CN109022368B - Porcine circovirus type 2 strain, vaccine composition, preparation method and application thereof - Google Patents

Porcine circovirus type 2 strain, vaccine composition, preparation method and application thereof Download PDF

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CN109022368B
CN109022368B CN201710433037.7A CN201710433037A CN109022368B CN 109022368 B CN109022368 B CN 109022368B CN 201710433037 A CN201710433037 A CN 201710433037A CN 109022368 B CN109022368 B CN 109022368B
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antigen
strain
porcine
virus
inactivated
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CN109022368A (en
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田克恭
姚亚丽
孙进忠
张许科
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Luoyang Huizhong Biotechnology Co Ltd
Pulaike Biological Engineering Co Ltd
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Pulaike Biological Engineering Co Ltd
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Abstract

The invention relates to a strain of a new gene subtype of porcine circovirus type 2, which has good and broad-spectrum immunogenicity. The prepared vaccine composition can prevent and/or treat related diseases caused by porcine circovirus type 2 infection, and has good immune protection efficacy on strains from different regional sources.

Description

Porcine circovirus type 2 strain, vaccine composition, preparation method and application thereof
Technical Field
The invention relates to a strain, a vaccine composition prepared from the strain, a preparation method and application of the strain, and belongs to the field of animal virology.
Background
Porcine Circovirus (PCV) is a single-stranded circular DNA virus with a genome length of approximately 1.7kb that is one of the smallest animal DNA viruses. Two types of PCV have been identified, porcine circovirus type 1(PCV 1) and porcine circovirus type 2(PCV 2). PCV1 was first identified in 1974 as a contaminant in PK cell cultures, which was not pathogenic to pigs. PCV2 was first reported in 1998 to cause Porcine circovirus-associated diseases (PCVAD) in pigs under clinical conditions, mainly causing postweaning multisystemic wasting syndrome, pneumonia, Porcine dermatitis and nephropathy syndrome and reproductive disorders, mainly manifested as respiratory, urinary, intestinal, lymphatic, cardiovascular, neurological, reproductive and cutaneous dysfunction, causing significant economic losses to live pig breeding worldwide.
PCV2 mainly has two gene subtypes, PCV2a and PCV2b, the gene group lengths of the two gene subtypes are 1767 and 1768 nucleotides, and PCV2c is only reported in Denmark. PCV2a was the main epidemic strain before 2003, after which PCV2b began to appear and expanded rapidly, and a new genetic subtype PCV2d was discovered in 2009. Clinically, with the widespread use of PCV2 vaccine, the mutation rate of PCV2 is accelerated under immune pressure.
In recent years, a new gene subtype strain between PCV2b and PCV2d has become prevalent, which is characterized by the presence of mutations in the ORF2 gene or recombination between different gene subtype genes. Because the new PCV2 gene subtype strain is epidemic and the antigen is different from the existing gene subtype, the existing commercial vaccines are all prepared by taking PCV2a or PCV2b as vaccine strains, and the newly epidemic strains can not be completely protected, so that the preparation of the new vaccine aiming at the newly epidemic strains is very important for controlling pig farm diseases.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a vaccine composition for preventing and/or treating new porcine circovirus type 2 infection, which can effectively protect the porcine circovirus type 2 virus and shows remarkable immunological properties.
Therefore, the invention aims to provide a porcine circovirus type 2 strain, which belongs to a new gene subtype circulating at present and has good and broad-spectrum immunogenicity.
Another object of the present invention is to provide a vaccine composition for the prevention and/or treatment of a novel porcine circovirus type 2 infection, said vaccine composition comprising an immunizing amount of an antigen of a porcine circovirus type 2 strain according to the invention and a pharmaceutically acceptable carrier.
Another objective of the invention is to provide a vaccine composition for preventing and/or treating porcine circovirus type 2 gene subtype infection and mixed infection thereof.
Another object of the present invention is to provide a vaccine composition for preventing and/or treating porcine circovirus type 2 gene subtype infections and mixed infections thereof from different geographical sources.
It is another object of the present invention to provide a method for preparing the vaccine composition, comprising: propagating the porcine circovirus type 2 strain; inactivating the porcine circovirus type 2 strain proliferated in the step (2), adding an adjuvant, and emulsifying.
The invention also aims to provide application of the vaccine composition in preparing a medicament for preventing and/or treating porcine circovirus type 2 related diseases.
The invention has the advantages that:
(1) the strain has good immunogenicity, can stimulate an organism to quickly generate immunity after immunization once, effectively protects the attack of the strain, has good protection effect, can achieve good immune protection effect by using lower antigen content, and further reduces the production cost;
(2) the vaccine composition prepared by the strain of the porcine circovirus type 2new gene subtype can not only protect the strain of the porcine circovirus type 2new gene subtype, but also prevent and/or treat the independent infection or mixed infection of the strains of different porcine circovirus type 2 gene subtypes;
(3) the vaccine can provide complete protection for porcine circovirus type 2 strains from different regional sources, and has broad-spectrum protection capability.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
The "porcine circovirus type 2new gene subtype" refers to a new PCV2 gene subtype, which has a mutation in ORF2 gene or recombination between different gene subtypes, has a PCV2b tag sequence, but forms an independent branch in genetic evolution analysis, and clinical characteristics appeared after pigs are only infected with a strain of the gene subtype are as follows: persistent high temperature, anorexia, mental depression, coarse and disordered hair, emaciation and slow growth speed, and lung consolidation, lymphadenectasis and kidney necrosis.
The invention relates to a Porcine Circovirus type 2HH3 strain (Porcine Circovirus type 2, strain HH3) which is preserved in China center for type culture Collection with the preservation number of CCTCC NO. V201726, the preservation date of 2017, 6 and 4 days, and the preservation address: wuhan, Wuhan university in China.
The porcine circovirus type 2HH3 strain belongs to a porcine circovirus type 2new gene subtype, and the immunogenicity of the porcine circovirus type 2HH3 strain is different from the PCV2 gene subtype in the prior art. The strain has good immunogenicity, and the prepared vaccine can effectively stimulate immune protection.
The new porcine circovirus type 2 genome provided by the invention has 1768 nucleotides in length and has a PCV2b tag sequence, but forms an independent branch in genetic evolution analysis, and the ORF2 gene has mutation or recombination among different gene subtypes, and is a new PCV2 gene subtype.
The invention also relates to a vaccine composition, wherein the vaccine composition contains the immunizing amount of the porcine circovirus type 2HH3 strain antigen and a pharmaceutically acceptable carrier.
The vaccine composition has good immunogenicity, can stimulate an organism to quickly generate immune efficacy after being immunized once, effectively protects the attack of a strain and realizes complete protection.
The porcine circovirus type 2HH3 strain antigen can be inactivated antigen or attenuated whole virus antigen of porcine circovirus type 2HH3 strain or culture thereof.
"cultures" are subcultures of different generations of the virus, and those skilled in the art know that only minor variations in the gene sequence are possible between generations.
"vaccine composition" refers to a pharmaceutical composition comprising porcine circovirus type 2 immunogenicity. The pharmaceutical composition can induce, stimulate or enhance the immune response of pigs against porcine circovirus type 2.
"inactivated antigen", also known as inactivated vaccine, refers to a suspension of inactivated virus used as an antigen to generate immunity. Examples of inactivated vaccines include whole virus vaccines and split vaccines. Inactivated vaccines can be readily produced using known methods. For example, whole virus inactivated vaccines can be obtained by treating the virus with formaldehyde solution. Split vaccines can be prepared from the viral envelope after treatment with ether.
