Porcine pseudorabies and porcine mycoplasma pneumonia combined inactivated vaccine and preparation method thereof
阅读说明：本技术 猪伪狂犬病、猪支原体肺炎二联灭活疫苗及其制备方法 (Porcine pseudorabies and porcine mycoplasma pneumonia combined inactivated vaccine and preparation method thereof ) 是由 贺笋 候凤 潘毅平 师小潇 李新苹 卞赛赛 姜峰 张锐 周婷婷 张艳盟 张飞 于 2021-06-04 设计创作，主要内容包括：本发明提供了一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗及其制备方法,涉及生物技术领域。本发明提供的猪伪狂犬病、猪支原体肺炎二联灭活疫苗,包括猪伪狂犬gB抗原、猪伪狂犬gD抗原和猪肺炎支原体CJ株抗原,抗原之间不存在干扰,免疫效力高,能够达到预防和治疗猪伪狂犬病和猪支原体肺炎的混合感染或继发感染的效果,显著降低了成本,节省了人力。(The invention provides a porcine pseudorabies and swine mycoplasma pneumonia combined inactivated vaccine and a preparation method thereof, and relates to the technical field of biology. The porcine pseudorabies and porcine mycoplasma pneumonia combined inactivated vaccine provided by the invention comprises a porcine pseudorabies gB antigen, a porcine pseudorabies gD antigen and a porcine mycoplasma pneumonia CJ strain antigen, interference does not exist among the antigens, the immune efficiency is high, the effect of preventing and treating mixed infection or secondary infection of the porcine pseudorabies and the porcine mycoplasma pneumonia can be achieved, the cost is obviously reduced, and the manpower is saved.)
1. The porcine pseudorabies and porcine mycoplasma pneumonia combined inactivated vaccine is characterized by comprising a porcine pseudorabies antigen and a porcine mycoplasma pneumonia antigen;
the porcine pseudorabies antigens comprise porcine pseudorabies gB antigens and porcine pseudorabies gD antigens;
the mycoplasma hyopneumoniae antigen comprises a mycoplasma hyopneumoniae CJ strain antigen.
2. The bivalent inactivated vaccine for porcine pseudorabies and porcine mycoplasmal pneumonia according to claim 1, wherein the content of the porcine pseudorabies gB antigen is 100-150 μ g per head, preferably 100 μ g per head.
3. The bivalent inactivated vaccine for porcine pseudorabies and porcine mycoplasmal pneumonia according to claim 1, wherein the content of the porcine pseudorabies gD antigen is 100-150 μ g per head, preferably 100 μ g per head.
4. The bivalent inactivated vaccine for porcine pseudorabies and porcine mycoplasmal pneumonia according to claim 1, wherein the antigen content of the porcine mycoplasma pneumoniae CJ strain is 109.0～1010.0CCU/first portion, preferably 7X 109.0CCU/first portion.
5. The bivalent inactivated vaccine against porcine pseudorabies and porcine mycoplasmal pneumonia according to claim 1, characterized in that the vaccine further comprises an adjuvant.
6. The porcine pseudorabies and porcine mycoplasmal pneumonia combined inactivated vaccine according to claim 5, characterized in that the adjuvant comprises an oil-in-water type adjuvant and aluminum hydroxide gel.
7. The porcine pseudorabies and porcine mycoplasmal pneumonia combined inactivated vaccine according to claim 6, characterized in that the oil-in-water adjuvant accounts for 12-18% of the total volume of the vaccine, preferably 15%.
8. The bivalent inactivated vaccine for porcine pseudorabies and porcine mycoplasmal pneumonia according to claim 6, wherein the aluminum hydroxide gel accounts for 12-18% of the total volume of the vaccine, preferably 15%.
9. The inactivated bivalent vaccine for porcine pseudorabies and porcine mycoplasmal pneumonia according to claim 1, wherein the immune dose of the vaccine is 1.5-2.5 mL/head, preferably 2 mL/head.
10. The method for preparing the porcine pseudorabies and porcine mycoplasmal pneumonia bivalent inactivated vaccine as claimed in any one of claims 1 to 9, characterized in that the porcine pseudorabies gB antigen, the porcine pseudorabies gD antigen and the porcine mycoplasma pneumonia CJ strain antigen are prepared into an antigen solution, and then mixed with an adjuvant to prepare the vaccine;
the volume ratio of the antigen solution to the adjuvant is 3: 1-3: 2, and preferably 7: 3.
Technical Field
The invention relates to the technical field of biology, in particular to a porcine pseudorabies and swine mycoplasma pneumonia combined inactivated vaccine and a preparation method thereof.
