CN112386685A - PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccine - Google Patents

Abstract

The invention belongs to the field of veterinary vaccines, and particularly relates to a porcine circovirus type2 baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccine, and further discloses a preparation method and application thereof. The tetrad inactivated vaccine contains Cap protein expressed by inactivated porcine circovirus type2 baculovirus, inactivated mycoplasma hyopneumoniae, inactivated swine influenza virus H1N1 subtype virus, inactivated haemophilus parasuis types 4, 5 and 13 and an adjuvant of the vaccine; the four antigens are not interfered, four kinds of protection of one-needle immunization can be realized, and four kinds of epidemic diseases can be prevented by one-time immunization; meanwhile, from the level of toxicity attacking protection and serum antibodies, the immune effect reaches or exceeds the level of single seedlings of each commodity, the immune duration is long, the efficacy is durable, and the preparation method has the advantages of high safety, simplicity in preparation method, convenience in immunization, reduction in immunization cost and the like.

Description

PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccine

Technical Field

The invention belongs to the field of veterinary vaccines, and particularly relates to a PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccine, and further discloses a preparation method and application thereof.

Background

Porcine circovirus, also known as porcine 'aids', completely destroys the immune system of infected pigs, loses immune response to other vaccines, leads to immune failure, and leads to secondary infection by other pathogens. Wherein, according to statistics, the secondary infection caused by the porcine circovirus clinically usually exceeds 50 percent, and the death rate reaches more than 40 percent. Among them, Porcine circovirus type2 (PCV 2) can cause piglet weaning multisystemic wasting syndrome (PMWS), Porcine respiratory disease syndrome (PRDC), Porcine skin and kidney disease syndrome (PDNS), sow reproductive disorders, etc., causing great economic loss to the swine industry all over the world. The domestic pig farm prevalence is mainly PCV2a subtype, and simultaneously, a partial PCV2b subtype strain exists, and certain cross protection exists between the two strains, but the two strains cannot be completely protected. PCV2 genome is 1767/1768bp in full length, and has 11 Open Reading Frames (ORFs), wherein ORF2 encodes the unique structural protein nucleocapsid protein (CAP) of virus, the protein consists of 234 amino acids, has the molecular weight of 28KD, can self-assemble into virus-like particles, is the main immunogenic protein and can induce specific PCV2 neutralizing antibody (Pogranchnyy et al, 2000), therefore, ORF2 gene is the preferred target gene for designing PCV2 novel vaccine.

The Mycoplasma hyopneumoniae is a contact chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae (Mycoplasma hyopneumaniae), is also called mycoplasmosis, has the characteristics of high morbidity and low fatality rate, belongs to two types of animal epidemic diseases in China, is widely popularized in the world, is difficult to control once infected in a pig farm, causes huge economic loss to the pig industry in China every year, and is one of important diseases causing economic loss of modern pig industry. The mycoplasma pneumonia of the pigs mainly shows symptoms of anorexia, fever, cough, asthma, dyspnea and the like, the sick pigs grow slowly, and the feed conversion rate is reduced. During dissection, the lung is mainly affected, especially the two lobes of the heart, the middle lobe and the tip lobe, which are characterized by pancreas-like and flesh-like changes. Mycoplasma hyopneumoniae is not only one of the important etiological agents of Porcine respiratory disease syndrome, and infection alone often causes mild chronic pneumonia, and when mixed with other etiological agents, respiratory disease becomes severe, causing Porcine respiratory disease syndrome (PRDC). Numerous studies have shown that mycoplasma hyopneumoniae has a synergistic effect with other pathogens in infection, and in most cases, the pathogen that synergizes mycoplasma hyopneumoniae infection can increase the severity and potential persistence of the associated disease.

The Swine Influenza (SI) is called swine influenza for short, and is an acute febrile and highly contact mass-produced respiratory infectious disease of swine caused by Swine Influenza Virus (SIV) of influenza virus of Orthomyxoviridae. Clinical symptoms are common, such as sudden cough, dyspnea, fever, anorexia, rhinorrhea and the like, the morbidity of the pig herds is extremely high, and can reach 100 percent at most, but the mortality is extremely low. The Swine influenza is mainly caused by Swine Influenza Virus (SIV) type A, and according to literature reports, domestic Swine influenza serum subtypes are mainly H1N1, H1N2, H1N7, H3N2, H3N6, H5N1 and H9N2, while H1N1 and H3N2 subtypes are more prevalent among a plurality of serotypes, but H1N1 is the most dominant. The swine influenza is a common epidemic disease in the world, and although the swine influenza can not cause large-area death of pigs, the swine influenza can cause the reduction of the production performance of the sick pigs, so that the breeding of the pigs is hindered, the weight gain of fattening pigs is slowed down, the feed conversion ratio is reduced, the time to market is delayed, and the economic benefit of farmers is influenced. In addition, researches show that the attack of swine influenza virus on respiratory epithelial cells of a host can cause the immunity to be reduced, and pigs are easily infected by other pathogens such as mycoplasma pneumoniae, plasmodium pleuropneumoniae, pasteurella, haemophilus parasuis, porcine reproductive and respiratory syndrome virus and secondary infection or mixed infection of porcine circovirus, so that the clinical symptoms are aggravated, and the death rate is increased.

Haemophilus parasuis (Hps) is also called pig Pirass disease, is a bacterial infectious disease of multiple serositis, arthritis and meningitis of pigs caused by Haemophilus parasuis, commonly exists in swine herds in China, and becomes a great important bacterial disease affecting the swine industry in the global range. The main clinical symptoms of the traditional Chinese medicine composition are fever, depression, appetite reduction or anorexia, dyspnea, emaciation, lameness, rough hair and the like; the autopsy lesions are mainly manifested by cellulosic pleurisy, pericarditis, peritonitis, arthritis, meningitis and the like. The disease is not obvious in seasonality and is mostly endemic, young pigs of 2 weeks to 4 months old are mainly damaged, the disease is mainly caused after weaning and in the stage of conservation, the disease death rate of the pigs of 5 to 8 weeks old is up to 50 percent in serious conditions, the disease rate of the pigs in the early stage of fattening is 20 percent, and the abortion or death can be induced by the attack of sows, so that serious economic loss can be caused. The haemophilus parasuis has a large number of heterologous genes, serology is relatively complex, at present, at least the haemophilus parasuis can be divided into 15 serotypes according to an agar diffusion serotyping method, and more than 20 percent of the serotypes can not be determined, wherein the serotypes 1, 5, 10, 12, 13 and 14 are the most virulent, the serotypes 2, 4 and 15 are the mesogenic, and the serotypes 3, 6, 7, 8, 9 and 11 are non-virulent. According to seroepidemiological investigations in countries such as the united states, germany, canada, japan and spain, type 4, type 5, and type 13 are most prevalent. The pathogenicity of HPS varies greatly between serotypes, and immune cross-protection is less between serotypes or strains. Therefore, the necessary requirement for preventing and controlling the haemophilus parasuis disease when the vaccine with high-efficiency cross protection is developed is met.

At present, the main methods for preventing and controlling porcine circovirus type2 (PCV 2) virus diseases, mycoplasma hyopneumoniae and haemophilus parasuis diseases and resisting swine influenza virus are vaccination, and the vaccines used in the domestic market are mainly PCV2 single vaccine, mycoplasma hyopneumoniae single vaccine, haemophilus parasuis single vaccine and swine influenza virus vaccine. Since swine influenza rarely causes death of swine when infected alone, it has not received sufficient attention in China.

At present, the porcine circovirus type2 vaccine is mainly PCV2 whole virus inactivated vaccine, PCV1-PCV2 mosaic virus inactivated vaccine, baculovirus expression PCV2 genetic engineering vaccine or Escherichia coli expression PCV2 genetic engineering vaccine. The whole virus inactivated vaccine has short stimulation time to organisms, needs 2 times of inoculation, and has low proliferation titer of PCV2 on cells and high preparation cost; compared with other vaccines, the genetic engineering subunit vaccine has the advantages that the vaccine antibody is generated earlier, the antibody level is stable, the replication of the virus can be effectively prevented, and the protective power is strong. In particular, the subunit vaccine of CAP protein expressed by baculovirus is a hot spot of domestic research, and the expressed protein is completely modified, is close to the natural protein of virus, and has good immunogenicity, so that the system becomes the first choice for developing the subunit vaccine of genetic engineering. However, the genetic engineering subunit vaccine has high requirements on antigens and adjuvants, so that the search for an adjuvant with lasting effectiveness and little side effect is very critical. In adjuvant materials in the prior art, water adjuvant vaccines are favored in the market, but the controlled and sustained release effect of the water adjuvant is not as good as that of an oil adjuvant, so that the matching of a long-acting controlled and sustained release immunopotentiator is very necessary.

At present, no combined vaccine commodities of porcine circovirus type2, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis exist in China, and the invention fills the blank of independent research and development of the porcine circovirus type2, mycoplasma hyopneumoniae, swine influenza virus (H1N 1 subtype) and haemophilus parasuis (type 4, type 5 and type 13) quadruple inactivated vaccine in China.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus (H1N 1 subtype), haemophilus parasuis (4 type, 5 type and 13 type) quadruple inactivated vaccine with high immunogenicity, no potential safety hazard and low immune cost, and the inactivated vaccine can be used for simultaneously preventing porcine circovirus disease, swine mycoplasma pneumonia, swine influenza virus and haemophilus parasuis disease;

the second technical problem to be solved by the present invention is to provide a method for preparing the PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis (type 13) quadruple inactivated vaccine.

In order to solve the technical problems, the invention provides a PCV2 type baculovirus vector, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccine, which is characterized by comprising an inactivated PCV2 type CAP protein antigen, an inactivated mycoplasma hyopneumoniae antigen, an inactivated swine influenza virus antigen, an inactivated haemophilus parasuis antigen and a vaccine adjuvant; wherein the content of the first and second substances,

the PCV2 type CAP protein antigen is a CAP protein antigen which is expressed efficiently by SF9 or highfive cell infected after being cloned and transfected in a pF astBac1 transfer vector, wherein the PCV2 type CAP protein antigen is a PCV2a and PCV2b isolate, codon is modified into a PCV2 ORF2 sequence of a bias codon of an insect cell baculovirus expression system by artificial synthesis codon optimization on the premise of keeping amino acid unchanged, EcoRI and XhoI enzyme cutting sites are introduced at two ends of the sequence, and the sequence is inserted into the pF astBac1 transfer vector by double enzyme cutting.

