Pseudorabies virus antigen of swine, and preparation method and application thereof

Abstract

The application provides a porcine pseudorabies virus antigen and a preparation method and application thereof, relates to the technical field of biotechnology, and the antigen comprises a porcine pseudorabies virus gD protein; the amino acid sequence of the porcine pseudorabies virus gD protein is shown as SEQ ID NO. 5; experiments prove that the porcine pseudorabies virus antigen has good antigenicity, is easy to express highly, and has good broad-spectrum property; and the antibody produced by the porcine pseudorabies virus antigen has high neutralization capacity for the currently popular strains. The porcine pseudorabies virus antigen provided by the application is easy to express highly, and the expression amount of the antigen in a fermentation liquor is significantly improved after fermentation culture of recombinant cells. The technical problem of low expression amount of antibodies generated by immunization of commercial vaccines in the prior art is solved.

Description

[0001] This application claims priority to Chinese patent application CN202411609766.X, the contents of which are incorporated in their entirety into and form part of the original description of this invention. The applicant further declares that it has the right to amend the description and claims of this invention based on that priority document. Technical Field

[0002] This invention relates to the field of biotechnology, and in particular to a porcine pseudorabies virus antigen, its preparation method, and its application. Background Technology

[0003] Porcine pseudorabies (PR) is a serious infectious disease caused by pseudorabies virus (PRV) that severely threatens the healthy development of my country's pig farming industry. This virus belongs to the linear double-stranded DNA virus of the subfamily Aherpesvirinae in the family Herpesviridae.

[0004] Pigs are important reservoir hosts and sources of infection for this virus, primarily causing abortion, stillbirth, and mummified fetuses in pregnant sows. Infected piglets mainly exhibit neurological symptoms, while adult pigs are predominantly latently infected. PRV possesses strong pantropic, neurotropic, and latent infection characteristics, allowing it to remain latent in the peripheral nervous system for extended periods. When the latent virus is activated and becomes infectious, the latently infected host will develop symptoms. Pseudorabies has a wide susceptibility range, and newborn piglets are often fatally infected, similar to other susceptible species, with death primarily due to central nervous system diseases.

[0005] Currently, isolated circulating strains have exhibited multiple signature mutations and continuity deletions in genes such as TK, gB, gE, and gC. These mutations and deletions may be a significant reason for the resurgence of pseudorabies in pigs. Currently available commercial vaccines produce antibodies with poor neutralizing ability against the circulating strains. The emergence of variant strains has caused adult pigs to exhibit severe symptoms similar to those in piglets, leading to mass mortality of sows, piglets, and finishing pigs in pig farms. This presents new challenges for disease control and causes severe economic losses to the pig industry.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide a porcine pseudorabies virus antigen to obtain a commercially available vaccine antigen with high expression and good immunogenicity.

[0008] The second objective of this invention is to provide a polynucleotide encoding the aforementioned porcine pseudorabies virus antigen.

[0009] The third objective of this invention is to provide a recombinant carrier.

[0010] The fourth objective of this invention is to provide a recombinant cell.

[0011] The fifth objective of this invention is to provide a method for constructing the above-mentioned recombinant cells.

[0012] The sixth objective of this invention is to provide a method for preparing porcine pseudorabies virus antigen.

[0013] The seventh objective of this invention is to provide the application of the above-mentioned porcine pseudorabies virus antigen or the porcine pseudorabies virus antigen prepared by the above-mentioned preparation method in the preparation of a vaccine against porcine pseudorabies virus.

[0014] The eighth objective of this invention is to provide a porcine pseudorabies virus vaccine.

[0015] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0016] In a first aspect, the present invention provides a porcine pseudorabies virus antigen, the antigen comprising porcine pseudorabies virus gD protein;

[0017] The amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO.5.

[0018] Secondly, the present invention provides a polynucleotide encoding the above-mentioned porcine pseudorabies virus antigen, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0019] Thirdly, the present invention provides a recombinant vector carrying the above-mentioned nucleotide sequence.

[0020] Fourthly, the present invention provides a recombinant cell that expresses the above-mentioned porcine pseudorabies virus antigen, or carries the above-mentioned polynucleotide, or carries the above-mentioned recombinant vector.

