CN115887636A - Porcine circovirus type 2, mycoplasma hyopneumoniae and streptococcus suis triple inactivated vaccine and preparation method thereof - Google Patents
Porcine circovirus type 2, mycoplasma hyopneumoniae and streptococcus suis triple inactivated vaccine and preparation method thereof Download PDFInfo
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- CN115887636A CN115887636A CN202210642333.9A CN202210642333A CN115887636A CN 115887636 A CN115887636 A CN 115887636A CN 202210642333 A CN202210642333 A CN 202210642333A CN 115887636 A CN115887636 A CN 115887636A
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- streptococcus suis
- mycoplasma hyopneumoniae
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Abstract
The invention discloses a triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and streptococcus suis 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 streptococcus suis antigen; the porcine circovirus type 2 antigen is PCV-2d-cap protein obtained by a genetic engineering means; the mycoplasma hyopneumoniae antigen is inactivated mycoplasma pneumoniae (ES-2) bacterial liquid; the streptococcus suis is inactivated SS2 and SS9 streptococcus suis bacterial liquid; the immune adjuvant is composed of a novel water adjuvant, and can improve cellular immunity and humoral immunity. The triple inactivated vaccine for the porcine circovirus type 2, the mycoplasma hyopneumoniae and the streptococcus suis provided by the invention has more remarkable advantages in preventing the three porcine diseases.
Description
Technical Field
The invention relates to the field of biological products for livestock, in particular to a porcine circovirus type 2, mycoplasma hyopneumoniae (ES-2) and streptococcus suis (SS 2 and SS 9) triple inactivated vaccine and a preparation method thereof.
Background
Porcine circovirus disease is an infectious disease caused by porcine circovirus type 2, and the virus attacks the immune system of pigs after entering the body, so that the immune capability is reduced, immune organs are damaged, and the infected pigs are leaned, hindered in growth and development, anaemia, diarrhea, jaundice and dyspnea, breeding disorders exist in sows, visceral organs and skins have wide pathological changes, and diseases such as mycoplasma hyopneumoniae, streptococcus suis and the like are secondary or concurrent.
The swine mycoplasmal pneumonia is a common respiratory infectious disease in the live pig breeding, mainly causes the disease symptoms of asthma of the live pigs, and in the actual breeding process, the death rate of singly infected swine mycoplasmal pneumonia diseases is relatively low, but the body immunity of the swine generally suffering from the mycoplasma pneumonia diseases is reduced, and the pathogens of porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, swine influenza virus, pasteurella multocida and the like are mixed to aggravate the disease condition, so that the death of the swine is caused.
The swine streptococcosis is an acute infectious zoonosis and is a two-class animal epidemic disease in China. Streptococcus suis belongs to the group of gram-positive cocci, which can be divided into 35 different serotypes according to the type of capsular polysaccharide, of which types 2 and 9 are pathogenic streptococci. Streptococcus is not obviously seasonal, wherein the incidence rate is higher in two seasons of summer and autumn, and the disease is more endemic and sometimes outbreak. Both morbidity and mortality are high. The sources of the swine streptococcosis are sick pigs, pigs died of illness and pigs with bacteria, and the swine streptococcosis is clinically manifested by septicemia, meningitis, arthritis and purulent lymphadenitis because of a large amount of bacteria in blood, muscle, viscera, saliva, excrement, urine, nasal discharge and the like.
Only single vaccine or double vaccine aiming at the three diseases exists in the market at present, the number of immunization times is excessive in the actual operation process, and the stress on pigs is large; meanwhile, the pig industry also has the problem of antibiotic abuse, which leads to the enhancement of the durability of pathogens, so that the curative effect of the medicament is basically lost. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae (ES-2) and streptococcus suis (SS 2 and SS 9) provided by the invention can meet the requirement of one injection for three prevention, reduce the frequency and stress of immunity and well solve the actual problems encountered in the pig industry.
Disclosure of Invention
In order to overcome the short plate of the existing vaccine, the invention provides a preparation method of a porcine circovirus type 2, mycoplasma hyopneumoniae (ES-2) and streptococcus suis (SS 2, SS 9) triple inactivated vaccine.
The technical scheme of the invention is as follows:
a triple inactivated vaccine of porcine circovirus type 2, mycoplasma hyopneumoniae and streptococcus suis comprises an inactivated vaccine and a vaccine adjuvant; the method is characterized in that: the inactivated vaccine contains an inactivated PCV2 protein antigen, and the inactivated PCV2 protein antigen has an inactivated preservation number of CCTCC NO: m2018570, the inactivated antigen of Mycoplasma hyopneumoniae ES-2 has a preservation number of CCTCC NO: m2011282 antigen of streptococcus suis 2-LT and inactivated collection number CCTCC NO: an antigen of Streptococcus suis 9-YT of M2022010; the immune adjuvant is composed of water-phase liquid nanoparticle adjuvant.
Preferably, the preparation process of the PCV-2d-cap protein antigen comprises the following steps: the method comprises the steps of replacing a promoter Pph of a skeleton vector with a promoter SV40 of a nucleotide sequence shown in SEQ ID No.1, inserting one or more nucleotide sequences of PCV2d cap protein shown in SEQ ID No.2 at a polyclonal site at the downstream of the skeleton vector, then transforming into a bacillus coli infected state DH10Bac, obtaining recombinant baculovirus plasmids through transposition recombination, coating kanamycin, tetracycline, gentamicin, IPTG and an X-gal flat plate with a recovered bacterial liquid, culturing at 37 ℃ in a dark place, selecting a white single colony for expanding culture, transfecting Sf21 insect cells to obtain P0 generation recombinant baculovirus, carrying out plaque purification, selecting a plaque to passage in Sf21 cells to obtain the recombinant baculovirus, culturing in a suspension cultured insect cell High Five infected with the recombinant baculovirus with the MOI of 0.01, centrifuging to obtain a supernatant, purifying through a nickel column affinity chromatography column to obtain PCV2d cap protein, wherein the skeleton vector is pFastBac AC.
Preferably, the adjuvant is an aqueous phase liquid nanoparticle adjuvant IMS251C.
