CN110812474A - Triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis and preparation method thereof - Google Patents
Triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine and a preparation method thereof, belonging to the technical field of biological products for livestock. The invention is composed of antigen and vaccine adjuvant; the antigen consists of porcine circovirus type 2 antigen, mycoplasma hyopneumoniae antigen and haemophilus parasuis antigen; the porcine circovirus type 2 antigen is cap protein obtained by pichia pastoris expression, and the protein content is more than or equal to 150 mug/mL; the mycoplasma hyopneumoniae antigen is inactivated mycoplasma hyopneumoniae bacterial liquid; the haemophilus parasuis is inactivated haemophilus parasuis bacterial liquid; the vaccine adjuvant is composed of an aqueous high molecular polymer adjuvant and a complex polysaccharide immunopotentiator. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis provided by the invention has obvious advantages in preventing the three porcine diseases and improving the production performance of pigs.
Description
Technical Field
The invention relates to the field of biological products for livestock, and in particular relates to a porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine and a preparation method thereof.
Background
Porcine circovirus disease is an infectious disease of pigs caused by porcine circovirus type 2, causes weaning syndrome of piglets, porcine dermatitis and nephrotic syndrome and sow dysgenesis, and has diversified clinical manifestations. After the pig is infected with the circovirus, serious immunosuppression can be caused, so that other infectious diseases such as mycoplasma hyopneumoniae, haemophilus parasuis, swine hemolytic streptococcus and the like are secondary or concurrent.
Mycoplasma hyopneumoniae pneumonia, also known as swine endemic pneumonia, is a respiratory disease caused by mycoplasma hyopneumoniae and mainly characterized by chronic, high infectivity, high morbidity and low mortality. Infected pigs have poor long-term development, low feed conversion rate and reduced daily gain, and the economic benefit of the pig industry is seriously influenced. The mycoplasma hyopneumoniae and other respiratory disease pathogens, such as porcine reproductive and respiratory syndrome virus, actinobacillus, pasteurella multocida, swine influenza virus and circovirus type 2 mixed infection, can aggravate the disease condition and cause death of pigs. The swine mycoplasmal pneumonia is a refractory chronic disease, the mycoplasma resistant medicine treatment can generally inhibit the disease but is difficult to remove, and cough and asthma symptoms disappear after the medicine is used, but the disease is easy to relapse after the medicine is stopped.
Haemophilus parasuis, which is a commensal bacterium of the upper respiratory tract of pigs, colonizes the upper respiratory tract of healthy pigs. The compound feed shows symptoms such as serositis and meningitis when the maternal antibody protection is lost after group transfer and weaning and the immunity is reduced due to other diseases, can affect suckling piglets of 2 weeks and young pigs of 4 months, mainly attack after weaning and during conservation, and are mostly seen in the pigs of 5-8 weeks. The porcine circus and mycoplasma hyopneumoniae belong to immunosuppressive diseases, so that the immunity of a swinery is reduced, conditions are created for the pathogenesis of haemophilus parasuis, the three diseases are mixed, the morbidity and the mortality of the pigs are caused, and the development of the pig raising industry in China is seriously influenced.
At present, only relevant single vaccine or bivalent vaccine exists in the commercial vaccines, the immunization frequency is too high, and the pigs are stressed. Meanwhile, due to the abuse of antibiotics, drug-resistant strains appear, so that the drug treatment tends to be ineffective; the triple vaccine of porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis provided by the invention can achieve three prevention effects by one injection, reduce the immunity times and stress, and practically solve the problem of the pig industry.
Although the invention also relates to a combined vaccine patent of the circovirus type 2 subunit vaccine, the circovirus type 2 Cap protein is obtained by using an Escherichia coli expression system. The escherichia coli expression system is a prokaryotic expression system and lacks the processing and modifying effect on protein after translation, so that the space configuration folding modification of the circular cap protein obtained by expression cannot be carried out, and the activity of VLP of the cap protein in the later period is difficult to ensure.
