Porcine pseudorabies virus antigen as well as preparation method and application thereof

Abstract

The invention provides a porcine pseudorabies virus antigen as well as a preparation method and application thereof, and relates to the technical field of biology, the antigen comprises 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 is good in antigenicity, easy to highly express and good in broad spectrum, and the generated antibody is high in neutralizing capacity for current epidemic strains. The porcine pseudorabies virus antigen provided by the invention is easy to highly express, and the expression quantity of the antigen in fermentation liquor after recombinant cells are subjected to fermentation culture is obviously improved. The technical problem that in the prior art, the expression quantity of antibodies generated by commercial vaccine immunization is low is solved.

Description

[0001] This application claims the priority of Chinese patent application CN202411609766.X, and the contents of the description and claims of the priority document are fully incorporated into the description of the present invention and are taken as part of the original description of the present invention. The applicant further declares that the applicant has the right to modify the description and claims of the present invention based on the priority document. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to a porcine pseudorabies virus antigen and a preparation method and application thereof. Background Art

[0003] Porcine pseudorabies (PR) is one of the important infectious diseases caused by pseudorabies virus (PRV), which seriously endangers the healthy development of my country's pig industry. The virus belongs to the linear double-stranded DNA virus of the alphaherpesvirus subfamily of the herpesviridae family.

[0004] Pigs are important reservoir hosts and sources of infection for the virus, which mainly causes abortion, stillbirth and mummification in pregnant sows. Infection in piglets is mainly neurological, while in adult pigs it is mainly latent infection. PRV has strong pan-tropism, neurotropism and latent infection characteristics. It can remain latent in the peripheral nervous system for a long time. When the latent virus is activated and becomes infectious, the latently infected host will become ill. Pseudorabies has a wide susceptibility range. Newborn piglets are mostly fatally infected. Similar to other susceptible species, most of them die from central nervous system diseases.

[0005] At present, the isolated epidemic strains have undergone multiple base mutations and continuous deletions in genes such as TK, gB, gE, and gC. These mutations and deletions may be an important reason for the current resurgence of pseudorabies in pigs. The antibodies produced by commercial vaccines have poor neutralization ability against the current epidemic strains. The emergence of mutant strains has caused adult pigs to show severe symptoms similar to those of piglets, and a large number of sows, piglets, and fattening pigs have died in pig farms, posing new challenges to the prevention and control of the disease and causing serious economic losses to the current pig farming industry.

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

[0007] One of the purposes of the present invention is to provide a porcine pseudorabies virus antigen to obtain a commercialized vaccine production antigen with high expression and good immunogenicity.

[0008] A second object of the present invention is to provide a polynucleotide encoding the above-mentioned porcine pseudorabies virus antigen.

[0009] The third object of the present invention is to provide a recombinant vector.

[0010] A fourth object of the present invention is to provide a recombinant cell.

[0011] A fifth object of the present invention is to provide a method for constructing the above-mentioned recombinant cell.

[0012] A sixth object of the present invention is to provide a method for preparing porcine pseudorabies virus antigen.

[0013] A seventh object of the present invention is to provide the use 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] An eighth object of the present invention is to provide a porcine pseudorabies virus vaccine.

[0015] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0016] In a first aspect, the present invention provides a porcine pseudorabies virus antigen, wherein the antigen comprises a 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] In a second aspect, the present invention provides a polynucleotide encoding the above-mentioned porcine pseudorabies virus antigen, and the nucleotide sequence of the polynucleotide is shown in SEQ ID NO.6.

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

[0020] In a fourth aspect, the present invention provides a recombinant cell, which expresses the aforementioned porcine pseudorabies virus antigen, or carries the aforementioned polynucleotide, or carries the aforementioned recombinant vector.