Preferably, the inactivated Mycoplasma hyopneumoniae (ES-2) bacterial liquid has the number of live bacteria before inactivation not less than 10 9 CCU/ml. Inactivated 2-LT and 9-YT streptococcus suis bacterial liquid, the number of viable bacteria before inactivation is not less than 3 x 10 9 CFU/ml。
The preparation method of the porcine circovirus type 2, mycoplasma hyopneumoniae and streptococcus suis triple inactivated vaccine comprises the following steps:
(1) Performing suspension culture on the expression system of the recombinant baculovirus and the insect cell by using a bioreactor to obtain the PCV-2d-cap protein of the porcine circovirus, and purifying and concentrating the protein; respectively culturing mycoplasma hyopneumoniae ES-2 and streptococcus suis 2-LT and 9-YT by using a fermentation tank;
(2) Respectively inactivating the mycoplasma hyopneumoniae ES-2 and the streptococcus suis 2-LT and 9-YT, and concentrating;
(3) Sterilizing adjuvant IMS 251C;
(4) Placing the PCV-2d-cap protein obtained in the step (1), the inactivated mycoplasma hyopneumoniae ES-2 obtained in the step (2) and the concentrated bacterial liquid of streptococcus suis 2-LT and 9-YT into an emulsification tank, slowly adding the prepared adjuvant while stirring, and uniformly stirring; and subpackaging to obtain the porcine circovirus type 2, mycoplasma hyopneumoniae ES-2, streptococcus suis 2-LT and 9-YT triple inactivated vaccine.
As a preferred scheme, the preparation method of the porcine circovirus type 2 antigen comprises the following steps:
(1) Constructing a recombinant plasmid pFastBacDual-PCV-2 d-cap;
(2) Transfecting a recombinant plasmid pFastBac Dual-PCV-2d-cap into sf21 insect cells by a liposome transfection method, and carrying out PCR to identify a recombinant baculovirus;
(3) The recombinant baculovirus was inoculated into suspension-cultured insect cells High Five (purchased from Invitrogen);
(4) Centrifuging after 3-4 days to obtain supernatant;
(5) Ultrafiltering, clarifying, and purifying with nickel column affinity chromatography column.
Preferably, the method for obtaining the mycoplasma hyopneumoniae (ES-2) antigen comprises the following steps:
(1) Inoculating mycoplasma hyopneumoniae (ES-2) into a liquid culture medium, and harvesting mycoplasma hyopneumoniae (ES-2) bacterial liquid when the culture medium turns yellow and is slightly turbid and the pH value is 6.8;
(2) Ultrafiltering and concentrating original bacterial liquid of mycoplasma hyopneumoniae (ES-2) to make viable count of mycoplasma hyopneumoniae reach 10 11 -10 12 Adding 1 XPBS buffer solution with the volume 0.5 times of the volume of the stock solution into the CCU/ml, performing ultrafiltration again, removing supernatant, and resuspending the thallus sediment to 1 percent of the original volume by using 1 XPBS;
(3) The concentrated Mycoplasma hyopneumoniae (ES-2) is subjected to inactivation treatment.
Preferably, the method for obtaining the streptococcus suis (2-LT and 9-YT) antigen comprises the following steps:
(1) Transferring the rejuvenated streptococcus suis 2-LT and 9-YT seed solutions into a TSB liquid culture medium according to the inoculation amount of 1%, respectively, setting the temperature of a fermentation tank at 37 ℃ and 200rpm, culturing for 18-20h, and harvesting a bacterial solution;
(2) Respectively counting the streptococcus suis 2-LT and 9-YT bacterial liquids by a dilution plate method;
(3) Inactivating the harvested bacteria liquid;
(4) And (3) adopting tangential flow ultrafiltration inactivated bacteria liquid, resuspending the bacteria by using 1 XPBS buffer solution with the same volume, performing ultrafiltration again to remove supernatant, repeating for 5 times, and finally resuspending the bacteria liquid to the required concentration by using 1 XPBS buffer solution with the proper volume.
As a preferred scheme, adding a thimerosal solution with the final concentration of 0.01 percent into the inactivation treatment of the mycoplasma hyopneumoniae (ES-2), fully and uniformly mixing, inactivating at 37 ℃ for 24 hours, and shaking the bottles for 4-5 times in the period; the inactivation treatment of Streptococcus suis (2-LT, 9-YT) is carried out by adding formaldehyde solution with final concentration of 0.3%, and inactivating at 37 deg.C for 20h, while shaking for 4-5 times.
The beneficial effects of the invention are as follows: the baculovirus expression system has post-translational modification, can correctly fold expressed protein, and has good biological safety, and in the triple inactivated vaccine of the present invention, the porcine circovirus type 2 antigen is PCV-2d-cap protein expressed and purified by baculovirus, and the protein has good immunogenicity. The porcine circovirus type 2 (PCV 2 d) in the triple inactivated vaccine is expressed by a baculovirus vector system, the baculovirus vector replaces a promoter Pph of the vector to be a promoter SV40, the expression level of PCV-2d-Cap protein reaches 0.15mg/mL, the expression level of target protein in the baculovirus vector is obviously improved, the expression content of Cap protein is improved to about 150ug/mL from 20-50ug/mL, and compared with a bacteria, yeast and mammal cell expression system, the baculovirus expression system has the characteristics of high expression yield and capability of processing after translation. The biological activity of the system expression product, such as antigenicity, immunogenicity and the like, is similar to that of natural protein, and a large amount of genetic engineering products can be extracted by infecting insect larvae. PCV2d is a variant strain of PCV2b discovered in 2012, shows higher infectivity and pathogenicity, although there are porcine circovirus type 2 and mycoplasma hyopneumoniae (ES-2) bivalent inactivated vaccines, the PCV2 type Cap protein antigens are mainly Cap protein antigens expressed after codon optimization of isolates of PCV2a and PCV2b, no combined vaccine aiming at PCV2d exists, and the existing porcine circovirus type 2 and mycoplasma hyopneumoniae (ES-2) bivalent inactivated vaccines mainly adopt aqueous high molecular polymer adjuvants Gel, ISA206, aluminum salt adjuvants, mineral oil adjuvants, water-soluble adjuvants and SP oil adjuvants, but are suitable for porcine circovirus type 2, mycoplasma hyopneumoniae (ES-2) and streptococcus suis (2-LT, 9-YT) bivalent inactivated vaccines. The key point is to find an adjuvant with lasting effect and small side effect, in the adjuvant material in the prior art, the side effect of the water adjuvant is small, the immune protection is early, the practicability and the convenience are favored, but the long-acting slow-release effect of the water adjuvant is not as good as that of the oil adjuvant, so that the long-acting slow-release immunopotentiator is very important to be matched. The PCV2d type Cap antibody level generated after the secondary immunization of the PCV2d type single vaccine with the application potential commercially recorded in CN110358742B is weaker than that of the triple vaccine, and the porcine piglet virus infection in the challenge experiment is 1/6 higher than that of the triple vaccine.
The porcine circovirus type 2, mycoplasma hyopneumoniae (ES-2) and streptococcus suis (2-LT, 9-YT) triple inactivated vaccine 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 Balb/c mice are subjected to intraperitoneal injection in an amount which is twice and excessive, the mice are continuously observed for one week, are in good mental state and 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 pig is immunized, the level of the circular antibody, the level of the mycoplasma antibody and the level of the streptococcus suis antibody can exceed the antibody titer level after single vaccine immunization, and the antigen components are not interfered with each other.