Disclosure of Invention
In order to make up the defects of the prior art, the invention provides a preparation method of a porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine.
The technical scheme of the invention is as follows:
a triple inactivated vaccine of porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis consists of an antigen and a vaccine adjuvant; the antigen consists of porcine circovirus type 2 antigen, mycoplasma hyopneumoniae antigen and haemophilus parasuis antigen; the porcine circovirus type 2 antigen is a cap protein obtained by expressing and purifying pichia pastoris, and the protein content is more than or equal to 150 microgram/mL; the mycoplasma hyopneumoniae antigen is inactivated mycoplasma hyopneumoniae broken bacterial liquid; the haemophilus parasuis is inactivated haemophilus parasuis crushed bacterial liquid; the vaccine adjuvant is composed of an aqueous high molecular polymer adjuvant and a complex polysaccharide immunopotentiator.
The pichia pastoris expression system is a eukaryotic expression system, and can carry out later-stage processing modification on the expressed protein, so that the target protein is closer to a natural space concept, and the protein activity is better. In the triple inactivated vaccine, the porcine circovirus type 2 antigen is a cap protein expressed and purified by pichia pastoris, and the cap protein has high activity and good immune effect.
Preferably, the aqueous polymer adjuvant is Montanide Gel.
Preferably, the complex polysaccharide immunopotentiator is a water-soluble complex of Chinese medicinal polysaccharide.
The water-based high molecular polymer adjuvant and the compound polysaccharide immunopotentiator form the adjuvant Montanide Gel-Max of the vaccine.
As a preferred scheme, the inactivated mycoplasma hyopneumoniae crushed bacterial liquid has the viable bacteria number not less than 10 before inactivation9CCU/mL。
Preferably, the number of viable bacteria before inactivation is not less than 2.5 multiplied by 109CCU/mL。
The preparation method of the porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine comprises the following steps:
1) culturing pichia pastoris by adopting a sterilized FBSM inorganic salt culture medium, and inducing and expressing the porcine circovirus type 2 cap protein by using methanol; respectively culturing mycoplasma hyopneumoniae and haemophilus parasuis by using a fermentation tank;
2) respectively inactivating the concentrated and purified solution of the circular cap protein and the concentrated solution of mycoplasma hyopneumoniae and haemophilus parasuis;
3) preparing an oil phase, and sterilizing the water-based high-molecular polymer adjuvant for later use;
4) preparing a water phase, dissolving the complex polysaccharide immunopotentiator by adopting a PBS solution, filtering and sterilizing, and adding a cap protein obtained and purified by pichia pastoris expression and an inactivated mycoplasma hyopneumoniae crushing liquid;
5) placing the water phase obtained in the step 4) into an emulsifying tank, slowly adding the oil phase prepared in the step 3) while stirring, and uniformly stirring;
6) adding inactivated haemophilus parasuis bacterial liquid, and uniformly stirring;
7) adding thimerosal solution, and stirring uniformly to obtain the porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine.
As a preferred scheme, the preparation method of the porcine circovirus type 2 antigen comprises the following steps:
① inoculating Pichia pastoris cell in culture medium for seed culture, inoculating the proliferated seed liquid in fermentation culture medium for fermentation, adding methanol for induction when OD600 of the liquid is 1.0, and collecting the liquid;
② centrifuging the yeast harvest solution continuously to remove supernatant, resuspending the precipitate with 0.01M PBS solution, and crushing;
③ ultrafiltering and concentrating the supernatant with 300KD to obtain concentrated solution of porcine circovirus type 2 antigen cap protein;
④ the cap protein concentrate is inactivated and purified by column chromatography.