Specifically, the PCV2 type CAP protein antigen comprises PCV2a type CAP protein and PCV2b type CAP protein;

the encoding gene of the PCV2a type CAP protein has a nucleotide sequence shown as SEQ ID No.1, and the PCV2a type CAP protein has an amino acid sequence shown as SEQ ID No. 2;

the encoding gene of the PCV2b type CAP protein has a nucleotide sequence shown as SEQ ID No.3, and the PCV2b type CAP protein has an amino acid sequence shown as SEQ ID No. 4.

Preferably, the concentration ratio of the PCV2a type CAP protein to the PCV2b type CAP protein is 1: 1.

specifically, the mycoplasma hyopneumoniae antigen is a mycoplasma hyopneumoniae DJ-166 virus strain with the preservation number of CGMCC NO.4545, and is obtained by proliferation, concentration, purification and inactivation of an improved CH culture medium.

Specifically, the swine influenza virus antigen is a swine influenza virus H1N1 subtype antigen obtained by propagating, concentrating, purifying and inactivating a swine influenza virus H1N1 subtype virus strain.

Specifically, the haemophilus parasuis antigen serum comprises haemophilus parasuis 4 type, 5 type and 13 type antigens obtained by proliferation, concentration, purification and inactivation of JS01 type 4 strain, FJ04 type 5 strain and HN01 type 13 strain respectively.

Specifically, the four-combined inactivated vaccine comprises:

the content of Cap protein expressed by the PCV2 type baculovirus is more than or equal to 8 mug/first part;

the content of mycoplasma hyopneumoniae antigen is more than or equal to 80 mug/part;

the swine influenza virus HA is more than or equal to 7log2 per head;

the content of antigens of the haemophilus parasuis serotype 4, 5 and 13 is more than or equal to 1.0 multiplied by 10⁸CFU per head.

Preferably, in the quadruple vaccine, the content of Cap protein expressed by PCV2 type baculovirus is 30-100 mug/head, and mycoplasma hyopneumoniaeThe body content is 80-200 mug/head, the swine influenza virus content is 7log2-10log2, and the haemophilus parasuis content is 1.0 x 10⁸-1.0×10¹⁰CFU per head.

Preferably, in the preparation method of the quadruple inactivated vaccine, the content of the PCV2 type Cap protein is at least 10 mug/head part, the content of the mycoplasma hyopneumoniae protein is at least 80 mug/head part, the content of the swine influenza virus HA is at least 8log 2/head part, and the content of the haemophilus parasuis is at least 1.0 x 10⁸CFU per head.

As a preferred embodiment, the vaccine comprises: the PCV2 type Cap protein content is 10 mug/head part, the mycoplasma hyopneumoniae protein content is 100 mug/head part, the swine influenza virus HA content is 8log 2/head part, and the haemophilus parasuis content is 2.0 x 10⁸CFU per head.

Most preferably, the quadruple inactivated vaccine contains 10 mug of PCV2 type Cap protein per head, 150 mug of mycoplasma hyopneumoniae protein per head, 9log2 of HA content of swine influenza virus H1N1 type, and 1.0 x 10 haemophilus parasuis⁹CFU/head, adjuvant 61VG, adjuvant 50% (V/V) of the total vaccine amount.

Specifically, the vaccine adjuvant comprises an aluminum salt adjuvant, a mineral oil adjuvant or a water-soluble adjuvant.

Specifically, the adjuvant comprises an alumina gel adjuvant, a 61VG adjuvant, a1313 adjuvant and an ISA251C adjuvant. Preferably, the vaccine adjuvant is 61VG adjuvant and/or ISA1313 adjuvant, more preferably 61VG, in an amount of 50% (V/V) of the total amount of the vaccine.

Preferably, the vaccine inactivator is BEI, thimerosal and formaldehyde solution.

Preferably, the inactivation agent of Cap protein expressed by the PCV2 type baculovirus is BEI (diethylene imine), the inactivation agent of Mycoplasma hyopneumoniae is thimerosal, the inactivation agent of the swine influenza virus is BEI (diethylene imine), and the inactivation agent of the haemophilus parasuis is formaldehyde solution.

In a preferred embodiment, the four-way inactivated vaccine comprises: 10 mug/head of Cap protein expressed by PCV2 type baculovirus, 150 mug/head of mycoplasma hyopneumoniae,9log2 of swine influenza virus and 1.0X 10 of haemophilus parasuis⁹CFU/head, 61AG adjuvant, to make up 50% (V/V) of the total vaccine.

The invention also discloses a method for preparing the quadruple inactivated vaccine, which comprises the steps of respectively inactivating and concentrating the PCV2 type CAP protein antigen, the mycoplasma hyopneumoniae antigen, the swine influenza virus antigen and the haemophilus parasuis antigen serum, and mixing the inactivated vaccine with the vaccine adjuvant according to a selected ratio.

Preferably, the vaccine also comprises a step of adding astragalus polysaccharide at the same time of adding the vaccine adjuvant, and the adding amount of the astragalus polysaccharide accounts for 0.5v/v% of the total amount of the vaccine.

Specifically, the preparation method of the quadruple vaccine further comprises a step of obtaining the PCV2 type CAP protein antigen, and specifically comprises the following steps:

(1) referring to the CAP protein sequence of the separated strains of the known PCV2a and PCV2b subtypes, under the premise of keeping amino acid unchanged, modifying codons into biased codons of an insect cell baculovirus expression system, introducing EcoRI and XhoI enzyme cutting sites at two ends of the sequence, and inserting the biased codons into a pF astBac1 transfer vector through double enzyme cutting to respectively obtain recombinant baculovirus transfer vectors pFastBac1-ORF2a and pFastBac1-ORF2 b;

(2) transforming the obtained recombinant baculovirus transfer vector into DH10 Bac. Coli, and carrying out homologous recombination to obtain recombinant baculovirus DNA;

(3) transfecting recombinant baculovirus DNA into SF9 cells, packaging to generate recombinant baculovirus DBN01a and DBN01b strains expressing PCV2 CAP protein;

(4) infecting SF9 or high five cells with the obtained recombinant baculovirus DBN01a and DBN01b strains to obtain recombinant PCV2 CAP protein virus-like particles, and inactivating the virus when the expression level is not lower than 200 mu g/ml through detecting the PCV2 CAP protein concentration;

(5) separating and purifying the recombinant PCV2 type CAP protein to obtain the recombinant PCV2 type CAP protein.

Specifically, in the step (4), SF9 or high five is suspension cultured in a bioreactor serum-free culture mediumCells, cell density 2.0X 10⁶-2.5×10⁶And/ml, inoculating the recombinant baculovirus in the step (3) according to the inoculation amount with the multiplicity of infection (MOI) of 0.5-5.0, and harvesting after culturing for 5-7 days.

Preferably, the culture parameters of the bioreactor are controlled to be set to be pH6.2-7.2, the temperature is 27 ℃, the dissolved oxygen is 20% -60%, and the stirring speed is 90-160 rpm.

Specifically, the bioreactor comprises 5L, 10L, 50L and 500L culture modes, and adopts a batch culture method, a fed-batch culture method or a combination of the batch culture method and the fed-batch culture method.

Specifically, in the step (4), the PCV2 CAP protein concentration is determined, the harvested cell culture is subjected to SDS-PAGE detection with standard protein (BSA), and the Cap protein concentration is determined by gray scale analysis according to the standard protein concentration.

Specifically, BEI is used in the inactivation step, the concentration is 0.1% -5%, the inactivation temperature is 30-37 ℃, the inactivation time is 24-48 h, and after inactivation, excessive BEI is neutralized by sodium thiosulfate. Preferably, the BEI inactivation concentration is 0.1%, and the BEI inactivation is carried out for 24 hours at 37 ℃.

Specifically, the preparation method of the tetrad vaccine comprises the following steps:

the method also comprises a step of obtaining the mycoplasma hyopneumoniae antigen, and specifically comprises the following steps: inoculating the mycoplasma hyopneumoniae virus strain into an improved CH liquid culture medium, culturing for 3-4 days under the condition of 20% oxygen introduction in a fermentation tank at 37 ℃, and determining the titer of viable bacteria to be not less than 10 by harvesting and determining¹¹CCU/ml, and purifying to obtain;

and/or the presence of a gas in the gas,

the method also comprises a step of obtaining the haemophilus parasuis antigen, and specifically comprises the following steps: respectively inoculating the haemophilus parasuis JS01 strain, FJ04 strain and HN01 strain into TBS liquid culture medium, culturing for 12-15h under the condition of a fermentation tank at 37 ℃ and 150rpm, harvesting bacterial liquid when the number of viable bacteria is not less than 50 hundred million by harvesting and measuring, and purifying to obtain the haemophilus parasuis strain;

and/or the presence of a gas in the gas,

the method also comprises a step of obtaining the swine influenza virus antigen, and specifically comprises the following steps: respectively inoculating the H1N1 subtype and H3N2 subtype virus strains of the swine influenza virus to a TPCK-pancreatin serum-free suspension culture medium, culturing at 33 ℃, at the rotating speed of 100r/min and at the Dissolved Oxygen (DO) value of 50%, harvesting bacterial liquid, and purifying to obtain the swine influenza virus.

The CH liquid culture medium comprises the following specific components:

solution A: mixing brain and heart leachate 2.0g, PPLO broth 5.0g, and deionized water 300ml, stirring to dissolve completely, autoclaving at 116 deg.C for 20 min, and cooling;

and B, liquid B: 5.0ml of 10 xHank's solution, 1.0g of lactalbumin hydrolysate, 5.0g of yeast extract, 0.8g of sodium pyruvate, 3.0g of peptone, 0.1g of sodium thiosulfate, 0.1% phenol red, 400U/ml of penicillin and 545ml of deionized water; mixing the above components, stirring, filtering with 0.22um filter membrane for sterilization, and storing at 4 deg.C;

and C, liquid C: 140ml of healthy horse serum;

and (3) fully and uniformly mixing the solution A, the solution B and the solution C, and adjusting the pH value to 7.6 by using 1mol/L sodium hydroxide to obtain the required mycoplasma hyopneumoniae modified CH liquid culture medium.