Drawings
In order to clarify the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below. However, the drawings described below are merely examples of the present invention, and it will be apparent to those skilled in the art that other information can be derived from the drawings without inventive exercise.
FIG. 1 shows the cap protein after purification;
FIG. 2 shows Western Blot assay;
FIG. 3 is a scanning electron microscope identification of Cap protein VLP;
FIG. 4 shows CCU changes during the culture of Mycoplasma hyopneumoniae (ES-2);
FIG. 5 shows the growth curves of Streptococcus suis SS2 and SS 9;
FIG. 6 is a flow chart of a triple inactivated seedling preparation process;
FIG. 7 is a metabolic inhibition assay;
FIG. 8 is the viral copy number;
FIG. 9 is a graph showing the survival rate of mice challenged with Streptococcus suis SS 2;
FIG. 10 is a graph showing the survival rate of mice challenged with Streptococcus suis SS 9;
FIG. 11 is a picture of lung after challenge.
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 should be apparent 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 sources and characteristics of porcine circovirus type 2 Cap protein, mycoplasma hyopneumoniae (ES-2), streptococcus suis (SS 2, SS 9)
1. Porcine circovirus type 2 Cap protein source and characteristics
(1) Porcine circovirus type 2 Cap protein source
Artificially synthesizing a fragment (709 bp) shown in SEQ ID NO.6 (PCV 2d-ORF 2-1) described in the patent CN110358742B according to a preparation method of the patent CN110358742B, using the target gene as a template to clone the target gene corresponding to a nucleotide sequence shown in SEQ ID NO.2, and performing an amplification procedure at 94 ℃ for 3min; 30s at 94 ℃,30 s at 52 ℃, 40s at 72 ℃ and 35 cycles; 10min at 72 ℃. Recovering PCR product, connecting with vector pMD19-T at 4 deg.C overnight, transforming into Trans5 alpha competent cell by heat shock method, selecting several single colonies for PCR identification, and identifying correct recombinant plasmid named pMD19-T-PCV2d-ORF2-1.
The expression vector pFastBac Dual promoter P PH Replacing by SV40 promoter, wherein SV40 promoter fragment is artificially synthesized, such as SEQ ID NO.1;
pMD19-T-PCV2d-ORF2-1 and the modified expression vector pFastBac Dual vector were double digested with BamHI and EcoRI restriction enzymes, 10 XBuffer 2. Mu.L, 1. Mu.L each of restriction enzymes BamHI and EcoRI, 1. Mu.L template, ddH 2 Adding O to 20 mu L, reacting for 3 hours at 37 ℃, carrying out electrophoresis on the enzyme digestion product by using 1% agarose gel, then recovering a DNA fragment and a pFastBac Dual vector skeleton by using an agarose gel recovery kit, connecting the enzyme digestion product overnight at 16 ℃ by using T4DNA ligase, then transforming the enzyme digestion product into 100ul escherichia coli competent DH10Bac, carrying out ice bath for 20min, carrying out heat excitation for 90s at 42 ℃, carrying out ice bath for 3min, adding 500ul of nonresistant LB (lysozym) for 1h at 37 ℃, coating the mixture on a three-antibody (kanamycin, gentamicin and tetracycline) LB plate, carrying out culture for 24h at 37 ℃, screening and purifying positive bacterial colonies by using a blue-white spot, aseptically selecting positive white bacterial colonies in a three-antibody LB liquid culture medium, carrying out culture for 12-16h, and carrying out extraction on recombinant bacmid-PCV 2d ORF 2;
transfecting recombinant bacmid rBac-PCV2d ORF2 into Sf21 insect cells by adopting a liposome transfection method, culturing at 25 ℃ for 4d, taking supernatant for plaque purification, selecting 3 plaques for passage in the Sf21 cells respectively, extracting the baculovirus DNA after passage, and carrying out PCR identification. Storing the identified recombinant baculovirus for later use;
infecting suspension-cultured insect cells High Five with recombinant baculovirus with multiplicity of infection (MOI) of 0.01, performing suspension culture in 250mL Grace culture medium at 25 ℃ for 4 days, centrifuging, taking supernatant, and purifying by a nickel column affinity chromatography column;
SDS-PAGE verification is carried out on the purified Cap protein, and the result shows that the purity of the Cap protein after purification is more than 90%, the concentration is 0.15mg/mL, and the purity is improved by 0.05mg/mL compared with the Cap protein before optimization, and the result is shown in figure 1.
The purified Cap protein was subjected to Western Blot validation, wherein the primary antibody was a murine PCV-2d-Cap monoclonal antibody (dilution ratio 1.
The purified Cap protein was identified by VLP (Virus-like particle) scanning electron microscope, and the result is shown in FIG. 3.
2. Mycoplasma hyopneumoniae (ES-2) origin and characteristics
CCU and time relation of ES-2 strain in same generation
Taking out Mycoplasma hyopneumoniae (ES-2) frozen at-20 deg.C, standing at room temperature, and recovering to room temperatureWarm inoculating into Friss medium, and adding 5% of CO 2 The culture box (2) was revived at 37 ℃ for 3 days, transferred to a Friss medium according to 1 2 The constant temperature incubator (2) is used for static culture. The CCU was measured every 12 h. Mycoplasma hyopneumoniae (ES-2) was deposited in the China center for type culture Collection (address: china, wuhan, university of Wuhan) at 28.8.2018, and was classified and named Mycoplasma hyopneumoniae ES-2 Mycoplasma hyopneumaniae ES-2 with the deposition number of CCTCC NO: and M2018570.
The results showed that the amount of Mycoplasma hyopneumoniae (ES-2) in the medium was gradually increased from day 0 to day 2.5, and reached a peak by day 2.5, and then the amount showed a straight-line decrease, as shown in FIG. 4.
3. Streptococcus suis (SS 2, SS 9) origin and characteristics
Growth curves and LD50 values for SS2 and SS9, respectively
Thawing Streptococcus suis SS2 and SS9 frozen at-20 deg.C, inoculating to TSB culture medium according to 1% inoculum size after the streptococcus suis is thawed to optimal growth state, performing shaking culture at 37 deg.C and 200rpm, and measuring bacterial liquid OD every 2h 600 . Streptococcus suis 2-LT (Streptococcus suis 2-LT), abbreviated as Streptococcus suis SS2, is deposited in China center for type culture Collection (address: china, wuhan university) at 8/9.2011 with the deposition number of CCTCC NO:
m2011282. Streptococcus suis type 9SS9YT Streptococcus suis serotyp 9SS9YT, abbreviated as Streptococcus suis SS9, was deposited in the China center for type culture Collection (address: china, wuhan university) at 1 month and 5 days 2022 with the deposition number of CCTCC NO: m2022010.
