CN117070476A - Bovine herpesvirus 4 strain and application thereof in preparation of inactivated vaccine - Google Patents
Bovine herpesvirus 4 strain and application thereof in preparation of inactivated vaccine Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly discloses a bovine herpesvirus 4 strain and application thereof in preparation of inactivated vaccines. The invention provides a bovine herpes virus 4-type strain BHV-4/CH/JY/2022, and the preservation number is CGMCC No.26500. The bovine herpes virus 4-type strain BHV-4/CH/JY/2022 provided by the invention has strong toxicity and good immunogenicity, is suitable for producing inactivated vaccines, and the prepared HBV-4 inactivated vaccine has high safety and strong immune efficacy, is suitable for industrialized mass production, and is favorable for preventing and controlling various diseases caused by BHV-4.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a bovine herpesvirus 4 strain and application thereof in preparation of inactivated vaccines.
Background
Bovine herpesvirus type 4 (Bovine herpesvirus, BHV-4) belongs to the family of herpesviridae c. BHV-4 infects cattle, sheep, goats, guinea pigs, cats, and the like. Cattle suffering from the disease are mostly recessive infections, often cause reproductive disorders, and after combining with other pathogens, clinical symptoms may be aggravated. At present, BHV-4 has been found to be directly associated with a variety of diseases, such as: peritonitis, infertility, abortion, metritis, colpitis, orchitis, mastitis, pneumonia, calf eye diseases, encephalitis, synovitis, and foodleaf inflammation. Based on the premise that respiratory and reproductive problems can cause huge losses to the cattle industry, the development of BHV-4 vaccine would benefit the cattle industry directly.
The safe and effective BHV-4 vaccine is an important means for preventing and controlling various diseases caused by BHV-4, however, no commercial BHV-4 inactivated vaccine is available for the diseases, so that further research on vaccine development is necessary.
Disclosure of Invention
The invention aims at providing a bovine herpesvirus 4 strain suitable for producing inactivated vaccines and application thereof.
In order to achieve the object, the technical scheme of the invention is as follows:
in a first aspect, the invention provides a bovine herpes virus type 4 strain BHV-4/CH/JY/2022, which has a preservation number of CGMCC No.26500.
In a second aspect, the invention provides an application of the bovine herpes virus type 4 strain BHV-4/CH/JY/2022 in preparing bovine herpes virus type 4 inactivated vaccine.
The BHV-4 strain BHV-4/CH/JY/2022 strain has strong toxicity and good immunogenicity, and the bovine herpesvirus 4 type inactivated vaccine prepared by the method can effectively prevent various respiratory and reproductive diseases caused by bovine herpesvirus 4 type.
In a third aspect, the invention provides a method for preparing an inactivated bovine herpes virus type 4 vaccine, which comprises the steps of mixing and emulsifying an aqueous phase containing an inactivated antigen with a vaccine adjuvant; the preparation raw materials of the inactivated antigen comprise bovine herpes virus type 4 strain BHV-4/CH/JY/2022.
In the preparation method of the invention, the inactivated antigen is obtained by the following steps:
s1: culturing the bovine herpes virus strain 4 BHV-4/CH/JY/2022 to obtain BHV-4 virus liquid;
s2: clarifying the BHV-4 virus solution obtained in the step S1, and concentrating to obtain BHV-4 antigen concentrate;
s3: purifying the BHV-4 antigen concentrate obtained in the step S2 to obtain BHV-4 purified antigen solution;
s4: inactivating the BHV-4 purified antigen solution obtained in the step S3 to obtain the inactivated antigen.
In the preparation method step S1, the bovine herpes virus 4-type strain BHV-4/CH/JY/2022 is cultured in a suspension mode by adopting suspension MDBK cells, and when the cell activity rate reaches 30% -40%, the culture is obtained and used as BHV-4 virus liquid.
Preferably, in said suspension culture, when said bovine herpes virus type 4 strain BHV-4/CH/JY/2022 is inoculated, the cell density of said suspended MDBK cells is 1.5-2.5X10 6 Individual/ml;
and/or inoculating the suspension virus liquid of bovine herpesvirus type 4 strain BHV-4/CH/JY/2022 into the suspension MDBK cells for suspension culture, wherein the virus content of the suspension virus liquid is more than or equal to 10 8.0 TCID 50 The inoculation proportion is 0.1% -1% (v/v), more preferably 0.2% -0.5% (v/v);
and/or, when the suspension culture is performed, the culture pH is controlled to 7.4-7.6.
And/or, when the suspension culture is carried out, the culture temperature is 35.5-37 ℃, the rotating speed is 40-60rpm, and the DO value is 40-60%.
The preparation method of the BHV-4 inactivated vaccine provided by the invention preferably utilizes a large-scale suspension culture process of a bioreactor to culture bovine herpesvirus 4 type BHV-4/CH/JY/2022, and can obtain bovine herpesvirus 4 type antigen with high titer by controlling culture parameters (including cell density during virus inoculation, virus inoculation proportion, culture rotating speed, culture temperature, dissolved oxygen amount, pH and the like).