An "attenuated whole virus antigen" refers to a virus that has attenuated virulence but can still replicate in or on a host. The term "attenuated" as used herein is intended to mean that the virulence of a pathogen is artificially reduced by mutating the gene in such a way that the pathogen loses pathogenicity, but remains immunogenic. Attenuation is typically achieved by UV irradiation, chemical treatment or in vitro sequential high-order subculture. Or artificial genetic alteration, such as deletion of specific nucleotides in known sequences to attenuate virulence.
In one embodiment of the invention, the antigen of porcine circovirus type 2HH3 strain is a whole virus inactivated antigen of porcine circovirus type 2HH3 strain or a culture thereof.
In one embodiment of the invention, the antigen of porcine circovirus type 2HH3 strain is a split antigen of porcine circovirus type 2HH3 strain or a culture thereof.
The gene sequence of different generations of subcultures of the virus only has slight variation, the same immunogenicity can be ensured, and the inactivated vaccine prepared by the method has similar immune efficacy.
In one embodiment of the invention, the culture of the porcine circovirus type 2HH3 strain is a culture of more than or equal to 1 generation.
In a preferred embodiment of the present invention, the culture of porcine circovirus type 2 strain HH3 is a culture of not less than 5 generations.
In a more preferred embodiment of the present invention, the culture of porcine circovirus type 2HH3 strain is a culture of 5 to 48 generations.
The amount of an ingredient or component of the composition of the present invention is preferably a therapeutically effective amount. The therapeutically effective amount refers to the amount necessary to exert their immunological effects in the host to which the composition is administered without causing undue side effects. The precise amounts of the ingredients used and the composition to be administered will vary depending on a variety of factors such as the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.
In one embodiment of the present invention, the inactivated whole virus antigen content of the porcine circovirus type 2HH3 strain or the culture thereof is not less than 10 before inactivation5.0TCID50/ml。
The inactivated whole virus antigen of the porcine circovirus type 2HH3 strain in the vaccine composition has good immunogenicity, can stimulate an organism to quickly generate immunity, and is 10 percent before inactivation5.0TCID50At the content of/ml, good immune protection effect can be achieved, and 100% protection rate is realized.
In a preferred embodiment of the present invention, the inactivated whole virus antigen content of the porcine circovirus type 2 strain HH3 or the culture thereof is 10 before inactivation5.0～107.0TCID50/ml。
In a more preferred embodiment of the present invention, the inactivated whole virus antigen content of the porcine circovirus type 2 strain HH3 or the culture thereof is 10 before inactivation6.0TCID50/ml。
In the vaccine composition of the present invention, the inactivated whole virus antigen content range of the porcine circovirus type 2HH3 strain or the culture thereof can be further selected from 105.0～106.0TCID50Per ml, or 106.0～107.0TCID50/ml。
As an embodiment of the present invention, the pharmaceutically acceptable carrier is an adjuvant, which includes white oil, derek oil, and animal, vegetable or mineral oil; or aluminum hydroxide, aluminum phosphate and metal salts; or montanide tmgel, carbomer, squalane or squalene, ISA206 adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
As a preferred embodiment of the present invention, the adjuvant is montanide tmgel.
In one embodiment of the present invention, the adjuvant content is 5 to 20V/V%.
As a preferred embodiment of the present invention, the adjuvant content is 10V/V%.
The vaccine compositions of the present invention may be formulated using available techniques, preferably together with a pharmaceutically acceptable carrier. For example, the oil may help stabilize the formulation and additionally serve as a vaccine adjuvant. The oil adjuvant can be natural source or obtained by artificial synthesis.
The term "adjuvant" refers to a substance added to the composition of the present invention to increase the immunogenicity of the composition. Known adjuvants include, but are not limited to: (1) aluminium hydroxide, saponins (saponin) (e.g. QuilA), alfuzidine, DDA, (2) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivatives, (3) vaccines can be made in the form of oil-in-water, water-in-oil or water-in-oil-in-water emulsions, or (4) montanide tmgel.
In particular, the emulsion may be based on light liquid paraffin oil, isoprenoid oil, such as squalane or squalene; oils resulting from the oligomerization of olefins, in particular isobutene or decene, esters of acids or alcohols with linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of polyoxyethylated fatty acids (e.g.oleic acid), sorbitan, mannitol (e.g.anhydromannitol oleate), glycerol, polyglycerol, propylene glycol and optionally ethoxylated oleic acid, isostearic acid, ricinoleic acid, hydroxystearic acid, ethers of fatty alcohols and polyols (e.g.oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic R, especially L121 (see Hunter et al, 1995, "The Theory and Practical applications of Advances" (Steward-Tull, D.E.S. eds.) John Wiley and sons, NY, 51-94; Todd et al, Vaccine, 1997, 15, 564 + 570).
In particular, the acrylic or methacrylic acid polymers are crosslinked by polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers.
Preferably, the adjuvant selected by the invention is MontanideTMGel.
The amount of adjuvant suitable for use in the compositions of the present invention is preferably an effective amount. By "effective amount" is meant the amount of adjuvant necessary or sufficient to exert their immunological effect in a host when administered in combination with the antigen of the invention without causing undue side effects. The precise amount of adjuvant to be administered will vary depending on a variety of factors such as the ingredients used and the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.
The invention also relates to a method for preparing the vaccine composition, wherein the method comprises the following steps: step (1) propagation culturing of the porcine circovirus type 2HH3 strain or a culture thereof; and (2) inactivating the porcine circovirus type 2HH3 strain propagated and cultured in the step (1) or a culture thereof, and (3) adding an adjuvant into the inactivated porcine circovirus type 2HH3 strain or the culture thereof and emulsifying the mixture.
The vaccine compositions of the invention further comprise other pathogen or antigen compositions for use in the preparation of combination or composite vaccines against diseases including porcine circovirus type 2 infection.
The term "combination vaccine" refers to a vaccine prepared from a mixture of viral antigens of porcine circovirus type 2 and at least one different virus according to the invention. The term "composite vaccine" refers to a vaccine prepared from the porcine circovirus type 2 and bacterial antigens of the invention. For example, porcine circovirus type 2 of the invention can be combined with antigens of: hog cholera virus, porcine pseudorabies virus, swine influenza virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine encephalitis B virus and/or haemophilus parasuis, Streptococcus suis, Bordetella suis, porcine contagious pleuropneumonia, Pasteurella multocida, Salmonella choleraesuis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, and a mixture or combination thereof.
As an embodiment of the invention, the vaccine composition further comprises one or more of the group consisting of an immunizing amount of: classical swine fever virus antigen, porcine pseudorabies virus antigen, porcine influenza virus antigen, porcine reproductive and respiratory syndrome virus antigen, porcine parvovirus antigen, porcine encephalitis B virus antigen, haemophilus parasuis antigen, streptococcus suis antigen, bordetella suis antigen, porcine infectious pleuropneumonia antigen, porcine pasteurella multocida antigen, salmonella choleraesuis antigen, mycoplasma hyopneumoniae antigen, mycoplasma hyorhinis antigen.
The porcine circovirus type 2 antigen can be used together with a vaccine composition consisting of a plurality of antigen components, the antigens are not interfered with each other, and the antigen components can generate complete protection against respective challenge strains.
As a preferred embodiment of the invention, the vaccine composition further comprises one or more of the group consisting of an immunizing amount of: the antigen comprises a classical swine fever virus attenuated antigen, a porcine pseudorabies virus attenuated antigen, a porcine reproductive and respiratory syndrome virus attenuated antigen, a porcine parvovirus inactivated antigen, a haemophilus parasuis inactivated antigen and a mycoplasma hyopneumoniae inactivated antigen.
