CN112891525A

CN112891525A - Application of mycoplasma capricolum subspecies of capricolum in preparing vaccine for contagious pleuropneumonia of capricolum

Info

Publication number: CN112891525A
Application number: CN202110102772.6A
Authority: CN
Inventors: 储岳峰; 郝华芳; 陈胜利; 颜新敏; 马丽娜; 刘永生
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2021-06-04
Anticipated expiration: 2041-01-26
Also published as: CN112891525B

Abstract

The invention provides application of mycoplasma capricolum subspecies capricolum pneumonia Mcpcp 1801 in preparation of a capricolumniferous pleuropneumonia vaccine, and relates to the technical field of biology. The invention also provides a vaccine based on the M1801 strain and a preparation method thereof, wherein the vaccine can improve the immune protection effect, reduce the immune side reaction and provide a good immune protection effect; meanwhile, the M1801 strain has strong toxicity, good immunogenicity and excellent growth performance, the vaccine preparation of the strain can induce an organism to generate high-level antibodies, the antibody generation time is short, and the titer is high; the vaccine is high in safety; the immune sheep 4/5 has effects of relieving body temperature rise, cough, sneeze, and mental depression, and alleviating pathological changes of tissues.

Description

Application of mycoplasma capricolum subspecies of capricolum in preparing vaccine for contagious pleuropneumonia of capricolum

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of mycoplasma capricolum subspecies pneumonia Mcpc 1801 in preparation of a contagious pleuropneumonia vaccine of goats.

Background

Contagious caprine pleuropneumonia (CCPP) is a contact respiratory infectious disease of goats, is caused by Mycoplasma caprine subsp. The contagious caprine pleuropneumonia is mainly transmitted by contact and spray, the incidence rate of epidemic diseases in a new epidemic area is up to 100 percent, and the death rate is up to 80 percent. The regions of Africa, middle east, Asia and the like are the main epidemic regions of the disease, in recent years, the host range of the disease is wider and wider, and the disease becomes an important epidemic disease which seriously jeopardizes the health of the world sheep raising industry and wild ruminants such as wild goats, Tibetan antelopes, Arab antelopes and the like.

The prevention and control of goat contagious pleuropneumonia mainly comprises vaccines, medicines and the like, and clinical medicines have limited effect and are difficult to completely eliminate pathogens. The prevention of immunity is an important measure for preventing and controlling goat contagious pleuropneumonia. At present, the vaccine of the goat infectious pleuropneumonia is mainly an inactivated vaccine, including a thallus inactivated vaccine and a tissue inactivated vaccine. The goat infectious pleuropneumonia tissue inactivated vaccine has the problems of great biological safety risk, product standardization, unstable pathogen content, easy existence of other pathogen pollution, complex process, high requirement on test plant sites, limited yield, great side effect and the like. The thallus inactivated vaccine can overcome partial defects of the tissue inactivated vaccine, such as unstable content of vaccine pathogen, possible pollution of other pathogen and the like, and plays an important role in preventing and controlling goat infectious pleuropneumonia. At present, a vaccine-making strain is a strain which is popular for more than ten years or earlier, and is prepared by inactivating inactivators such as formaldehyde and matching with adjuvants such as an oil adjuvant, so that part of sheep has large side reaction after immunization, the problem of effective antigen destruction or antigen epitope inactivation existing in the inactivators such as formaldehyde and the like is solved, and the problems of limited protective effect on virulent mycoplasma capriae and goat pneumonia subspecies infection, high cost and the like are solved.

Disclosure of Invention

In view of the above, the invention aims to provide an application of the mycoplasma capricolum subsp pneumoniae Mcpcp 1801 in the preparation of the contagious pleuropneumonia capricolumnae vaccine, and the vaccine created by the Mcpcp 1801 can induce an organism to generate high-level antibodies, so that the antibody generation time is short, the titer is high, and the vaccine is safe and efficient.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of Mycoplasma capricolum subsp. caprepneumoniae (Mycoplasma capropolium subsp. caprepneumoniae) Mcp 1801 in preparation of a goat infectious pleuropneumonia vaccine.

Preferably, the type of vaccine comprises an inactivated vaccine.

The invention also provides a goat infectious pleuropneumonia vaccine which comprises an inactivated antigen of mycoplasma capricolum subspecies of pneumonia Mcpcp 1801.