The virus maintenance solution used for the suspension culture is preferably purchased from the inner Mongolia Jin Yuankang biological engineering Co., ltd (product number: CM-VA 001), and can improve the virus titer more efficiently than the virus maintenance solution of the Jianshun biological technology Co., ltd (product number: 11801-211) and the virus maintenance solution of Shanghai ao Pu Mai biological technology Co., ltd (product number: V006201-010).
In the preparation method, in the step S2, a continuous flow centrifugation or 0.45 mu m filter element positive pressure filtration mode is adopted to clarify the BHV-4 virus liquid obtained in the step S1;
and/or concentrating in step S2 by using a hollow fiber column with 500KD, preferably by 5-7 times.
In the preparation method step S3, PEG is used for purification, wherein the PEG is selected from PEG6000 or PEG8000;
and/or, the purifying operation in step S3 includes: mixing the BHV-4 antigen concentrate obtained in the step S2 with PEG at 2-8deg.C, standing overnight (12-16 h), centrifuging at 6000-10000 rpm for more than 15 min, discarding supernatant, collecting precipitate, and re-suspending with PBS to obtain the BHV-4 purified antigen solution;
and/or the antigen content of the BHV-4 purified antigen solution is 10 8.00 TCID 50 Preferably 10 or more per ml 8.5 TCID 50 Preferably at least/ml, more preferably at least 10 9.00 TCID 50 And/or above.
In the method, most of the impurity proteins can be removed after the concentration and PEG purification by the hollow fiber column, so that purer antigen liquid for preparing vaccine can be obtained, and the use safety of the prepared inactivated vaccine is improved.
The inactivating operation in the step S4 of the preparation method comprises the following steps: mixing beta-propiolactone liquid with BHV-4 purified antigen liquid according to the proportion of 1:2000-1:4000 (v/v), inactivating for 20-28h at 2-8 ℃, and hydrolyzing for 2-3h at 35-37 ℃ to obtain an inactivated antigen solution; the solution of the inactivated antigen is the aqueous phase.
In the preparation method of the invention, the vaccine adjuvant comprises 206 adjuvant;
and/or the mass ratio of the water phase to the vaccine adjuvant is 1:1, and the virus content before the inactivation of the inactivated antigen in the water phase is 10 7.00 TCID 50 /ml -10 9.50 TCID 50 /ml；
And/or, the operation of mixing and emulsifying comprises: mixing the water phase with vaccine adjuvant, and emulsifying at 30-33deg.C for 30-40 min.
In a fourth aspect, the invention provides an inactivated bovine herpes virus type 4 vaccine comprising an inactivated bovine herpes virus type 4 strain BHV-4/CH/JY/2022 or prepared by the preparation method; the bovine herpes virus type 4 strain BHV-4/CH/JY/2022 is described above.
The invention has the advantages that:
the bovine herpes virus type 4 BHV-4/CH/JY/2022 strain provided by the invention has strong toxicity and good immunogenicity, so that the strain is suitable for producing inactivated vaccines with wide protection range and good immune effect.
The preparation method of the BHV-4 inactivated vaccine provided by the invention can obtain the bovine herpesvirus 4 antigen with high titer, and the inactivated vaccine has high use safety, the preparation method has high degree of automation, can maximally reduce the influence of human factors, has small batch-to-batch difference, is easy to amplify, is convenient for realizing industrial production, can reduce the risk of mixing exogenous factors, and is more favorable for matching with the existing epidemic prevention policy.
The safety test and efficacy test of the finished vaccine show that the BHV-4 inactivated vaccine prepared by the invention can not cause obvious local reaction or systemic adverse reaction due to injection, and can realize 100% protection in a toxicity attack test, so the BHV-4 inactivated vaccine also has the characteristics of high safety and high immune efficacy, and can be used for preventing and controlling various respiratory and reproductive diseases caused by bovine herpesvirus type 4.
Drawings
FIG. 1 is an electrophoresis chart of a conventional PCR assay for BHV-4 in example 1 of the present invention.
FIG. 2 is a photograph showing a lesion of BHV-4 cultured in suspension in example 2 of the present invention.
FIG. 3 shows TCID at different cell densities at the time of virus inoculation in example 3 of the present invention 50 And a culture time relation curve.
FIG. 4 is a TCID under different pH culture conditions in example 3 of the present invention 50 And a culture time relation curve.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the examples below, unless otherwise indicated, are all those available commercially or may be prepared by methods conventional in the art.
The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
EXAMPLE 1 isolation and identification of BHV-4 Virus strains
In this example, a strain of BHV-4 virus (designated BHV-4/CH/JY/2022) was isolated from a disease stock of cattle from Anhui, china, and the specific isolation method included the following steps:
1.1 isolation of BHV-4 Virus strains
(1) Isolation of BHV-4 Virus strains: collecting testis of sick cattle, freeze thawing for three times, sufficiently grinding at 4deg.C, freeze thawing for three times, centrifuging at 5000rpm for 10min, filtering supernatant with 0.45 μm and 0.22 μm filter membrane, and collecting the filtered liquid. Selecting 70% confluent MDBK adherent cells (T25), discarding the stock culture, adding 10ml DMEM containing 1% FBS into each bottle, adding 1ml of the above filtered liquid, standing at 37deg.C and 5% CO 2 Culturing, freezing and thawing for 3 times when cytopathy reaches more than 80%, and harvesting virus liquid, which is marked as F1 generation.