Preferably, the method for obtaining the mycoplasma hyopneumoniae antigen comprises the following steps:
A) inoculating the mycoplasma hyopneumoniae into a mycoplasma liquid culture medium, and when the color of the culture medium turns yellow and is slightly turbid and the pH value is reduced to 6.8, obtaining mycoplasma hyopneumoniae bacterial liquid;
B) the mycoplasma hyopneumoniae harvest liquid is concentrated by ultrafiltration, so that the viable count of the mycoplasma hyopneumoniae reaches 1012-1013Adding 0.01M PBS buffer solution with the volume of 1/2 of the stock solution into the CCU/ml for ultrafiltration again, removing supernatant, crushing thalli precipitates, and finally suspending the thalli precipitates to 1 percent of the original volume by using 0.01M PBS;
C) the concentrated mycoplasma hyopneumoniae is crushed several times and then subjected to inactivation treatment.
Preferably, the method for obtaining the haemophilus parasuis antigen comprises the following steps:
a) inoculating qualified Haemophilus parasuis type 4 and 5 seeds in a TSB liquid culture medium according to the amount of 1%, culturing for 16-18h at 37 ℃ at 250 rpm in a fermentation tank, and harvesting a bacterial liquid;
b) adopting tangential flow ultrafiltration thallus fermentation liquor; resuspending the thallus precipitate with an equal volume of 0.01M PBS solution containing 1% reagent A, ultrafiltering again to remove supernatant, and finally, resuspending and counting with the PBS solution;
c) and (4) inactivating the thalli obtained by ultrafiltration concentration.
Preferably, the formaldehyde solution with the final concentration of 0.4% is added in the inactivation treatment, and the inactivation treatment is carried out for 24 hours at the temperature of 2-8 ℃, and the mixture is shaken up once every 2 hours.
The invention has the beneficial effects that:
the triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis provided by the invention has obvious advantages in preventing the three porcine diseases and improving the production performance of pigs. Safety tests show that after the guinea pigs are subjected to intraperitoneal injection in an amount which is twice the amount and is excessive, the guinea pigs are continuously observed for one week, the mental states of the guinea pigs are good, and the guinea pigs do not die; after the pig is subjected to the excess immunity test, the mental state is good, and abnormal clinical reactions such as vomit, red swelling of injection parts and the like do not exist. The efficacy test shows that after the triple vaccine of the immunized pig, the level of the circular antibody, the level of the mycoplasma antibody and the level of the haemophilus parasuis antibody can reach the level of a single vaccine, and the antigen components are not interfered with each other.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is an electrophoretogram of cap protein; wherein, 1: marker; 2:28kd standard protein; 3-7 are five batches of purified cap proteins.
FIG. 2 shows the reduction rate of lung lesions in each group after the triple vaccine of the present invention was subjected to mycoplasma intensity challenge;
FIG. 3 is a comparison graph of mean daily gain of triple vaccine immunized piglets with non-immunized groups;
FIG. 4 is a comparison graph of survival rate of immunized piglets and non-immunized groups transformed with triple vaccine of the present invention;
FIG. 5 is a process flow chart for preparing the triple inactivated vaccine of porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the implementations of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of triple inactivated vaccine against porcine circovirus type 2, mycoplasma hyopneumoniae, haemophilus parasuis, as shown in figure 5:
1. preparation of semi-finished product antigen of porcine circovirus type 2
1) A preferred eukaryotic expression plasmid containing the circovirus type 2 cap protein was electro-transformed into Pichia pastoris cells (X-33, available from Invitrogen, USA). Screening out positive clone seeds, inoculating the positive clone seeds into 250ml of BMGY liquid culture medium, and carrying out shaking culture at 30 ℃ at 250r/min for 16-24 h; the grown 250ml seed solution was then added to sterilized FBSM mineral salts medium and expanded in a fermentor. And when the OD600 of the bacterial liquid is 1.0, adding 1% methanol for inducing for 80-96 h, and harvesting the bacterial liquid.