The results show that SS9 has a higher growth rate in the logarithmic growth phase than SS2 and enters stationary phase at 8h, while SS2 enters stationary phase at 10h, the results are shown in FIG. 5.
LD50 values (semi-lethal amounts) of SS2 and SS9 were 2.0X 10, respectively 8 CFU，3.0×10 7 CFU。
Example 2 preparation of triple inactivated vaccines against porcine circovirus type 2, mycoplasma hyopneumoniae (ES-2), streptococcus suis (SS 2, SS 9):
1. preparation of semi-finished product antigen of porcine circovirus type 2
(1) Recombinant baculovirus-infected suspension-cultured insect cells were transferred to a 500L suspension cell culture reactor for suspension culture, and the objective protein PCV-2d-cap was obtained as described in example 1.
(2) Total concentration of expressed protein was determined by BCA method after purification:
(1) Preparing a working solution: an appropriate amount of BCA working solution was prepared according to standard and sample number as 50 volumes of BCA reagent a plus1 volume of BCA reagent B reagent (50.
(2) Diluting a standard product: a10 μ LBSA standard was diluted to 100 μ L with PBS to give a final concentration of 0.5mg/mL. Adding the standard substance into protein standard substance wells of a 96-well plate according to the proportion of 0,1,2,4,8, 12, 16 and 20 mu L, and adding PBS to make up to 20 mu L;
(3) Diluting the sample to a suitable concentration with a total volume of 20 μ L;
(4) 200 μ LBCA working solution was added to each well, and the mixture was left at 37 ℃ for 15 to 30 minutes. And measuring the absorbance value under 562nm by using an enzyme labeling instrument, and calculating the total protein concentration according to a standard curve, wherein the concentration is 1.0mg/mL.
2. Preparation of semi-finished antigen of mycoplasma hyopneumoniae (ES-2)
(1) A liquid medium of Mycoplasma hyopneumoniae (ES-2) was prepared according to the Friss medium recipe (1L) (1 Xhanks 282mL, 376mL of base solution, 235mL of pig serum, 47mL of 50% (w/v) glucose, 47mL of 10% (w/v) lactoprotein hydrolysate, 8.46mL of 0.4% (w/v) phenol red, 4.54mL of 1mol/LNaOH, pH 7.5).
(2) Mycoplasma hyopneumoniae (ES-2) was inoculated into Friss liquid medium according to a ratio of 1 (v: v) and cultured in a 37 ℃ fermentor for 48h, and the bacterial solution was harvested when the medium turned yellow, appeared slightly turbid and the pH dropped to 6.8.
(3) Ultrafiltering and concentrating the harvest solution of Mycoplasma hyopneumoniae (ES-2) to make the viable count of Mycoplasma hyopneumoniae reach 10 12 - 10 13 CCU/ml; adding 1 XPBS buffer solution with 1/2 of the volume of the original solution for ultrafiltration again, removing the supernatant, crushing the bacterial pellet, and finally resuspending the bacterial pellet to 1 percent of the original volume by using 1 XPBS.
(4) Adding thimerosal solution with final concentration of 0.01%, inactivating at 2-8 deg.C for 24 hr, and shaking every 2 hr to ensure complete inactivation.
3. Preparation of semi-finished product antigen of streptococcus suis (SS 2, SS 9)
(1) Inoculating Streptococcus suis SS2 and SS9 to TSB liquid culture medium according to 1% inoculum size, culturing at 37 deg.C and 250rpm for 18h in a fermentation tank, and collecting bacterial liquid.
(2) Respectively counting the SS2 type and SS9 type bacteria liquid of the streptococcus suis by a dilution plate method;
(3) Inactivating the harvested bacterium solution by using 0.3% of formaldehyde;
(4) And (3) adopting tangential flow ultrafiltration inactivated bacteria liquid, resuspending the bacteria by using 1 XPBS buffer solution with the same volume, performing ultrafiltration again to remove supernatant, repeating for 5 times, and finally resuspending the bacteria liquid to the required concentration by using 1 XPBS buffer solution with the proper volume.
4. Triple inactivated vaccine preparation
The preparation of the triple inactivated vaccine is carried out according to the flow chart of the preparation process of the triple inactivated vaccine shown in figure 6.
(1) The adjuvant is steam sterilized at 121 deg.C for 20 min.
(2) Purified Cap protein (final concentration of 20. Mu.g/ml), mycoplasma hyopneumoniae (ES-2) (final concentration of 1X 10) 9 CCU/head), streptococcus SS2 and SS9 (final concentrations 2X 10 each) 9 CFU/ml) in an emulsification tank, and slowly adding the prepared adjuvant while stirring, wherein the stirring parameter is set to be 0.5Kr/min and 30min.
(3) Obtaining the triple inactivated vaccine of porcine circovirus type 2, mycoplasma hyopneumoniae (ES-2) and streptococcus suis (SS 2, SS 9).
Example 3 screening of porcine triple inactivated vaccine adjuvants
1. Alternative adjuvants
The invention mainly selects 8 adjuvants of Summit Poly Solution (Sps, water adjuvant), cpc (water adjuvant), ISA201 (double-phase oil emulsion adjuvant), GEL02 (water-soluble polymeric adjuvant), IMS1313 (water-soluble nano adjuvant), IMS251C (water-phase liquid nano particle adjuvant), aluminum hydroxide adjuvant (colloidal aluminum adjuvant) and white oil 15A adjuvant (mineral oil adjuvant). The ISA201 adjuvant is a novel water-in-oil-in-water (W/O/W) biphasic oil emulsion adjuvant, the main component of the adjuvant is highly refined light mineral oil, and simultaneously contains a small amount of mannitol and oleic acid which are derived from plants, the vaccine prepared by the adjuvant changes the water-in-oil (O/W) formulation formed by the original mineral oil vaccine, so that the viscosity of the vaccine is reduced, the injection is easy, the side reaction is reduced, and the antigen delivery efficacy of the oil adjuvant is kept; the GEL02 water-soluble polymer adjuvant (GEL for short) mainly comprises sodium polyacrylate, has the advantages of large antigen loading capacity, stable property and simple emulsification process, can induce an organism to generate humoral immunity and can also obviously promote the proliferation and differentiation of T lymphocytes so as to generate higher cellular immunity level; IMS1313 water-soluble nanometer adjuvant, gather the lymphocyte active cell through the micro-infiltration of nanometer microgranule to the lymph system, promote antigen presentation cell to the uptake of antigen, act as the antigen presenting cell finally; montanide adjuvant is developed by Seppic company of France, and has Montanide ISA series adjuvant and Montanide IMS series adjuvant, and Montanide IMS series adjuvant is a water-soluble compound containing immunocompetent organic compound and special excipient, and can improve the poor safety of traditional oil adjuvant and aluminium salt adjuvant.