As a preferred embodiment of the invention, the vaccine composition further comprises one or more of the group consisting of an immunizing amount of: classical swine fever virus attenuated antigen, porcine pseudorabies virus inactivated antigen, porcine reproductive and respiratory syndrome virus attenuated antigen, porcine parvovirus inactivated antigen, haemophilus parasuis inactivated antigen, and mycoplasma hyopneumoniae inactivated antigen.
As a more preferable embodiment of the present invention, in the vaccine composition of the present invention, the attenuated antigen of the classical swine fever virus is a lapinized attenuated strain of classical swine fever virus, the attenuated antigen of the pseudorabies virus is a low virulent strain of HN1201-R strain, the inactivated antigen of the pseudorabies virus is an inactivated whole virus antigen of HN1201 strain, the attenuated antigen of the porcine reproductive and respiratory syndrome virus is an attenuated strain of JXA1-R strain, the inactivated antigen of the porcine parvovirus is an inactivated whole virus antigen of HN-2011 strain, the inactivated antigen of the haemophilus parasuis is an inactivated whole virus antigen of JS strain serotype 4 or ZJ strain 5 of haemophilus parasuis, and the inactivated antigen of the mycoplasma hyopneumoniae is an inactivated whole virus antigen of HN0613 strain.
In one embodiment of the present invention, the inactivated whole virus antigen content of the porcine circovirus type 2HH3 strain or the culture thereof in the vaccine composition of the present invention is 10 before inactivation5.0～107.0TCID50The attenuated antigen content of the hog cholera virus is 10ml4.0～106.0TCID50Per ml, the content of the porcine pseudorabies virus attenuated antigen is 106.0～107.0TCID50The inactivated antigen content of the porcine pseudorabies virus is 10 before inactivation6.0～107.0TCID50The porcine reproductive and respiratory syndrome virus attenuated antigen content is 105.0～107.0TCID50The inactivated antigen content of the porcine parvovirus is 10 before inactivation6.0～108.0TCID50The content of the inactivated antigen of the haemophilus parasuis is 10 before inactivation8.0～1010.0CFU/ml, the content of the mycoplasma hyopneumoniae inactivated antigen is 10 before inactivation8.0～1010.0CCU/ml。
The antigen in the vaccine composition can effectively stimulate immune response within the content range, resist the attack of respective pathogen, do not generate interference among antigen components in the vaccine composition, and can be completely protected, and the protection rate is 100 percent.
In a preferred embodiment of the present invention, the inactivated whole virus antigen content of the porcine circovirus type 2HH3 strain or the culture thereof in the vaccine composition of the present invention is 10 before inactivation5.0～107.0TCID50The attenuated antigen content of the hog cholera virus is 10ml5.0TCID50Per ml, the content of the porcine pseudorabies virus attenuated antigen is 106.0TCID50The inactivated antigen content of the porcine pseudorabies virus is 10 before inactivation6.0TCID50The porcine reproductive and respiratory syndrome virus attenuated antigen content is 106.0TCID50The inactivated antigen content of the porcine parvovirus is 10 before inactivation7.0TCID50The content of the inactivated antigen of the haemophilus parasuis is 10 before inactivation9.0CFU/ml, the content of the mycoplasma hyopneumoniae inactivated antigen is 10 before inactivation9.0CCU/ml。
The vaccine composition of the present invention may further comprise other agents added to the composition of the present invention. For example, the compositions of the present invention may also comprise agents such as: drugs, immunostimulants (e.g., alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2(IL2)), antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives. To prepare such compositions, methods well known in the art may be used.
The vaccine composition according to the present invention may be prepared in oral or non-oral dosage forms.
Preferred are non-oral dosage forms that can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or epidural routes.
The invention also relates to application of the vaccine composition in preparing a medicament for preventing and/or treating related diseases caused by porcine circovirus type 2 infection, wherein the related diseases caused by porcine circovirus type 2 infection are related diseases caused by single infection or mixed infection of different gene subtypes of PCV 2.
The porcine circovirus type 2new gene subtype antigen of the vaccine composition can not only carry out immune protection on a porcine circovirus type 2new gene subtype strain, but also carry out immune protection on other gene subtype strains of PCV2, and can also effectively protect mixed infection among different PCV2 gene subtypes.
The porcine circovirus type 2 antigen of the vaccine composition has broad antigenic spectrum, and can provide complete protection for porcine circovirus type 2 strains from different regional sources.
As an embodiment of the invention, the related diseases caused by the porcine circovirus type 2 infection are related diseases caused by PCV2a, PCV2b, PCV2d and PCV2new gene subtype single infection or mixed infection thereof.
As an embodiment of the present invention, the porcine circovirus type 2-associated disease includes postweaning multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, reproductive disorders, and cardiac and multisystemic inflammatory responses.
The term "porcine circovirus type 2 associated disease" as used herein refers to a disease caused by infection with porcine circovirus type 2. Non-exhaustive examples include, but are not limited to, postweaning multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, reproductive disorders, and cardiac and multisystemic inflammatory responses.
The term "preventing and/or treating" when referring to a porcine circovirus type 2 infection means inhibiting replication of porcine circovirus type 2, inhibiting transmission of porcine circovirus type 2 or preventing colonization of porcine circovirus type 2 in its host, and alleviating a symptom of a disease or disorder of porcine circovirus type 2 infection. Treatment is considered to be therapeutically effective if the viral load is reduced, the condition is reduced and/or the food intake and/or growth is increased.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group.
In order that the invention may be more readily understood, reference will now be made to the following examples. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 isolation and characterization of porcine circovirus type 2
1. Origin of disease material
In a domestic pig farm for immunizing commercial PCV2 vaccine, the phenomena of sow abortion and mummy fetus increase occur sporadically. Affected sows showed anorexia and contain mummified fetuses of different gestational ages in the litters of the miscarriage, consistent with symptoms of PCV 2-related miscarriage. PCV2 was positive as detected by immunohistochemistry and quantitative PCR. PCV2 is still detected from diseased pig tissues after immunization of PCV2 commercial vaccine, the reason is worthy of deep study, and in order to further clarify the reason, each tissue pathogen is selected for pathogen isolation.
2. Isolation and culture of viral strains
The disease material is mixed according to the proportion of 1: 10 (volume ratio) adding DMEM culture solution, grinding and preparing tissue suspension; repeatedly freezing and thawing the tissue suspension for 3 times, centrifuging at 12000r/min for 15min, and collecting supernatant; filtering the supernatant with 0.22 μm filter membrane filter; the filtrate was passaged on PK15 cells, cultured at 37 ℃ for 1 hour, and DMEM medium containing 2% calf serum was added instead, and cultured at 37 ℃ for 5 days. And (4) harvesting a culture solution containing the virus, and after the culture solution is frozen and thawed for 2 times, harvesting the virus.
3. Identification of viruses by PCR and sequencing analysis
And (3) taking the virus culture harvested in the step (1), extracting nucleic acid of a virus sample by using a nucleic acid extraction kit, and performing PCR amplification identification by using PCV2 specific primers, wherein the result shows that a 1.7kb band is amplified by PCR.
And sending the PCR product to a sequencing company for nucleotide sequence determination, and carrying out genetic evolution analysis on the sequence determination result. The results show that the whole genome sequence of the strain has less than 96 percent of homology with other reported PCV2, the amino acid sequence is less than 94 percent, the strain is further analyzed to be between PCV2b and PCV2d, and the ORF2 gene has mutation or recombination of different gene subtypes, and belongs to a new PCV2 gene subtype in genetic evolution analysis.