2) Centrifuging the yeast harvest liquid by continuous flow to remove supernatant, reselecting the precipitate by using 0.01M PBS solution, and then crushing the precipitate; centrifuging the crushing liquid to remove precipitates, and harvesting a supernatant;
3) and (3) carrying out ultrafiltration concentration on the supernatant by 300KD to obtain a concentrated solution of porcine circovirus type 2 antigen cap protein.
4) And inactivating the cap protein concentrated solution by formaldehyde, and purifying by column chromatography. PAGE gel identification is carried out on the eluted protein purification solution, and the result shows that the purity of the cap protein after purification is more than 90%, and the result is shown in figure 1.
5) The protein concentration of the purified cap protein is measured by a BCA method, and a formaldehyde solution with the final concentration of 0.4 percent is added and is inactivated at the temperature of 2-8 ℃.
2. Preparation of semi-finished antigen of mycoplasma hyopneumoniae
1) Preparing a mycoplasma hyopneumoniae liquid culture medium according to the existing laboratory process formula (4-8 g/L of MEM culture medium, 3-7g/L of yeast powder, 15-20g/L of peptone, 5-12g/L of glucose, 6-10g/L of horse serum protein freeze-dried powder, 30-100 mu g/L of transferrin, 0.6-2.0g/L of sodium pyruvate, 3-7g/L of sodium chloride, 0.1-0.4g/L of potassium dihydrogen phosphate, 0.5-2.5g/L of dipotassium hydrogen phosphate and 0.005-0.02g/L of phenol red), adding 2% of pig serum, and adjusting the pH of the culture solution to be about 7.5 by using 1M sodium hydroxide;
2) inoculating 10% of Mycoplasma hyopneumoniae WH-39 strain (epidemic strain isolated from Shandong Binzhou in 2016) into Mycoplasma liquid culture medium, culturing in 37 deg.C fermentation tank for 36-48h, and collecting bacterial liquid when the culture medium turns yellow and slightly turbid, and the pH value is reduced to 6.8;
3) the mycoplasma hyopneumoniae harvest fluid obtained by ultrafiltration concentration enables the viable count of the mycoplasma hyopneumoniae to reach 1012-1013CCU/ml; adding 0.01M PBS buffer solution with the volume of 1/2 of the stock solution for ultrafiltration again, removing supernatant, crushing thallus precipitates, and finally resuspending to 1 percent of the original volume by using 0.01M PBS;
4) adding 0.2% formaldehyde solution, inactivating at 2-8 deg.C for 24 hr while shaking every 2 hr to ensure complete inactivation.
3. Preparation of Haemophilus parasuis semi-finished product antigen
1) Inoculating qualified Haemophilus parasuis type 4 (BZ strain, obtained by separating Shandong Binzhou) and 5 (LZ strain, from Shandong province farm) seeds in a TSB liquid culture medium according to the amount of 1%, culturing at 37 ℃ and 250 rpm for 16-18h in a fermentation tank, and harvesting a bacterial solution;
2) adopting tangential flow ultrafiltration thallus fermentation liquor; resuspending the thallus precipitate with an equal volume of 0.01M PBS solution containing 1% reagent A, ultrafiltering again to remove supernatant, and finally, resuspending and counting with the PBS solution;
3) the cells obtained by the ultrafiltration and concentration were inactivated with 0.4% formaldehyde.