2. Dominant adjuvant screening
(1) The triple inactivated vaccine was formulated using the above 8 alternative adjuvants, respectively, with reference to example 2.
90 BALB/C mice, 18-22g, were fed for one week for acclimation and then divided into 9 groups, i.e., 8 adjuvant candidates and 1 control group. Each mouse was injected intramuscularly with 0.2ml, and the control group was injected with the same dose of PBS solution, and after 7 days, the mice were immunized twice according to the same method, with 0.2ml dose.
(2) After secondary immunization, 14d,8 kinds of alternative adjuvant groups and a control group are respectively subjected to retroorbital venous plexus blood sampling, then blood in a centrifuge tube is placed in a thermostat at 37 ℃ for 1 hour and then is placed at 4 ℃ overnight, after blood coagulation blood clots shrink, the centrifuge is carried out at 4,000rpm for 10 minutes, supernatant is taken out and put in a clean centrifuge tube, namely serum to be detected, and the serum is stored at-20 ℃.
(3) The indirect Elisa method is adopted to detect the serum titer, the Cap protein and the SS2 are respectively coated on the plates, wherein the coating amount of each hole of the Cap protein is 50ng, the coating amount of each hole of the SS2 is 300ng of the total protein concentration after the fragmentation, the serum to be detected is respectively diluted by 40 times, then enzyme-labeled secondary antibody (goat anti-mouse) is added, finally TMB is added for color development, and the light absorption value at 450nm is measured.
The results show that the corresponding SS2 antibody levels are ISA201, sps, cpc, white oil 15A, IMS251C, aluminum hydroxide, GEL02 and IMS1313 in sequence from high to low; the corresponding Cap protein antibody levels are aluminum hydroxide, ISA201, sps, IMS251C, white oil 15A, GEL02, cpc and IMS1313 in sequence from high to low, and the results are shown in Table 1.
TABLE 1 results of antibody detection of Streptococcus SS2 and Cap proteins in mice of different adjuvant groups at 14d post-immunization
(4) After 14 days of second immunization, SS2 challenge was performed with a dose of 4.54 × 10 8 Survival of mice was observed for 5 consecutive days for CFU/mouse, and survival rate was counted.
The result shows that the IMS251C has the best protective potency on the streptococcus suis SS2 challenge, and the potency reaches 80%, and the result is shown in the table 2.
TABLE 2 protective effect of different adjuvant groups on Streptococcus suis SS2 challenge
And combining the antibody level and the toxicity attacking protection effect, and primarily selecting IMS251C as an adjuvant for subsequently preparing the triple inactivated vaccine.
Example 4 safety testing and immune efficacy evaluation of porcine triple vaccine
Safety test of pig triple vaccine in mice
20 BALB/C mice, 18-22g, were fed for one week for acclimation and then divided into 2 groups, i.e., triple vaccine group (adjuvant IMS251C, the same below) and control group. Each mouse is injected with 0.2ml of intramuscular injection, a control group is injected with PBS solution with the same dose, and after continuous observation for 14 days, two groups of mice are all healthy and have no local or systemic adverse reaction, and the result shows that the porcine triple vaccine prepared in the example 2 is safe for the mice.
Antibody production level of pig triple vaccine in mice
70 BALB/C mice (18-22 g) were fed for one week and then divided into 7 groups, i.e., group A (commercial porcine circulant monoprop group (i.e., ketonin, supplied by probiotics of Wuhan Ke Co., ltd.)), group B (Streptococcus suis SS2+ SS9 inactivated vaccine group (SS 2, SS9, 2X 10, respectively) 9 CFU/mL, the same below)), group C (PCV 2dCap (20. Mu.g/mL) + SS2+ SS 9), group D (PCV 2dCap (20. Mu.g/mL) + Mycoplasma hyopneumoniae (ES-2) (1X 10) 9 CCU/head)), group E (SS 2+ SS9+ Mycoplasma hyopneumoniae (ES-2) (1X 10) 9 CCU/head)), group F (pig triple vaccine group) and group G (control group), each of which was 10 mice, each of which was injected intramuscularly with 0.2ml, and the control group was injected with an equal dose of PBS solution, and after 7 days, secondary immunization was performed according to the same method, with a dose of 0.2ml and 14d after secondary immunization, and the antibody production in each group was determined with reference to example 3.
TABLE 3 antibody levels corresponding to PCV2dCap protein, SS2 and SS9 produced by different groups
The results show that after the serum to be tested is diluted by 40 times, the antibody level corresponding to PCV2D Cap protein generated by the triple vaccine group (group F) is slightly higher than that of the porcine circovirus single vaccine group (group A) and the double vaccine group (group C and group D), while the antibody corresponding to SS2 and the antibody corresponding to SS9 generated by the triple vaccine group (group F) are both equivalent to that of the SS2+ SS9 inactivated vaccine group (group B) and the double vaccine group (group C and group E), and the results are shown in Table 3.
Evaluation of immune effect of porcine triple vaccine in New Zealand white rabbits
25 New Zealand white rabbits (1-1.2 kg) were fed for one week and randomly divided into 5 groups, group a (i.e., group a)Commercial mycoplasma inactivated vaccine group (Kechuanning, supplied by probion GmbH of Wuhan Ke), group D (PCV 2D Cap (20. Mu.g/ml) + Mycoplasma hyopneumoniae (ES-2) (1X 10) 9 CCU/head)), group E (SS 2+ SS9+ Mycoplasma hyopneumoniae (ES-2) (1X 10) 9 CCU/head)), group F (triple), and group G (control). Each rabbit was injected intramuscularly with 0.5ml, and the control group was injected with the same dose of PBS solution, and after 7 days, secondary immunization was performed according to the same method, with a dose of 0.5ml. At 30d after immunization, blood was collected from the marginal vein of the ear, and serum was separated and subjected to IHA (Indirect hemagglutination assay) to determine the antibody titer against Mycoplasma hyopneumoniae (ES-2).
The IHA comprises the following specific steps:
(1) Taking 1mL of 10% dialdehyde-fixed sheep red blood cell suspension, centrifuging at 3000r/min, discarding the supernatant, washing once with 0.1mol/LpH4.0 acetic acid buffer solution, centrifuging at 3000r/min, discarding the supernatant, adding 0.5mL of concentrated lysis mycoplasma hyopneumoniae (ES-2) and 9.5mL of mol/L pH4.0 acetic acid buffer solution into the precipitated red blood cells, carrying out water bath at 37 ℃ for 1h, centrifuging at 3000r/min, discarding the supernatant, washing 5 times with 0.1mol/L of phosphate buffer solution with pH7.2, centrifuging at 3000r/min, discarding the supernatant, adding 10mL of 5% small phosphate buffer solution to obtain sensitized bovine serum, and placing at 4 ℃ for later use.