(2) Plaque clone purification: the method adopts plaque clone purification to obtain BHV-4 virus strain, and specifically comprises the following steps:
(2.1) plaque clone: diluting virus solution containing F1 virus with DMEM 10 times gradient to 10 -1 ～10 -7 After each dilution gradient was inoculated into 6-well plates (1 ml/well, preferably just confluent, 3 wells per dilution) of well-grown MDBK cells, and after adsorption at 37℃for 1h, the virus solution was discarded and a first layer of agar of about 3mm thickness containing 1% low melting agarose and DMEM with 2% FBS at a final concentration was applied at 37℃with 5% CO 2 Culturing under inverted condition, observing cytopathy every day, spreading a layer of agar when cytopathy appears, culturing at 37deg.C with 5% CO 2 The inversion culture is continued under the condition.
(2.2) picking spots: after plaque formation, the plaque was aspirated with a 10 μl gun head, placed in 1.0ml serum-free DMEM together with agarose, repeatedly freeze-thawed 3 times to allow sufficient release of virus, and inoculated in new MDBK adherent cells (T25)Placing at 37deg.C, 5% CO 2 Culturing, freezing and thawing for 3 times when cytopathy reaches more than 80%, and harvesting virus liquid, which is marked as F2 generation.
(2.3) cloning and purification: the cloning passages were repeated according to the methods described in "2.1", "2.2" and in the order described.
1.2 identification of BHV-4 Virus strains
And (2) carrying out PCR detection on the strain obtained by the separation in the step (1.1), wherein the method comprises the following steps:
1.2.1 Bovine herpesvirus 4-type PCR detection method
Sample processing and detection by conventional PCR methods were performed according to the documents Epidemiology and genetic characterization of BVDV, BHV-1, BHV-4, BHV-5 and Brucella spp. Infections in cattle in Turkey.
1.2.1.1 Primer: the primer sequences were as follows, using a concentration of 10. Mu. Mol/L:
F：5’- CCC TTC TTT ACC ACC ACC TAC A-3’（SEQ ID No.1）；
R：5’- TGC CAT AGC AGA GAA ACA ATG A-3’（SEQ ID No.2）。
1.2.1.2 Method of
1.2.1.2.1 Viral DNA extraction: the procedure was performed using Promega DNA extraction kit or other commercial kits according to the kit instructions.
1.2.1.2.2 And (3) PCR amplification: reaction system (25 μl): 1. Mu.l each of the upstream primer and the downstream primer, 12.5. Mu.l of 2 XPromix Ex Taq buffer, 2. Mu.l of template DNA, and 8.5. Mu.l of sterile deionized water.
Reaction conditions: the reaction was terminated by a pre-denaturation at 95℃for 2 minutes, followed by a denaturation at 95℃for 30 seconds, an annealing at 56℃for 45 seconds and an extension at 72℃for 30 seconds for 35 cycles.
1.2.1.2.3 Agarose gel electrophoresis: after 1.5% agarose gel (1.5 g agarose was weighed and 100ml of 1 XTAE buffer was added), 10. Mu.l of nucleic acid dye was added, and the mixture was poured into a gel dish (gel thickness: about 5 mm). Selecting proper comb according to the number of samples, extracting the comb (forming sample adding hole in the gel) after the gel is cooled and solidified, placing the gel into an electrophoresis tank, and adding 1 xTAE buffer solution (submerged gel surface) into the electrophoresis tank. Mu.l of the PCR amplification product was mixed with 1. Mu.l of a Loading Buffer (6 Xloading Buffer) and spotted. Constant pressure of 125V/cm, electrophoresis for 20-30 min, and analysis by gel imaging system.
1.2.1.3 And (3) result judgment: the negative sample control (MDBK cell freeze-thawing liquid without toxicity) has no specific amplification band, the positive control has a specific amplification band with the size of 615bp, and the test is established. If the detected sample has no specific amplified band, the detection is negative, if the detected sample has a specific amplified band, the size of the detected sample is 615bp identical to that of a positive control sample, the detected sample is positive.
The PCR detection results are shown in FIG. 1, and the information of each lane is shown in Table 1. The BHV-4 strain was isolated in this example.
TABLE 1
1.3 Determination of viral content
1.3.1 Virus passage proliferation method: taking 70% -80% of MDBK adherent cells (T25) converged, discarding the original culture solution, adding 10ml of DMEM containing 1% FBS into each bottle, adding the BHV-4 virus solution obtained by the identification in the step 1.2 according to the proportion of 1% (v/v), and placing at 37 ℃ and 5% CO 2 Culturing, freezing and thawing for 3 times when cytopathy reaches more than 80%, and harvesting virus liquid once for one generation.