4. Vaccine formulation
1) Preparing an oil phase, subpackaging the water-based high polymer Montanide Gel into a sterilization bottle, and carrying out high-pressure moist heat sterilization at 121 ℃ for 30 min;
2) preparing water phase, dissolving Chinese medicinal polysaccharide complex (one or more of Astragalus polysaccharides, Ganoderma polysaccharides, and rhizoma Polygonati Odorati polysaccharides) in 0.01M PBS solution, sterilizing with 0.45um and 0.22um filter core, and filtering to obtain water phase A solution; adding inactivated yeast into the solution A to express the cap protein, so that the content of the cap protein is more than or equal to 150 mu g/ml, and the concentration of the mycoplasma hyopneumoniae protein is more than or equal to 150 mu g/ml;
3) adding the water phase into an emulsification tank, stirring for 30min at 200 rpm; slowly adding the oil phase to make the final concentration of the adjuvant be 10 percent, rotating/min at 300, and stirring for 30 minutes; then adding the inactivated thallus of the haemophilus parasuis to ensure that the thallus content of the 4 type haemophilus parasuis is more than or equal to 2.5 multiplied by 109The thallus content of the CFU/ml and the Haemophilus parasuis type 5 is more than or equal to 2.5 multiplied by 109CFU/ml, 180 r/min, stirring for 30 min;
4) adding a thimerosal solution to ensure that the final concentration of the thimerosal is 0.01 percent, and uniformly stirring to obtain the porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine.
Example 2 safety test of triple inactivated vaccine against porcine circovirus type 2, Mycoplasma hyopneumoniae, Haemophilus parasuis
1. Safety inspection of guinea pigs: the 180-220g guinea pigs were divided into two groups of 8 animals each. In the test group, 5ml of pig triple vaccine is injected into the abdominal cavity of each guinea pig; in the control group, each guinea pig was intraperitoneally injected with 5ml of physiological saline and continuously observed for 7 days, and both groups of guinea pigs were all kept healthy without local or systemic adverse reactions, and the results are shown in table 1.
TABLE 1 safety test results of the triple pig vaccine of the present invention in guinea pigs
2. And (3) piglet safety inspection: three litters of healthy piglets are screened for 14-21 days, 6 piglets are selected in each litter, 2ml of the pig triple vaccine is injected into the neck muscle, and abnormal clinical reactions such as vomit, tachypnea and the like are not found after 2 hours of continuous observation. After 14 days of continuous observation, no adverse reaction is observed.
Example 3 Effect of porcine triple vaccine on challenge protection of circovirus type 2, Haemophilus parasuis in mice
1. Protective effect of triple vaccine on virus attack of circovirus type 2
30 BALB/C mice, 18-22g, were fed for one week for acclimation and immediately divided into 3 groups, i.e., triple vaccine group, single circular vaccine group and blank control group. Each mouse was injected with 0.5ml of the vaccine intraperitoneally, and the control group was injected with the same dose of PBS solution, and after 14 days, the mice were boosted by the same method at a dose of 0.5 ml/mouse.
The DBN-SX07 strain was used to test the toxicity (. gtoreq.10) 21 days after immunization of BALB/c mice, together with 10 control mice7.0TCID50And/ml), injecting 0.5ml into the abdominal cavity of each mouse, killing the spleen 21 days after virus killing, and performing virus separation, wherein the virus is judged to be positive after separation. And (3) inoculating Dulac cells after spleen grinding treatment, judging whether the cells are infected by immunofluorescence, continuously carrying out passage for three times, and judging a final result.
The results show that the positive rate 1/10(9/10 negative) of the PCV2 virus separation of the pig triple challenge group and the positive rate 9/10 of the control group have better protection effect according to the standard (the positive rate of the virus separation of the control group 7/10 is positive, the negative rate of the virus separation of the immune group 7/10 is negative), and the pig triple vaccine has better protection effect on mice, and the results are shown in table 2.
TABLE 2 Positive rate of virus isolation in various groups of mice after challenge with PVC2
Note: -represents a virus isolation negative; + positive for virus isolation;
2. toxin attacking and protecting effect of triple vaccine on haemophilus parasuis
30 BAL of 18-22gThe B/C mice were fed for one week and immediately divided into 3 groups, i.e., triple vaccine group, Haemophilus parasuis vaccine group, and placebo group. Each mouse was injected with 0.5ml of the intraperitoneal injection, and the control group was injected with the same amount of PBS solution, and after 14 days, the booster immunization was performed according to the same method, with the dose of 0.5 ml. The drug is attacked 14 days after the immunization, and the dose of the drug attacked by each mouse is 5 multiplied by 109Mice were observed for survival for 7 consecutive days in CFU/mouse.