Preferably, the vaccine also comprises saponin and ISA201 VG double adjuvant;

preferably, the preparation method of the inactivated antigen comprises: (1) inoculating the recovered strain of mycoplasma capricolum subspecies of pneumonia Mcpcp 1801 in an improved MTB culture medium, and performing activation culture and amplification culture to obtain mycoplasma bacterial liquid; the modified MTB medium comprises the following components in concentration: 2g/L of glucose, 2g/L, PPLO g/L of sodium pyruvate and 21g/L of broth, 100ml/L of yeast extract with the mass percentage of 25%, 2.5ml/L of phenol red with the mass percentage of 1%, 200ml/L of horse serum and 10 ten thousand IU of penicillin, and the pH value is 7.4-7.6;

(2) centrifuging the mycoplasma bacterial liquid, collecting bacterial precipitates, and carrying out heavy suspension to obtain a mycoplasma antigen solution with the concentration of 100-400 mug/ml;

(3) and mixing the mycoplasma antigen solution with saponin, and inactivating at 4 ℃ for 24-48 h to obtain an inactivated antigen.

Preferably, the activation culture and expansion culture of step (1) comprises: culturing at 37 ℃ for 12-24 h, carrying out continuous amplification culture for 3-6 times by using the inoculum size of 5-10% when the pH value of the culture is reduced to 6.5-6.8, and harvesting the mycoplasma bacterial liquid when the pH value of the culture is reduced to 6.5-6.8.

Preferably, the centrifugation in the step (2) is performed at 4 ℃, the rotation speed of the centrifugation is 12000rpm, the centrifugation time is 20-40 min, and the preferred centrifugation time is 30 min; the collection of the pellet also included 3 washes with PBS buffer.

Preferably, the mass-to-volume ratio of the saponin to the mycoplasma antigen solution in the step (3) is 1-4 mg/ml.

The invention also provides a preparation method of the vaccine, which comprises the following steps: 1) mixing the inactivated antigen with a thimerosal solution with the mass percentage of 1% to obtain a liquid phase component;

2) mixing ISA201 VG adjuvant with the liquid phase component, and emulsifying for 10-30 min to obtain a water-in-oil-in-water type emulsified inactivated vaccine; the volume ratio of the ISA201 VG adjuvant to the liquid phase component is (50-55): (45-50).

Preferably, the volume of the thimerosal solution of step 1) is 1% of the volume of the inactivated antigen.

The invention provides an application of Mycoplasma capricolum subsp. caprepneumoniae (Mycoplasma caprophyllum subsp. caprepneumoniae) Mcp 1801(M1801 for short) in preparation of a goat infectious pleuropneumonia vaccine, wherein the M1801 strain has stronger toxicity, better immunogenicity and excellent growth performance than the existing vaccine strains M1601 and C87001, and the strain can induce an organism to generate high-level antibodies, so that the antibody generation time is short, the titer is high, and the maintenance time is long; the vaccine is high in safety; the immune sheep 4/5 has effects of relieving body temperature rise, cough, sneeze, and mental depression, and tissue pathological changes of lung.

The invention also provides a preparation method of the vaccine based on the M1801 strain, in particular to a preparation method of an inactivated vaccine, the M1801 strain has high antigen purity, the saponin is used as an inactivator and an adjuvant, the saponin can destroy and dissolve mycoplasma cell membranes and effectively retain effective components of the antigen, and the problem of effective antigen destruction or antigen epitope inactivation existing in the prior inactivators such as formaldehyde and the like is solved. The invention uses saponin and ISA201 VG double adjuvant; the saponin can enhance immune response, stimulate body cell and humoral immune response, and has long immune duration; the ISA201 VG adjuvant is a novel adjuvant, is a water-in-oil-in-water emulsion, has good immune effect, is stable, has low viscosity, is easy to inject, has small stimulation reaction, can improve immune response when being used, improves immune protection effect, reduces immune side reaction, and can provide good immune protection effect.

Drawings

FIG. 1 shows goat serum antibody levels after vaccine immunization;

FIG. 2 is a lung dissecting lesion; in the figure, a is a vaccine group, and b is a control group;

FIG. 3 shows the histopathological changes of lung, wherein a is vaccine group and b is control group.