1.3.2 Virus content determination method and results: serial 10 times dilution of BHV-4 virus solution with DMEM solution, 10 times dilution -5 ～10 -10 Respectively inoculating 4 holes of 96-well plate single-layer MDBK adherent cells at each dilution, adsorbing at 37deg.C for 1 hr, adding 100 μl of DMEM culture solution containing 2% new born calf serum into each hole, simultaneously adding 100 μl of DMEM into each hole, adding 100 μl of DMEM culture solution containing 2% new born calf serum into each hole, adding 5% CO, and adding 37 deg.C 2 Culturing in incubator for 4-5 days, observing cytopathy under microscope, recording the number of cytopathy holes of each dilution, and calculating TCID 50 。
In the embodiment, a strain with the highest virus content and stable proliferation cycle is selected, and MDBK cells are used for continuous passage to obtain the purified bovine herpesvirus 4, which is named as BHV-4/CH/JY/2022 strain. BHV-4/CH/JY/2022 strain was deposited at China general microbiological culture Collection center (CGMCC, address: north Chen Xie Lu No. 1, 3, china academy of sciences of China, post code 100101) at month 08 and 16 of 2023, and was classified and named bovine herpes virus type 4 Bovine herpesvirus, with the deposit number: CGMCC No.26500.
The specific results of the determination of the BHV-4/CH/JY/2022 strain virus content are shown in Table 2. As can be seen from Table 2 below, the virus content of each generation was not less than 10 8.5 TCID 50 /ml。
TABLE 2 summary of viral content of different generations of BHV-4 viral fluids
EXAMPLE 2 preparation of inactivated BHV-4 vaccine
The BHV-4/CH/JY/2022 strain separated in the above example 1 is used for preparing bovine herpes virus type 4 inactivated vaccine, which specifically comprises the following steps:
2.1 cell culture
2.1.1 MDBK suspension cell preparation: collecting 1-2 MDBK working seed cells (Jin Yubao, bio-medicine Co., ltd.) for resuscitation, gradually expanding culture (MDBK Medium A, available from Jian Shun Biotechnology Co., ltd.), culturing in suspension, sampling and counting after 36-72 h cell suspension culture, and collecting the culture Medium when the density reaches 6×10 6 More than one/ml, and when the activity rate reaches 90%, the mixture is transferred into a 5L reactor.
2.1.2 MDBK suspension cells 5L reactor culture: the satisfactory MDBK suspension cells cultured in step 2.1.1 (density up to 6X 10) 6 More than one per ml) is transferred to a sterile transfer bottle and transferred to a reactor, and the same cell culture solution is added to the specified volume of the reactor at the same time, the cell density is as follows: 0.5-1.0X10 6 The temperature is 37 ℃, the pH value is 7.2, the dissolved oxygen value is 40-60%, the rotating speed is 80-120 rpm, and each timeCell growth was examined daily as shown in Table 3 below and was practically extended to 50L reactor culture or to bovine herpes virus type 4 BHV-4/CH/JY/2022.
TABLE 3 growth of MDBK suspension cells cultured in 5L reactor
2.1.3 MDBK suspension cells 50L reactor culture: the satisfactory MDBK suspension cells cultured in step 2.1.2 (density up to 6X 10 6 More than one per ml) was transferred to a 50L reactor while the same cell culture broth was fed to the reactor at the specified volume at a cell density of: 0.5-1.0X10 6 Each ml of the culture medium was subjected to temperature 37 ℃, pH 7.2, dissolved oxygen value 40% -60%, rotational speed 40-60rpm, cell growth conditions were examined daily, as shown in Table 4 below, and the culture medium was expanded to 500L reactor culture or used for culturing bovine herpes virus type 4 BHV-4/CH/JY/2022 according to practical conditions.
TABLE 4 50L reactor culture MDBK suspension cell growth
2.1.4 MDBK suspension cells 500L reactor culture: the satisfactory MDBK suspension cells cultured in step 2.1.3 (density up to 6X 10 6 More than one per ml) was transferred to a 500L reactor while the same cell culture broth was fed to the reactor at the same time at the specified volume, cell density: 0.5-1.0X10 6 Each ml of the cells were tested daily at 37℃pH 7.2, dissolved oxygen 40% -60% and rotational speed 40-60rpm, as shown in Table 5 below, and cultured in either expanded form or as actual conditions for bovine herpes virus type 4 BHV-4/CH/JY/2022.
TABLE 5 growth of MDBK suspension cells cultured in 500L reactor
2.2 cultivation of bovine herpes virus 4 type BHV-4/CH/JY/2022
2.2.1 Preparation of BHV-4/CH/JY/2022 base seed virus: taking MDBK adherent cells growing to a good monolayer, discarding the original culture solution, adding a proper amount of maintenance solution (the maintenance solution is DMEM culture medium added with 0.5% -1% (v/v) of newborn bovine serum), adding the virus solution of BHV-4/CH/JY/2022 according to the proportion of 0.1% -1% (v/v), culturing at 37 ℃, and when cytopathy is more than or equal to 80%, harvesting the virus solution and freezing and thawing for 3 times to obtain basic seed viruses.