Preferably, the inoculation proportion of the mycoplasma hyopneumoniae is 8% -10%.

Specifically, clarified cell cultures were 3-5 fold concentrated and purified using 50-100kD ultrafiltration membrane packs.

Specifically, the preparation method of the PCV2 baculovirus and Mycoplasma hyopneumoniae bivalent inactivated vaccine comprises the following purification steps:

(1) collecting fermentation liquid, concentrating to 1/10 with 50KD membrane package, centrifuging at 10000r/min at 4 deg.C for 60 min, suspending the precipitated bacteria with Tris-NaCl buffer solution with pH of 7.2-7.4, centrifuging and washing for 3 times to obtain 1/100 bacteria suspension with original culture volume;

(2) adding the concentrated and purified bacterial suspension into a thimerosal solution with the concentration of 1.0 percent to ensure that the final concentration is 0.01 percent, uniformly mixing, and inactivating for 12 hours at the temperature of 2-8 ℃;

(3) and (4) measuring the protein concentration by using a BCA kit, wherein the protein concentration of 1/100 concentrated bacterial liquid is more than or equal to 4 mg/ml.

PCV2 typeBaculovirus vectors, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccines contain Cap protein expressed by inactivated PCV2 type baculovirus, inactivated mycoplasma hyopneumoniae, inactivated swine influenza virus H1N1 subtype virus, inactivated haemophilus parasuis type 4, 5 and 13 and adjuvants of the vaccines; the PCV2 type baculovirus vector is PCV2 type isolate, comprises PCV2a type and PCV2b type subtype strains, and CAP protein codon is optimized by insect cell baculovirus codon, artificially synthesized PCV2 ORF2 sequence with optimized codon, SF9 cell is cloned and transfected to obtain recombinant baculovirus, and then SF9 or highfive cell is infected to express CAP protein virus-like particles (VLPs) with high efficiency, thus improving the immunogenicity of protein; the protein expression level is not lower than 200 mu g/ml, the protein expression amount is high, and the lowest use amount of each head part is 8 mu g/head part; the mycoplasma hyopneumoniae is a DJ-166 strain, the immunogenicity is good, and the content of the mycoplasma hyopneumoniae is not less than 80 mug/part; the swine influenza virus HAs an HA content of at least 7log2 per serving; the content of Haemophilus parasuis is at least 1.0 x 10⁸CFU per head. The four antigens of the quadruple vaccine are free of interference, four kinds of protection of one-needle immunization can be realized, four kinds of epidemic diseases such as porcine circovirus disease, swine mycoplasmal pneumonia, swine influenza virus (H1N 1 subtype) and swine haemophilus parasuis disease (type 3, type 5 and type 13) can be prevented by one-time immunization, the labor, the material resources and the financial resources are saved, the stress response of animals is effectively reduced, and the immune paralysis and the immune failure caused by frequent immunization are avoided; meanwhile, from the level of toxicity attacking protection and serum antibodies, the immune effect reaches or exceeds the level of each single seedling of each commodity, and the immune dose is 2.0 ml/head, so that the 2-time immune effect of each single seedling can be achieved; moreover, the immune duration is long, the efficacy is lasting for at least 4 months aiming at PCV2 type, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis, and the immune preparation method has the advantages of good safety, simple preparation method, convenient immunization, reduction of immunization cost and the like.

The PCV2 type baculovirus vector, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccine can effectively enhance the immune efficacy and the controlled-release performance by adding the astragalus polysaccharide on the basis of the water-soluble nano adjuvant so as to promote the generation of antibodies and enhance the immune system. Astragalus polysaccharide is used as an interferon inducer, can increase the number of E-ring forming cells by stimulating the functions of macrophages and T cells, induce cytokines and promote interleukin induction to enable an animal body to generate endogenous interferon so as to achieve the aim of resisting virus, can be used as an immune promoter or a regulator to enhance the nonspecific immune function and the humoral immune function, remarkably enhance the phagocytic function of mouse macrophages, increase the number of E-ring forming cells, induce cytokines and promote interleukin induction to enable the animal body to generate endogenous interferon, and further achieve the aim of resisting virus. According to the tetravaccine disclosed by the invention, the water adjuvant is screened and compounded with the astragalus polysaccharide, so that the problem that the controlled-release effect of the water adjuvant is not as good as that of an oil adjuvant is effectively solved, the generation time, the antibody level and the duration time of the antibody of the vaccine are greatly improved, the antibody level is obviously improved, the time for generating a protective antibody is one week earlier than that for not adding, and the tetravaccine has a good prevention effect on the infection of PCV2 type, mycoplasma hyopneumoniae, H1N1 subtype swine influenza virus and Haemophilus parasuis types 4, 5 and 13.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 is a diagram showing the identification of the recombinant baculovirus transfer vector pFastBac1-ORF2b of the present invention;

FIG. 2 shows PCR identification of recombinant bacmid;

FIG. 3 is a diagram of the lesion map of SF9 and the map of healthy SF9 cells after infection with recombinant baculovirus;

FIG. 4 is an electron micrograph of virus-like particles (VLPs) formed by CAP protein expressed in the present invention;

FIG. 5 is an SDS-PAGE grayscale quantification and specificity detection graph of PCV2 b-type CAP protein;

FIG. 6 is a flow chart of a method for preparing the quadruple inactivated vaccine according to the present invention;

FIG. 7 shows the difference between the PCV2 type protein which is genetically modified and genetically codon modified according to the invention and the conventional PCV2 type protein which is not genetically modified and genetically codon modified.

Detailed Description

Description of the sources of the strains

The mycoplasma hyopneumoniae strain is DJ-166 strain with the preservation number of CGMCCNo.4545.

The swine influenza virus H1N1 subtype is DBN-HB2 strain, and is separated and identified by Beijing Dabei agricultural technology group member company.

The haemophilus parasuis serotype 4 strain was JS01 strain, serotype 5 was FJ04 strain, serotype 13 was HN01 strain, and isolated and identified by beijing north agrotechnology group ltd.

Example 1 construction of recombinant baculovirus of PCV2 type

Optimization and synthesis of target gene

The nucleotide sequence of PCV2 type ORF2b gene is designed by referring to PCV2b subtype DBN-SX07 strain, and the original nucleotide sequence of PCV2 subtype strain ORF2b gene is shown as SEQ ID No.5 (702 bp), and the original amino acid sequence of PCV2b subtype strain Cap protein is shown as SEQ ID No.6 (233 aa).

In the present example, the codon optimization process of PCV2b subtype strain ORF2b gene is described in detail, and the optimization of PCV2a subtype strain ORF2a gene is performed according to the process.

On the premise of keeping the amino acid of ORF2b of PCV2b subtype strain unchanged, modifying the codon into a biased codon of an insect cell baculovirus expression system, introducing EcoRI and XhoI enzyme cutting sites at two ends of a sequence, and inserting the biased codon into a pFastBac transfer vector through double enzyme cutting to obtain a recombinant baculovirus transfer vector which is named as pFastBac1-ORF2 b.

Amplification and identification of recombinant baculovirus transfer vectors

Transforming the obtained recombinant baculovirus transfer vector pFastBac1-ORF2 into DH5 alpha escherichia coli for amplification, and extracting the recombinant baculovirus transfer vector by adopting a conventional plasmid extraction method; the recombinant baculovirus transfer vector was identified by EcoRI and XhoI double digestion, and as shown in FIG. 1, the 737bp size of the target band and 4775bp of pFastBac1 two bands were visualized by electrophoresis.

Obtaining DNA of recombinant baculovirus

pFastBac1-ORF2b was transformed into DH10 Bac. E. Coli, homologous recombination was performed, the DNA fragment was spread on LB triple-antibody selection plates (containing 50. mu.g/ml kanamycin, 7. mu.g/ml gentamicin, 10. mu.g/ml tetracycline, 100. mu.g/ml Bluo-gal and 40. mu.g/ml IPTG), and after 48h of culture at 37 ℃, white colonies were selected and identified by the following general primer M13PCR, and a band of about 3000bp was observed in the nucleic acid electrophoresis of the positive colonies.

M13 Forward (-40)：5′GTTTTCCCAGTCACGAC3′ ；

M13 Reverse：5′CAGGAAACAGCTATGAC3′。

Selecting and marking out the colonies which are identified to be positive, purifying again, identifying the positive colonies through PCR, and freezing at-20 ℃ for later use. The results of the identification are shown in FIG. 2.

The 50ul PCR system used (fine tuning) included:

10X Taq Buffer（appropriate for enzyme） 5ul；

dNTP Mix 4ul；

M13 Forward(-40)（10nm） 2ul；

M13 Reverse（10nm） 2ul；

Taq polymerase（5units/μl） 1ul；

Sterile Water 35ul；

bacterial liquid or extracted recombinant bacmid DNA (100 ng) 1 ul.

The PCR program was set up (25-35 cycles in steps 2-4) as follows: 3min at 93 ℃, 45s at 94 ℃, 45s at 55 ℃, 5min at 72 ℃ and 7min at 72 ℃.

Obtaining of recombinant baculovirus

Extracting recombinant baculovirus DNA, OD by isopropanol precipitation method₂₆₀/OD₂₈₀Between 1.8 and 2.0, at higher concentrations (500 ng/ul or above), sf9 cells were transfected with Cellfunction transfection reagent (from Iinvitrogen) according to the instructions, incubated at 27 ℃ and developed significant cytopathic events from 72h to 96 h. It is clear that the cells become large,the appearance of particulate matter, the cell vesiculation, harvest of the recombinant baculovirus DBN01 strain, and the disease profile of SF9 and the profile of healthy SF9 cells after infection with recombinant baculovirus are shown in figure 3.