Background
Pseudorabies (PR) is an important infectious disease caused by Pseudorabies virus (PRV) and mainly characterized by fever, extreme itching (except for pigs), nervous system symptoms and encephalomyelitis in various domestic animals (pigs, cattle, sheep, etc.) and wild animals. Mycoplasma hyopneumoniae (Swine enzootic hyopneumoniae), commonly known as mycoplasmosis, is a chronic, contact, respiratory infectious disease caused by Mycoplasma hyopneumoniae. Because of the interference or influence of multiple antigens in the combined vaccine (vaccine composition), such as the combined vaccine or the triple vaccine, the products of the combined vaccine of the porcine pseudorabies and the porcine mycoplasmal pneumonia are not seen in the market at present. Therefore, for preventing the co-infection or the secondary infection of the porcine circovirus and the mycoplasma hyopneumoniae, the porcine pseudorabies vaccine and the mycoplasma hyopneumoniae vaccine can only be used for respectively carrying out secondary immunization for preventing and controlling, so that the immunization program is complex, the immunization times are increased, the immunization cost is increased, the immunization stress times of the pigs are increased, and the growth performance of the pigs is influenced; or, the 2 vaccines are mixed and injected for prevention and control, so that the lack of immune efficiency is caused, the sterility cannot be ensured in the vaccine mixing process, and the operation process is complicated.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first objective of the present invention is to provide a porcine pseudorabies and porcine mycoplasmal pneumonia combined inactivated vaccine to solve at least one of the above problems.
The second purpose of the invention is to provide a preparation method of the porcine pseudorabies and mycoplasma hyopneumoniae bivalent inactivated vaccine, which is simple and rapid.
In a first aspect, the application provides a porcine pseudorabies and porcine mycoplasmal pneumonia combined inactivated vaccine, wherein the vaccine comprises a porcine pseudorabies antigen and a porcine mycoplasmal pneumonia antigen;
the porcine pseudorabies antigens comprise porcine pseudorabies gB antigens and porcine pseudorabies gD antigens;
the mycoplasma hyopneumoniae antigen comprises a mycoplasma hyopneumoniae CJ strain antigen.
As a further technical scheme, the content of the porcine pseudorabies gB antigen is 100-150 mu g/head part, and preferably 100 mu g/head part.
As a further technical scheme, the content of the porcine pseudorabies gD antigen is 100-150 mu g/head part, and preferably 100 mu g/head part.
As a further technical scheme, the content of the antigen of the Mycoplasma hyopneumoniae CJ strain is 109.0～1010.0CCU/first portion, preferably 7X 109.0CCU/first portion.
As a further technical scheme, the vaccine also comprises an adjuvant.
As a further technical scheme, the adjuvant comprises an oil-in-water adjuvant and aluminum hydroxide gel;
as a further technical scheme, the oil-in-water adjuvant accounts for 12% -18% of the total volume of the vaccine, and is preferably 15%;
as a further technical scheme, the aluminum hydroxide gel accounts for 12% -18% of the total volume of the vaccine, and preferably accounts for 15%.
As a further technical scheme, the immunization dose of the vaccine is 1.5-2.5 mL/head, and preferably 2 mL/head.
In a second aspect, the application provides a preparation method of a porcine pseudorabies and porcine mycoplasma pneumonia combined inactivated vaccine, which comprises the steps of preparing an antigen solution from a porcine pseudorabies gB antigen, a porcine pseudorabies gD antigen and a porcine mycoplasma pneumonia CJ strain antigen, and mixing the antigen solution with an adjuvant to prepare the vaccine;
the volume ratio of the antigen solution to the adjuvant is 3: 1-3: 2, and preferably 7: 3.
Compared with the prior art, the invention has the following beneficial effects:
the porcine pseudorabies and porcine mycoplasma pneumonia combined inactivated vaccine provided by the invention comprises a porcine pseudorabies gB antigen, a porcine pseudorabies gD antigen and a porcine mycoplasma pneumonia CJ strain antigen, interference does not exist among the antigens, the immune efficiency is high, the effect of preventing and treating mixed infection or secondary infection of the porcine pseudorabies and the porcine mycoplasma pneumonia can be achieved, the cost is obviously reduced, and the manpower is saved.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the application provides a porcine pseudorabies and porcine mycoplasmal pneumonia combined inactivated vaccine, wherein the vaccine comprises a porcine pseudorabies antigen and a porcine mycoplasmal pneumonia antigen;
the porcine pseudorabies antigens comprise porcine pseudorabies gB antigens and porcine pseudorabies gD antigens;
the mycoplasma hyopneumoniae antigen comprises a mycoplasma hyopneumoniae CJ strain antigen.