Detailed Description

The Mcp 1801(M1801) disclosed in the invention is a target for identifying and applying mycoplasma capricolum goat pneumonia subspecies specific molecule detection targets (Wu ya Qin, Liu Bao hong, Yuan Ting, etc.. China veterinary science 2020,50(10): 1257-1262), and is stored in veterinary research in Lanzhou of Chinese academy of sciences. The strain M1801 is preferably obtained by separating the lung of a diseased goat, has stronger toxicity, better immunogenicity and excellent growth performance compared with the prior vaccine strains M1601 and C87001, can induce an organism to generate high-level antibodies by vaccine preparation of the strain, has the advantages of early antibody generation time, high titer, high safety and high efficiency, and can be used for preparing infectious caprine pleuropneumonia vaccines. The type of vaccine according to the invention preferably comprises an inactivated vaccine.

The preparation method of the inactivated antigen of the present invention preferably includes: (1) inoculating the recovered strain of mycoplasma capricolum subspecies of pneumonia Mcpcp 1801 in an improved MTB culture medium, and performing activation culture and amplification culture to obtain mycoplasma bacterial liquid; the modified MTB medium comprises the following components in concentration: 2g/L of glucose, 2g/L, PPLO g/L of sodium pyruvate and 21g/L of broth, 100ml/L of yeast extract with the mass percentage of 25%, 2.5ml/L of phenol red with the mass percentage of 1%, 200ml/L of horse serum and 10 ten thousand IU of penicillin, and the pH value is 7.4-7.6;

In the invention, when the improved MTB medium in the step (1) is prepared, 2g of glucose and 2g of sodium pyruvate are preferably dissolved by adding 25ml of water respectively, and are filtered and sterilized by a 0.22 mu m microporous filter membrane for later use; dissolving 21g of PPLO broth in 650ml of deionized water, adding 100ml of 25% (m/m) yeast extract and 2.5ml of 1% (m/m) phenol red, mixing uniformly, carrying out autoclaving at 121 ℃ for 20min, cooling, adding 200ml of sterile healthy horse serum and 25ml of the filtered and sterilized 8% (m/m) glucose, 25ml of 8% (m/m) sodium pyruvate solution and 1ml of 10 ten thousand IU/ml penicillin, adjusting the pH value to 7.4-7.6 by using sterilized 1mol/L NaOH, carrying out sterile subpackaging, and placing at 2-8 ℃ for later use, namely the improved MTB culture medium. The invention inoculates the recovered M1801 strain on the improved MTB culture medium, and the recovery and inoculation method is not particularly limited, and the recovery and inoculation can be carried out by utilizing the conventional technical means in the field. The improved MTB culture medium solves the problems of slow growth speed, low growth yield and the like of Mcp pathogen, reduces the production cost and can be produced in a large scale. The inoculated M1801 strain is subjected to activation culture and expansion culture, the culture mediums of the activation culture and the expansion culture are the improved MTB culture mediums, the culture temperature is preferably 37 ℃, the activation culture time is 12-24 hours, and the expansion culture is performed when the pH value of the culture is reduced to 6.5-6.8. The inoculation amount in the amplification culture is 5-10%, preferably 10%, and the continuous amplification culture is carried out for 3-6 times until the pH value of the culture is reduced to 6.5-6.8, so as to obtain mycoplasma bacteria liquid.

The centrifugation in the step (2) of the invention is preferably performed at 4 ℃, the rotation speed of the centrifugation is preferably 12000rpm, the centrifugation time is 20-40 min, preferably 30min, the thallus precipitate is collected and washed for 3 times by using PBS buffer solution, the concentration of the PBS buffer solution is preferably 0.01M, and the pH value is 7.2-7.4. After the bacterial precipitation is obtained, the method preferably further comprises the steps of measuring the concentration of the bacterial protein by using a BCA protein quantitative kit, and adjusting the concentration of the mycoplasma antigen to be 100-400 mu g/ml by using the PBS buffer solution.

The mass-volume ratio of the saponin to the mycoplasma antigen solution in the step (3) is preferably 1-4 mg/ml. The inactivation is preferably carried out at 4 ℃, the inactivation time is preferably 24-48 h, and the inactivation time is preferably carried out once every 2-4 h. After the inactivation is finished, the method preferably further comprises an inactivation test, and more preferably comprises sucking inactivated M1801 bacterial liquid 0.2ml, inoculating 0.1ml of modified MTB culture medium, and diluting to 10 times of gradient concentration^-6While simultaneously performing control on the modified MTB medium, culturing at 37 deg.C for 7d, inoculating 0.1ml modified MTA solid medium (phenol red removed in modified MTB medium, and agar powder 1.2 g/L), and placing at 37 deg.C with 5% CO₂And (5) culturing in an incubator for 7d, observing whether mycoplasma grows, and if the mycoplasma does not grow, inactivating to reach the standard. The vaccine of the invention has high antigen purity, and the saponin is used as an inactivator and an adjuvant, can destroy and dissolve mycoplasma cell membranes, effectively retains the effective components of the antigen, and solves the problem of effective antigen destruction or antigen epitope inactivation existing in the existing inactivators such as formaldehyde, sodium hypochlorite and the like.