Identification of expression products

Inoculating virus seed with virus infection index MOI (appendix 4) =0.5-5.0 to well-grown cell density of 2.0 × 10⁶-2.5×10⁶SF9 cells with the activity of more than or equal to 95 percent are cultured for 96 to 120 hours at the temperature of 27 ℃, cell cultures are harvested and identified by Western Blot, and specific bands with the molecular weight of 28KD should appear. The obtained PCV2 CAP protein has an amino acid sequence shown as SEQ ID No.4, and a coding gene thereof has a nucleotide sequence shown as SEQ ID No. 3.

Example 2 expression of CAP protein in insect cell bioreactor in serum-free suspension culture

Preparation of seed poison for production

Inoculating basic virus seed at MOI =0.01-5.0, and culturing at cell density of 2.0 × 10⁶-2.5×10⁶Culturing Sf9 cells at 27 deg.C for 72-96 hr, collecting virus liquid, quantitatively packaging, noting name, harvesting date, and virus generation, detecting virus content, specifically detecting, and detecting purity to obtain Sf9 cells with virus content of 1.0 × 10⁸PFU/ml, stored at 2-8 ℃ for further use.

Serum-free suspension culture of SF9 cells in bioreactor and CAP protein expression quantification

The suspension cell culture method is used. Culturing 800ml SF9 cells in 2000ml shake flask with 95% cell viability and cell density of 2.0 × 10⁶-2.5×10⁶At a cell/ml, according to 0.5X 10⁶Transferring the cells/ml to a 5L bioreactor, and culturing for 3-4 days until the cell density reaches 2.0 × 10⁶-2.5×10⁶When the cell viability is 95% or more per cell/ml, the cell viability is 0.5X 10⁶The individual cells/ml were transferred to a 50L bioreactor and the culture continued.

Wherein, the shake flask culture condition is 27 ℃, 120prm/min, and the bioreactor parameters are set as follows: pH6.2-7.2, temperature 27 deg.C, dissolved oxygen 20% -60%, stirring speed 90-160 rpm. When the cell density reaches 2.5 multiplied by 10⁶The number of the cells per ml is increased,the recombinant baculovirus DBN01b strain was inoculated at MOI =2, cultured at 27 ℃ for 96-120 hours, and the cell culture was harvested, and stored at 2-8 ℃ with the name, harvest date, lot number, and the like noted. An electron micrograph of the virus-like particles (VLPs) formed by CAP protein expressed in the present invention is shown in FIG. 4.

Quantification of proteins

The harvested cell culture and a standard protein (BSA) are subjected to SDS-PAGE detection, the concentration of Cap protein is determined by gray scale analysis according to the concentration of the standard protein, and should not be lower than 200. mu.g/ml, and a band of about 28KD appears in Western-blot detection, and the result is shown in FIG. 5.

Inactivation of proteins

Filtering the harvested cell culture by using an ultrafiltration system with the aperture of 10-15 mu m to remove cell fragments, then adding a 4% BEI solution into the cell culture to enable the final concentration of the BEI solution to be 0.1%, inactivating the BEI solution at 37 ℃ for 24 hours, after inactivation is finished, adding a 50% sodium thiosulfate solution into the cell culture to enable the final concentration of the BEI solution to be 0.2%, stirring the mixture for 1 hour to terminate inactivation, performing 10-15 times of concentration and purification on the clarified cell culture by using a 50-100kD ultrafiltration membrane package after inactivation, and storing the cell culture at 2-8 ℃ for no more than 35 days.

Detection and identification of protein

The inactivated cell culture was inoculated with Sf9 cells and subjected to MTT inactivation assay. Meanwhile, Sf9 cells inoculated with the inactivated recombinant baculovirus DBN01 strain are used as a positive control, and normal Sf9 cells are used as a negative control. And when the reading of the detection hole of the positive control group is lower than the comparison standard value, the reading of the detection hole of the negative control and the reading of the detection hole of the inactivated sample are not lower than the comparison standard value, and the inactivation is judged to be complete. Results inactivated samples were tested for wells with readings below the alignment standard.

The concentration of PCV2 type protein is detected by an ELISA method and should not be lower than 400 mug/ml.

The inactivated PCV2 type Cap protein is taken and checked according to 2015 edition of Chinese animal pharmacopoeia, and the result shows that the inactivated antigen grows aseptically.

And diluting the inactivated PCV2b type Cap protein culture to 250 mu g/ml by using Tris-NaCl buffer solution, and preparing the seedling for later use.

Example 3

In this example, the nucleotide sequence of PCV2 ORF2a gene was designed with reference to PCV2a subtype strain, and the original nucleotide sequence of PCV2a subtype strain ORF2a gene was shown as SEQ ID No.7 (702 bp), and the original amino acid sequence of PCV2a subtype strain Cap protein was shown as SEQ ID No.8 (233 aa).

Similarly, the above-mentioned PCV2a subtype strain ORF2a gene optimization process was carried out by referring to the method of the above-mentioned example 1-2, and the obtained PCV2b type CAP protein had the amino acid sequence shown in SEQ ID No.2, and the encoding gene thereof had the nucleotide sequence shown in SEQ ID No. 1.

Example 4 preparation of a bacterial suspension of Mycoplasma hyopneumoniae DJ-166 Strain by growth

In the embodiment, the mycoplasma hyopneumoniae DJ-166 strain is separated from the lung of a Shanxi diseased pig and is obtained by 2 times of subcloning, the immunogenicity of the strain is good, the protection rate of the immune pig after vaccine preparation is over 80 percent, and the strain is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms; the preservation address is: the Beijing West Lu No.1 Hospital No.3 of Chaoyang district, the preservation number is: CGMCC No. 4545.

In this embodiment, the CH liquid medium includes the following specific components:

solution A: mixing brain and heart leachate 2.0g, PPLO broth 5.0g, and deionized water 300ml, stirring to dissolve completely, autoclaving at 116 deg.C for 20 min, and cooling;

and B, liquid B: 5.0ml of 10 xHank's solution, 1.0g of lactalbumin hydrolysate, 5.0g of yeast extract, 0.8g of sodium pyruvate, 3.0g of peptone, 0.1g of sodium thiosulfate, 0.1% phenol red, 400U/ml of penicillin and 545ml of deionized water; mixing the above components, stirring completely, filtering with 0.22um filter membrane for sterilization, and storing at 4 deg.C:

and C, liquid C: 140ml of healthy horse serum;

and (3) fully and uniformly mixing the solution A, the solution B and the solution C, and adjusting the pH value to 7.6 by using 1mol/L sodium hydroxide to obtain the required mycoplasma hyopneumoniae modified CH liquid culture medium.

First-order seed propagation: inoculating the freeze-dried strain into an improved CH liquid culture medium according to the proportion of 5-10%, culturing for 3-4 days at 37 ℃, and harvesting a bacterial liquid as a first-level seed when the color of the culture medium turns yellow and is slightly turbid and the pH value is reduced to about 6.8-7.0.

And (3) secondary seed propagation: inoculating 5-10% of the first-stage seeds into an improved CH liquid culture medium, culturing at 37 deg.C for 3-4 days, harvesting bacterial liquid when the culture medium turns yellow and is slightly turbid, and the pH value is reduced to about 6.8-7.0, and using the obtained product as second-stage seeds after pure inspection.

Culturing by fermentation culture method. Inoculating the second-level seeds to an improved CH liquid culture medium according to the proportion of 5-10%, culturing in a fermentation tank with oxygen introduction of 20% at 37 ℃ for 4-5 days, and harvesting a bacterial solution when the culture medium turns yellow and is slightly turbid and the pH value is reduced to 6.5-6.8. Storing at 2-8 deg.C for no more than 15 days.

Taking 12 sterile test tubes, each tube containing 4.5ml CH liquid culture medium, adding 0.5ml culture into the 1 st tube, mixing well, adding 0.5ml into the 2 nd tube, and continuously diluting by 10 times to 10^-11Meanwhile, the CH liquid culture medium without the added bacteria liquid is set as a negative control. Culturing at 37 + -1 deg.C for 14 days, and culturing in the final 1 tube with pH value lower than 0.5 as CCU of bacterial liquid. The detection and identification show that the bacterial liquid is pure and free from foreign bacteria, and the viable count CCU is 10¹¹ml。

Example 5 concentration, purification and inactivation of Mycoplasma hyopneumoniae DJ-166 Strain antigen and protein quantification

Concentrating and purifying

The harvested bacteria liquid is sampled and is subjected to pure inspection according to the addendum of 2010 version of Chinese veterinary pharmacopoeia, and the sampling is performed to perform viable bacteria titer determination. Concentrating the harvested bacterium liquid with a 30KD-50KD membrane package to 1/10 of the original volume, centrifuging at 10000r/min at 4 ℃ for 60 minutes, suspending the precipitated bacterium with Tris-NaCl (pH 7.2-7.4) buffer solution, centrifuging and washing for 3 times to prepare 1/100 bacterium suspension of the original culture volume, and storing at-40 ℃.

Inactivating

Subjecting the concentrated purified bacteria to ultrasonic treatment in ice water bath for 4 min, outputting power of 250-.

Inactivation test

Inoculating 5.0ml of inactivated bacteria liquid45ml of modified CH liquid medium was incubated at 37 ℃ for 14 days, and the color change of the medium was observed. Meanwhile, inactivated bacteria liquid is used as a positive control, and a CH liquid culture medium is improved as a negative control. Taking out 0.2ml of inoculated solid culture medium, and placing in 5% CO₂The culture was carried out at 37 ℃ for 10 days under ambient conditions, and the presence or absence of growth of mycoplasma colonies was observed. When the positive control has obvious color change (the pH value is reduced by below 0.5), the negative control has no color change correspondingly, the inactivated bacteria liquid has no color change, and the solid culture medium does not see the typical bacterial colony of the mycoplasma hyopneumoniae and is judged to be completely inactivated. If the liquid culture medium has constant pH change and the solid culture medium has typical mycoplasma hyopneumoniae bacterial colony, the inactivation is judged to be incomplete.