The amino acid sequence of the porcine pseudorabies gB antigen (SEQ ID NO.1) is as follows:
MPAGGGLWRGPRGHRPGHHGGAGLGRLWPAPHHAAAARGAVALALLLLALAATPTCGAAAVTRAASASPAPGTGATPDGFSAEESLEEIDGAVSPGPSDAPDGEYGDLDARTAVRAAATERDRFYVCPPPSGSTVVRLEPEQACPEYSQGRNFTEGIAVLFKENIAPHKFKAHIYYKNVIVTTVWSGSTYAAITNRFTDRVPVPVQEITDVIDRRGKCVSKAEYVRNNHKVTAFDRDENPVEVDLRPSRLNALGTRGWHTTNDTYTKIGAAGFYHTGTSVNCIVEEVEARSVYPYDSFALSTGDIVYMSPFYGLREGAHGEHIGYAPGRFQQVEHYYPIDLDSRLRASESVTRNFLRTPHFTVAWDWAPKTRRVCSLAKWREAEEMIRDETRDGSFRFTSRALGASFVSDVTQLDLQRVHLGDCVLREASEAIDAIYRRRYNNTHVLAGDKPEVYLARGGFVVAFRPLISNELAQLYARELERLGLAGVVGPASPAAARRARRSPGPAGTPEPPAVNGTGHLRITTGSAEFARLQFTYDHIQAHVNDMLSRIAAAWCELQNKDRTLWGEMSRLNPSAVATAALGQRVSARMLGDVMAISRCVEVRGGVYVQNSMRVPGERGTCYSRPLVTFEHNGTGVIEGQLGDDNELLISRDLIEPCTGNHRRYFKLGGGYVYYEDYSYVRMVEVPETISTRVTLNLTLLEDREFLPLEVYTREELADTGLLDYSEIQRRNQLHALKFYDIDRVVKVDHNVVLLRGIANFFQGLGDVGAAVGKVVLGATGAVISAVGGMVSFLSNPFGALAIGLLVLAGLVAAFLAYRHISRLRRNPMKALYPVTTKALKEDGVEEDDVDEAKLDQARDMIRYMSIVSALEQQEHKARKKNSGPALLASRVGAMATRRRHYQRLENEDPDAP(SEQ ID NO.1)。
the amino acid sequence of the porcine pseudorabies gD antigen (SEQ ID NO.2) is as follows:
MLLAALLAALVARTTLGADVDAVPAPTFPPPAYPYTESWQLTLTTVPSPFVGPADVYHTRPLEDPCGVVALISDPQVDRLLNEAVAHRRPTYRAHVAWYRIADGCAHLLYFIEYADCDPRQIFGRCRRRTTPMWWTPSADYMFPTEDELGLLMVAPGRFNEGQYRRLVSVDGVNILTDFMVALPEGQECPFARVDQHRTYKFGACWSDDSFKRGVDVMRFLTPFYQQPPHREVVNYWYRKNGRTLPRAYAAATPYAIDPARPSAGSPRPRPRPRPRPRPKPEPAPATPAPPGRLPEPATRDHAAGGRPTPRPPRPETPHRPFAPPAVVPSGWPQPAEPFPPRTTAAPGVSRHRSVIVGTGTAMGALLVGVCVYIFFRLRGAKGYRLLGGPADADELKAQPGP(SEQ ID NO.2)。
the mycoplasma hyopneumoniae CJ strain is selected from Xinjiang Uygur autonomous region Changji city by a separating screen, and the classification and the designation of the strain are as follows: mycoplasma hyopneumoniae, latin literature name: mycoplasma hyopneumoniae, deposited in China general microbiological culture Collection center, with a deposition address: china general microbiological culture Collection center, preservation date: 11/h 06/2014, deposit number: CGMCC NO: 9099.
the porcine pseudorabies and porcine mycoplasma pneumonia combined inactivated vaccine provided by the invention comprises a porcine pseudorabies gB antigen, a porcine pseudorabies gD antigen and a porcine mycoplasma pneumonia CJ strain antigen, interference does not exist among the antigens, the immune efficiency is high, the effect of preventing and treating mixed infection or secondary infection of the porcine pseudorabies and the porcine mycoplasma pneumonia can be achieved, the cost is obviously reduced, and the manpower is saved.
In some preferred embodiments, the porcine pseudorabies gB antigen is present in an amount of 100 to 150. mu.g per head, for example, but not limited to 100. mu.g, 110. mu.g, 120. mu.g, 130. mu.g, 140. mu.g or 150. mu.g, preferably 100. mu.g per head.
In some preferred embodiments, the porcine pseudorabies gD antigen is present in an amount of 100 to 150. mu.g per head, for example, but not limited to 100. mu.g, 110. mu.g, 120. mu.g, 130. mu.g, 140. mu.g or 150. mu.g, preferably 100. mu.g per head.
In some preferred embodiments, the mycoplasma hyopneumoniae CJ strain has an antigen content of 109.0～1010.0CCU/head, for example, but not limited to, 1X 109.0CCU/first 2X 109.0CCU/first, 4X 109.0CCU/first, 6X 109.0CCU/first portion, 8X 109.0CCU/first or 1X 1010.0CCU/first portion, preferably 7X 109.0CCU/first portion.
In the invention, the content of each antigen in the vaccine is appropriate by further optimizing and adjusting the content of the porcine pseudorabies gB antigen, the porcine pseudorabies gD antigen and the porcine mycoplasma pneumoniae CJ strain antigen in the vaccine, so that the immunity to the antigen is better realized.
In some preferred embodiments, the vaccine further comprises an adjuvant. The addition of the adjuvant helps to improve the stability of the vaccine and improve the immune efficacy of pathogen infection.
In some preferred embodiments, the adjuvant comprises an oil-in-water adjuvant and an aluminum hydroxide gel. Wherein the oil-in-water adjuvant includes, but is not limited to SEPPIC ISA 11R, SEPPIC ISA 15A, SEPPIC ISA 28 or SEPPIC ISA 28R. In the invention, the vaccine prepared by matching the oil-in-water adjuvant and the aluminum hydroxide gel has good penetration, and has the characteristics of quick immune response, lasting immune effect and less stimulation to animal organisms. After the vaccine is injected, the aluminum hydroxide gel adjuvant can quickly start immunity, and the oil-in-water adjuvant prolongs the immunity time. Firstly, the antigen released by the aluminum hydroxide gel of the water phase part of the adjuvant stimulates to generate strong humoral immune response; then, the antigen on the surface of the large oil drop stimulates the generation of cellular immune response; finally, the large oil drop releases the antigen of the small oil drop to stimulate the generation of lifelong compound immunity.