2.2.2 Preparation of suspension seed virus for production: taking the MDBK suspension cells meeting the requirements prepared in step 2.1, centrifuging at 1000 rpm for 5 min, discarding supernatant, supplementing virus maintenance solution (available from Nemongolian Jin Yuankang biological engineering Co., ltd., product number: CM-VA 001), and diluting the cells to 1.5-2.5X10 6 Adding the basic seed toxin prepared in the step 2.2.1 into the seed toxin per ml according to the toxin receiving proportion of 0.1% -1% (v/v), culturing at 37 ℃ and 100-110 rpm, harvesting the virus liquid and freezing and thawing for 3 times when the cell viability reaches 30% -40%, sampling and detecting TCID 50 The virus content of each 1ml should be more than or equal to 10 8.0 TCID 50 . The suspension seed toxin for production is preserved at minus 20 ℃ or below. According to different production requirements, the BHV-4/CH/JY/2022 suspension seed virus is cultivated by using reactors with different specifications and volumes. FIG. 2 shows a photograph of a lesion of BHV-4/CH/JY/2022 in suspension culture.
2.2.3 Preparation of antigen: taking MDBK suspension cells meeting the requirements prepared in step 2.1, discarding stock culture solution, supplementing virus maintenance solution (purchased from inner Mongolian bioengineering Co., ltd.) and diluting the cells to 1.5-2.5X10 6 Adding the suspension seed toxin for production prepared in the step 2.2.2 according to the toxin receiving proportion of 0.1% -1% (v/v) per ml, and adjusting parameters: when the temperature is 37 ℃, the rotating speed is 40-60rpm, the pH is 7.35, the DO value is 40-60%, and the cell viability reaches 30-40%, the virus liquid is harvested and frozen and thawed 3 times. Sampling detection TCID 50 The virus content should be more than or equal to 10 8.00 TCID 50 /ml. Namely, the antigen solution for preparing the seedlings is preserved at minus 20 ℃ or below.
2.3 concentration and purification of antigen
2.3.1 Antigen concentration: taking the antigen solution for preparing the vaccine prepared in the step 2.2, thawing, centrifuging in a continuous flow centrifuge to obtain clear supernatant, and concentrating the antigen solution by 5 times by using a hollow fiber column with 500KD (in the concentration process, PBS is required to be used for washing and filtering to replace virus maintenance solution), thereby obtaining antigen concentrate. Table 6 below shows that three times (NS 22001-NS 22003) of antigen solution for vaccine preparation is concentrated by a 500KD hollow fiber column, and the antigen solution for vaccine preparation is concentrated by a 500KD hollow fiber column, so that the virus antigen is not basically lost, and the concentration effect is good.
TABLE 6 Concentration of vaccine antigen solution by 500KD hollow fiber column
2.3.2 Antigen purification: taking the antigen concentrate prepared in the step 2.3.1, adding 1% (v/v) of PEG6000, uniformly mixing for 3 hours at 2-8 ℃, standing overnight, centrifuging at 8000rpm for 15 min, discarding the supernatant, collecting the precipitate, and re-suspending with PBS (pH 7.4, concentration of 0.01 mol/L) to obtain purified antigen liquid for preparing seedlings. The conditions of antigen loss and foreign protein removal after PEG purification of the antigen concentrate are shown in the following table 7, and the foreign protein removal rate can reach more than 84% after PEG purification of the antigen concentrate in three parallel experiments, so that the foreign protein in the antigen concentrate can be effectively removed, and the purified antigen protein has low content, thereby being beneficial to the safety of vaccine use.
TABLE 7 antigen loss and removal of proteins after PEG purification of antigen concentrate
2.4 antigen inactivation
2.4.1 Antigen inactivation: and (3) carrying out an inactivation procedure on the purified antigen liquid for preparing the seedlings, which is prepared in the step (2.3.2). Inactivating beta-propiolactone at a ratio of 1:3000 (v/v) at 4deg.C for 28 hr, and hydrolyzing at 37deg.C for 2 hr to obtain inactivated antigen solution.
2.4.2 Inactivation test): inoculating 3 MDBK good monolayer cells (T25 cell bottle per time) to 2.4.1 of inactivated antigen solution at 25% (v/v)2.5ml was added to the flask), adsorbed for 1h at 37℃and then the inactivated antigen solution was discarded, and 10 ml/flask of cell maintenance solution (DMEM, brand: gibco, cat No.: 10569010; when in use, 1% FBS is added according to the volume ratio), two controls are set simultaneously, one bottle is normal cell group, the other bottle is maintenance solution containing beta-propiolactone and hydrolyzed for 2 hours at 37 ℃ (the content of beta-propiolactone is consistent with that of inactivated antigen solution), and the two controls are set at 37 ℃ and 5% CO 2 Culturing in an incubator for 5 days, observing whether cytopathic effect appears daily, recording the observation result, harvesting the culture after 5 days of culturing, inoculating MDBK cells for two generations in a blind way after freezing and thawing the harvested culture, observing whether cytopathic effect appears daily, and recording the observation result.