The results show that after the haemophilus parasuis attacks the poison of the mice, the attacking dose is 5 multiplied by 109CFU/control group is 100% dead, the protection effect of the haemophilus parasuis vaccine group is 80%, and the protection effect of the triple pig group is 80%, which indicates that the protection level of the triple pig vaccine against haemophilus parasuis is consistent with that of the single vaccine, and the results are shown in Table 3.
TABLE 3 results of efficacy determination of triple inactivated vaccine Haemophilus parasuis of the present invention
Example 4 protective Effect of the porcine triple vaccine against mycoplasma challenge on piglets
Screening 18 healthy piglets 14-21 days negative with mycoplasma antibody, and dividing into three groups, namely a triple vaccine group, a single mycoplasma vaccine group and a blank control group; each group was given 2ml of the corresponding vaccine intramuscularly, a blank was given 2ml of physiological saline, and the immunization was performed once in the same manner after 14 days. And (4) after the immunization, performing challenge on each group by using mycoplasma virulent CVCC354 (veterinary drug administration), observing for 14d, killing all test pigs without pain, and counting the reduction rate of lung lesions of each group.
The result shows that after the mycoplasma is strongly toxic and attacked, the lung of the pigs in the blank control group is completely diseased, and the reduction rate of the lung disease is 0%; the reduction rate of lung lesions of the mycoplasma single vaccine group and the pig triple vaccine group are similar, and are respectively 86.5% and 84.9%, which shows that the triple vaccine effect is equivalent to the mycoplasma single vaccine effect, and the results are shown in fig. 2.
Example 5 Effect of porcine circovirus type 2, Mycoplasma hyopneumoniae, Haemophilus parasuis triple inactivated vaccine on piglet Productivity
In a certain scale pig farm (100 sows in a stall) in Shandong, the strain is binary, 3 batches are randomly selected, about 200 pigs in each batch are respectively immunized round branch and auxiliary triple seedlings at the age of 14 days, a non-immunized group is arranged, and the weight (more than about 20 kg) of the pigs after finishing nursing is recorded when the pigs are transformed into fattening groups. The results show that the average daily gain and the survival rate of the group of the round-branch and auxiliary triple inactivated seedlings are higher than those of the non-immune group in the breeding period, as shown in figure 3 and figure 4.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (10)
1. A triple inactivated vaccine of porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis consists of an antigen and a vaccine adjuvant; the method is characterized in that: the antigen consists of porcine circovirus type 2 antigen, mycoplasma hyopneumoniae antigen and haemophilus parasuis antigen; the porcine circovirus type 2 antigen is a cap protein obtained by expressing and purifying pichia pastoris, and the protein content is more than or equal to 150 microgram/mL; the mycoplasma hyopneumoniae antigen is inactivated mycoplasma hyopneumoniae broken bacterial liquid; the haemophilus parasuis is inactivated haemophilus parasuis crushed bacterial liquid; the vaccine adjuvant is composed of an aqueous high molecular polymer adjuvant and a complex polysaccharide immunopotentiator.
2. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis according to claim 1, wherein: the water-based high molecular polymer adjuvant is Montanide Gel.
3. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis according to claim 1 or 2, wherein: the compound polysaccharide immunopotentiator is a water-soluble compound of Chinese medicinal polysaccharide.
4. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis according to claim 1, wherein: inactivated mycoplasma hyopneumoniae crushed bacterial liquid, the number of viable bacteria before inactivation is not less than 109CCU/mL。
5. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis according to claim 1, wherein: inactivated haemophilus parasuis crushed bacterial liquid, the number of viable bacteria before inactivation is not less than 2.5 multiplied by 109CCU/mL。
6. The method for preparing the triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis according to claim 1, comprising the steps of:
1) culturing pichia pastoris by adopting a sterilized FBSM inorganic salt culture medium, and inducing and expressing the porcine circovirus type 2 cap protein by using methanol; respectively culturing mycoplasma hyopneumoniae and haemophilus parasuis by using a fermentation tank;
2) respectively inactivating the concentrated and purified solution of the circular cap protein and the concentrated solution of mycoplasma hyopneumoniae and haemophilus parasuis;
3) preparing an oil phase, and sterilizing the water-based high-molecular polymer adjuvant for later use;
4) preparing a water phase, dissolving the complex polysaccharide immunopotentiator by adopting a PBS solution, filtering and sterilizing, and adding a cap protein obtained and purified by pichia pastoris expression and an inactivated mycoplasma hyopneumoniae crushing liquid;
5) placing the water phase obtained in the step 4) into an emulsifying tank, slowly adding the oil phase prepared in the step 3) while stirring, and uniformly stirring;
6) adding inactivated haemophilus parasuis bacterial liquid, and uniformly stirring;
7) adding thimerosal solution, and stirring uniformly to obtain the porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine.
7. The method for preparing the triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis according to claim 6, wherein the method for preparing the porcine circovirus type 2 antigen comprises the following steps:
① inoculating Pichia pastoris cell in culture medium for seed culture, inoculating the proliferated seed liquid in fermentation culture medium for fermentation, adding methanol for induction when OD600 of the liquid is 1.0, and collecting the liquid;
② centrifuging the yeast harvest solution continuously to remove supernatant, resuspending the precipitate with 0.01M PBS solution, and crushing;
③ ultrafiltering and concentrating the supernatant with 300KD to obtain concentrated solution of porcine circovirus type 2 antigen cap protein;
④ the cap protein concentrate is inactivated and purified by column chromatography.
8. The method for preparing the triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis according to claim 6, wherein the method for obtaining the mycoplasma hyopneumoniae antigen comprises the following steps:
A) inoculating the mycoplasma hyopneumoniae into a mycoplasma liquid culture medium, and when the color of the culture medium turns yellow and is slightly turbid and the pH value is reduced to 6.8, obtaining mycoplasma hyopneumoniae bacterial liquid;
B) the mycoplasma hyopneumoniae harvest liquid is concentrated by ultrafiltration, so that the viable count of the mycoplasma hyopneumoniae reaches 1012-1013Adding 0.01M PBS buffer solution with the volume of 1/2 of the stock solution into the CCU/ml for ultrafiltration again, removing supernatant, crushing thalli precipitates, and finally suspending the thalli precipitates to 1 percent of the original volume by using 0.01M PBS;
C) the concentrated mycoplasma hyopneumoniae is crushed several times and then subjected to inactivation treatment.
9. The method for preparing the triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis according to claim 6, wherein the method for obtaining the haemophilus parasuis antigen comprises the following steps:
a) inoculating qualified Haemophilus parasuis type 4 and 5 seeds in a TSB liquid culture medium according to the amount of 1%, culturing for 16-18h at 37 ℃ at 250 rpm in a fermentation tank, and harvesting a bacterial liquid;
b) adopting tangential flow ultrafiltration thallus fermentation liquor; resuspending the thallus precipitate with an equal volume of 0.01M PBS solution containing 1% reagent A, ultrafiltering again to remove supernatant, and finally, resuspending and counting with the PBS solution;
c) and (4) inactivating the thalli obtained by ultrafiltration concentration.
10. The method for preparing the triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and haemophilus parasuis according to any one of claims 7-9, wherein the triple inactivated vaccine comprises: and adding a formaldehyde solution with the final concentration of 0.4% in the inactivation treatment, and inactivating at the temperature of 2-8 ℃ for 24 hours while shaking up every 2 hours.
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