In some preferred embodiments, the oil-in-water adjuvant comprises 12% to 18% of the total volume of the vaccine, for example, but not limited to, 12%, 13%, 14%, 15%, 16%, 17% or 18%, preferably 15%;
in some preferred embodiments, the aluminum hydroxide gel comprises 12% to 18% of the total volume of the vaccine, for example, but not limited to, 12%, 13%, 14%, 15%, 16%, 17%, or 18%, preferably 15%.
The immune efficacy of the vaccine is improved by further optimizing and adjusting the dosage of the oil-in-water adjuvant and the aluminum hydroxide gel.
In some preferred embodiments, the vaccine is administered at an immunizing dose of 1.5-2.5 mL/head, for example, but not limited to, 1.5 mL/head, 1.7 mL/head, 1.9 mL/head, 2.1 mL/head, 2.3 mL/head, or 2.5 mL/head, preferably 2 mL/head.
In a second aspect, the application provides a preparation method of a porcine pseudorabies and porcine mycoplasma pneumonia combined inactivated vaccine, which comprises the steps of preparing an antigen solution from a porcine pseudorabies gB antigen, a porcine pseudorabies gD antigen and a porcine mycoplasma pneumonia CJ strain antigen, and mixing the antigen solution with an adjuvant to prepare the vaccine;
the volume ratio of the antigen solution to the adjuvant is 3: 1-3: 2, and preferably 7: 3.
The preparation method of the porcine pseudorabies and swine mycoplasma pneumonia combined inactivated vaccine is simple, quick and convenient.
The porcine pseudorabies and porcine mycoplasmal pneumonia combined inactivated vaccine contains a porcine pseudorabies gB antigen, a porcine pseudorabies gD antigen and a porcine mycoplasmal pneumonia CJ strain antigen, has high immune efficiency, and can be used for preventing and treating mixed infection or secondary infection of the porcine pseudorabies and the porcine mycoplasmal pneumonia.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Note that CHO cells used in the following examples contain genes encoding porcine pseudorabies gB, gD antigens, and are capable of expressing the porcine pseudorabies gB, gD antigens.
The improved Fris liquid culture medium is prepared from basic culture medium PPLO bouillon powder, brain heart extract powder, deionized water and agar powder, and auxiliary culture medium 10 XHank's balanced salt solution, yeast extract, penicillin, phenol red and pig serum.
Example 1 preparation of a bivalent inactivated vaccine against porcine pseudorabies and porcine mycoplasmal pneumonia
1 breeding and culturing porcine pseudorabies gB and gD antigens, and the specific method comprises the following steps:
1.1 cell Resuscitation
And taking out the freezing tube from the liquid nitrogen tank, quickly placing the freezing tube in a constant-temperature water bath kettle at 37 ℃, and slightly shaking to quickly melt the freezing tube. Sucking the cell suspension in the freezing tube into a centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, adding appropriate amount of fresh culture medium to resuspend the cells, placing at 37 deg.C and 5% CO2Culturing in an incubator.
1.2 cell passages
CHO cells were cultured in adherent medium at 37 ℃ with 5% CO2Culturing in an incubator. When the cells grow to reach the fusion degree of more than 80%, sucking out the culture medium, washing with PBS for 2 times, adding 1mL of 0.25% pancreatin solution for digestion, and gently shaking the cell bottle until the cell grows to be more than 80%The pancreatin soaks the bottle wall, partial pancreatin is sucked out, the remaining pancreatin is continuously digested by a small amount of residual pancreatin, the digestion is observed under an inverted microscope, the residual pancreatin solution is removed after the cells become round and bright, a fresh culture medium containing serum is added to stop the digestion, the bottle wall is lightly blown by a straw until the cells completely fall off, partial cell sap is discarded, the straw is lightly blown to prepare single cell suspension, a proper amount of culture medium is added, and the single cell suspension is placed into an incubator to be continuously cultured. And counting the cells in the logarithmic growth phase of the suspension cells, observing the cell state, calculating according to the required concentration and volume, reselecting by using a certain amount of fresh culture medium, uniformly blowing and mixing, and culturing in a constant-temperature shaking table at 37 ℃ at 100 rpm/min.
1.3 protein purification
Collecting cell culture solution, centrifuging at 4 deg.C and 8000g for 30min, collecting supernatant, filtering with 0.8um filter membrane, and purifying by nickel affinity chromatography. And pouring the imidazole eluent containing the target protein into a dialysis bag, dialyzing by 1 × PBS for at least 10 times, and detecting the residual sample. Determining the protein concentration by using a BCA method; the purity is detected by HPLC method, and the purity is required to be more than 90%. In a biological safety cabinet, a 0.22um vacuum filter is used, and a filtered protein solution sample is stored in a refrigerator at minus 80 ℃.
2 culturing the antigen of the mycoplasma hyopneumoniae CJ strain, which comprises the following steps:
2.1 seed preparation for production
Taking birth seeds from a strain library, dissolving the birth seeds in an improved Fris liquid culture medium, subculturing the birth seeds in the improved Fris liquid culture medium according to a ratio of 1: 10-1: 15, placing the cultivation medium in a constant-temperature oscillation incubator at 36-38 ℃, performing oscillation culture for 42-48 hours at a speed of 50-75 r/min, harvesting the strain liquid when the color of the strain liquid is changed and the pH value is reduced to 6.70-6.80, using the strain liquid as a primary seed, performing pure inspection according to the appendix of the current Chinese veterinary pharmacopoeia, and performing pure inspection. The product is stored at the temperature of 2-8 ℃, and the service life is not more than 14 days. The subculture should not exceed 5 generations.