2.5 preparation of vaccine
2.5.1 Aqueous phase preparation: taking the qualified inactivated antigen liquid prepared in the step 2.4.1 as an aqueous phase.
2.5.2 Oil phase preparation: 206 adjuvants (available from Saike (Shanghai) specialty Chemicals Co., ltd., product number: 36022E) were taken, sterilized by filtration and then preheated to 30℃to obtain an oil phase.
2.5.3 Emulsification): mixing the water phase and the oil phase at a mass ratio of 1:1 (adding the water phase into the oil phase), emulsifying at 30-33deg.C for 30min, packaging the emulsified vaccine according to standard mark amount, labeling, and storing at 2-8deg.C for use.
2.6 vaccine testing
2.6.1 Properties (1)
(1) Appearance: milky white or pale pink viscous emulsions.
(2) Dosage form: water-in-oil-in-water (W/O/W). A cleaning straw is taken, a small amount of vaccine is sucked and dripped on the surface of the cleaning cold water, and the vaccine is dispersed in a cloud form.
(3) Stability: 10.0ml of the extracted vaccine is added into a centrifuge tube, and the mixture is centrifuged at 3000r/min for 15 minutes, and the water precipitated at the bottom of the tube is not more than 0.5ml.
(4) Viscosity: the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the test should not exceed 200cP.
2.6.2 Inspection of the loading amount: the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the rule is met.
2.6.3 Sterility test): the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the bacteria should grow aseptically.
2.6.4 Endotoxin content detection: sucking 12ml vaccine, placing into a 15ml centrifuge tube, placing into 50+ -5deg.C water bath for 90 min, centrifuging at 15000g for 10min at 4deg.C, and taking 5.0ml water phase, and checking according to an annex of the current "Chinese animal pharmacopoeia". The endotoxin content of each vaccine should not exceed 20EU.
The test results of the vaccine prepared with the qualified inactivated antigen liquid prepared in step 2.4.1 are shown in table 8.
TABLE 8 vaccine trait testing
2.6.5 Security check)
(1) Test with small animals: 5 guinea pigs weighing 350-400g were used, each subcutaneously injecting 0.5ml of vaccine. After 7 days of daily observation, death or obvious local reaction or systemic adverse reaction caused by vaccine injection should not occur.
(2) The animal is tested: with 5 healthy susceptible cattle of 2-6 months of age, 4.0ml (2 parts) of vaccine is intramuscular injected into each head and neck, and the clinical symptoms caused by BHV-4 or obvious local reaction or systemic adverse reaction caused by vaccine injection are avoided when the eyes are observed for 14 days.
The results of the safety test of the vaccine prepared from the qualified inactivated antigen liquid prepared in step 2.4.1 are shown in the following table 9, and it can be seen that no death or obvious local reaction or systemic adverse reaction caused by vaccine injection occurs no matter the vaccine is tested by small animals or the animal is tested by the animal, which indicates that the BHV-4 inactivated vaccine prepared by the invention is safe.
TABLE 9 vaccine safety test cases
2.6.6 Efficacy test)
The immune toxin-attacking method for cattle: 10 healthy susceptible cattle with the age of 2-6 months are randomly divided into 2 groups, wherein 5 groups of immunization groups and 5 groups of virus attack control groups. The immunized group cattle were injected with 2.0ml (1 part) of vaccine per head and neck muscle, and the same method was used for boosting 1 time 21 days after immunization, and the BHV-4/CH/JY/2022 strain (virus content was diluted to 10) was used 21 days after the double immunization 7.0 TCID 50 Per ml) of the seed are subjected to detoxification, and each seed is inoculated with 5.0ml. After the challenge, the control group should have at least 4 diseases and the immunized group should have at least 4 protections, and the 14 days after the challenge are continuously observed. The efficacy test conditions of the vaccine of the invention are shown in the following table 10, and it can be seen that the BHV-4 inactivated vaccine prepared by the invention can achieve 100% protection.
Table 10 final vaccine efficacy test
EXAMPLE 3 optimization of BHV-4 antigen production conditions
In example 2, the high-density culture of BHV-4 was achieved by optimizing conditions such as cell density, pH, virus maintenance solution, etc. at the time of virus inoculation.
(1) Effect of cell Density on viral proliferation at the time of receiving the toxin
Virus-retaining liquid (available from Nemongolia Jin Yuankang Biotechnology Co., ltd., product number: CM-VA 001) was added according to the cell density so that the cell densities transferred into the virus culture reactor were 1.0X10, respectively 6 Per ml, 1.5X10) 6 Per ml, 2.0X10 6 Individual/ml and 2.5X10 6 And each ml. The production suspension seed virus prepared in step 2.2.2 was inoculated at an inoculation ratio of 0.2% (v/v). The virus culture parameters of the reactor are set as follows: the temperature is 37 ℃, the pH value is 7.2, the dissolved oxygen value is 45 percent, and the rotating speed is 100rpm. Culturing for 5 days, harvesting virus liquid, and determining TCID 50 As a result, as shown in Table 11 and FIG. 3 below, it was found that the optimal virus-receiving cell density was 1.5 to 2.5X10 6 Every one ml, 120 hours after virus inoculation, the virus liquid drop degree is the highest; 144h after virus inoculation, the virus droplet size is equal to or occasionally reduced from 120 h.