Example 2 screening of porcine circovirus type 2 vaccine strains
Specific primers are designed according to the separated porcine circovirus type 2, 42 suspected PCV2 positive samples collected from pig farms with sporadic incidence after the nationwide immune commercial PCV2 vaccine are analyzed through quantitative PCR, 16 PCV2 viruses are screened and separated, among the 16 viruses, the homology of the nucleotide sequence of the genome is up to 99.6-100%, the homology of the amino acid sequence of Cap protein is up to 99.5-100%, and the 16 viruses are further analyzed to belong to the same gene subtype. Through animal pathogenicity test and immunogenicity test, 1 PCV2 virus strain with strong pathogenicity, good immunogenicity and strong broad spectrum is finally screened out. The porcine circovirus type 2 virus is named porcine circovirus type 2HH3 strain and submitted for preservation.
Example 3 pathogenicity test of porcine circovirus type 2HH3 Strain
10 heads of healthy piglets which are detected by ELISA at the age of 28-30 days and are PCV2 and PCV3 antigens and negative by antibodies are randomly divided into two groups, 5 heads/group, and the 1 st group uses PCV2HH3 strain (containing 10 heads)5.0TCID50Head), intramuscular injection, a blank control group is inoculated with a DMEM medium, and piglets in each group are separately raised. After the challenge, each group of piglets is continuously observed and judged according to clinical symptoms, pathological changes and virus detection, and specific results are shown in table 1.
TABLE 1 pathogenicity test results of porcine circovirus type 2HH3 strain on piglets
Figure BDA0001317893440000111
Figure BDA0001317893440000121
The results show that all pigs in the challenge group have high temperature of more than 40.5 ℃ continuously for 3-5 days, anorexia, depression, rough hair, emaciation and slow growth speed, and have lung metaplasia, lymphadenectasis and kidney dead points of different degrees in autopsy, and porcine circovirus type 2 virus can be separated again through PCR detection of various organ tissues, while the blank control group has no abnormal condition, which indicates that the HH3 strain of porcine circovirus type 2 inoculated to piglets can cause piglet morbidity, and the clinical symptoms are typical porcine circovirus infection symptoms.
Example 4 preparation of porcine circovirus type 2HH3 strain antigen
The cultures of porcine circovirus type 2HH3 strain selected in example 2 in different generations were inoculated into monolayer-forming PK15 passaged cells at 1% (V/V) of the virus culture medium, adsorbed at 37 ℃ for 30 minutes, added with a cell-maintaining solution, and cultured at 37 ℃. Observing for 1-2 times every day, wherein the cells grow well, harvesting cell cultures after culturing for 4-7 days at 36-37 ℃, freezing and thawing the harvested cell cultures for 2-3 times, harvesting virus liquid, and determining virus titer in the virus liquid. Filtering the virus liquid by using a hollow fiber (0.5-2 mu m) filter column to remove cell fragments, adding 0.1-0.2% of formaldehyde solution to inactivate at 37 ℃ for 24h, and preparing the vaccine by using the virus antigen after complete inactivation.
Example 5 preparation of inactivated vaccine of porcine circovirus type 2HH3 strain
The inactivated antigen of porcine circovirus type 2HH3 strain prepared in example 4 is slowly added into a water-soluble adjuvant Gel adjuvant (French Saybolt Corp.) and stirred by an emulsifying machine with the rotation speed of 800rpm for 12min during the addition process, and then the mixture is uniformly mixed. The specific formulation of the vaccine is shown in table 2.
TABLE 2 porcine circovirus type 2HH3 inactivated vaccine formula and content
Figure BDA0001317893440000131
Example 6 immunogenicity test of inactivated vaccine of porcine circovirus type 2HH3 strain
25 healthy piglets which are detected by ELISA for PCV2 and PCV3 antigens and negative antibodies at the age of 28-30 days are randomly divided into 5 groups, 5 heads/group, and the porcine circovirus type 2HH3 inactivated vaccine prepared in the immune example 5 is immunized. The 3 rd to 6 th groups are immune vaccines 1 to 4 respectively, and the 7 th group is immune and used as a control group. Each immunization group was injected with 2 ml/head of vaccine, and the challenge control group was inoculated with 2 ml/head of DMEM medium. The virus is attacked 28 days after the immunization, and the toxin attacking dose is HH3 porcine circovirus 105.0TCID50And/or continuously observing each piglet after the virus attack, and judging according to the clinical symptoms, pathological changes and virus detection results of each piglet, wherein the specific results are shown in a table 3.
TABLE 3 immunogenicity test results of inactivated vaccine of porcine circovirus type 2HH3 strain
Figure BDA0001317893440000141
The result shows that the porcine circovirus type 2HH3 inactivated vaccine can provide 100 percent (5/5) protection for piglets after being immunized by the piglets, and the control piglets with the challenge virus are all attacked. The porcine circovirus type 2HH3 inactivated vaccine provided by the invention has good protective efficacy.
Example 7 comparative immunogenicity test for inactivated vaccine of porcine circovirus type 2HH3 strain
130 healthy piglets with 28-30 days old and negative in PCV2 and PCV3 antigens and antibodies detected by ELISA are randomly divided into 13 groups, 10 groups/group, 8-10 groups to immunize the porcine circovirus type 2HH3 inactivated vaccine 1 prepared in example 5, and 11-13 groups to immunize the commercial vaccine D1(PCV2a strain, the content is more than or equal to 10 before inactivation)5.0TCID50Ml), group 14-16 immune commercial vaccine D2(PCV2b strain, the content is more than or equal to 10 before inactivation5.0TCID50And/ml), the group 17-19 is used as a challenge control group, and the group 20 is used as a blank control group. 8 th to 10 th groups of immunizationGroup injection vaccine 2 ml/head, 1 times of immunity; 2 ml/head of vaccine is injected into the 11 th to 16 th immune groups, and 2 times of immunization is carried out; inoculating the 17 th group of the control group with 2ml of DMEM culture medium per head for 1 time; the 18 th to 20 th groups are inoculated with 2ml of DMEM medium per head of the control group for 2 times. Performing virus challenge 28 days after immunization, and performing virus challenge on 8 th group, 11 th group, 14 th group and 17 th group by using porcine circovirus type 2HH3 strain; the 9 th group, the 12 th group, the 15 th group and the 18 th group are attacked by virulent strains of porcine circovirus type 2a JX02 strains separated from Jiangxi of China; the 10 th group, the 13 th group, the 16 th group and the 19 th group are attacked by the virulent strain of the porcine circovirus type 2b SH03 strain separated from Shanghai in China; the dose of the antidote is 105.0TCID50And/or continuously observing each piglet after the challenge, and judging according to clinical symptoms, pathological changes and virus detection of each piglet, wherein specific results are shown in table 4.
TABLE 4 immunogenicity comparison of inactivated vaccine of porcine circovirus type 2HH3
Figure BDA0001317893440000151
Figure BDA0001317893440000161
The results show that the 17 th to 19 th groups of the control groups with the offensive toxin have different degrees of body temperature rise of more than 40.5 ℃ after offensive toxin, the clinical symptoms such as anorexia, depression, rough and disordered hair, emaciation, slow growth speed and the like are continued for 3 to 5 days, the pathological changes of lung excess change, lymphadenectasis and necrotic spots in the kidney with different degrees are all presented in the autopsy, and the porcine circovirus type 2 can be separated again by performing PCR detection on each organ tissue;
after being attacked by poison, the immune groups of 8-10 groups, 12-13 groups and 15-16 groups have no abnormal clinical symptoms, tissues and organs are subjected to autopsy without abnormality, and PCR detection is carried out on the tissues of organs, so that PCV2 is negative; the body temperature of 4 groups of 11 and 3 groups of 14 is raised by more than 40.5 ℃ to different degrees, the clinical symptoms such as anorexia, mental depression, rough and disorderly hair, emaciation, slow growth speed and the like are kept for 3-5 days, pathological changes such as lung consolidation, lymphadenectasis and kidney necrosis points to different degrees are detected by a autopsy, PCR detection is carried out on each organ tissue, and the porcine circovirus type 2 virus can be separated again;
the 20 th placebo group had no abnormalities. The porcine circovirus type 2HH3 inactivated vaccine provided by the invention is immunized once, so that effective and complete immune protection can be provided for pigs only aiming at PCV2a and PCV2b, and the attacked PCV2 strain cannot be detected from organ tissues; however, the commercial PCV2a vaccine or PCV2b vaccine can only provide complete protection against the existing gene subtypes PCV2a and PCV2b virus strains, and cannot provide complete protection against the new porcine circovirus type 2 epidemic strain.