2.2 preparation of bacterial liquid for preparing seedlings
The culture medium for preparing the seedlings is an improved Fris liquid culture medium. Adding the seed solution into the improved Fris liquid culture medium according to the ratio of 1: 10-1: 15, sealing the opening of a fermentation tank, introducing sterile air, closing all inlets and outlets on the fermentation tank when the pressure in the fermentation tank reaches 0.08MPa, maintaining the pressure, stirring at 50-100 r/min, culturing at 36-38 ℃ for 42-48 hours, sampling when the pH value is reduced to 6.70-6.80, and carrying out pure inspection and viable count.
2.3 inactivation
Preparing 0.1mol/L BEI solution, filtering and sterilizing, and storing at 2-8 ℃ for later use. Adding 0.1mol/L BEI solution into the bacterial liquid, inactivating the bacterial liquid at the final concentration of 2mmol/L for 24 hours at 37 ℃, and stirring at 50-100 r/min. And after inactivation, adding 1mol/L sodium thiosulfate with the final concentration of 20mmol/L, reacting at 37 ℃ for 1 hour, storing at 2-8 ℃, and performing inactivation inspection.
3 vaccine preparation
Firstly, respectively sterilizing an aluminum hydroxide gel adjuvant and an oil-in-water type oil adjuvant for later use. Mixing porcine pseudorabies gB and gD antigens and mycoplasma hyopneumoniae antigens according to a ratio of 15: 30 (volume ratio) to prepare a mixed antigen, then stirring and mixing 64.3% of the mixed antigen and an aluminum hydroxide gel adjuvant at a low speed according to a ratio of 45: 15, stirring and mixing 35.7% of the mixed antigen and an oil adjuvant at a low speed according to a ratio of 25: 15, and finally stirring and mixing the 2 mixed solutions at a low speed. Packaging under aseptic condition, and sealing with cover.
Example 2 verification of immune effect of bivalent inactivated vaccine of porcine pseudorabies and porcine mycoplasmal pneumonia with different antigen contents
1 Material
The following three vaccines were prepared according to the procedure of example 1.
The gB and gD antigen contents of the vaccine 1 porcine pseudorabies are respectively 50 mu g/head; the content of mycoplasma hyopneumoniae antigen is 4 × 109.0CCU/head, 2mL immunization dose.
Vaccine 2 porcine pseudorabies gB and gD antigen contents are respectively 100 mu g/head part; the content of mycoplasma hyopneumoniae antigen is 7 × 109.0CCU/head, 2mL immunization dose.
The gB and gD antigen contents of the vaccine 3 porcine pseudorabies are respectively 150 mu g/head part; the content of mycoplasma hyopneumoniae antigen is 10 × 109.0CCU/head, 2mL immunization dose.
2 method
2.1 safety test
15 healthy susceptible piglets of 2-3 weeks old are injected with 2-fold doses (4mL) of immune vaccines 1, 2 and 3 through deep muscles of the neck behind each head of the healthy susceptible piglets, and the abnormal conditions and death conditions of body temperature, appetite, mental state, clinical reaction and the like do not appear after continuous observation for 14 days. Details are given in table 1.
TABLE 1 vaccine safety test conditions
Grouping
Body temperature
Appetite stimulation
Mental state
Clinical response
Death was caused by death
Vaccine 1
Is normal
Is normal
Is normal
Is free of
Is free of
Vaccine 2
Is normal
Is normal
Is normal
Is free of
Is free of
Vaccine 3
Is normal
Is normal
Is normal
Is free of
Is free of
2.2 test of effectiveness
2.2.1 partial efficacy test for porcine pseudorabies
Screening 20 healthy susceptible piglets (negative to porcine pseudorabies virus antigen and antibody) with the age of 3-4 weeks, and randomly dividing into 4 groups with 5 piglets in each group. Wherein, 2mL of 3 vaccines are respectively immunized by 1-3 groups, and the 4 th group is not injected as a control and is separately fed under the same condition. At 28 days after immunization, together with control pigs, each nasal drop was inoculated with classical virulent virus (porcine pseudorabies virus SC strain), and observed for 14 days. Control pigs should have at least 4 outbreaks and immunized pigs should have at least 4 protections.
2.2.2 partial efficacy test for Mycoplasma hyopneumoniae
20 healthy susceptible piglets (negative to mycoplasma hyopneumoniae antigen antibody) of 3-4 weeks old are screened and randomly divided into 4 groups, and 5 piglets in each group are selected. Wherein, 2mL of 3 vaccines are respectively immunized by 1-3 groups, and the 4 th group is not injected as a control and is separately fed under the same condition. On 28 days after immunization, 5mL of mycoplasma hyopneumoniae CJ strain tissue virus (containing 0.01g of lung tissue virus prepared from diseased lungs after virus attack by the mycoplasma hyopneumoniae CJ strain) is injected into each trachea together with control pigs, 28 days of continuous observation are carried out, the dissection is carried out, the pneumonia lesions of each pig are scored according to a 28-score method, and the pneumonia lesion reduction rate is calculated.