TABLE 11 results of Virus content determination at different cell densities at different virus-receiving times
(2) Optimization of pH: the virus maintenance solution (available from Nemongolia Jin Yuankang Bioengineering Co., ltd., cat# CM-VA 001) was added according to the cell density so that the cell density transferred into the virus culture reactor was 1.5-2.5X10, respectively 6 Inoculating the production suspension seed toxin prepared in the step 2.2.2 according to the toxin receiving proportion of 0.2 percent per ml. The virus culture parameters of the reactor are set as follows: the temperature was 37℃and the pH was set at 7.2, 7.4, 7.6, 7.8, dissolved oxygen value 45% and rotational speed 100rpm, respectively. Culturing for 5 days, collecting virus solution, and measuring TCID 50 As shown in Table 12 and FIG. 4 below, it was found that the virus content was relatively low at pH 7.2 and 7.8, and therefore it was suggested that the pH was controlled to 7.4-7.6 and that the virus titer could be 10 after 120 hours of cultivation 7.50 TCID 50 /ml and above.
TABLE 12 results of virus content assay at different pH' s
EXAMPLE 4 neutralizing antibody Condition of BHV-4 inactivated vaccine
The vaccine was prepared by the method described in example 2, 5 healthy susceptible cattle of 2-6 months of age were selected, each cattle was intramuscular injected with 2.0ml (1 part) of vaccine, and the vaccine was boosted 1 time in the same way 21 days after immunization, and serum was collected for cell neutralization test for the first 2 days, the first 7 days, the first 14 days, the first 21 days (before the second), the second 7 days, the second 14 days, and the second 21 days, respectively, to determine the antibody value.
Wherein the cell neutralization assay is: the neutralization with BHV-4 virus was diluted to 200 TCID with DMEM medium containing 2% neonatal calf serum 50 0.1ml; next, the negative serum was diluted 1:2, 1:4, 1:8 with DMEM medium containing 2% of the new born calf serum, and the positive serum was serially diluted 2-fold to 1:256 (dilution was performed according to this standard in this experiment, and the person skilled in the art could appropriately increase the dilution in accordance with the actual situation)Each serum to be tested was serially diluted 2-fold to 1:1024, and each dilution of serum to be tested and positive and negative serum was added to a 96-well cell culture plate, each dilution was inoculated with 4 wells, 50 μl per well. Setting 4 holes of normal cell control and neutralization virus control, and not adding sample; then, 200 TCID was added to each of the serum to be tested, the virus-neutralizing control, the negative serum and the positive serum 50 0.1ml of virus-neutralizing working fluid 50. Mu.l/well. Adding 50 μl/hole of DMEM culture solution containing 2% of new born calf serum into each neutralized virus control hole, adding 100 μl/hole of DMEM culture solution containing 2% of new born calf serum into normal cell control holes, shaking, mixing uniformly, neutralizing at 37deg.C for 1h, and shaking for 2-3 times; finally, 100. Mu.l/well of MDBK cell suspension containing 2% neo-bovine serum at a concentration of about 20 ten thousand cells/ml was added to all wells and the wells were incubated at 37℃with 5% CO 2 Culturing in incubator for 4-6 days, observing cytopathy condition with inverted microscope every day, and calculating antibody value.
The research conclusion shows that the antibody value of the cattle after immunization is gradually increased, the antibody value is obviously increased 14 days after the first immunization, and the antibody value is directly increased to 1:256 after 14 days after the second immunization, which shows that the inactivated vaccine prepared by the strain and the method has obvious effect. The results are shown in Table 13.
TABLE 13 detection results of cell neutralizing antibodies at different immunization times
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A bovine herpes virus 4-type strain BHV-4/CH/JY/2022 is characterized in that the preservation number is CGMCC No.26500.
2. Use of bovine herpes virus type 4 strain BHV-4/CH/JY/2022 as claimed in claim 1 in the preparation of inactivated bovine herpes virus type 4 vaccine.
3. The preparation method of the bovine herpes virus type 4 inactivated vaccine is characterized by comprising the steps of mixing and emulsifying an aqueous phase containing an inactivated antigen and a vaccine adjuvant; the preparation raw materials of the inactivated antigen comprise bovine herpes virus type 4 strain BHV-4/CH/JY/2022 as claimed in claim 1.