Taking inactivated mycoplasma hyopneumoniae, and inspecting according to 2015 edition of Chinese veterinary pharmacopoeia, so that the inactivated antigen grows aseptically.

Determination of mycoplasma antigen concentration

The concentration of the inactivated mycoplasma hyopneumoniae antigen protein is not lower than 4mg/ml by the quantification of the BCA protein quantitative kit. And diluting the inactivated mycoplasma hyopneumoniae to 1500 mug/ml by using Tris-NaCl buffer solution to prepare for seedling preparation.

Example 6 preparation of Swine influenza Virus antigen

The swine influenza virus strain selected in the embodiment is DBN-HB2 strain.

Preparing swine influenza virus seeds: washing full single layer MDCK cells with cell culture medium (70% MEM +30% serum-free suspension medium) containing TPCK-trypsin at final concentration of 2 μ g/ml for 3 times, inoculating virus at a ratio of 1 ‰, standing at 33 deg.C and 5% CO₂Culturing for 48-72 hours under the condition, harvesting virus liquid when more than 50% -60% of cells have CPE, and taking the virus liquid as a production virus seed after various tests are qualified.

Preparing a swine influenza virus liquid: when the MDCK suspension cell density reaches 2.5 multiplied by 10⁶-3.5×10⁶When the strains are cultured per ml, respectively inoculating qualified production strains of the swine influenza virus H1N1 subtype and the swine influenza virus H3N2 subtype according to 1/1000-1/10000 (V/V), adding TPCK-pancreatin serum-free suspension culture medium with the final concentration of 5 mug/ml, and continuously culturing under the conditions of 33 ℃, the rotation speed of 100r/min and the Dissolved Oxygen (DO) value of 50%. To be aliveAnd when the rate reaches about 50-60%, the virus liquid is harvested.

And (3) determining the content of the swine influenza virus: diluting virus solution with culture medium by 10 times, respectively taking 10 times^-4、10^-5、10^-6And 10^-74 dilutions were used to inoculate MDCK cell monolayers in well-grown 96-well cell culture plates, 6 wells (0.1 ml per well) were inoculated per dilution, and 6 wells were used as normal cell control and placed at 33 deg.C with 5% CO₂Culturing in incubator for 3-4 days, and observing cytopathic effect. TCID is calculated according to Reed-Muench method₅₀The virus content should be more than or equal to 10^6.0TCID₅₀/ml。

Determination of HA titer of swine influenza virus: and (3) measuring the HA titer of the virus liquid by adopting an erythrocyte agglutination test method (HA), wherein the HA titer is more than or equal to 8log 2.

Swine influenza virus liquid: the harvested cell culture is filtered and clarified by a clarification system, and then the culture is concentrated by 20-25 times by an ultrafiltration concentration system.

Inactivation of swine influenza virus (subtype H1N 1): adding the concentrated and purified virus solution into 4% BEI solution to make its final concentration be 0.1%, inactivating at 37 deg.C for 24 hr, adding 50% sodium thiosulfate solution into cell culture to make its final concentration be 0.2% after inactivation is finished, stirring for 1 hr to terminate inactivation, and storing at 2-8 deg.C.

Inactivation test of swine influenza virus (subtype H1N 1): well-growing MDCK cell monolayers (25 cm) were inoculated with the inactivated H1N1 DBN-HB2 strain and H3N2 DBN-HN3 strain antigens²Cell culture bottle) 2 bottles, 1.0ml of each bottle, simultaneously adding cell maintenance liquid containing TPCK pancreatin with the final concentration of 2 mug/ml, setting 1 bottle of cell negative control and 1 bottle of virus positive control respectively, culturing for 72 hours at 33 ℃, repeatedly freezing and thawing for 2 times to obtain cell culture, taking supernatant after centrifugation, performing blind passage for 2 generations according to the method, and performing HA titer determination. The sample to be detected and the cell control should not generate erythrocyte agglutination reaction, and the HA titer of the positive control should be more than or equal to 4log 2.

And (4) sterile inspection: taking the inactivated swine influenza virus liquid, and checking according to 2015 edition of Chinese veterinary pharmacopoeia, wherein the inactivated antigens grow aseptically.

Determination of HA titer of swine influenza virus: the inactivated swine influenza virus H1N1 antigen HAs an HA titer no less than 13log2 as detected by an erythrocyte agglutination test.

Inactivated swine influenza virus was diluted to 13log2 with Tris-NaCl buffer.

Example 7 preparation of bacterial solution of Haemophilus parasuis (type 13)

First-order seed propagation: the freeze-dried strains of the haemophilus parasuis type 4 JS01 strain, the type 5 FJ04 strain and the type 13 HN01 strain are streaked and inoculated on a Tryptic Soy Agar (TSA) plate containing 0.005% Nicotinamide Adenine Dinucleotide (NAD) and 5% newborn bovine serum, the plate is cultured for 18 to 24 hours at 37 ℃, 5 single typical colonies are selected, a Tryptic Soy Agar (TSA) slant containing 0.005% Nicotinamide Adenine Dinucleotide (NAD) and 5% newborn bovine serum is inoculated, the plate is cultured for 18 to 24 hours at 37 ℃, and the obtained product is taken as a primary seed after pure inspection is qualified.

And (3) secondary seed propagation: inoculating single colony selected from the first-stage seed into Tryptic Soy Broth (TSB) culture medium containing 0.005% Nicotinamide Adenine Dinucleotide (NAD) and 5% newborn bovine serum, shake-culturing at 37 deg.C and 200rpm for 12 hr, sampling gram stain, observing bacterial morphology under microscope, conforming to morphological characteristics of Haemophilus parasuis, and growing without hetero-bacteria as second-stage seed.

Culturing by fermentation culture method. Respectively inoculating qualified Haemophilus parasuis 4 type JS01 strain, 5 type FJ04 strain and 13 type HN01 strain into TBS liquid culture medium according to the proportion of 1% (V/V), culturing at 37 ℃ and 150rpm for 12-15 hours under stirring, and harvesting bacterial liquid when the number of viable bacteria reaches 50 hundred million.

Diluting the harvested bacterial liquid by 10 times, and taking 10 times^-6、10^-7And inoculating 3 dilutions in total to each TSA solid culture medium, inoculating 0.1ml of each plate, shaking the plates to uniformly spread the surface of the bacterial liquid, placing the plates at 37 ℃ for 60 minutes, performing inverted culture on the plates for 24 hours, and calculating the average colony number of each dilution, namely the viable count of the bacterial liquid, multiplied by the dilution factor.

Haemophilus parasuis (JS 01 type 4, FJ04 type 5, and HN01 type 13) strain liquid treatment: cultured Haemophilus parasuis (JS 01 type 4, FJ04 type 5, and HN01 type 13) was centrifuged at 10000rpm in a continuous flow, and then recovered to the original volume with Tris-NaCl buffer (pH 7.2-7.4).

Inactivation of Haemophilus parasuis (type 13 HN01 Strain)

Adding 0.3% (V/V) formaldehyde solution into the prepared bacterial liquid (JS 01 type 4, FJ04 type 5, and HN01 type 13), inactivating at 37 deg.C for 24 hr while shaking 1 time every 2 hr, and preserving at 2-8 deg.C for no more than 3 months after inactivation.

Mycoplasma hyopneumoniae inactivation assay

0.2ml of the inactivated bacterial liquid of the JS01 strain type 4, FJ04 strain type 5 and HN01 strain type 13 is inoculated into 2 TSA solid culture medium plates, after streaking by using an inoculating loop, the bacterial colony growth is observed after culturing for 24-48 hours at 37 ℃, and 2 non-inoculated bacterial liquids of the solid culture medium plates of the same TSA batch are set as a control. Results 4 plates should have no bacterial growth and complete inactivation.

Taking the inactivated haemophilus parasuis bacterial solution, and checking according to 2015 edition of Chinese veterinary pharmacopoeia, so that the inactivated antigens grow aseptically. The viable bacteria titer of the concentrated and purified haemophilus parasuis bacterial liquid is not less than 2 multiplied by 10¹⁰CFU/ml。

The inactivated haemophilus parasuis bacterial liquid (JS 01 type 4, FJ04 type 5 and HN01 type 13) was diluted with Tris-NaCl buffer to 1.0X 10 before inactivation¹⁰CFU/ml, standby and seedling preparation.

Example 8 seedling preparation

As shown in the process flow chart of FIG. 6, the required PCV2a type CAP protein antigen, PCV2b type CAP protein antigen, Mycoplasma hyopneumoniae antigen, swine influenza virus antigen and Haemophilus parasuis antigen are obtained according to the methods of the foregoing examples 1-7, respectively, and are ready for use.

Adjuvant formulation

61VG adjuvant was autoclaved at 121 ℃ for 20 minutes, cooled and then placed at room temperature for use.

Mixing the components according to the proportion shown in the following table 1, aseptically mixing qualified PCV2 type Cap protein antigen (the adding proportion of two subtypes of CAP protein is controlled to be 1: 1), mycoplasma hyopneumoniae antigen, swine influenza virus H1N1 subtype antigen, Haemophilus parasuis type 4 JS01 strain, type 5 FJ04 strain and type 13 HN01 strain antigen, adding astragalus polysaccharide with the final concentration of 0.5%, and emulsifying with the adjuvant to produce the quadruple inactivated vaccine.

In the embodiment, the content of PCV2 type Cap protein is 10 mug/first part; the content of mycoplasma hyopneumoniae antigen is 150 mug/head part; the content of swine influenza virus antigen is 9log2 per head; the content of Haemophilus parasuis antigens (type 4 JS01 strain, type 5 FJ04 strain and type 13 HN01 strain) before inactivation is 1.0 x 10⁹CFU per head.

TABLE 1 Components and proportions of the quadruple vaccine

Subpackaging 500 ml/bottle, 250 ml/bottle, 100 ml/bottle or 20 ml/bottle under an aseptic condition, covering a bottle stopper, pressing an aluminum-plastic cover to obtain the quadruple inactivated vaccine, wherein the content of the PCV2 type Cap protein is 10 mug/head; the content of mycoplasma hyopneumoniae antigen is 150 mug/head part; the HA content of the swine influenza virus H1N1 subtype is 9log2 per head; the content of the haemophilus parasuis antigen type 4 JS01 strain before inactivation is 1.0 x 10⁹The contents of CFU/head and 5-type FJ04 strain before inactivation are respectively 1.0X 10⁹CFU/head, 13 type HN01 strain content before inactivation is 1.0X 10⁹CFU per head.

