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.7. Experiments prove that the optimized porcine pseudorabies virus antigen is good in antigenicity, high in stability and easy to store for a long time. The technical problem that in the prior art, the stability of the porcine pseudorabies virus gD protein is poor is solved.

Description

Technical Field

[0001] 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

[0002] 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.

[0003] 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.

[0004] Although the currently disclosed porcine pseudorabies virus gD protein has certain immunogenicity, it has the problem of poor antigenic stability in practical applications.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a porcine pseudorabies virus antigen to at least solve the technical problem of poor stability of porcine pseudorabies virus gD protein in the prior art.

[0007] A second object of the present invention is to provide a polynucleotide encoding the above-mentioned porcine pseudorabies virus antigen.

[0008] The third object of the present invention is to provide a recombinant vector.

[0009] A fourth object of the present invention is to provide a recombinant cell.

[0010] A fifth object of the present invention is to provide a method for constructing the above-mentioned recombinant cell.

[0011] A sixth object of the present invention is to provide a method for preparing porcine pseudorabies virus antigen.

[0012] 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.

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

[0014] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0015] In a first aspect, the present invention provides a porcine pseudorabies virus antigen, wherein the antigen comprises a porcine pseudorabies virus gD protein;

[0016] The amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO.7.

[0017] 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.8.

[0018] In a third aspect, the present invention provides a recombinant vector carrying the above-mentioned nucleotide sequence.

[0019] 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.

[0020] 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;

[0021] Preferably, the host cell is a mammalian cell.

[0022] 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.

[0023] 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;

[0024] Preferably, the inoculation amount is 0.5 to 1.5×10 5 / ml, preferably 1.0×10 5 Pieces / ml;

[0025] Preferably, the constant temperature fermentation step further comprises constant temperature culturing in an environment containing 4.5-5.5% CO2, preferably 5%;

[0026] Preferably, the constant temperature fermentation step further comprises a shaker with a reactor speed of 150 to 350 r / min, preferably 250 r / min during the fermentation process;

[0027] 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;

[0028] Preferably, 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The invention provides a porcine pseudorabies virus antigen. Experiments have shown that the porcine pseudorabies virus antigen obtained in the invention has good immunogenicity, is suitable for the preparation of porcine pseudorabies virus vaccine, has high stability, and is easy to store for a long time. DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] 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.7.

[0036] Experiments have shown that the optimized porcine pseudorabies virus antigen has good antigenicity and high stability and is easy to store for a long time, thus solving the technical problem of the poor stability of the porcine pseudorabies virus gD protein antigen before vaccination in the prior art.

[0037] 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.8.

[0038] 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.

[0039] According to another aspect of the present invention, a recombinant vector is provided, wherein the recombinant vector carries the above-mentioned nucleotide sequence.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] In a specific embodiment, the host cell is a mammalian cell, such as a CHO cell.

[0045] 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.

[0046] By testing the antigen content in the fermentation broth stored for different periods of time, it was found that the porcine pseudorabies virus antigen maintained a high level for a long time, showing good stability.

[0047] 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.

[0048] In a specific embodiment, the inoculum size is 0.5 to 1.5×10 5 / ml, preferably 1.0×10 5 Pieces / ml.

[0049] 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%.

[0050] In a specific embodiment, the constant temperature fermentation step also includes a reactor rotation speed of 150 to 350 r / min, preferably 250 r / min during the fermentation process.

[0051] 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.

[0052] 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.

[0053] 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 a vaccine against porcine pseudorabies virus.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] Example 1 Construction of a cell line expressing porcine pseudorabies virus antigen

[0058] 1. gD gene synthesis

[0059] 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.

[0060] 2. Vector Construction

[0061] 2.1 Construction of recombinant plasmid

[0062] 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, pcDNA3.1-gD was constructed as a positive control, and the amino acid sequence of gD of the positive control is shown in SEQ ID NO.1, and the nucleotide sequence of gD is shown in SEQ ID NO.2.

[0063] 2.1 Cell transfection

[0064] 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).

[0065] 2.2 Monoclonal cell screening

[0066] 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.

[0067] Example 2 Determination of the stability of pseudorabies virus antigen expression

[0068] 1. Resuscitation and expansion

[0069] 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.

[0070] 2. Fermentation Broth Preparation

[0071] Remove cells from the 5% CO2 incubator and count them according to the density of 1.0×10 5 The cells were inoculated with 100 μg / ml of culture medium in a bioreactor and cultured at a constant temperature of 37°C and 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 starting from the 5th day of fermentation. On the 12th day, the cell fermentation liquid was collected.

[0072] 3. Determination of the expression of pseudorabies virus gD protein in fermentation broth at different storage times

[0073] The cell fermentation broth collected in step 2 was divided into 12 portions, and the protein content in the cell fermentation broth was detected at different times according to the following steps.

[0074] 1.1 Clarification

[0075] Wash the hollow fiber ultrafiltration system with 1×PBS, add 1000 ml of the cell fermentation liquid collected in step 2 to the liquid exchange tank, turn on the agitator and circulation pump, and set the shear rate to 4000 s -1 After circulating for a certain period of time, it was filtered through a 0.2μm hollow fiber microfiltration column and 900ml of the permeate was collected.

[0076] 1.2 Diafiltration of the retentate

[0077] Add an equal volume of 1× PBS to the remaining 100 ml of cell fermentation liquid in the liquid exchange tank, turn on the agitator and circulation pump, and set the shear rate to 4000 s. -1 , collect the permeate to obtain 100 ml of washing filtrate for standby use; repeat this step twice. Mix the permeate and washing filtrate evenly to obtain 1200 ml of clarified cell supernatant.

[0078] 1.3 Medium replacement and determination

[0079] The harvested cell supernatant was concentrated and replaced with 30 kDa hollow fiber membrane. The replacement buffer was PBS and the replacement was performed by continuous flow equal volume replacement. After the replacement was completed, the antigen solution was collected and stored at 2-8°C. The protein content was determined according to the plan, as shown in Table 1.

[0080] Table 1 Detection results of the gD protein content of pseudorabies virus in fermentation broth stored at 2-8℃

[0081]

[0082]

[0083] As shown in Table 1, although the initial expression of gD-2 is high, its protein content is lower than that of gD-3 when stored for more than 7 months, and the protein content of gD-2 decreases rapidly compared with the initial content. The protein content of gD-3 maintains a high level for a long time, showing good stability, and its stability is better than that of pcDNA3.1-gD protein. The amino acid sequence of gD-1 protein is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4. The amino acid sequence of gD-2 protein is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.6. The amino acid sequence of gD-3 protein is shown in SEQ ID NO.7, and its nucleotide sequence is shown in SEQ ID NO.8. The amino acid sequence of gD-4 protein is shown in SEQ ID NO.9, and its nucleotide sequence is shown in SEQ ID NO.10.

[0084] Example 3 Immune effect and safety evaluation

[0085] This example uses the gD protein expressed by the gD-3 strain cells with the best stability screened in Example 2 to perform immunological effect and safety evaluation. The operation is specifically performed according to the following steps:

[0086] 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.

[0087] 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 the attack of both the classic strong virus (SC strain) and the current epidemic strain (JS strain), and the protection rate can reach 100%. All immunized pigs did not have elevated body temperature and abnormal clinical symptoms.

[0088] Table 2 Experimental groups and results

[0089]

[0090]

[0091] Among them, "N / A" (Not Available) means not applicable.

[0092] 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.

7.

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.

8.

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 culturing and fermenting the reactor at a speed of 150 to 350 r / min, preferably 250 r / min; 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 a vaccine against porcine pseudorabies virus.

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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