4. A method of preparation according to claim 3, wherein the inactivated antigen is obtained by:
s1: culturing the bovine herpes virus strain 4 BHV-4/CH/JY/2022 to obtain BHV-4 virus liquid;
s2: clarifying the BHV-4 virus solution obtained in the step S1, and concentrating to obtain BHV-4 antigen concentrate;
s3: purifying the BHV-4 antigen concentrate obtained in the step S2 to obtain BHV-4 purified antigen solution;
s4: inactivating the BHV-4 purified antigen solution obtained in the step S3 to obtain the inactivated antigen.
5. The preparation method according to claim 4, wherein in step S1, the bovine herpes virus strain 4 BHV-4/CH/JY/2022 is cultured in suspension by using suspension MDBK cells until the cell viability reaches 30% -40%, and the culture is harvested as BHV-4 virus liquid;
and/or, in step S1, suspension culture is carried out on the bovine herpes virus 4-type strain BHV-4/CH/JY/2022 by adopting suspension MDBK cells, and the cell density of the suspension MDBK cells is 1.5-2.5X10 when the bovine herpes virus 4-type strain BHV-4/CH/JY/2022 is inoculated in the suspension culture 6 Individual/ml;
and/or inoculating the suspension virus liquid of bovine herpesvirus type 4 strain BHV-4/CH/JY/2022 into the suspension MDBK cells for suspension culture, wherein the virus content of the suspension virus liquid is more than or equal to 10 8.0 TCID 50 The inoculation proportion is 0.1% -1% (v/v)；
And/or, when the suspension culture is performed, the culture pH is controlled to 7.4-7.6.
6. The preparation method of claim 4, wherein in step S2, the BHV-4 virus solution obtained in step S1 is clarified by continuous flow centrifugation or positive pressure filtration of a 0.45 μm filter element;
and/or concentrating in step S2 by using a hollow fiber column with 500 KD;
and/or, the concentration in step S2 is 5-7 times.
7. The method according to claim 4, wherein the purification is performed in step S3 using PEG selected from PEG6000 or PEG8000;
and/or, the purifying operation in step S3 includes: mixing the BHV-4 antigen concentrate obtained in the step S2 with PEG at 2-8 ℃, standing for 12-16 h, centrifuging at 6000-10000 rpm for more than 15 min, discarding supernatant, collecting precipitate, and re-suspending with PBS to obtain the BHV-4 purified antigen solution;
and/or the antigen content of the BHV-4 purified antigen solution is 10 8.00 TCID 50 And/ml or more.
8. The method according to claim 4, wherein the inactivating operation in step S4 comprises: mixing beta-propiolactone liquid with BHV-4 purified antigen liquid according to the proportion of 1:2000-1:4000 (v/v), inactivating for 20-28h at 2-8 ℃, and hydrolyzing for 2-3h at 35-37 ℃ to obtain an inactivated antigen solution; the solution of the inactivated antigen is the aqueous phase.
9. The method of preparation of any one of claims 3-8, wherein the vaccine adjuvant comprises 206 adjuvant;
and/or the mass ratio of the water phase to the vaccine adjuvant is 1:1, and the virus content before the inactivation of the inactivated antigen in the water phase is 10 7.00 TCID 50 /ml -10 9.50 TCID 50 /ml；
And/or, the operation of mixing and emulsifying comprises: mixing the water phase with vaccine adjuvant, and emulsifying at 30-33deg.C for 30-40 min.
10. An inactivated bovine herpes virus type 4 vaccine comprising an inactivated bovine herpes virus type 4 strain BHV-4/CH/JY/2022 or prepared by the method of any one of claims 3-9; the bovine herpes virus type 4 strain BHV-4/CH/JY/2022 is described in claim 1.
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| WO1994028177A1 (en) * | 1993-05-28 | 1994-12-08 | Cornell Research Foundation, Inc. | Bovine herpesvirus 1 detection |
| CN102337250A (en) * | 2011-05-24 | 2012-02-01 | 深圳市青珊瑚科技有限公司 | Infectious bovine rhinotracheitis virus strain and preparation method and application thereof |
| CA3174826A1 (en) * | 2021-01-19 | 2022-07-28 | Katsuro HAGIWARA | Virus stabilizer, gelatin hydrolysate for virus stabilizer, and virus-containing composition |
| CN116042536A (en) * | 2022-08-02 | 2023-05-02 | 大能生物科技(南京)有限公司 | Bovine herpesvirus 4 strain and culture method and application thereof |
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|---|---|---|---|---|
| WO1994028177A1 (en) * | 1993-05-28 | 1994-12-08 | Cornell Research Foundation, Inc. | Bovine herpesvirus 1 detection |
| CN102337250A (en) * | 2011-05-24 | 2012-02-01 | 深圳市青珊瑚科技有限公司 | Infectious bovine rhinotracheitis virus strain and preparation method and application thereof |
| CA3174826A1 (en) * | 2021-01-19 | 2022-07-28 | Katsuro HAGIWARA | Virus stabilizer, gelatin hydrolysate for virus stabilizer, and virus-containing composition |
| CN116042536A (en) * | 2022-08-02 | 2023-05-02 | 大能生物科技(南京)有限公司 | Bovine herpesvirus 4 strain and culture method and application thereof |
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