Example 9

This example compares the PCV2 type baculovirus vector, Mycoplasma hyopneumoniae, swine influenza virus, Haemophilus parasuis quadruple inactivated vaccine prepared in example 8 with PCV2 type baculovirus vector, Mycoplasma hyopneumoniae, swine influenza virus, Haemophilus parasuis quadruple inactivated vaccine and commercial vaccine prepared with 61VG adjuvant without astragalus polysaccharide.

The PCV2 type baculovirus vector, mycoplasma hyopneumoniae, swine influenza virus, and haemophilus parasuis quadruple inactivated vaccine prepared in example 8 was recorded as group a;

PCV2 type baculovirus vector, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccine prepared by 61VG adjuvant without astragalus polysaccharide is marked as group B;

the commercial PCV2 type baculovirus vector inactivated vaccine (batch No. 309-909A) was designated as group C;

a commercial Mycoplasma hyopneumoniae inactivated vaccine (strain J) (batch No. 273-;

a commercial swine influenza virus H1N1 subtype inactivated vaccine (lot No. 150301) designated as group E;

a commercial inactivated vaccine against Haemophilus parasuis (type 4, type 5) (batch No. 150914) was assigned as group F.

Design of animal experiments

Immunization: selecting 90 healthy susceptible pigs of 3-4 weeks old, randomly dividing into 7 groups (experimental groups shown in table 2), and 30 pigs in group 1, and injecting group A vaccine 2.0ml into head and neck muscle; group 2, 30 heads, each head and neck intramuscular injection of group B vaccine 2.0 ml; in group 3, 2.0ml of group C vaccine is injected into the muscle of the head and neck; group 4, 5 heads, each head and neck intramuscular injection of group D vaccine 2.0 ml; group 5, 2.0ml of group E vaccine is injected into each head and neck muscle; group 6, 10 heads, each head and neck intramuscular injection of group F vaccine 2.0ml, 3 weeks after initial immunization 2 immunizations at the same dose and route; no.5 pigs in group 6 were kept as blank control, and were kept in isolation under the same conditions without any vaccine.

TABLE 2 vaccine test groups

And (3) measuring the antibody titer: after each group of vaccine inoculation, blood is collected before inoculation, 7 days, 14 days, 28 days, 2 months, 3 months and 4 months after inoculation, and PCV2 type, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis serum 4 type, 5 type and 13 type antibody titer is detected.

Determination of serum antibody titer

PCV2 type serum antibody determination: the S/P value is more than or equal to 0.4, and the test result is positive; the S/P value is less than 0.3, and the result is negative; the S/P value is more than or equal to 0.3 and less than 0.4, and the product is judged to be suspicious;

determination of mycoplasma hyopneumoniae serum antibodies: the S/P value is more than or equal to 0.4, and the test result is positive; the S/P value is more than or equal to 0.3 and less than or equal to 0.4, and the result is judged to be suspicious; the S/P value is less than 0.3, and the result is negative;

and (3) judging swine influenza virus serum antibodies: HI is less than or equal to 1:10, and the sample is judged to be negative; HI is more than 1:10, the product is judged to be positive;

hps serum antibody determination: the IHA is more than 1:4, the result is judged to be positive; the IHA is less than or equal to 1:4, and the result is negative.

The results of the serum antibody titer measurement are shown in tables 3-5, table 3 shows PCV2 and Mhy serum antibody titers at different times after vaccine immunization, table 4 shows SIV serum antibody titers at different times after vaccine immunization, and table 5 shows Hps serum antibody titers at different times after vaccine immunization.

PCV2 type serum antibody titer assay: PCV2 was detected using PCV2 ELISA antibody detection kit (Kino Korea). The serum antibodies of the group A vaccine are all converted into positive antibodies 2 weeks after immunization, the positive antibodies are superior to those of the group B vaccine, the serum antibodies are still at a higher level 4 months after immunization, and the serum antibodies of the group B vaccine and the group C vaccine are gradually reduced; the blank control serum antibodies remained negative at all times. The antibody level of the A group vaccine PCV2 added with astragalus polysaccharide is better than that of the B group and C group vaccines, and the positive transfer time of the A group vaccine antibody is 1 week earlier than that of the B group and C group.

Determination of mycoplasma hyopneumoniae serum antibody titer: mycoplasma hyopneumoniae was detected using a Mycoplasma hyopneumoniae ELISA antibody detection kit (IDEXX). The serum antibody of the vaccine of the group A turns positive after 2 weeks of immunization, while the serum antibody of the vaccine of the group B and the vaccine of the group D turns positive gradually after 3 weeks of immunization, 4 months after immunization, the serum antibody of the vaccine of the group A is still in a higher level, and the vaccine of the group B and the vaccine of the group D are positive, but are obviously lower than the vaccine of the group A; the blank control serum antibodies remained negative at all times. The antibody level of the A group vaccine mycoplasma hyopneumoniae added with astragalus polysaccharide is better than that of B group and D group vaccines, and the positive transfer time of the A group vaccine antibody is 1 week earlier than that of the B group and D group.

TABLE 3PCV2, Mhy serum antibody detection results at different times after vaccine immunization

Determination of swine influenza virus serum antibody titer: the hemagglutination inhibition test method is adopted for detection. The serum antibody of each group of vaccines gradually turns positive after immunization, the serum antibody reaches the peak 2-3 months after immunization, then slowly decreases, the antibody level of the group A vaccine is obviously higher than that of the group B vaccine 4 months after immunization, and the antibody level of the group A vaccine is not obviously different from that of the group E vaccine (2-needle immunization); the blank control serum antibodies remained negative at all times. The results show that the group A vaccine animals can stimulate the organism to produce SIV specific antibody after immunization.

TABLE 4 Swine influenza virus H1N1 serum antibody test results at different times after vaccine immunization

And (3) measuring the serum antibody titer of the haemophilus parasuis: the detection is carried out by adopting an indirect hemagglutination test method. In the group A vaccine, serum antibodies of Hps serum 4, 5 and 13 are all converted to be positive 2 weeks after immunization, individual pigs of the group B vaccine and the group F vaccine are converted to be positive, the serum antibodies of each group vaccine are converted to be positive 3 weeks after immunization, the serum antibody titer reaches a peak 2-3 months after immunization and then shows a slow descending trend, and the serum antibody titer of the group A vaccine serum 4, 5 and 13 is obviously higher than that of the group B vaccine 4 months after immunization and is basically consistent with the antibody level of the group F vaccine (2-needle immunization). The vaccine shows that the A group vaccine animal can stimulate an organism to generate Hps specific antibody after being immunized, the duration of the antibody in the organism is obviously superior to that of the B group vaccine, and the immunization effect is exact.

TABLE 5 Hps serum antibody test results at different times after vaccine immunization (Indirect hemagglutination assay)

Example 10 evaluation of safety

The PCV2 type baculovirus vector, Mycoplasma hyopneumoniae, swine influenza virus, Haemophilus parasuis quadruple inactivated vaccine prepared in example 8 was subjected to safety evaluation with a commercial vaccine PCV2 type baculovirus vector inactivated vaccine, a Mycoplasma hyopneumoniae inactivated vaccine (strain J), a swine influenza virus H1N1 subtype inactivated vaccine and a Haemophilus parasuis inactivated vaccine.

Taking the PCV2 type baculovirus vector, the mycoplasma hyopneumoniae, the swine influenza virus and the haemophilus parasuis quadruple inactivated vaccine prepared in the example 8; PCV 2-type baculovirus vector inactivated vaccine (batch No. 309-; mycoplasma hyopneumoniae inactivated vaccine (strain J) (batch No. 273-; a commercial swine influenza virus H1N1 subtype inactivated vaccine (lot No. 150301); inactivated haemophilus parasuis vaccine (type 4, type 5) (batch 150914) was used.

Design of animal experiments

The four-combined inactivated vaccine prepared in example 8 was administered to pigs 5 at 3 weeks old, 4.0ml each, through neck intramuscular injection, and fed under the same conditions as the healthy control pigs 5. The observation was continued for 14 days and the clinical health of the test pigs was recorded.

90 healthy susceptible pigs of 3-4 weeks old are selected, randomly divided into 7 groups (shown in a test group shown in a table 6) and 30 groups of 1, and 2.0ml of PCV2 type baculovirus vector, mycoplasma hyopneumoniae, swine influenza virus (H1N 1 subtype) and haemophilus parasuis quadruple inactivated vaccine is injected into each head and neck muscle; in group 2, 2.0ml of mycoplasma hyopneumoniae inactivated vaccine (J strain) is injected into each head and neck muscle of 5 heads; group 3, 5 heads, 2.0ml of PCV2 type baculovirus vector inactivated vaccine is injected into each head and neck muscle; in group 4, 2.0ml of swine influenza virus H1N1 subtype inactivated vaccine is injected into each head and neck muscle, and 2-immunization is carried out 3 weeks after the first-immunization in the same dose and way; in group 5, 10 heads are injected with 2.0ml of haemophilus parasuis disease inactivated vaccine per head and neck muscle, and 2-stage immunization is carried out 3 weeks after the first immunization in the same dose and way; group 6 was a control group of 30 vaccinations; group 7 is a blank control of 5 pigs, which are not vaccinated or detoxified, and the pigs of each group are separately fed under the same conditions.

TABLE 6 vaccine test groups

The safety evaluation results are shown in table 7 below, and it can be seen that when the vaccine prepared in example 8 is over-dosed to healthy susceptible pigs of 3 weeks of age, the experimental vaccine pigs and the healthy control pigs are 5/5 healthy and alive within the observation period of 14 days, the experimental vaccine pigs and the healthy control pigs are not different from the healthy control group in terms of mental state, body temperature, body weight, food intake and drinking conditions, no abnormal reaction occurs in local injection and whole body, and no abnormality occurs in parenchymal organs and cavitary organs after caesarean examination.

