The vaccine comprises the recombinant protein according to claim 1, as well as an adjuvant, a stabilizer and a buffer.

7. The method for preparing a vaccine for preventing and treating ovine echinococcosis infection according to claim 6, characterized in that: The following steps are involved: Cloning the coding gene of the recombinant protein according to claim 1 into a eukaryotic expression vector to obtain a recombinant vector; The recombinant vector is used to transfect host cells, and host cells that stably and efficiently express the recombinant protein in suspension are screened out, and then cultured at a lower temperature, and then the recombinant protein is separated and purified from the cell supernatant.

8. The method for preparing a vaccine for preventing and treating ovine echinococcosis infection according to claim 6, characterized in that: The eukaryotic expression vector includes: one of the pEE series vectors.

9. Use of the recombinant protein according to claim 1 or the immune composition according to claim 6 in the preparation of a genetically engineered subunit vaccine against sheep echinococcosis infection.

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