[0021] Fifthly, the present invention provides a method for constructing the above-mentioned recombinant cells, comprising the following steps: converting the above-mentioned recombinant vector into host cells to obtain recombinant cells;

[0022] Preferably, the host cell is a mammalian cell.

[0023] Sixthly, the present invention provides a method for preparing porcine pseudorabies virus antigen, comprising the following steps: fermenting and culturing the above-mentioned recombinant cells or recombinant cells constructed using the above-mentioned method, collecting the fermentation broth, and separating and purifying it to obtain porcine pseudorabies virus antigen.

[0024] Furthermore, the fermentation culture step involves isothermal fermentation of the culture medium inoculated with recombinant cells at a temperature of 36–38°C, followed by a temperature reduction to 31–33°C on the 5th day of fermentation, and continued culture for another 7 days.

[0025] Preferably, the inoculation amount is 0.5–1.5 × 10⁻⁶. 5 The number of cells / ml is preferably 1.0 × 10⁻⁶. 5 pcs / ml;

[0026] Preferably, the isothermal fermentation step further includes isothermal fermentation in an environment containing 4.5-5.5% CO2, preferably 5%;

[0027] Preferably, the isothermal fermentation step further includes a reactor rotation speed of 150-350 r / min during fermentation, preferably 250 r / min;

[0028] Preferably, when the glucose concentration in the culture medium is <2.5 g / L, glucose is supplemented to 3-4 g / L;

[0029] Preferably, the culture medium is added on the 4th and 9th days of fermentation, respectively, and the amount of culture medium added is 10% of the original amount of culture medium added.

[0030] In a seventh aspect, the present invention provides the use of the above-described porcine pseudorabies virus antigen or the porcine pseudorabies virus antigen prepared by the above-described preparation method in the preparation of a vaccine against porcine pseudorabies virus.

[0031] Eighthly, the present invention provides a porcine pseudorabies virus vaccine comprising the above-mentioned porcine pseudorabies virus antigen or porcine pseudorabies virus antigen prepared by the above-mentioned preparation method and an adjuvant.

[0032] Furthermore, the content of the porcine pseudorabies virus antigen is 30-125 μg / ml, preferably, the content of the porcine pseudorabies virus antigen is 40 μg / ml.

[0033] This invention provides a porcine pseudorabies virus antigen. Experiments have shown that this porcine pseudorabies virus antigen has good antigenicity, is easily highly expressed, and has good broad-spectrum activity. The antibodies produced by it have high neutralizing ability against currently circulating strains. The porcine pseudorabies virus antigen provided by this invention is easily highly expressed, and the antigen expression level in the fermentation broth of recombinant cells is significantly increased after fermentation culture. Detailed Implementation

[0034] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0035] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0036] The present invention provides a porcine pseudorabies virus antigen, the antigen comprising porcine pseudorabies virus gD protein; the amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO.5.

[0037] Experiments have demonstrated that this porcine pseudorabies virus antigen possesses high antigenicity and is easily highly expressed; it also exhibits broad-spectrum activity, and the antibodies produced have a high neutralizing capacity against currently circulating strains. This solves the technical problem of low antibody expression levels produced by commercially available vaccines in existing technologies.

[0038] The aforementioned porcine pseudorabies virus antigen can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically. According to another aspect of the present invention, a polynucleotide encoding the aforementioned porcine pseudorabies virus antigen is also provided, the nucleotide sequence of which is shown in SEQ ID NO. 6.

[0039] The aforementioned porcine pseudorabies virus antigens are derived from the gD gene of classic porcine pseudorabies virus strains and currently circulating porcine pseudorabies virus strains.

[0040] According to another aspect of the present invention, a recombinant vector is also provided, the recombinant vector carrying the above-described nucleotide sequence;

[0041] The vector is known to those skilled in the art and includes, but is not limited to, plasmids, bacteriophages, granules, Ti plasmids, or viral vectors. Specifically, it can be pcDNA3.1, pEE6.4, pEE12.4, or pGL4.13, preferably pcDNA3.1.

[0042] According to another aspect of the present invention, a recombinant cell is also provided, which expresses the above-mentioned porcine pseudorabies virus antigen, or carries the above-mentioned polynucleotide, or carries the above-mentioned recombinant vector.