TABLE 7 safety test results

Example 11 Immunopotentiality evaluation experiment

The four-combined inactivated vaccine prepared in example 8 was evaluated for immunopotency with commercial four inactivated single vaccines, i.e., PCV2 type baculovirus vector inactivated vaccine, swine mycoplasma pneumonia inactivated vaccine, swine influenza virus H1N1 subtype inactivated vaccine, and haemophilus parasuis disease inactivated vaccine (type 4, type 5).

In this example, the quadruple inactivated vaccine prepared in example 8 was used in the experiment after passing the sterility test, and the experimental design of the animal was the same as that in example 10, that is, as shown in the above table 6.

The results of the above-described immunopotency evaluation are shown in tables 8 to 10 below.

As can be seen from the results of PCV2 immune challenge protection in table 8 below, PCV2 challenge and viremia detection are performed 28 days after vaccine immunization, the quadruple inactivated vaccine serum prepared in example 8 is negative by PCR detection 5/5, the commercial PCV2 baculovirus vector inactivated vaccine group is negative by 4/5, and the challenge control group is positive by 5/5; the relative daily gain of the tetrad inactivated vaccine immunity group and the commercial PCV2 type baculovirus vector inactivated vaccine group is not significantly different (P =0.397 and 0.284 > 0.05) compared with the blank control group, and the difference of the challenge control group and the blank control group is extremely significant (P =0.003 < 0.01); and (3) immunohistochemical detection, wherein the tetrad inactivated vaccine group 5/5 is negative, the commercial PCV2 type baculovirus vector inactivated vaccine group 4/5 is negative, and the challenge control group 5/5 is positive. By combining the detection results, the protective rate of a quadruple inactivated vaccine PCV2 immune group is 100%, the protective rate of a commercial PCV2 type baculovirus vector inactivated vaccine immune group is 100%, the disease incidence of a challenge control group is 100%, and blank control groups are all normal.

TABLE 8 PCV2 immune challenge protection results

As can be seen from the results of Mhy immune challenge protection in table 9 below, after 28 days of vaccine immunization, Mhy challenge was reduced by 87.6% in the immunization group of the quadruple inactivated vaccine prepared in example 8, and 70.5% in the single vaccine immunization group of the commercial mycoplasma hyopneumoniae inactivated vaccine.

TABLE 9 protection results of Mhy immune challenge

Note: "/" indicates that this is not done.

As can be seen from the results of the SIV (H1N 1) immunization challenge protection in table 10 below, in the quadruple inactivated vaccine prepared in example 8, the swine influenza virus H1N1 subtype challenge was performed, the immunization group 5/5 was protected, the commercial swine influenza virus H1N1 subtype inactivated vaccine 5/5 was protected, and the challenge control group 5/5 developed. The quadruple inactivated vaccine prepared in example 1 is shown to have basically the same immune effect with the commercial swine influenza virus H1N1 subtype inactivated vaccine.

TABLE 10 SIV immune challenge protection results

As can be seen from the Hps immune challenge protection results in table 11 below, when Hps challenge is performed 28 days after vaccine immunization, the protective rate of the immune group is 100%, the protective rate of the commercial haemophilus parasuis inactivated vaccine is 100%, and the morbidity of the challenge control group is 80%, in the haemophilus parasuis serum type 4 challenge of the quadruple inactivated vaccine prepared in example 1; the protective rate of a serum 5 type challenge group and a quadruple inactivated vaccine immunity group is 100 percent, the protective rate of a commercial haemophilus parasuis inactivated vaccine is 80 percent, and the morbidity of a challenge control group is 100 percent; the protective rate of a serum 13 type challenge and quadruple inactivated vaccine immune group is 100 percent, and the morbidity of a challenge control group is 100 percent.

TABLE 11 Hps immune challenge protection results

Note: "/" indicates no such item; the denominator is the total number of the detected pigs, and the numerator is the number of the pigs with diseases

Example 12 duration of immunization test

Duration of immunization test for PCV2 type baculovirus vector, Mycoplasma hyopneumoniae, swine influenza virus, Haemophilus parasuis quadruple inactivated vaccine prepared in example 8.

Materials: the inactivated vaccine against PCV2 type baculovirus vector, Mycoplasma hyopneumoniae, swine influenza virus, Haemophilus parasuis tetravaccine, and PCV2 type baculovirus vector in example 8 (batch No. 309-; mycoplasma hyopneumoniae inactivated vaccine (strain J) (batch No. 273-; inactivated haemophilus parasuis vaccine (type 4, type 5) (batch 150914) was used.

Design of animal experiments

Immunization: selecting 85 healthy susceptible pigs of 3-4 weeks old, randomly dividing into 6 groups (experimental groups are shown in table 12), and 30 pigs in group 1, and injecting 2.0ml of quadruple inactivated vaccine into each head and neck muscle; in group 2, 2.0ml of mycoplasma hyopneumoniae inactivated vaccine (J strain) is injected into each head and neck muscle of 5 heads; group 3, 5 heads, 2.0ml of PCV2 type baculovirus vector inactivated vaccine is injected into each head and neck muscle; in group 4, 5 heads and neck muscles are injected with swine influenza virus H1N1 subtype inactivated vaccine 2.0m, and 2-immunization is carried out 3 weeks after the first-immunization in the same dose and way; in group 5, 10 heads are injected with 2.0ml of haemophilus parasuis disease inactivated vaccine per head and neck muscle, and 2-stage immunization is carried out 3 weeks after the first immunization in the same dose and way; group 6 was a control group of 30 vaccinations; group 6 was a blank control of 5 pigs, which were not vaccinated or detoxified, and the pigs of each group were kept separately under the same conditions.

And (3) toxin counteracting protection: 4 months after immunization, PCV2 virulent DBN-SX07 strain, Mhy virulent HB strain, SIV virulent DBN-FJ strain, Hps serum 4 JS01 strain, serum 5 FJ04 strain and serum 13 HN01 strain are respectively used for virus challenge of each group of test pigs, and the test pigs are killed by dissection 28 days after virus challenge and the results are recorded.

TABLE 12 groups of challenge test and challenge dose 4 months after immunization

Note: A. group 1 is the quadruple inactivated vaccine prepared in example 1; group 2 mycoplasma hyopneumoniae inactivated vaccine (strain J); group 3 is a PCV2 type baculovirus vector inactivated vaccine; group 4 is swine influenza virus H1N1 subtype inactivated vaccine; the 5 th group is inactivated vaccine of haemophilus parasuis (type 4, type 5). B. PCV2, SIV and Hps were not sensitive to older pigs, PCV2, SIV and Hps required increased challenge doses at 4 months, PCV2 was 8.0ml (containing 10)^7.0TCID₅₀Per ml) for counteracting toxic substances; SIV at 12.0ml (containing 10)^8.0EID₅₀Per ml); hps serotype 4 was measured at 6.0ml (viable count 9.0X 10)⁹CFU), Hps type 5 serum in 6.0ml (containing viable count 6.0 × 10)⁹CFU), Hps serotype 13 (6.0 ml, viable count 6.0X 10)⁹CFU)。

The results of the above-described evaluation of the sustained potency of immunity are shown in tables 13 to 16 below.

As shown in table 13, the PCV2 challenge protection results show that PCV2 challenge protection is performed after PCV2 challenge 4 months after single vaccine immunization of the quadruple inactivated vaccine prepared in example 8 and the commercial PCV2, the quadruple inactivated vaccine serum is negative by PCR detection 5/5, the commercial PCV2 type baculovirus vector inactivated vaccine group 4/5 is negative, and the challenge control group 4/5 is positive; the relative daily gain of the quadruple inactivated vaccine immunization group and the commercial PCV2 type baculovirus vector inactivated vaccine group is not significantly different (P is more than 0.05) compared with that of a blank control group, and the difference of the challenge control group and the blank control group is not significant (P is more than 0.05); and (3) immunohistochemical detection, wherein the tetrad inactivated vaccine group 5/5 is negative, the commercial PCV2 type baculovirus vector inactivated vaccine group 4/5 is negative, and the challenge control group 5/5 is positive. By combining the detection results, the protective rate of a quadruple inactivated vaccine PCV2 immune group is 100%, the protective rate of a commercial PCV2 type baculovirus vector inactivated vaccine immune group is 80%, the morbidity of a challenge control group is 80%, and a blank control group is normal.

TABLE 13PCV2 protection results of 4-month challenge

The results of protection against Mhy immunization shown in table 14 indicate that the four-combined inactivated vaccine prepared in example 8 and the commercial mycoplasma hyopneumoniae inactivated vaccine were challenged 4 months after single vaccine immunization, pneumonia lesions of the Mhy immunized group of the four-combined inactivated vaccine were reduced by 76.2%, and pneumonia lesions of the commercial single vaccine immunized group were reduced by 69.0%.

TABLE 14 Mhy immunization 4 months protective outcome of challenge

As shown in table 15, the SIV challenge protection results include that the four-combined inactivated vaccine prepared in example 8 and the commercial SIV single vaccine are challenged 4 months after immunization, the SIV challenge in the four-combined inactivated vaccine is 100% protected, and the commercial SIVH1N1 subtype single vaccine is 80% protected.

TABLE 15 SIV immunization 4 months protection results against challenge

As shown in table 16, the protection results of the Hps immune challenge, the Hps challenge 4 months after the immunization of the four-way inactivated vaccine prepared in example 8, the Hps serum type 4 challenge, the protection rate of the immune group is 100%, the protection rate of the commercial haemophilus parasuis inactivated vaccine is 80%, and the morbidity of the challenge control group is 80%; the protective rate of a serum 5 type challenge group and a quadruple inactivated vaccine immunity group is 100 percent, the protective rate of a commercial haemophilus parasuis inactivated vaccine is 80 percent, and the morbidity of a challenge control group is 80 percent; the protective rate of a serum 13 type challenge and quadruple inactivated vaccine immune group is 80 percent, and the morbidity of a challenge control group is 80 percent. The quadruple inactivated vaccine prepared in example 8 is superior to the commercial vaccine.