A sample of diluted solution is added into each hole of a V-shaped micro hemagglutination plate with 96 holes, 25 mu L/hole, equal amounts of negative serum, positive serum and serum to be detected are respectively added into the 1 st hole of each row for multiple serial dilution, the last hole is reserved for blank control, 1% sensitized erythrocyte suspension is added into each hole, 25 mu L/hole is placed at 37 ℃,1h is carried out, and the result is observed. The serum titer is expressed by the highest dilution of serum when the sensitized red blood cells in the hole agglutinate more than 50%, and the test determines that the titer is more than 1:5 as positive.
The results show that the antibody levels produced by mycoplasma inactivated vaccine group (group a) were consistent with triple vaccine group (group F), with triple vaccine group (group F) titers greater than 1:20, the triple vaccine is proved to have protective power on pigs, and the results are shown in table 4.
TABLE 4 antibody levels corresponding to Mycoplasma produced by different groups
Metabolic inhibition assay
Taking 7 sterile vials containing 2mL Fris liquid medium, adding 0.4mL (10 in sequence) of Mycoplasma hyopneumoniae ES-2 strain culture into the first 3 vials (5, 4, 3) 5 ，10 4 ，10 3 CCU) and separated serum which is the serum of the inactivated single seedling group or triple seedling group, and 0.4ml (10 in sequence) of mycoplasma hyopneumoniae ES-2 strain culture is respectively added into the last 3 (5-1, 4-1 and 3-1) of the inactivated single seedling group or triple seedling group 5 ，10 4 ，10 3 CCU), 0.4ml 1 × PBS was added to the last (negative) as a negative control. After culturing at 37 ℃ for 15 days, the color change of the medium was observed.
The result shows that the color of the culture medium is unchanged and is consistent with that of the negative control when the serum of the inactivated single-seedling group and the serum of the triple-seedling group are added, but only the mycoplasma hyopneumoniae ES-2 is added, the color of the culture medium is changed from red to yellow, which shows that the antibody generated by the inactivated single-seedling group and the triple-seedling group has a metabolic inhibition effect on the mycoplasma hyopneumoniae ES-2, and the result is shown in figure 7.
Protective effect of porcine triple vaccine on porcine circovirus PCV-2d, streptococcus suis SS2 and streptococcus suis SS9 challenge
The porcine triple vaccine has the protective effect on the PCV-2d virus attack of porcine circovirus: 30 BALB/C mice, 18-22g, were fed for one week for acclimation and immediately divided into 3 groups, i.e., a commercial porcine circus single-shoot group, a triple-shoot group, and a control group. Each mouse was injected intramuscularly with 0.2ml, and the control group was injected with the same dose of PBS solution, and after 7 days, secondary immunization was performed according to the same method, with a dose of 0.2ml. 14d after immunization, PCV-2d strain challenge (more than or equal to 10) 6.5 TCID 50 mL), 0.5mL was intraperitoneally injected into each mouse, and blood was collected from the retroorbital venous plexus 21 days after challenge, and serum was separated to determine the virus copy number.
The virus copy number is measured, and fluorescence quantitative PCR is firstly carried out to obtain a Ct value. The fluorescent quantitative reaction system comprises: 2 XSuperRealPreMixGlus 10. Mu.L, upstream and downstream primers and probes 0.5. Mu.L each, serum 2. Mu.L, ddH 2 And O is supplemented to 20 mu L. Wherein the upstream guideThe substance sequence is PCV2-F2:
5 'CGGATATTGTAGTCCTGGTCGTA-3', the downstream primer sequence is PCV2-R2:
5' CCTGTGTCCCTATTGATT-:
FAM-5 'CTAGGCCTACTGTGGGTCTACATTTC-3' -TAMRA. The fluorescent quantitative reaction program comprises: 2min at 95 ℃;95 ℃ 10s,60 ℃ 35s,40 cycles, and a dissolution curve analysis from 60 ℃ to 95 ℃. Wherein, the relation between the Ct value and the copy number is as follows: ct = -2.3693 XLog 10 (copy number) +33.628 (R) 2 ＝0.9999)。
The results show that the corresponding virus copy numbers of the commercial porcine circovirus single-vaccine group and the commercial porcine tripartite vaccine group are obviously lower than those of the control group, and the porcine tripartite vaccine has a good protection effect on PCV-2d virus challenge, and the results are shown in FIG. 8.
Protective effect of swine triple vaccine on streptococcus suis SS2 challenge
30 BALB/C mice (18-22 g) were fed for one week and then divided into 3 groups, i.e., streptococcus suis SS2+ SS9 inactivated vaccine groups (SS 2, SS9 2X 10, respectively) 9 CFU/mL), triple shoot group and control group. Each mouse was injected intramuscularly with 0.2ml, and the control group was injected with the same dose of PBS solution, and after 7 days, the mice were immunized twice according to the same method, with 0.2ml dose. 14 days after immunization, SS2 challenge was performed, and the challenge dose per mouse was 5.38X 10 8 Mice were observed for survival for 5 consecutive days in CFU/mouse.
The results show that the toxic dose of the streptococcus suis SS2 after challenge of mice is 5.38 multiplied by 10 8 The control group died 100% after 3 days, the protective effect of the SS2+ SS9 inactivated vaccine group was 40%, and the protective effect of the pig triple group was 60%, which indicates that the protective level of the pig triple vaccine against Streptococcus suis SS2 challenge is good, and the results are shown in FIG. 9.
Protective effect of swine triple vaccine on streptococcus suis SS9 virus challenge
30 BALB/C mice, 18-22g, were fed for one week and then divided into 3 groups, i.e., S.suis SS2+ SS9 inactivated vaccine groups (SS 2, SS9, 2X 10, respectively) 9 CFU/mL), triple-vaccinated group and control group. Each mouse was injected intramuscularly with 0.2ml, the control group was injected with the same dose of PBS solution, 7 days later, as per the sameThe method is used for secondary immunization, and the dosage is 0.2ml. 14 days after immunization, SS9 challenge was performed, and the challenge dose per mouse was 9.6X 10 7 Mice were observed for survival 5 consecutive days CFU/mouse.
The results show that the toxin-attacking dose is 9.6 multiplied by 10 after the streptococcus suis SS9 toxin-attacking mice 7 The control group died 90% after 2 days, the protective effect of the SS2+ SS9 inactivated vaccine group was 70%, and the protective effect of the swine triple combination group was 90%, which indicates that the swine triple combination vaccine has a good protective level against SS9 virus challenge of streptococcus suis, and the results are shown in FIG. 10.