The vaccine composition has good immunogenicity, and can provide complete protection for PCV2 of different gene subtypes.
Example 8 broad-spectrum protection test of inactivated vaccine of porcine circovirus type 2HH3 strain
50 healthy piglets which are 28-30 days old and have PCV2 and PCV3 antigen detected by ELISA and have negative antibody are randomly divided into 10 groups, 5 groups/group, 21-25 groups immunize the porcine circovirus type 2HH3 inactivated vaccine 1 prepared in the example 5, and 26-30 groups are not immunized and serve as challenge control groups. Each immunization group was injected with 2 ml/head of vaccine, and the challenge control group was inoculated with 2 ml/head of DMEM medium. Performing virus challenge 28 days after immunization, wherein the 21 st group and the 26 th group use a virulent strain HN06 strain of porcine circovirus 2a gene subtype newly separated from Henan province of China to challenge the virus; the 22 nd group and the 27 th group use a virulent strain JS04 strain of porcine circovirus 2b gene subtype which is newly separated from Jiangsu province in China to challenge; the 23 rd group and the 28 th group are attacked by virulent strains of porcine circovirus 2d gene subtype JL13 strain newly separated from Jilin province in China; the 24 th group and the 29 th group use a virulent strain CQ14 strain of a porcine circovirus 2new gene subtype newly separated from Chongqing city of China to challenge; the 25 th group and the 30 th group are attacked by virulent strains of porcine circovirus 2new gene subtype GD15 strain newly separated from Guangdong province in China; the dose of the antidote is 105.0TCID50And/or continuously observing each piglet after the challenge, and judging according to clinical symptoms, pathological changes and virus detection of each piglet, wherein specific results are shown in table 5.
TABLE 5 broad-spectrum protection test results of inactivated vaccine of porcine circovirus type 2HH3 strain
Figure BDA0001317893440000171
Figure BDA0001317893440000181
The results show that the 26 th to 30 th groups of the virus attacking control groups have different degrees of body temperature rise of more than 40.5 ℃ after virus attacking, the clinical symptoms such as anorexia, depression, rough and disordered hair, emaciation, slow growth speed and the like are continued for 3 to 5 days, pathological changes of lung consolidation, lymphadenectasis and necrotic spots in kidney are all detected by autopsy, and porcine circovirus type 2 virus can be separated again by performing PCR detection on each organ tissue; the 21 st to 25 th immune groups have no abnormal clinical symptoms after virus attack, have no abnormality in tissues and organs after autopsy, and show PCV2 negativity by PCR detection of organ tissues.
The porcine circovirus type 2HH3 inactivated vaccine provided by the invention can provide effective and complete immune protection for the porcine circovirus type 2 virus of different gene subtypes from different regional sources, and the PCV2 of the virus can not be detected from organ tissues. The vaccine composition has broad-spectrum immunogenicity, and can completely protect porcine circovirus type 2 from different genetic subtypes from different regions.
Example 9 preparation of classical swine fever virus antigen
The ST cells growing into a good single layer and highly sensitive to the hog cholera lapinized virus strain are digested and dispersed by a digestive juice containing 0.125 percent pancreatin and 0.03 percent EDTA, the cells are counted after dispersion, a cell culture bottle is inoculated, a DMEM cell culture solution containing 3 percent calf serum is added, and meanwhile, hog cholera virus seed viruses (purchased from Zhongzhu institute, the accession number of AVCC NO. AV1412) are added according to the quantity of M.O.I. ═ 0.1 inoculation agents and are placed in a 37 ℃ incubator for culture. The first detoxification is carried out after three days of culture, cell maintenance liquid containing 1.5 percent of calf serum is supplemented after detoxification, and the detoxification is carried out once every 2 days and can be continuously carried out for 5 times. Finally, mixing the detoxified antigens in batches and storing at-20 ℃.
Example 10 preparation of attenuated antigen of porcine pseudorabies virus
Inoculating 1% (V/V) of porcine pseudorabies virus HN1201-R strain (disclosed in Chinese patent application CN105087506A) culture into ST cell culture forming a single layer according to the virus culture fluid volume, placing the ST cell culture in a rotary culture at 37 ℃, harvesting a virus-containing cell culture solution when the lesion reaches 80%, freezing and thawing the cell culture solution for 2 times, collecting the virus, measuring the virus price, and storing at low temperature.
Example 11 preparation of inactivated porcine pseudorabies Virus antigen
Inoculating the gE gene deleted strain (disclosed in Chinese patent application CN103923884A) culture of porcine pseudorabies virus HN1201 strain into an ST cell culture forming a single layer according to 1% (V/V) of the virus culture solution, placing the ST cell culture in a rotary culture at 37 ℃, harvesting a virus-containing cell culture solution when the lesion reaches 80%, freezing and thawing the harvested cell culture solution for 2 times, collecting the virus, and determining the virus price. Adding 10% (v/v) formaldehyde solution to the virus solution to make the final concentration of formaldehyde be 0.2% (v/v), inactivating at 37 deg.C for 18 hr, stirring every 4 hr for 1 time, stirring for 10min each time, and inactivating completely for use.
Example 12 preparation of attenuated antigens of porcine reproductive and respiratory syndrome Virus
Selecting a required number of Marc-145 cells (cell spinner flasks which have grown into a monolayer), discarding growth liquid, inoculating a production virus strain JXA1-R strain (disclosed in Chinese patent application CN101307305A) according to 1% (V/V), adding DMEM cell culture liquid containing 2% fetal calf serum, rotating (9-12R/h) for culturing at 37 ℃, observing cytopathic effect (CPE) caused by viruses under a microscope, harvesting cell cultures when the CPE reaches more than 70%, freeze-thawing the cell cultures for 1 time, removing cell fragments by centrifugation or filtration, and freezing and storing at the temperature of below-40 ℃.
Example 13 preparation of porcine parvovirus inactivated antigen
Inoculating a culture of porcine parvovirus HN-2011 (disclosed in Chinese patent application CN102886043A) into an ST cell culture forming a monolayer according to the inoculation dose of M.O.I. ═ 0.01, placing the ST cell culture at 37 ℃ for rotary culture, harvesting a cell culture solution containing virus when lesion reaches 80%, freezing and thawing the harvested cell culture solution for 2 times, harvesting the virus, and measuring the virus price. Adding 10% (V/V) formaldehyde solution to the virus solution to make the final concentration of formaldehyde be 0.2% (V/V), inactivating at 37 deg.C for 18 hr, stirring every 4 hr for 1 time, stirring for 10min each time, and inactivating completely.