Details of vaccine effectiveness testing are shown in table 2.
TABLE 2 vaccine effectiveness test results
Note: "/" indicates not applicable.
The experimental data in table 2 show that after 3 prepared porcine pseudorabies and mycoplasma pneumonia bivalent inactivated vaccines with different antigen contents are used for immunizing piglets, the piglets can be respectively protected by 60% (3/5), 100% (5/5) and 100% (5/5) when being inoculated with the current classical virulent virus (porcine pseudorabies virus SC strain) for attack, and the piglets are attacked by 100% after being attacked by the control piglets. The results show that the vaccine 2 has good immune protection effect.
The experimental data in table 2 show that after 3 kinds of combined inactivated vaccines with different antigen contents for porcine pseudorabies and swine mycoplasma pneumonia are prepared to immunize piglets, the combined inactivated vaccines are inoculated with mycoplasma hyopneumoniae CJ strain tissue virus to attack, and can respectively provide 56.06%, 75.76% and 84.85% protection for the piglets. The results show that the vaccine 2 has good immune protection effect.
In conclusion, the gB and gD antigen contents of the porcine pseudorabies are respectively 100 mu g/head, and the mycoplasma hyopneumoniae antigen content is 7 multiplied by 109.0The CCU/head combined inactivated vaccine for porcine pseudorabies and porcine mycoplasmal pneumonia has the best immune effect.
Example 3 antigen interference test of Components of bivalent inactivated vaccine against porcine pseudorabies and porcine Mycoplasma pneumoniae
1 Material
The following 2 vaccines were prepared according to the procedure of example 1.
The gB and gD antigen contents of the vaccine 4 porcine pseudorabies are respectively 100 mu g/head, and the immunizing dose is 2 mL.
Vaccine 5 mycoplasma hyopneumoniae antigen content of 7 × 109.0CCU/head, 2mL immunization dose.
2 method
2.1 porcine pseudorabies partial antigen interference test
Screening 15 healthy susceptible piglets (negative to porcine pseudorabies virus antigen and antibody) with the age of 3-4 weeks, and randomly dividing the piglets into 3 groups, wherein each group comprises 5 piglets. Wherein 2mL of 2 vaccines are respectively immunized by 1-2 groups, and the 3 rd group is not injected as a control and is separately fed under the same condition. At 28 days after immunization, together with control pigs, each nasal drop was inoculated with classical virulent virus (porcine pseudorabies virus SC strain), and observed for 14 days. Control pigs should have at least 4 outbreaks and immunized pigs should have at least 4 protections.
2.2 Mycoplasma hyopneumoniae partial antigen interference assay
Screening 15 healthy susceptible piglets (negative to mycoplasma hyopneumoniae antigen and antibody) with the age of 3-4 weeks, and randomly dividing into 3 groups, wherein each group comprises 5 piglets. Wherein 2mL of 2 vaccines are respectively immunized by 1-2 groups, and the 3 rd group is not injected as a control and is separately fed under the same condition. On 28 days after immunization, together with control pigs, 5mL of mycoplasma hyopneumoniae CJ strain tissue virus (containing 0.01g of lung tissue virus) was injected into each trachea, and 28 days of continuous observation were performed to perform a dissection examination, and pneumonia lesions of each pig were scored by 28-point scoring method to calculate the pneumonia lesion reduction rate.
Details of antigen interference assays are shown in tables 3 and 4.
TABLE 3 partial antigen interference test results of porcine pseudorabies
Note: "/" indicates not applicable.
TABLE 4 partial antigen interference test results for Mycoplasma hyopneumoniae
Note: "/" indicates not applicable.
The experimental data in tables 3 and 4 show that the prepared porcine pseudorabies, porcine mycoplasma pneumonia combined inactivated vaccine and porcine pseudorabies inactivated vaccine can respectively provide 100% (5/5) and 100% (5/5) protection for piglets after being inoculated with the current classical virulent virus (porcine pseudorabies virus SC strain) attack after being immunized for piglets, and the control piglets are attacked by 100%. The result shows that the mycoplasma hyopneumoniae antigen has no influence on the immune protection of the porcine pseudorabies and the porcine mycoplasma pneumoniae bivalent inactivated vaccine.
Experimental data in tables 3 and 4 show that the prepared porcine pseudorabies, the prepared porcine mycoplasma pneumonia combined inactivated vaccine and the prepared porcine mycoplasma pneumonia inactivated vaccine can respectively provide 78.26 percent and 79.71 percent protection for piglets after being inoculated with the porcine mycoplasma pneumonia CJ strain tissue virus attack after immunizing the piglets. The result shows that the porcine pseudorabies antigen has no influence on the immune protection of the porcine pseudorabies and porcine mycoplasma pneumonia combined inactivated vaccine.