[0043] According to another aspect of the present invention, a method for constructing the above-described recombinant cells is also provided, comprising the following steps: converting the above-described recombinant vector into host cells to obtain recombinant cells.

[0044] The transformation can be any known transformation method, such as chemical transformation or electroporation, to transform the vector carrying the above-mentioned nucleotide sequence into the host cell. Specifically, it can be the integration of the foreign gene into the host chromosome, or it can be expressed extrachromosomally by a plasmid.

[0045] In a specific implementation, the host cell is a mammalian cell, such as a CHO cell.

[0046] According to another aspect of the present invention, a method for preparing porcine pseudorabies virus antigen is also provided, comprising the following steps: fermenting and culturing the above-mentioned recombinant cells or recombinant cells constructed using the above-mentioned method, collecting the fermentation broth, and obtaining porcine pseudorabies virus antigen after separation and purification.

[0047] Porcine pseudorabies virus antigen is easily overexpressed, and the antigen expression level in the fermentation broth of recombinant cells was significantly increased after fermentation culture.

[0048] In a specific implementation, the fermentation culture step involves isothermal fermentation of the culture medium inoculated with recombinant cells at a temperature of 36–38°C, followed by a temperature reduction to 31–33°C on the 5th day of fermentation, and continued culture for another 7 days.

[0049] In a specific implementation, the inoculation amount is 0.5–1.5 × 10⁻⁶. 5 The number of cells / ml is preferably 1.0 × 10⁻⁶. 5 per ml.

[0050] In a specific implementation, the isothermal fermentation step further includes isothermal fermentation in an environment containing 4.5-5.5% CO2, preferably 5%.

[0051] In a specific implementation, the isothermal fermentation step further includes a reactor rotation speed of 150-350 r / min, preferably 250 r / min, during the fermentation process.

[0052] In a specific implementation, when the glucose concentration in the culture medium is <2.5g / L, glucose is supplemented to 3-4g / L.

[0053] In a specific implementation, culture medium is added on the 4th and 9th days of fermentation, respectively, and the amount of culture medium added is 10% of the original amount of culture medium added.

[0054] According to another aspect of the present invention, the use of the above-described porcine pseudorabies virus antigen or porcine pseudorabies virus antigen prepared by the above-described preparation method in the preparation of an anti-porcine pseudorabies virus vaccine is also provided.

[0055] According to another aspect of the present invention, a porcine pseudorabies virus vaccine is also provided, comprising the porcine pseudorabies virus antigen described above or the porcine pseudorabies virus antigen prepared by the above preparation method and an adjuvant.

[0056] In some specific embodiments, the content of the porcine pseudorabies virus antigen is 30-125 μg / ml, and in some specific embodiments, the content of the porcine pseudorabies virus antigen is preferably 40 μg / ml.

[0057] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] Example 1: Construction of a cell line expressing porcine pseudorabies virus antigen

[0059] 1. gD gene synthesis

[0060] The gD gene of classic strains and currently popular strains were selected from GenBank as research objects. Through codon optimization and modification, different signal peptide sequences were added to the N-terminus of the optimized gD sequence and tagged with His. Finally, four gD gene sequences were obtained and synthesized, and labeled as gD-1, gD-2, gD-3 and gD-4 respectively.

[0061] 2. Carrier Construction

[0062] 2.1 Construction of Recombinant Plasmids

[0063] Positive plasmids pcDNA3.1-gD-1, pcDNA3.1-gD-2, pcDNA3.1-gD-3, and pcDNA3.1-gD-4 were constructed using standard recombinant plasmid construction procedures. Simultaneously, plasmid pcDNA3.1-gD was constructed as a control. The amino acid sequence of control gD is shown in SEQ ID NO.1, and the nucleotide sequence of gD is shown in SEQ ID NO.2.