Example 5 rescreening of porcine triple inactivated vaccine adjuvant
(1) The 3 kinds of adjuvants (IMS 251C, white oil 15A, GEL 02) obtained by the preliminary screening in example 3 were prepared as triple inactivated vaccines with reference to example 2.
(2) 85 healthy susceptible piglets of 14-21 days old are screened and immediately divided into 4 groups, namely 20 triple-seedling IMS251C adjuvant groups, 20 triple-seedling white oil 15A adjuvant groups, 20 triple-seedling white oil GEL02 adjuvant groups, 20 toxicity attack control groups and 5 circular blank control groups. Each piglet was injected with 2ml of neck muscle, and the control group was injected with the same amount of PBS solution. After 21 days, a second immunization was carried out in an equivalent manner at a dose of 2ml.
(3) At 14d after the second immunization, 5 of each group were randomly selected for collecting blood from the anterior vena cava, and antibody levels corresponding to Cap protein, SS2, SS9, and Mycoplasma (detected according to the instruction manual of the kit of IDEXX) were determined. When the corresponding antibody titer of the Cap protein is determined, the Cap protein is diluted by 40, 80, 160, 320, 640, 1280, 2560 and 5120 times.
The results show that the average water levels of Cap protein, SS2, SS9 and mycoplasma antibodies generated by the IMS251C adjuvant group are superior to those of the white oil 15A adjuvant group and the GEL02 adjuvant group, so the IMS251C is selected as an adjuvant for the subsequent preparation of the triple inactivated vaccine, and the results are shown in Table 5.
TABLE 5 levels of antibodies corresponding to Cap protein, SS2, SS9 and Mycoplasma produced by different groups
At 14d after the second immunization, 5 piglets of the triple vaccine group and the challenge control group were simultaneously challenged, and 2.5ml of PCV2WH strain virus solution (the virus content is 1.0 multiplied by 107.0 TCID50/ml) was injected into each piglet through nasal drip and muscle meat injection. All piglets were weighed on the day of infection. Weighing all piglets 28 days after infection; collecting blood of all piglets, separating serum, detecting viremia by using porcine circovirus type 2 specific PCR, killing all piglets, taking inguinal lymph node and mesenteric lymph node, performing histological and immunohistochemical detection (IHC), and counting the morbidity of each test group.
Relative weight gain rate = (average daily gain of control group-average daily gain of challenge test piglet)/average daily gain of triple-bred group piglet;
average daily gain of control group = (sum of piglet weights of control group at 28 days after challenge-sum of piglet weights of control group at the same day of challenge)/(28 days × 5 heads);
average daily gain of the triple-bred group piglets = (the sum of weights of the triple-bred group piglets at 28 days after challenge-the sum of weights of the triple-bred group piglets at the same day of challenge)/(28 days × 5 piglets);
average daily gain of the challenge trial piglets = (the weight of the piglet in 28 days after challenge-the weight of the piglet in the day of challenge)/28 days.
The results show that the incidence rate of the IMS251C adjuvant group is obviously lower than that of the challenge control group, and is lower than that of the white oil 15A adjuvant group and the white oil GEL02 adjuvant group, and the IMS251C adjuvant group has the best effect of challenge protection on the PCV2WH strain (see Table 6).
TABLE 6 PCV2WH strain challenge protection test results 14 days after secondary immunization
At 14d after the second immunization, simultaneously performing virus attack on 5 piglets of the triple vaccine group and the virus attack control group, performing tracheal injection on each piglet by 10ml of mycoplasma hyopneumoniae ES-2, observing 28-day section killing after virus attack, scoring the pneumonia lesions of the test pigs according to a Goodwin 55 division method, and calculating the pneumonia lesion reduction rate;
the result shows that the reduction rate of pneumonia lesions is the highest in the IMS251C adjuvant group, and that the IMS251C adjuvant group has the best virus attack protection effect on the mycoplasma hyopneumoniae ES-2 (see Table 7).
TABLE 7 protective test results against challenge with Mycoplasma hyopneumoniae ES-2 14 days after secondary immunization
Performing SS2 and SS9 challenge 14d after the second immunization, wherein the SS2 challenge dose is 1.638 multiplied by 10 7 CFU/head, SS9 challenge dose 4X 10 9 CFU/head, growth status of piglets was observed for 14 consecutive days.
The results show that the IMS251C has the best protective titer against the streptococcus suis SS2 challenge, the immune protection is 100%, meanwhile, the IMS251C has the best protective effect against the streptococcus suis SS9 challenge, the immune protection is 60%, and the results are shown in tables 8 and 9.
TABLE 8 protective effect of different adjuvant groups on Streptococcus suis SS2 challenge
TABLE 9 protective effect of different adjuvant groups on Streptococcus suis SS9 challenge
Example 6 evaluation of safety and immune Effect of the porcine triple vaccine in pigs
Safety test of pig triple vaccine in piglet body
10 healthy susceptible piglets of 14-21 days old are screened and immediately divided into 2 groups, namely a triple-bred group and a control group. 4ml of neck muscle injection is carried out on each piglet, a PBS solution with the same dose is injected into a control group, and the piglet is normal in spirit, breath and ingestion after continuous observation for 14 days; the vaccine injection part has no red swelling and induration, and has no local inflammatory reaction.
The body temperature of the piglets after the triple-vaccine injection has no abnormal change, and only slightly rises 1 day after the immunization (not more than 0.5 ℃ before the inoculation), and the piglets recover to the basal body temperature from the 2 nd day (see table 10). The average daily gain of piglets 14 days after inoculation is 0.2kg (see table 11), which indicates that the pig triple vaccine has good safety for piglets.
TABLE 10 body temperature measurements of piglets by one overdose vaccination of vaccines
TABLE 11 piglet weight gain results 14 days post-inoculation
Evaluation of antibody level immune effect of pig triple vaccine in piglet body
Screening 30 healthy susceptible piglets of 14-21 days old, and immediately dividing into 6 groups, namely a commercial porcine circovirus single vaccine group (Kechuanning) (5 heads), a commercial mycoplasma inactivated vaccine group (Kechuanning) (5 heads), a commercial streptococcus inactivated vaccine group (Kechuanning) (5 heads), a streptococcus suis SS2+ SS9 inactivated vaccine group (5 heads), a triple vaccine group (5 heads) and a control group (5 heads). 2ml of the control group was injected with PBS solution at the same dose in the neck muscle of each piglet, and after 21 days, secondary immunization was carried out at 2ml in the same manner, and blood samples before and after immunization (14 days, 21 days, 28 days, 35 days, and 48 days) were taken, respectively, and antibody levels corresponding to PCV2d Cap protein, SS2, SS9, and Mycoplasma were measured with reference to example 5.