TABLE 16 Hps immunization 4 months challenge protection results

The test results show that the quadruple inactivated vaccine prepared by the invention is equivalent to a single commercial vaccine in the market from the serum antibody and the virus attack protection results 35 days after immunization, the antigens are not interfered, and the number of viable bacteria used by each head of Hps vaccine in the quadruple inactivated vaccine is 1.0 multiplied by 10⁹CFU, lower than the antigen content of the commercial single seedling; from the immunity duration, the quadruple inactivated vaccine is subjected to one-time immunity, the immunity period is 4 months, the single vaccine of the commercial PCV2 is subjected to one-time immunity, the immunity period is 4 months, the single vaccine of the commercial Mhy is subjected to one-time immunity, the immunity period is 4 months, the single vaccine of the commercial SIV is subjected to 2 times of immunity, and the immunity period is 4 months; the Hps single seedling of the commodity is immunized for 2 times, and the immunization period is 4 months. No matter from the immune period, the virus attack protection and the antibody level, the quadruple inactivated vaccine is superior to the single vaccine of the commercial SIV and Hps, the quality of the PCV2 and Mhy vaccine is reached or exceeded, and one injection is used for preventing 4 diseases, the stress response of animals is reduced, and the cost is reduced.

Example 13PCV2 type protein selection differences

In the scheme of the application, the Cap protein of PCV2 type forming the triple vaccine comprises two subtypes of PCV2b and PCV2a recombinant proteins, and the protection of the PCV2 type diseases is more comprehensive. In the scheme of the invention, the PCV2 type Cap protein is subjected to gene modification and gene codon modification, and the expression yield is not lower than 200 mu g/ml. When the triple vaccine is prepared, the PCV2 type Cap protein is 4 ug/head, and the optimal adjuvant proportion is adopted, so that 100% protection can be realized.

The performance advantage analysis of the quadruple vaccine of the present embodiment and the existing quadruple vaccine is shown in table 17 below. The traditional triple vaccine contains only one subtype of Cap protein or whole virus inactivated protein.

TABLE 17 advantages of the quadruple seedlings of the present invention over the currently available related quadruple seedlings

Example 14PCV2 type protein differences

The PCV2a protein which is subjected to gene modification and gene codon modification in the embodiment of the invention and the traditional PCV2a protein which is not subjected to gene modification and gene codon modification in the prior art are added with a melittin signal peptide sequence before the PCV2a gene sequence and codon optimization of the gene to the codon of an insect baculovirus expression system is carried out. The results of SDS-PAGE electrophoresis of the two samples are shown in FIG. 7, in which lane 1 is 250ug/ml BSA; lane 2 is 125ug/ml BSA; lane 3 is 62.5ug/ml BSA; lane 4 at 31.25ug/ml BSA, lane 5 at Protein Marker (14 KD-120 KD); lane 6 electrophoresis band of the supernatant of PCV2a protein of the invention; lane 7 is the electrophoretic band of the supernatant of conventional PCV2a protein.

It can be seen that the expression level of PCV2 type Cap protein modified by gene modification and gene codon modification is up to more than 200ug/ml and the protein is virus-like particles (lane 6), while the expression level of PCV2 type Cap protein constructed by traditional unmodified and optimized gene in the prior art is generally 50ug/ml (lane 7). The vaccine provided by the invention has higher protein expression amount, can achieve the same immune effect with lower dosage, and effectively reduces the production cost from the production of finished products.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Sequence listing

<110> Applicant: beijing Kogayof Biopharmaceutical Co., Ltd, Mtoyowa Biotechnology (Nanjing) Co., Ltd, Beijing Biomedicine technology center, Mtoyowa Biotechnology (Fuzhou) Co., Ltd

<120> a four-linked inactivated vaccine of PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis

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Claims (10)

1. A PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and Haemophilus parasuis quadruple inactivated vaccine is characterized in that the vaccine contains inactivated PCV2 type CAP protein antigen, inactivated mycoplasma hyopneumoniae antigen, inactivated swine influenza virus antigen, inactivated Haemophilus parasuis antigen and a vaccine adjuvant; wherein the content of the first and second substances,

the PCV2 type CAP protein antigen is a CAP protein antigen which is expressed efficiently by SF9 or High Five cell after being cloned and transfected in a pF astBac1 transfer vector, wherein the PCV2 type CAP protein antigen is a PCV2a and PCV2b isolate, the PCV2 ORF2 sequence of a partial codon of an insect cell baculovirus expression system is modified by artificially synthesizing codon optimization on the premise of keeping amino acid unchanged, EcoRI and XhoI enzyme cutting sites are introduced at two ends of the sequence, the recombinant baculovirus is inserted into the pF astBac1 transfer vector by double enzyme cutting, and the recombinant baculovirus is infected with SF9 or High Five cell.

2. The quadruple inactivated vaccine according to claim 1, wherein the PCV2 type CAP antigen comprises PCV2a type CAP protein and PCV2b type CAP protein;

the encoding gene of the PCV2a type CAP protein has a nucleotide sequence shown as SEQ ID No.1, and the PCV2a type CAP protein has an amino acid sequence shown as SEQ ID No. 2;

the encoding gene of the PCV2b type CAP protein has a nucleotide sequence shown as SEQ ID No.3, and the PCV2b type CAP protein has an amino acid sequence shown as SEQ ID No. 4.

3. The quadruple inactivated vaccine according to claim 2, wherein the mycoplasma hyopneumoniae antigen is a mycoplasma hyopneumoniae DJ-166 virus strain with the preservation number of CGMCC No.4545, and is obtained by proliferation, concentration, purification and inactivation in an improved CH medium.

4. The quadruple inactivated vaccine according to claim 3, wherein the swine influenza virus antigen is a swine influenza virus H1N1 subtype antigen obtained by propagating, concentrating, purifying and inactivating a swine influenza virus H1N1 subtype virus strain.

5. The quadruple inactivated vaccine according to claim 4, wherein the Haemophilus parasuis antigen serum comprises the Haemophilus parasuis type 4, type 5 and type 13 antigens obtained by proliferation, concentration, purification and inactivation of JS01 type 4, FJ04 type 5 and HN01 type 13, respectively.

6. The quadruple inactivated vaccine according to any one of claims 1 to 5, wherein in the vaccine:

the content of Cap protein expressed by the PCV2 type baculovirus is more than or equal to 8 mug/first part;

the content of mycoplasma hyopneumoniae antigen is more than or equal to 80 mug/part;

the swine influenza virus HA is more than or equal to 7log2 per head;

the content of antigens of the haemophilus parasuis serotype 4, 5 and 13 is more than or equal to 1.0 multiplied by 10⁸CFU per head.

7. The quadruple inactivated vaccine according to claim 6, wherein the vaccine adjuvant comprises an aluminum salt adjuvant, a mineral oil adjuvant or a water-soluble adjuvant.

8. A method for preparing a inactivated vaccine for PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple according to any one of claims 1 to 7, comprising the steps of inactivating and concentrating the CAP protein antigen of PCV2 type, the mycoplasma hyopneumoniae antigen, the swine influenza virus antigen and the haemophilus parasuis antigen serum respectively, and mixing the inactivated vaccine with the vaccine adjuvant according to a selected ratio.

9. The method for preparing the quadruple inactivated vaccine according to claim 8, further comprising a step of obtaining the PCV2 type CAP protein antigen, which specifically comprises:

(1) referring to the CAP protein sequence of the separated strains of the known PCV2a and PCV2b subtypes, under the premise of keeping amino acid unchanged, modifying codons into biased codons of an insect cell baculovirus expression system, introducing EcoRI and XhoI enzyme cutting sites at two ends of the sequence, and inserting the biased codons into a pF astBac1 transfer vector through double enzyme cutting to respectively obtain recombinant baculovirus transfer vectors pFastBac1-ORF2a and pFastBac1-ORF2 b;

(2) transforming the obtained recombinant baculovirus transfer vector into DH10 Bac. Coli, and carrying out homologous recombination to obtain recombinant baculovirus DNA;

(3) transfecting recombinant baculovirus DNA into SF9 cells, packaging to generate recombinant baculovirus DBN01a and DBN01b strains expressing PCV2 CAP protein;

(4) infecting SF9 or highfive cells with the obtained recombinant baculovirus DBN01a and DBN01b strains to obtain recombinant PCV2 CAP protein virus-like particles, and inactivating the virus when the concentration of PCV2 CAP protein is detected and the expression level is not lower than 200 mug/ml;

(5) separating and purifying the recombinant PCV2 type CAP protein to obtain the recombinant PCV2 type CAP protein.

10. The method for preparing the quadruple inactivated vaccine according to claim 8 or 9, wherein:

the method also comprises a step of obtaining the mycoplasma hyopneumoniae antigen, and specifically comprises the following steps: inoculating the mycoplasma hyopneumoniae virus strain into an improved CH liquid culture medium, culturing for 3-4 days under the condition of 20% oxygen introduction in a fermentation tank at 37 ℃, and determining the titer of viable bacteria to be not less than 10 by harvesting and determining¹¹CCU/ml, and purifying to obtain;

and/or the presence of a gas in the gas,

the method also comprises a step of obtaining the haemophilus parasuis antigen, and specifically comprises the following steps: respectively inoculating the haemophilus parasuis JS01 strain, FJ04 strain and HN01 strain into TBS liquid culture medium, culturing for 12-15h under the condition of a fermentation tank at 37 ℃ and 150rpm, harvesting bacterial liquid when the number of viable bacteria is not less than 50 hundred million by harvesting and measuring, and purifying to obtain the haemophilus parasuis strain;

and/or the presence of a gas in the gas,

the method also comprises a step of obtaining the swine influenza virus antigen, and specifically comprises the following steps: respectively inoculating the H1N1 subtype and H3N2 subtype virus strains of the swine influenza virus to a TPCK-pancreatin serum-free suspension culture medium, culturing at 33 ℃, at the rotating speed of 100r/min and at the Dissolved Oxygen (DO) value of 50%, harvesting bacterial liquid, and purifying to obtain the swine influenza virus.

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