Example 14 preparation of inactivated antigen of Haemophilus parasuis
Propagation of first-order seeds: haemophilus parasuis serum 4 type JS strain and 5 type ZJ strain (disclosed in Chinese patent application CN102908615A) freeze-dried strains are respectively streaked and inoculated on a TSA/NAD (TSA is produced by BD company, NAD is produced by Roche company) flat plate, the flat plate is placed at 37 ℃ for culturing for 18-24 hours, colonies meeting the requirement are selected and inoculated in a TSB/NAD (TSB is produced by BD company, NAD is produced by Roche company) liquid culture medium, and the liquid culture medium is cultured for 12-16 hours at 37 ℃ to serve as a first-grade seed;
and (3) propagation of secondary seeds: adding 1% of the prepared primary seed culture of the JS strain 4 and ZJ strain 5 of the haemophilus parasuis serum into a TSB/NAD liquid culture medium, culturing at 37 ℃ for 12-16 hours, and checking to obtain pure secondary seeds;
adding 0.01-0.05% NAD, 5-10% calf serum (Hangzhou Biotechnology limited company in Zhejiang and 0.1-5% glucose into a TSB culture medium, adding the prepared secondary seed bacterial liquid of the haemophilus parasuis serum 4 type JS strain and 5 type ZJ strain into the culture medium according to the amount of 1% respectively for culture, uniformly mixing, placing at 37 ℃ for culture for 16-18 hours, and stopping culture when the concentration OD600 of the bacterial liquid reaches more than 2.5, the DO value begins to rise and the pH value is reduced to below 6.5.
After the culture is subjected to viable count, adding a 37% formaldehyde solution (Nicoti city Shuangshu chemical Co., Ltd.) according to 0.2% (V/V) of the total amount, inactivating the mixture at 37 ℃ for 24 hours, stirring the mixture for 3-5 times during the inactivation, and keeping the mixture for later use after complete inactivation.
Example 15 preparation of inactivated Mycoplasma hyopneumoniae antigen
Propagation of first-order seeds: after the freeze-dried strain HN0613 (disclosed in the Chinese patent application CN103031258A) is unsealed, a liquid culture medium is inoculated according to the inoculum size of 10 percent, the liquid culture medium is subjected to shaking culture at 37 ℃ for 3-7 days, a culture is harvested when the pH value is reduced from 7.5 to 6.8, and the culture is used as a primary seed after pure inspection;
and (3) propagation of secondary seeds: inoculating the prepared primary seeds into a liquid culture medium according to the inoculation amount of 5%, placing the liquid culture medium at 37 ℃ for shaking culture for 3-7 d, harvesting a culture when the pH value is reduced from 7.5 to 6.8, and taking the culture as secondary seeds after pure inspection;
formulation of liquid medium (1065 ml): 300ml of bovine heart extract, 360ml of ddH2O (redistilled water), pH adjusted to 7.4, and sterilized at 121 ℃ for 15 minutes. The following filter sterilized components were added: 40ml of Hank's balanced salt solution (10X) (10 times concentrated), 10ml of 0.25% phenol red, 200ml of horse serum, 100ml of 5% hydrolyzed milk protein, 20ml of 25% yeast extract, 10ml of 10000IU/ml penicillin, and 25m1 of 1% thallium acetate solution;
inoculating the secondary seed solution into a liquid culture medium at 5% (v/v), placing the secondary seed solution at 37 ℃ for shake culture for 3-7 days, and harvesting the bacterial solution when the pH value is reduced from 7.5 to 6.8.
Taking the qualified mycoplasma hyopneumoniae bacterial liquid, slowly adding a formaldehyde solution (v/v) with the final concentration of 0.2% according to the total volume of the bacterial liquid, placing the mixture at 37 ℃ for inactivation, stirring the mixture once every 3-4 h, taking the mixture out after 24h, and completely inactivating the mixture for later use.
EXAMPLE 16 preparation of vaccine composition containing porcine circovirus type 2HH3 strain
The attenuated swine fever virus antigen prepared in example 9, the attenuated pseudorabies virus antigen prepared in example 10, and the attenuated porcine reproductive and respiratory syndrome virus antigen prepared in example 12 were mixed with a heat-resistant protective agent (2 wt% gelatin aqueous solution and 15 wt% lactose aqueous solution at a ratio of 1:1 (v/v)) at a ratio of 1:1(v/v), and then mixed well, quantitatively packaged, and rapidly subjected to freeze vacuum drying, thereby obtaining a live swine fever virus vaccine part, a live porcine pseudorabies virus vaccine part, and a live porcine reproductive and respiratory syndrome virus vaccine part.
The porcine circovirus type 2HH3 inactivated antigen prepared in the example 4 is slowly added into a water-soluble adjuvant Gel adjuvant (French Saybolt Corp.) and is continuously stirred for 12min by an emulsifying machine with the rotating speed of 800rpm in the adding process, and the mixture is uniformly mixed, so that the porcine circovirus type 2 inactivated vaccine is obtained. In use, the live vaccine portion was diluted with the inactivated vaccine portion, and the ratio of the two antigens was as shown in table 6.
TABLE 6 ingredient ratio of vaccine composition containing porcine circovirus type 2HH3 strain (live vaccine part)
Figure BDA0001317893440000221
The inactivated antigen of porcine circovirus type 2HH3 strain prepared in example 4, the inactivated antigen of porcine pseudorabies virus prepared in example 11, the inactivated antigen of porcine parvovirus prepared in example 13, the inactivated antigen of Haemophilus parasuis prepared in example 14 and the inactivated antigen of Mycoplasma hyopneumoniae prepared in example 15 were respectively prepared according to the final antigen content of the composition in Table 7, added into a water-soluble adjuvant Gel adjuvant (Saebik, France), and stirred by an emulsifying machine with the rotation speed of 800rpm for 12min during the adding process and mixed evenly. The specific formulation and content of the vaccine composition are shown in table 7.
TABLE 7 ingredient ratio of vaccine composition containing porcine circovirus type 2HH3 strain (inactivated vaccine part)
Figure BDA0001317893440000222
Figure BDA0001317893440000231
Example 17 immunogenicity testing of vaccine compositions containing porcine circovirus type 2HH3 Strain antigen
1. Partial immunogenicity assay for porcine circovirus type 2
45 healthy piglets which are detected by ELISA for PCV2 and PCV3 at the age of 28-30 days and are negative to antibodies are randomly divided into 9 groups and 5 groups, and the vaccine composition prepared in the example 16 is immunized. The 31 th to 38 th groups are respectively immune vaccines 5 to 12, and the 39 th group is not immune and is used as a challenge control group. Each immunization group was injected with 2 ml/head of vaccine, and the control group was inoculated with 2 ml/head of DMEM medium. 28 days after immunizationPerforming virus attack with the virus attack dose of HH3 porcine circovirus 105.0TCID50And/or continuously observing each piglet after the virus attack, and judging according to the clinical symptoms, pathological changes and virus detection results of each piglet, wherein the specific results are shown in a table 8.
TABLE 8 partial immunogenicity test results for vaccine composition PCV2 containing porcine circovirus type 2HH3 strain antigen
Figure BDA0001317893440000232
Figure BDA0001317893440000241
The result shows that the vaccine composition containing the HH3 strain of porcine circovirus type 2 antigen can provide 100 percent (5/5) protection for piglets against porcine circovirus type 2 infection after one-time immunization of the piglets, and the piglets in the challenge control group are all attacked by the virus. The porcine circovirus type 2 antigen part in the vaccine composition containing the porcine circovirus type 2HH3 strain provided by the invention has good protective effect, can provide 100% protection for pigs, and virus detection is negative. The porcine circovirus type 2HH3 strain does not interfere with a plurality of viral antigens and bacterial antigens, and can jointly form a vaccine composition for later administration.
2. Partial immunogenicity test for hog cholera
The 20-day-old healthy piglets tested for PCV2, PCV3, CSFV antigen, antibody negative by ELISA were randomly divided into 2 groups of 8, 4/group. Group 40 immunization of vaccine 5 prepared in example 16, and group 41 immunization as a challenge control group. Each 2ml vaccine 5 is diluted 3000 times, intramuscular injection is carried out, each 1ml, and a virus challenge control group is inoculated with 1ml of DMEM culture medium per head. After 10-14 days, 4 control groups with the same conditions were injected with 1.0 ml/head (10) of HCG-GIT5MLD), observed for 16 days. The results are shown in Table 9.