The results show that the antibody titers corresponding to PCV2d Cap protein, SS2 and SS9 produced by the triple vaccine group are increased along with the time after immunization, the average antibody titer to 48 days is 512 +/-175.27, 0.866 +/-0.133 and 0.733 +/-0.152 respectively, the antibody titer corresponding to mycoplasma reaches the maximum value (1.022 +/-0.051) at 35 days, and the average antibody titer produced by the triple vaccine group is superior to that produced by the inactivated vaccine group compared with the commercial porcine circovirus single vaccine group, the commercial mycoplasma inactivated vaccine group and the streptococcus suis SS2+ SS9 inactivated vaccine group, and the results are shown in tables 12, 13, 14 and 15.
TABLE 12 mean antibody titers corresponding to PCV2d Cap protein at different times
TABLE 13 mean antibody titers corresponding to Streptococcus SS2 at different times
TABLE 14 mean antibody titers corresponding to different time streptococci SS9
TABLE 15 mean antibody titers corresponding to Mycoplasma at different times
Porcine triple vaccine for virus challenge protection effect on PCV2WH strain
The results show that the morbidity of the triple seedling group is obviously lower than that of the control group, the morbidity is 0 and is equivalent to that of a commercial single seedling, and the effects of virus attack protection of the triple seedling on the PCV2WH strain are better (see table 16).
TABLE 16 PCV2WH strain challenge protection test results 14 days after secondary immunization
Protective effect of pig triple vaccine on virus attack of mycoplasma hyopneumoniae (ES-2)
the result shows that the pneumonia lesion reduction rate of the triple vaccine group is more than 60 percent and is equivalent to that of the commercialized single vaccine (see table 17), and compared with the control group, the triple vaccine group has no obvious lesion (see figure 11), and the virus attack protection effect on the mycoplasma hyopneumoniae ES-2 by the triple vaccine is better.
TABLE 17 protection of Mycoplasma hyopneumoniae ES-2 challenge 14 days after II immunization test results
Protective effect of swine triple vaccine on attack of streptococcus suis type 2
The results show that the virus challenge protection of the triple vaccine on the streptococcus suis SS2 is equivalent to that of the commercial single vaccine, the immune protection is 100%, and the results are shown in table 18.
TABLE 18 protective effect of different adjuvant groups on Streptococcus suis SS2 challenge
Protective effect of swine triple vaccine on virus attack of streptococcus suis 9
The results show that the protective effect of the triple vaccine on the streptococcus suis SS9 virus challenge is the best, the immune protection is 60%, and the results are shown in table 19.
TABLE 19 protective effect of different adjuvant groups on Streptococcus suis SS9 challenge
Sequence listing
<110> Wuhan Keshi Probiotics GmbH
<120> triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and streptococcus suis and preparation method thereof
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ccctgctccc caatcaccca gggtgacagg ggagtgggct ccactgctgt tattctagat 360
gataactttg taacaaaggc caatgcccta acctatgacc cctatgtaaa ctactcctcc 420
cgccatacca taacccagcc cttctcctac cactcccggt actttacccc gaaacctgtc 480
cttgatagga caatcgatta cttccaaccc aataacaaaa gaaatcaact ctggctgaga 540
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Claims (6)
1. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and streptococcus suis consists of an inactivated vaccine and a vaccine adjuvant; the method is characterized in that: the inactivated vaccine contains an inactivated PCV2 protein antigen, and the inactivated PCV2 protein antigen has an inactivated preservation number of CCTCC NO: m2018570, the inactivated antigen of Mycoplasma hyopneumoniae ES-2 has a preservation number of CCTCC NO: m2011282 antigen of streptococcus suis 2-LT and inactivated collection number CCTCC NO: an antigen of Streptococcus suis 9-YT of M2022010; the immunologic adjuvant is composed of an aqueous phase liquid nanoparticle adjuvant.
2. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and streptococcus suis according to claim 1, wherein the PCV-2d-cap protein antigen is prepared by a process comprising: replacing a promoter Pph of a skeleton vector with a promoter SV40 of a nucleotide sequence shown as SEQ ID No.1, inserting one or more nucleotide sequences of PCV2d cap protein shown as SEQ ID No.2 into a multiple cloning site at the downstream of the skeleton vector, then transforming into escherichia coli competent DH10Bac, recombining by transposition to obtain recombinant baculovirus plasmids, coating a recovered bacterial solution with kanamycin, tetracycline, gentamicin, IPTG and an X-gal plate, culturing at 37 ℃ in a dark place, selecting a white single colony for amplification culture, transfecting Sf21 insect cells to obtain P0 generation recombinant baculovirus, carrying out plaque purification, subculturing hollow plaques in Sf21 cells to obtain recombinant baculovirus, infecting the suspension cultured insect cells with the recombinant baculovirus with the infection complex MOI of 0.01 in High Five, centrifuging to take supernatant, and purifying through a nickel column affinity chromatography column to obtain the PCV2d cap protein, wherein the skeleton vector is pFastBac.
3. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae, and streptococcus suis of claim 2, wherein: the adjuvant is an aqueous phase liquid nanoparticle adjuvant IMS251C.
4. The triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae, and streptococcus suis of claim 3, wherein: the number of viable bacteria before the inactivation of mycoplasma hyopneumoniae is not less than 10 9 CCU/ml。
5. The porcine circovirus type 2, mycoplasma hyopneumoniae of claim 4The streptococcus suis triple inactivated vaccine is characterized in that: the number of live bacteria before inactivation of streptococcus suis 2-LT and 9-YT is not less than 3 × 10 9 CFU/ml。
6. The method for preparing the triple inactivated vaccine for porcine circovirus type 2, mycoplasma hyopneumoniae and streptococcus suis according to any one of claims 2 to 5, comprising the steps of:
(1) Performing suspension culture on the expression system of the recombinant baculovirus and the insect cell by using a bioreactor to obtain the PCV-2d-cap protein of the porcine circovirus, and purifying and concentrating the protein; respectively culturing mycoplasma hyopneumoniae ES-2 and streptococcus suis 2-LT and 9-YT by using a fermentation tank;
(2) Respectively inactivating the mycoplasma hyopneumoniae ES-2 and the streptococcus suis 2-LT and 9-YT, and concentrating;
(3) Sterilizing the water-phase liquid nanoparticle adjuvant IMS251C for later use;
(4) Placing the PCV-2d-cap protein obtained in the step (1), the inactivated mycoplasma hyopneumoniae ES-2 obtained in the step (2) and the concentrated bacterial liquid of streptococcus suis 2-LT and SS9 in an emulsification tank, slowly adding the prepared adjuvant while stirring, and uniformly stirring to obtain the porcine circovirus type 2, mycoplasma hyopneumoniae ES-2, streptococcus suis 2-LT and 9-YT triple inactivated vaccine.
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