[0064] 2.1 Cell transfection

[0065] The specific steps are as follows, referring to the Lipofectamine™ 3000 product instruction manual. One day before transfection, adjust the CHO cell density to 1.5 × 10⁻⁶ cells / day. 5Cells were seeded at a density of 2.0 ml / well in 6-well plates and incubated overnight at 36-38°C in a 5% CO2 incubator. Transfection was initiated when cell confluence reached 80%-90%. Positive plasmids were diluted in OPTI-MEM medium to prepare a DNA premix, which was then supplemented with Lipofectamine. TM 3000 reagent (mix thoroughly); dilute Lipofectamine with OPTI-MEM medium. TM 3000 reagent (mix thoroughly); add to each tube of diluted Lipofectamine TM Add diluted DNA (molar ratio 1:1) to 3000 reagent, incubate at room temperature for 10-15 minutes, and then add it dropwise evenly to a 6-well plate; incubate at 36℃-38℃ in a cell culture incubator containing 5% CO2 for 48 hours, and then replace with DMEM complete medium (containing 0.8 mg / ml G418).

[0066] 2.2 Monoclonal cell screening

[0067] Selection was performed using DMEM complete medium (containing 0.8 mg / ml G418) under pressure. The viable cell density was adjusted to 1.0 cells / well, and 200 μl was seeded into each well of a 96-well plate. The plates were incubated at 37°C. Once the single cell line in the 96-well plate had expanded to over 80%, the protein expression yield of the monoclonal cell line was detected by ELISA. Cells with high expression levels were digested and transferred to 24-well plates. After the 24-well plates reached confluence, the supernatant was collected for analysis, and the protein expression yield of the monoclonal cell line was detected by ELISA. Monoclonal cells with expression yields above 1 g / L were stored in liquid nitrogen.

[0068] Example 2: Expression of porcine pseudorabies virus antigen

[0069] 1. Recovery and expansion

[0070] Monoclonal cells were removed from the liquid nitrogen tank and rapidly thawed in a water bath at 36–38°C. The cells were then suspended in culture medium at a density of 1.0 × 10⁻⁶. 5 Cells were seeded at 1 / ml in CHO serum-free medium (SPM) and cultured in suspension at 36–38°C in a shaker containing 5% CO2 for 72 hours. The cells were then passaged to expand the culture of single clones.

[0071] 2. Fermentation

[0072] Cells were removed from a 5% CO2 constant-temperature shaker and counted, at a density of 1.0 × 10⁻⁶. 5Cells were inoculated at a density of 1 / ml in the bioreactor and cultured at a constant temperature of 37℃ with a rotation speed of 250 r / min. Cell density, viability, and glucose content were measured every 24 hours. When the glucose concentration was lower than 2.5 g / L, glucose was added to 3-4 g / L. Feeding was performed on days 4 and 9 of fermentation (using Cell Boost 7A), with the added volume being 10% of the initial culture medium volume. The temperature was adjusted to 31-33℃ starting from day 5 of fermentation. On day 12, the cell fermentation broth was collected, and the protein content was determined. The experimental results are shown in Table 1.

[0073] Table 1. Results of cell line fermentation supernatant protein content on day 12.

[0074] cell lines Fermentation yield (g / L) gD-1 1.68 gD-2 4.58 gD-3 1.14 gD-4 1.98 pcDNA3.1-gD 0.95

[0075] As shown in Table 2, the gD-2 cell line exhibited the highest gD protein expression level, significantly superior to that of the control pcDNA3.1-gD cell line. The amino acid sequence of the gD gene in gD-1 is shown in SEQ ID NO. 3, and its nucleotide sequence is shown in SEQ ID NO. 4. The amino acid sequence of the gD gene in gD-2 is shown in SEQ ID NO. 5, and its nucleotide sequence is shown in SEQ ID NO. 6. The amino acid sequence of the gD gene in gD-3 is shown in SEQ ID NO. 7, and its nucleotide sequence is shown in SEQ ID NO. 8. The amino acid sequence of the gD gene in gD-4 is shown in SEQ ID NO. 9, and its nucleotide sequence is shown in SEQ ID NO. 10.

[0076] Example 3: Evaluation of Immunization Efficacy and Safety

[0077] In this embodiment, the gD protein expressed by cell line gD-2 obtained in Example 1 was used to evaluate its immunogenicity and safety. The specific steps are as follows:

[0078] After decomposing and quantifying the expressed protein, it was mixed with ISA201 adjuvant to prepare the vaccine. The aqueous and oil phases were emulsified at a 1:1 mass ratio. The oil phase was first poured into a beaker, stirred slowly, and then the aqueous phase was added slowly. After the addition was complete, the mixture was sheared and emulsified for 5 minutes. The vaccine was aseptically aliquoted, sealed, and stored at 2–8°C to obtain the porcine pseudorabies virus subunit vaccine.