In conclusion, the antigens of the components of the bivalent inactivated vaccine for the porcine pseudorabies and the porcine mycoplasmal pneumonia are determined to be mutually free of interference.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Tiankang pharmaceutical (Suzhou) Co., Ltd
<120> bivalent inactivated vaccine for porcine pseudorabies and swine mycoplasmal pneumonia and preparation method thereof
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 914
<212> PRT
<213> Artificial sequence
<400> 1
Met Pro Ala Gly Gly Gly Leu Trp Arg Gly Pro Arg Gly His Arg Pro
1 5 10 15
Gly His His Gly Gly Ala Gly Leu Gly Arg Leu Trp Pro Ala Pro His
20 25 30
His Ala Ala Ala Ala Arg Gly Ala Val Ala Leu Ala Leu Leu Leu Leu
35 40 45
Ala Leu Ala Ala Thr Pro Thr Cys Gly Ala Ala Ala Val Thr Arg Ala
50 55 60
Ala Ser Ala Ser Pro Ala Pro Gly Thr Gly Ala Thr Pro Asp Gly Phe
65 70 75 80
Ser Ala Glu Glu Ser Leu Glu Glu Ile Asp Gly Ala Val Ser Pro Gly
85 90 95
Pro Ser Asp Ala Pro Asp Gly Glu Tyr Gly Asp Leu Asp Ala Arg Thr
100 105 110
Ala Val Arg Ala Ala Ala Thr Glu Arg Asp Arg Phe Tyr Val Cys Pro
115 120 125
Pro Pro Ser Gly Ser Thr Val Val Arg Leu Glu Pro Glu Gln Ala Cys
130 135 140
Pro Glu Tyr Ser Gln Gly Arg Asn Phe Thr Glu Gly Ile Ala Val Leu
145 150 155 160
Phe Lys Glu Asn Ile Ala Pro His Lys Phe Lys Ala His Ile Tyr Tyr
165 170 175
Lys Asn Val Ile Val Thr Thr Val Trp Ser Gly Ser Thr Tyr Ala Ala
180 185 190
Ile Thr Asn Arg Phe Thr Asp Arg Val Pro Val Pro Val Gln Glu Ile
195 200 205
Thr Asp Val Ile Asp Arg Arg Gly Lys Cys Val Ser Lys Ala Glu Tyr
210 215 220
Val Arg Asn Asn His Lys Val Thr Ala Phe Asp Arg Asp Glu Asn Pro
225 230 235 240
Val Glu Val Asp Leu Arg Pro Ser Arg Leu Asn Ala Leu Gly Thr Arg
245 250 255
Gly Trp His Thr Thr Asn Asp Thr Tyr Thr Lys Ile Gly Ala Ala Gly
260 265 270
Phe Tyr His Thr Gly Thr Ser Val Asn Cys Ile Val Glu Glu Val Glu
275 280 285
Ala Arg Ser Val Tyr Pro Tyr Asp Ser Phe Ala Leu Ser Thr Gly Asp
290 295 300
Ile Val Tyr Met Ser Pro Phe Tyr Gly Leu Arg Glu Gly Ala His Gly
305 310 315 320
Glu His Ile Gly Tyr Ala Pro Gly Arg Phe Gln Gln Val Glu His Tyr
325 330 335
Tyr Pro Ile Asp Leu Asp Ser Arg Leu Arg Ala Ser Glu Ser Val Thr
340 345 350
Arg Asn Phe Leu Arg Thr Pro His Phe Thr Val Ala Trp Asp Trp Ala
355 360 365
Pro Lys Thr Arg Arg Val Cys Ser Leu Ala Lys Trp Arg Glu Ala Glu
370 375 380
Glu Met Ile Arg Asp Glu Thr Arg Asp Gly Ser Phe Arg Phe Thr Ser
385 390 395 400
Arg Ala Leu Gly Ala Ser Phe Val Ser Asp Val Thr Gln Leu Asp Leu
405 410 415
Gln Arg Val His Leu Gly Asp Cys Val Leu Arg Glu Ala Ser Glu Ala
420 425 430
Ile Asp Ala Ile Tyr Arg Arg Arg Tyr Asn Asn Thr His Val Leu Ala
435 440 445
Gly Asp Lys Pro Glu Val Tyr Leu Ala Arg Gly Gly Phe Val Val Ala
450 455 460
Phe Arg Pro Leu Ile Ser Asn Glu Leu Ala Gln Leu Tyr Ala Arg Glu
465 470 475 480
Leu Glu Arg Leu Gly Leu Ala Gly Val Val Gly Pro Ala Ser Pro Ala
485 490 495
Ala Ala Arg Arg Ala Arg Arg Ser Pro Gly Pro Ala Gly Thr Pro Glu
500 505 510
Pro Pro Ala Val Asn Gly Thr Gly His Leu Arg Ile Thr Thr Gly Ser
515 520 525
Ala Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asp His Ile Gln Ala His
530 535 540
Val Asn Asp Met Leu Ser Arg Ile Ala Ala Ala Trp Cys Glu Leu Gln
545 550 555 560
Asn Lys Asp Arg Thr Leu Trp Gly Glu Met Ser Arg Leu Asn Pro Ser
565 570 575
Ala Val Ala Thr Ala Ala Leu Gly Gln Arg Val Ser Ala Arg Met Leu
580 585 590
Gly Asp Val Met Ala Ile Ser Arg Cys Val Glu Val Arg Gly Gly Val
595 600 605
Tyr Val Gln Asn Ser Met Arg Val Pro Gly Glu Arg Gly Thr Cys Tyr