[0021] In a fifth aspect, the present invention provides a method for constructing the above-mentioned recombinant cell, comprising the following steps: transforming the above-mentioned recombinant vector into a host cell to obtain a recombinant cell;

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

[0023] In a sixth aspect, the present invention provides a method for preparing a porcine pseudorabies virus antigen, comprising the following steps: fermenting and culturing the above-mentioned recombinant cells or the recombinant cells constructed using the above-mentioned method, collecting the fermentation broth, and obtaining the porcine pseudorabies virus antigen after separation and purification.

[0024] Furthermore, the fermentation culture step is to perform constant temperature fermentation of the culture medium inoculated with the recombinant cells at a temperature of 36 to 38° C., reduce the temperature to 31 to 33° C. on the fifth day of fermentation, and continue to culture for 7 days;

[0025] Preferably, the inoculation amount is 0.5 to 1.5×10 5 / ml, preferably 1.0×10 5 Pieces / ml;

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

[0027] Preferably, the constant temperature fermentation step further comprises the step of rotating the reactor at a speed of 150 to 350 r / min, preferably 250 r / min, during the fermentation process;

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

[0029] Preferably, culture medium is added on the 4th day and the 9th day 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 use of the above-mentioned porcine pseudorabies virus antigen or the porcine pseudorabies virus antigen prepared using the above-mentioned preparation method in the preparation of a vaccine against porcine pseudorabies virus.

[0031] In an eighth embodiment, the present invention provides a porcine pseudorabies virus vaccine, comprising the porcine pseudorabies virus antigen described above or the porcine pseudorabies virus antigen prepared using the above preparation method and an adjuvant.

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

[0033] The present invention provides a porcine pseudorabies virus antigen, which has good antigenicity, is easy to be highly expressed, and has good broad spectrum, and the antibodies produced by the porcine pseudorabies virus antigen have high neutralizing ability against currently prevalent strains. The porcine pseudorabies virus antigen provided by the present invention is easy to be highly expressed, and the antigen expression amount in the fermentation liquid is significantly improved after the recombinant cell is fermented and cultured. DETAILED DESCRIPTION

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

[0035] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0036] In one aspect, the present invention provides a porcine pseudorabies virus antigen, which comprises a 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 shown that the porcine pseudorabies virus antigen has good antigenicity, is easy to express at a high level, and has a good broad spectrum, and the antibodies produced by it have high neutralizing ability against the currently prevalent strains, thus solving the technical problem of low antibody expression produced by commercial vaccine immunization in the prior art.

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

[0039] The above-mentioned porcine pseudorabies virus antigen is derived from the gD gene of the classic porcine pseudorabies virus strain and the currently prevalent porcine pseudorabies virus strain.

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

[0041] The vector is well known to those skilled in the art, including but not limited to: plasmid, phage, cosmid, Ti plasmid or viral vector, and 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 provided, wherein the recombinant cell expresses the porcine pseudorabies virus antigen, or carries the polynucleotide, or carries the recombinant vector.

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

[0044] The transformation may be any known transformation method such as chemical transformation or electroporation transformation, wherein the vector carrying the nucleotide sequence is transformed into the host cell. Specifically, the foreign gene may be integrated into the host chromosome, or expressed outside the chromosome by a plasmid.

[0045] In a specific embodiment, 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 provided, comprising the following steps: fermenting and culturing the above-mentioned recombinant cells or the recombinant cells constructed using the above-mentioned method, collecting the fermentation broth, and obtaining the porcine pseudorabies virus antigen after separation and purification.

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

[0048] In a specific embodiment, the fermentation culture step is to ferment the culture medium inoculated with the recombinant cells at a constant temperature of 36-38° C., reduce the temperature to 31-33° C. on the 5th day of fermentation, and continue to culture for 7 days.

[0049] In a specific embodiment, the inoculum size is 0.5 to 1.5×10 5 / ml, preferably 1.0×10 5 Pieces / ml.

[0050] In a specific embodiment, the constant temperature fermentation step further comprises constant temperature fermentation in an environment containing 4.5-5.5% CO2, preferably 5%.

[0051] In a specific embodiment, the constant temperature fermentation step also includes a reactor speed of 150 to 350 r / min, preferably 250 r / min during the fermentation process.