TABLE 9 partial immunogenicity test results for vaccine composition CSFV comprising porcine circovirus type 2HH3 strain antigen
Group of Immune head Rate of protection Clinical symptoms
40 1/3000 100％(4/4) No body temperature reaction
41 DMEM Medium 1ml 0％(0/4) All 4 deaths
According to the standard of Chinese veterinary drug, the immune efficacy of the classical swine fever virus vaccine is detected, the vaccine needs to be diluted by more than 300 times and then is detected, and the result of the embodiment shows that after 1/3000 heads (diluted by 3000 times) of the vaccine composition containing the porcine circovirus type 2HH3 strain antigen are used for immunizing piglets, 100% (4/4) protection can be provided for the piglets aiming at the infection of the classical swine fever virus, no body temperature reaction exists, and all the piglets are attacked and die after the control piglets attacked by the attacked virus. The hog cholera virus antigen part in the vaccine composition containing the porcine circovirus type 2HH3 strain antigen provided by the invention has good protective effect and safety.
3. Partial immunogenicity test for porcine pseudorabies virus
15 healthy piglets which are 9 days old and are detected to be PCV2, PCV3, PRV antigen and antibody negative by ELISA are randomly divided into 3 groups,5 heads/group, group 42 immunization vaccine 6 prepared in example 16, group 43 immunization vaccine 8 prepared in example 16, and group 44 immunization as challenge control group. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. The virus is attacked 21 days after the immunization, and the attacking dose is 10 of porcine pseudorabies virus HN1201 strain7.0TCID50The body temperature of piglets is measured every day after the challenge, and the clinical symptoms and death condition are observed, and the specific results are shown in a table 10.
TABLE 10 partial immunogenicity test results for vaccine compositions containing porcine circovirus type 2HH3 strain antigen
Figure BDA0001317893440000251
The result shows that the vaccine composition containing the porcine circovirus type 2HH3 strain antigen can block virus infection (clinical symptoms) after immunizing piglets, can provide 100 percent (5/5) protection for the piglets aiming at the porcine pseudorabies virus infection, and all the piglets die 4 days after the challenge of the control group of the challenged piglets. The inactivated antigen or the live virus antigen part of the porcine pseudorabies virus in the vaccine composition containing the porcine circovirus type 2HH3 strain antigen has good protective effect.
4. Partial immunogenicity test for porcine reproductive and respiratory syndrome virus
10 healthy piglets which are 4-6 weeks old and are detected to be PCV2, PCV3, PRRSV antigen and antibody negative by ELISA are randomly divided into 2 groups, 5 groups/group, the 45 th group is used for immunizing the vaccine 7 prepared in the example 16, and the 46 th group is used as a challenge control group. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. The virus is attacked 28 days after the immunization, and the attacking dose is 10 of porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain5.0TCID50Head, daily thermometry and observation of clinical symptoms and mortality. The specific results are shown in Table 11.
TABLE 11 partial immunogenicity test results for vaccine compositions PRRSV containing porcine circovirus type 2HH3 strain antigen
Figure BDA0001317893440000261
The results show that the vaccine composition containing the porcine circovirus type 2HH3 strain antigen can provide 100 percent (5/5) protection for piglets against porcine reproductive and respiratory syndrome virus infection after immunization of the piglets, and the piglets in a challenge control group are all attacked and die for 3 times after challenge. The vaccine composition containing the porcine circovirus type 2HH3 strain antigen has good protective effect on the porcine reproductive and respiratory syndrome virus antigen part.
5. Partial immunogenicity assay for porcine parvovirus
10-20 kg of healthy piglets which are detected to be PCV2, PCV3, PPV antigen and antibody negative by ELISA are randomly divided into 2 groups, 5 groups/group, 47 groups are used for immunizing the vaccine 9 prepared in the example 16, and 48 groups are used for immunizing as blank control groups. The immunization group was injected with 2 ml/head of vaccine, and the blank control group was inoculated with 2 ml/head of DMEM medium. And (3) collecting blood together with a blank control group 28 days after immunization, and measuring the antibody, wherein the blank control group is negative (HI antibody titer is less than or equal to 1:8), at least 4 heads of the immunized group have antibody reaction, and the HI antibody titer is more than or equal to 1: 64. The specific results are shown in Table 12.
TABLE 12 partial immunogenicity test results for vaccine compositions PPV containing porcine circovirus type 2HH3 strain antigen
Figure BDA0001317893440000271
The results show that the HI antibodies of PPV are more than 1:64 after the piglet is immunized by the vaccine composition containing the porcine circovirus type 2HH3 strain antigen, and can provide 100% (5/5) protection for the piglet against porcine parvovirus infection. The porcine parvovirus antigen part in the vaccine composition containing the porcine circovirus type 2HH3 strain antigen provided by the invention has good protective efficacy.
6. Partial immunogenicity assay for haemophilus parasuis
30 healthy piglets which are 14-21 days old and have PCV2, PCV3 and PCV HPs antigen and antibody negativity detected by ELISA are randomly divided into 6 groups, 5 groups, 49 groups and 50 groups16, group 51 and group 52 immunization of vaccine 12 prepared in example 16, and group 53 and group 54 immunization as challenge control. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. 35 days after immunization, 49 th group and 51 th group of the immunization group and 53 th group of the challenge control group are taken, 4 type JS strain is used for challenge, 3ml bacterial liquid is injected into the abdominal cavity, and the challenge dosage is 9.0 multiplied by 109CFU/head; collecting the 50 th group, the 52 th group and the 54 th group of the challenge control group of the immunization group, performing challenge by using the 5 type ZJ strain, injecting 3ml of bacterial liquid into the abdominal cavity, wherein the dose of the challenge is 6.0 multiplied by 109CFU/head; the clinical manifestations of the pigs were observed after the challenge, and the pigs were killed 14 days later for pathological observation. The results are shown in Table 13.
TABLE 13 partial immunogenicity test results for vaccine composition HPs containing porcine circovirus type 2HH3 strain antigen
Figure BDA0001317893440000272
Figure BDA0001317893440000281
Note: the pathogenic standard of haemophilus parasuis disease: the sick pigs have clinical symptoms of fever (body temperature of more than 40.5 ℃ and lasting for 1-5 days), listlessness, cough, dyspnea, emaciation, lameness, rough fur and the like. The autopsy of the dying pig shows the pathological changes of multiple serositis (pleuritis, pericarditis and peritonitis), arthritis, meningitis and the like, and serous or cellulosic exudates appear on each serosal surface (joint capsule, pericardium, pleura and peritoneum).
The result shows that the vaccine composition containing the porcine circovirus type 2HH3 strain antigen can provide 100 percent (5/5) protection for piglets infected by haemophilus parasuis type 4 and type 5 bacteria after immunizing the piglets, and the piglets in a control group with the virus challenge are all attacked. The result shows that the haemophilus parasuis antigen part in the vaccine composition containing the porcine circovirus type 2HH3 strain antigen has good protective effect.