[0079] After immunizing 21-day-old piglets with this vaccine, blood samples were collected 21 days later to test for neutralizing antibodies. The results showed that the neutralizing antibody level was not lower than 1:256 on day 21. Furthermore, the challenge results showed that the vaccine could effectively prevent both the classic virulent strain (SC strain) and the currently circulating strain (JS strain) from attack, with a protection rate of 100%. No fever or abnormal clinical symptoms were observed in any of the immunized pigs.

[0080] Table 2 Experimental Groups and Results

[0081]

[0082] "N / A" (Not Available) indicates that the application is not applicable.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A porcine pseudorabies virus antigen, characterized in that, The antigen is the porcine pseudorabies virus gD protein; The amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO.

5.

2. A polynucleotide encoding the porcine pseudorabies virus antigen of claim 1, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.

6.

3. A recombinant vector, characterized in that, The recombinant vector carries the polynucleotide of claim 2.

4. A recombinant cell, characterized in that, The recombinant cells express the porcine pseudorabies virus antigen of claim 1, or carry the polynucleotide of claim 2, or carry the recombinant vector of claim 3.

5. A method for constructing the recombinant cells of claim 4, characterized in that, The procedure includes the following steps: converting the recombinant vector according to claim 3 into a host cell to obtain recombinant cells.

6. The method according to claim 5, characterized in that, The host cell is a mammalian cell.

7. A method for preparing porcine pseudorabies virus antigen, characterized in that, Includes the following steps: The recombinant cells of claim 4 or the recombinant cells constructed using the method of claim 5 are fermented and cultured. The fermentation broth is collected, and after separation and purification, porcine pseudorabies virus antigen is obtained.

8. The preparation method according to claim 7, characterized in that, The fermentation culture steps are as follows: the culture medium inoculated with recombinant cells is fermented at a constant temperature of 36~38℃. On the 5th day of fermentation, the temperature is lowered to 31~33℃ and cultured for another 7 days.

9. The preparation method according to claim 8, characterized in that, The inoculation dose is 0.5~1.5×10⁻⁶. 5 per ml.

10. The preparation method according to claim 9, characterized in that, The inoculation dose was 1.0 × 10⁻⁶. 5 per ml.

11. The preparation method according to claim 9, characterized in that, The isothermal fermentation step also includes isothermal fermentation in an environment containing 4.5~5.5% CO2.

12. The preparation method according to claim 11, characterized in that, The constant temperature fermentation step also includes constant temperature fermentation in an environment containing 5% CO2.

13. The preparation method according to claim 9, characterized in that, The isothermal fermentation step also includes a reactor rotation speed of 150~350 r / min during the fermentation process.

14. The preparation method according to claim 13, characterized in that, The isothermal fermentation step also includes a reactor rotation speed of 250 r / min during the fermentation process.

15. The preparation method according to claim 9, characterized in that, When the glucose concentration in the culture medium is <2.5g / L, supplement glucose to 3~4g / L.

16. The preparation method according to claim 9, characterized in that, Culture medium was added on day 4 and day 9 of fermentation, respectively, and the amount of culture medium added was 10% of the amount of the original culture medium.

17. The use of the porcine pseudorabies virus antigen of claim 1 or the porcine pseudorabies virus antigen prepared by any one of claims 7 to 16 in the preparation of an anti-porcine pseudorabies virus vaccine.

18. A porcine pseudorabies virus vaccine, characterized in that, It comprises the porcine pseudorabies virus antigen as described in claim 1 or the porcine pseudorabies virus antigen prepared by the preparation method described in any one of claims 7 to 16 and an adjuvant.

19. The porcine pseudorabies virus vaccine according to claim 18, characterized in that, The content of the porcine pseudorabies virus antigen is 30~125μg / ml.

20. The porcine pseudorabies virus vaccine according to claim 19, characterized in that, The content of the porcine pseudorabies virus antigen is 40 μg / ml.

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