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

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

[0054] According to another aspect of the present invention, there is also provided the use 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 an anti-porcine pseudorabies virus vaccine.

[0055] According to another aspect of the present invention, a porcine pseudorabies virus vaccine is provided, comprising the porcine pseudorabies virus antigen 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. 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 in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 genes of classic strains and current epidemic 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 a His tag was added. Finally, four gD gene sequences were obtained and synthesized, labeled as gD-1, gD-2, gD-3 and gD-4 respectively.

[0061] 2. Vector Construction

[0062] 2.1 Construction of recombinant plasmid

[0063] The positive plasmids pcDNA3.1-gD-1, pcDNA3.1-gD-2, pcDNA3.1-gD-3 and pcDNA3.1-gD-4 were constructed by conventional recombinant plasmid construction process. At the same time, the plasmid pcDNA3.1-gD was constructed as a control, the amino acid sequence of the 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 instruction manual of LipofectamineTM3000. One day before transfection, the CHO cells were cultured at a density of 1.5×10 5 / ml, inoculate 6-well plates, 2.0ml per well, incubate overnight in a cell culture incubator at 36℃-38℃, 5% CO2; start transfection when the cell confluence reaches 80%-90%; dilute the positive plasmid in OPTI-MEM medium, prepare DNA premix, and then add Lipofectamine TM 3000 reagent (mix thoroughly); dilute Lipofectamine with OPTI-MEM medium TM 3000 reagent (mix thoroughly); add the diluted Lipofectamine TM Add diluted DNA (molar ratio 1:1) to 3000 reagent, incubate at room temperature for 10-15 minutes, and then add dropwise evenly to a 6-well plate; culture in a cell culture incubator at 36°C-38°C with 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] Use DMEM complete medium (containing 0.8mg / ml G418) for pressure screening. Adjust the live cell density to 1.0 / well, inoculate 96-well plates, 200μl per well, and place at 37°C. When the single cell line in the 96-well plate is amplified to more than 80%, the expression yield of the monoclonal cell line protein is detected by ELISA. Digest the cells with higher expression values ​​and transfer them to 24-well plates. When the 24-well plates are full, take the supernatant for detection, and detect the expression yield of the monoclonal cell line protein by ELISA. The screened monoclonal cells with an expression yield of more than 1g / L are stored in liquid nitrogen.

[0068] Example 2 Expression of Pseudorabies Virus Antigen in Porcine

[0069] 1. Resuscitation and expansion

[0070] Take out the monoclonal cells from the liquid nitrogen tank, thaw them quickly in a 36-38℃ water bath, suspend the cells in culture medium, and prepare the cells at a density of 1.0×10 5 The monoclonal cells were inoculated into CHO serum-free medium (SPM) at 1000 μg / ml and placed in a shaker at 36-38°C with 5% CO2 for suspension culture for 72 hours, and then continued to be subcultured and expanded.

[0071] 2. Fermentation

[0072] Remove cells from the 5% CO2 incubator and count them according to the density of 1.0×10 5The cells were inoculated with 100 μg / ml of culture medium in a bioreactor and cultured at 37°C at a rotation speed of 250 r / min. The cell density, activity and glucose content were detected every 24 hours. When the glucose concentration was lower than 2.5 g / L, glucose was added to 3-4 g / L. Feed (Cell Boost 7A) was added on the 4th and 9th days of fermentation, and the added volume was 10% of the initial culture medium volume. The temperature was adjusted to 31-33°C from the 5th day of fermentation. On the 12th day, the cell fermentation broth was collected and the protein content was determined. The test results are shown in Table 1.

[0073] Table 1 Detection results of protein content in supernatant of cell lines on day 12 of fermentation

[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 expression of gD protein in gD-2 strain cells is the highest, and is much better than the expression of gD protein in the control pcDNA3.1-gD cell strain. The amino acid sequence of the gD gene of 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 of 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 of 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 of gD-4 is shown in SEQ ID NO.9, and its nucleotide sequence is shown in SEQ ID NO.10.