7. Partial immunogenicity assay for Mycoplasma hyopneumoniae
15 healthy piglets which are 14-21 days old and have PCV2, PCV3, Mhp antigen and negative antibody detected by ELISA are randomly divided into 3 groups, 5 groups/group, 55 groups of vaccine 11 prepared in the immunization example 16, 56 groups of vaccine 12 prepared in the immunization example 16 and 57 groups of non-immunized animals as an attack control group. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. 35 days after immunization, the 55 th group and the 56 th group of the immune group and the 57 th group of the virus attack control group are taken, 5 ml/head (100MID) is injected by CVCC354 strain (purchased from Chinese veterinary medicine institute, the strain is strain for mycoplasma hyopneumoniae vaccine efficacy test stored in Chinese veterinary medicine institute) through a trachea, 28 days after virus attack is observed, lungs are cut and taken, the pneumonopathy and the pneumonia lesion of the test pig are scored according to the 28-division method, and the pneumonia lesion reduction rate is calculated according to the following formula. The specific results are shown in Table 14.
The pneumonia lesion reduction rate is (average score of virus attacking control pig pneumonia lesions-average score of immune pig pneumonia lesions)/average score of virus attacking control pig pneumonia lesions is multiplied by 100%
TABLE 14 partial immunogenicity test results for vaccine compositions containing porcine circovirus type 2HH3 strain antigen
Group of Number of piglets Mean lung lesion index ± standard deviation Reduction rate of pneumonia lesions
55 5 1.8±1.14b 84.6％
56 5 2.0±0.76b 85.4％
57 5 11.7±3.94a /
Note: in the difference statistical analysis, compared among groups, the difference is not significant when the letters are the same, and the difference is significant when the letters are different (P < 0.05)
The results show that after the piglet is immunized by the vaccine composition containing the porcine circovirus type 2HH3 strain, the average lung lesion of each immune group is remarkably different from that of an attack control group in comparison with the infection control group aiming at the mycoplasma hyopneumoniae infection. The mycoplasma hyopneumoniae antigen part in the vaccine composition containing the porcine circovirus type 2HH3 strain antigen provided by the invention has good protective effect.
In conclusion, from the results of immunogenicity tests of the different vaccine compositions against PCV2, CSFV, PRV, PRRSV, PPV, HPs type 4 bacteria, HPs 5 type bacteria and Mhp, the vaccine compositions can achieve good protection effects, and the different antigen components in the vaccine composition containing the porcine circovirus type 2HH3 strain antigen provided by the invention have good protection effects. The inactivated antigen of the porcine circovirus type 2HH3 strain antigen can be jointly applied with different pathogenic antigen components to form an immune composition.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (17)

1. Porcine Circovirus type 2HH3 strain (Porcine Circovirus type 2, strain HH3) which is preserved in China center for type culture Collection with the preservation number of CCTCC NO. V201726, the preservation date of 2017, 6 months and 4 days, the preservation address: wuhan, Wuhan university in China.
2. A vaccine composition comprising an immunizing amount of the antigen of porcine circovirus type 2HH3 according to claim 1 and a pharmaceutically acceptable carrier; the porcine circovirus type 2HH3 strain antigen is porcine circovirus type 2HH3 strain or whole virus inactivated antigen of a culture thereof, and the porcine circovirus type 2HH3 strain culture is a culture of 5-48 generations.
3. The vaccine composition according to claim 2, wherein the inactivated whole virus antigen content of the porcine circovirus type 2HH3 strain or culture thereof is not less than 10 before inactivation5.0TCID50/ml。
4. The vaccine composition according to claim 2, wherein the inactivated whole virus antigen content of the porcine circovirus type 2HH3 strain or culture thereof is 10 before inactivation5.0～107.0TCID50/ml。
5. The vaccine composition according to claim 2, wherein the inactivated whole virus antigen content of the porcine circovirus type 2HH3 strain or culture thereof is 10 before inactivation6.0TCID50/ml。
6. The vaccine composition of claim 2, wherein the pharmaceutically acceptable carrier is an adjuvant comprising Gel adjuvant; the content of the adjuvant is 5-20V/V%.
7. The vaccine composition of claim 2, wherein the adjuvant is a Gel adjuvant.
8. The vaccine composition according to claim 2, wherein the adjuvant content is 10V/V%.
9. The vaccine composition according to claim 2, wherein the vaccine composition further comprises one or more of the group consisting of an immunizing amount of: the antigen comprises a classical swine fever virus attenuated antigen, a porcine pseudorabies virus attenuated antigen, a porcine reproductive and respiratory syndrome virus attenuated antigen, a porcine parvovirus inactivated antigen, a haemophilus parasuis inactivated antigen and a mycoplasma hyopneumoniae inactivated antigen.
10. The vaccine composition according to claim 2, wherein the vaccine composition further comprises one or more of the group consisting of an immunizing amount of: the antigen comprises a classical swine fever virus attenuated antigen, a porcine pseudorabies virus inactivated antigen, a porcine reproductive and respiratory syndrome virus attenuated antigen, a porcine parvovirus inactivated antigen, a haemophilus parasuis inactivated antigen and a mycoplasma hyopneumoniae inactivated antigen.
11. The vaccine composition according to claim 10, wherein the classical swine fever virus attenuated antigen is a classical swine fever virus lapinized attenuated strain, the porcine pseudorabies virus inactivated antigen is an HN1201 strain inactivated whole virus antigen, the porcine reproductive and respiratory syndrome virus attenuated antigen is an attenuated strain of JXA1-R strain, the porcine parvovirus inactivated antigen is an HN-2011 strain inactivated whole virus antigen, the haemophilus parasuis inactivated antigen is an inactivated whole virus antigen of JS strain type 4 or ZJ strain type 5 of haemophilus parasuis serum, and the porcine mycoplasma pneumoniae inactivated antigen is an HN0613 strain inactivated whole virus antigen.
12. The vaccine composition according to claim 9, wherein the porcine pseudorabies virus attenuated antigen is an attenuated strain of HN1201-R strain.
13. The vaccine composition according to claim 9, wherein the inactivated whole virus antigen content of the porcine circovirus type 2HH3 strain or culture thereof is 10 before inactivation5.0～107.0TCID50The attenuated antigen content of the hog cholera virus is 10ml4.0～106.0TCID50Per ml, the content of the porcine pseudorabies virus attenuated antigen is 106.0～107.0TCID50The porcine reproductive and respiratory syndrome virus attenuated antigen content is 105.0～107.0TCID50The inactivated antigen content of the porcine parvovirus is 10 before inactivation6.0～108.0TCID50Per ml, the content of the inactivated antigen of the haemophilus parasuis is 10 before inactivation8.0～1010.0CFU/ml, the content of the mycoplasma hyopneumoniae inactivated antigen is 10 before inactivation8.0～1010.0CCU/ml。
14. The vaccine composition according to claim 10, wherein the content of the inactivated antigen of the porcine pseudorabies virus is 10 before inactivation6.0～107.0TCID50/ml。
15. The vaccine composition according to claim 9, wherein the inactivated whole virus antigen content of the porcine circovirus type 2HH3 strain or culture thereof is 10 before inactivation5.0～107.0TCID50The attenuated antigen content of the hog cholera virus is 10ml5.0TCID50Per ml, the content of the porcine pseudorabies virus attenuated antigen is 106.0TCID50The porcine reproductive and respiratory syndrome virus attenuated antigen content is 106.0TCID50The inactivated antigen content of the porcine parvovirus is 10 before inactivation7.0TCID50The content of the inactivated antigen of the haemophilus parasuis is 10 before inactivation9.0CFU/ml, the content of the mycoplasma hyopneumoniae inactivated antigen is 10 before inactivation9.0CCU/ml。
16. The vaccine composition according to claim 10, wherein the content of the inactivated porcine pseudorabies virus antigen is 10 before inactivation6.0TCID50/ml。
17. Use of a vaccine composition according to any one of claims 2 to 16 in the preparation of a medicament for the prevention of diseases associated with porcine circovirus type 2 infection, wherein the diseases associated with porcine circovirus type 2 include postweaning multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, reproductive disorders, and cardiac and multisystemic inflammatory responses.
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