610 615 620
Ser Arg Pro Leu Val Thr Phe Glu His Asn Gly Thr Gly Val Ile Glu
625 630 635 640
Gly Gln Leu Gly Asp Asp Asn Glu Leu Leu Ile Ser Arg Asp Leu Ile
645 650 655
Glu Pro Cys Thr Gly Asn His Arg Arg Tyr Phe Lys Leu Gly Gly Gly
660 665 670
Tyr Val Tyr Tyr Glu Asp Tyr Ser Tyr Val Arg Met Val Glu Val Pro
675 680 685
Glu Thr Ile Ser Thr Arg Val Thr Leu Asn Leu Thr Leu Leu Glu Asp
690 695 700
Arg Glu Phe Leu Pro Leu Glu Val Tyr Thr Arg Glu Glu Leu Ala Asp
705 710 715 720
Thr Gly Leu Leu Asp Tyr Ser Glu Ile Gln Arg Arg Asn Gln Leu His
725 730 735
Ala Leu Lys Phe Tyr Asp Ile Asp Arg Val Val Lys Val Asp His Asn
740 745 750
Val Val Leu Leu Arg Gly Ile Ala Asn Phe Phe Gln Gly Leu Gly Asp
755 760 765
Val Gly Ala Ala Val Gly Lys Val Val Leu Gly Ala Thr Gly Ala Val
770 775 780
Ile Ser Ala Val Gly Gly Met Val Ser Phe Leu Ser Asn Pro Phe Gly
785 790 795 800
Ala Leu Ala Ile Gly Leu Leu Val Leu Ala Gly Leu Val Ala Ala Phe
805 810 815
Leu Ala Tyr Arg His Ile Ser Arg Leu Arg Arg Asn Pro Met Lys Ala
820 825 830
Leu Tyr Pro Val Thr Thr Lys Ala Leu Lys Glu Asp Gly Val Glu Glu
835 840 845
Asp Asp Val Asp Glu Ala Lys Leu Asp Gln Ala Arg Asp Met Ile Arg
850 855 860
Tyr Met Ser Ile Val Ser Ala Leu Glu Gln Gln Glu His Lys Ala Arg
865 870 875 880
Lys Lys Asn Ser Gly Pro Ala Leu Leu Ala Ser Arg Val Gly Ala Met
885 890 895
Ala Thr Arg Arg Arg His Tyr Gln Arg Leu Glu Asn Glu Asp Pro Asp
900 905 910
Ala Pro
<210> 2
<211> 402
<212> PRT
<213> Artificial sequence
<400> 2
Met Leu Leu Ala Ala Leu Leu Ala Ala Leu Val Ala Arg Thr Thr Leu
1 5 10 15
Gly Ala Asp Val Asp Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala
20 25 30
Tyr Pro Tyr Thr Glu Ser Trp Gln Leu Thr Leu Thr Thr Val Pro Ser
35 40 45
Pro Phe Val Gly Pro Ala Asp Val Tyr His Thr Arg Pro Leu Glu Asp
50 55 60
Pro Cys Gly Val Val Ala Leu Ile Ser Asp Pro Gln Val Asp Arg Leu
65 70 75 80
Leu Asn Glu Ala Val Ala His Arg Arg Pro Thr Tyr Arg Ala His Val
85 90 95
Ala Trp Tyr Arg Ile Ala Asp Gly Cys Ala His Leu Leu Tyr Phe Ile
100 105 110
Glu Tyr Ala Asp Cys Asp Pro Arg Gln Ile Phe Gly Arg Cys Arg Arg
115 120 125
Arg Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro
130 135 140
Thr Glu Asp Glu Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe Asn
145 150 155 160
Glu Gly Gln Tyr Arg Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu
165 170 175
Thr Asp Phe Met Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala
180 185 190
Arg Val Asp Gln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser Asp
195 200 205
Asp Ser Phe Lys Arg Gly Val Asp Val Met Arg Phe Leu Thr Pro Phe
210 215 220
Tyr Gln Gln Pro Pro His Arg Glu Val Val Asn Tyr Trp Tyr Arg Lys
225 230 235 240
Asn Gly Arg Thr Leu Pro Arg Ala Tyr Ala Ala Ala Thr Pro Tyr Ala
245 250 255
Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro Arg Pro Arg
260 265 270
Pro Arg Pro Arg Pro Arg Pro Lys Pro Glu Pro Ala Pro Ala Thr Pro
275 280 285
Ala Pro Pro Gly Arg Leu Pro Glu Pro Ala Thr Arg Asp His Ala Ala
290 295 300
Gly Gly Arg Pro Thr Pro Arg Pro Pro Arg Pro Glu Thr Pro His Arg
305 310 315 320
Pro Phe Ala Pro Pro Ala Val Val Pro Ser Gly Trp Pro Gln Pro Ala
325 330 335
Glu Pro Phe Pro Pro Arg Thr Thr Ala Ala Pro Gly Val Ser Arg His
340 345 350
Arg Ser Val Ile Val Gly Thr Gly Thr Ala Met Gly Ala Leu Leu Val
355 360 365
Gly Val Cys Val Tyr Ile Phe Phe Arg Leu Arg Gly Ala Lys Gly Tyr
370 375 380
Arg Leu Leu Gly Gly Pro Ala Asp Ala Asp Glu Leu Lys Ala Gln Pro
385 390 395 400
Gly Pro
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一种纳米佐剂及其制备方法和应用