[0076] Example 3 Immune effect and safety evaluation

[0077] This example uses the gD protein expressed by the cell strain gD-2 obtained by screening in Example 1 to perform immune effect and safety evaluation. The operation is specifically performed according to the following steps:

[0078] After removing the cell fragments and quantifying the expressed protein, it was mixed with ISA201 adjuvant to prepare the vaccine. The emulsification ratio of the water phase to the oil phase was 1:1 by mass. The oil phase was first introduced into the beaker, stirred slowly, and the water phase was slowly added. After the addition, shear emulsification was performed for 5 minutes. Sterile quantitative packaging was performed, and the pseudorabies virus subunit vaccine was obtained and stored at 2-8°C.

[0079] After immunizing 21-day-old piglets with the vaccine, blood was collected 21 days after immunization to measure neutralizing antibodies. The results showed that the neutralizing antibody on the 21st day was not less than 1:256. In addition, the challenge results showed that the vaccine can effectively prevent attacks from both classic virulent strains (SC strain) and currently prevalent strains (JS strain), with a protection rate of 100%. All immunized pigs did not have elevated body temperatures or abnormal clinical symptoms.

[0080] Table 2 Experimental groups and results

[0081]

[0082] Among them, "N / A" (Not Available) means not applicable.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 comprises porcine pseudorabies virus gD protein; The amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO.

5.

2. The polynucleotide encoding the porcine pseudorabies virus antigen according to 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 nucleotide sequence of claim 2.

4. A recombinant cell, characterized in that The recombinant cell expresses the porcine pseudorabies virus antigen according to claim 1, or carries the polynucleotide according to claim 2, or carries the recombinant vector according to claim 3.

5. A method for constructing the recombinant cell according to claim 4, characterized in that: The method comprises the following steps: transforming the recombinant vector according to claim 3 into a host cell to obtain a recombinant cell; Preferably, the host cell is a mammalian cell.

6. A method for preparing porcine pseudorabies virus antigen, characterized in that: The following steps are involved: The recombinant cell according to claim 4 or the recombinant cell constructed by the method according to claim 5 is fermented and cultured, the fermentation liquid is collected, and the porcine pseudorabies virus antigen is obtained after separation and purification.

7. The preparation method according to claim 6, characterized in that: The fermentation culture step comprises: subjecting the culture medium inoculated with the recombinant cells to constant temperature fermentation at a temperature of 36 to 38° C., lowering the temperature to 31 to 33° C. on the fifth day of fermentation, and continuing to culture for 7 days; Preferably, the inoculum size is 0.5 to 1.5 × 10 5 / ml, preferably 1.0×10 5 Pieces / ml; Preferably, the constant temperature fermentation step further comprises constant temperature fermentation in an environment containing 4.5-5.5% CO2, preferably 5%; Preferably, the constant temperature fermentation step further comprises the step of rotating the reactor at a speed of 150 to 350 r / min, preferably 250 r / min, during the fermentation process; Preferably, when the concentration of glucose in the culture medium is less than 2.5 g / L, glucose is supplemented to 3-4 g / L; Preferably, culture medium is added on the 4th day and the 9th day of fermentation, respectively, and the amount of culture medium added is 10% of the original amount of culture medium added.

8. Use of the porcine pseudorabies virus antigen according to claim 1 or the porcine pseudorabies virus antigen prepared by the preparation method according to claim 6 or 7 in preparing an anti-porcine pseudorabies virus vaccine.

9. A porcine pseudorabies virus vaccine, characterized in that: The invention comprises the porcine pseudorabies virus antigen according to claim 1 or the porcine pseudorabies virus antigen prepared by the preparation method according to claim 6 or 7 and an adjuvant.

10. The porcine pseudorabies virus vaccine according to claim 9, characterized in that: The content of the porcine pseudorabies virus antigen is 30-125 μg / ml, and preferably, the content of the porcine pseudorabies virus antigen is 40 μg / ml.

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