The vaccine disclosed by the invention comprises an adjuvant, preferably a double adjuvant, and specifically comprises saponin and an ISA201 VG double adjuvant; the saponin can enhance immune response, stimulate body cell and humoral immune response, and has long immune duration; the ISA201 VG adjuvant is a novel adjuvant, is a water-in-oil-in-water emulsion, has good immune effect, is stable, has low viscosity, is easy to inject, has small stimulation reaction, can improve immune response, improves immune protection effect, reduces immune side reaction, and can provide good immune protection effect. The vaccine is safe for goats, and has immunoprotection rate of more than 4/5 for goat contagious pleuropneumonia caused by mycoplasma capricolum subspecies pneumonia.

The invention mixes the inactivated antigen with a thimerosal solution with the mass percentage of 1% to obtain a liquid phase component, and the volume of the thimerosal solution is preferably 1% of the volume of the inactivated antigen.

After a liquid phase component is obtained, mixing ISA201 VG adjuvant with the liquid phase component, and emulsifying for 10-30 min to obtain the water-in-oil-in-water type emulsified inactivated vaccine; the volume ratio of the ISA201 VG adjuvant to the liquid phase component is (50-55): (45-50). During the emulsification, the adjuvant is preferably added into a sterilization container, then the liquid phase component is added, and the mixture is emulsified for 10-30 min by an emulsifying machine to form a slightly viscous water-in-oil-in-water (w/o/w) emulsified inactivated vaccine. The vaccine provided by the invention is milky in appearance and is water-in-oil-in-water (w/o/w).

After the vaccine is prepared by the preparation method, the vaccine is preferably detected, and more preferably, the detection comprises physical property detection, sterility detection, safety detection and efficacy detection. The physical property test of the invention comprises the following steps: the vaccine is milky white in appearance, slightly viscous emulsion and water-in-oil-in-water (w/o/w); centrifuging at 3000rpm for 15min without layering; the sterility test comprises: the test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the sterile growth is required; the security detection includes: the inactivated vaccine of mycoplasma capricolum goat pneumonia subspecies is inoculated to healthy goats (Mccp antibody, antigen negative), each goat is injected with 2ml of vaccine in neck muscle, and the control group is injected with PBS with the same amount, and the continuous observation is carried out for 14 days. As a result, the goats in the vaccine group and the control group are healthy and alive, and the goats have no symptoms of cough, sneeze and the like, eat,Drinking water is not abnormal, obvious adverse reaction does not exist, and the vaccine is safe; the efficacy test comprises: dividing healthy goats (Mccp antibody and antigen negative) into a vaccine group and a control group, wherein each group comprises 5 goats, 2ml of vaccine is subcutaneously injected into the neck of each goat in the immune group, 2ml is injected again after two weeks, PBS is injected into the control group in the same mode and dosage, 35d after first immunization, the goats in the immune group and the control group are subjected to mycoplasma caprium subspecies challenge, M1801 bacterial liquid is used, and the challenge dosage is 10⁸～10⁹CCU/mL, the mode of counteracting toxic substance adopts nasal cavity spraying 2mL for two consecutive days, and tracheal injection 6mL after 2d interval. The control sheep with offensive toxin is completely attacked, the immune sheep can protect at least 4 sheep, or the control sheep can be attacked, the immune sheep can be completely protected or vaccine immune, and the symptoms such as body temperature rise, cough, sneeze, mental depression and the like or the pathological lesions and pathological damages of lung tissues of the immune sheep are obviously relieved.

The following examples are provided to illustrate the application of the mycoplasma capricolum subsp pneumoniae Mccp1801 in the preparation of contagious pleuropneumonia capricolum vaccine, but they should not be construed as limiting the scope of the present invention.

And (3) reagent sources: the mycoplasma capricolum goat pneumonia subspecies M1801 strain is from Lanzhou veterinary research institute of Chinese academy of agricultural sciences; PPLO broth was purchased from BD, sodium pyruvate was obtained from AMRESCO, horse serum was obtained from Gibco, phenol red and saponin were obtained from Sigma, and ISA201 VG adjuvant was purchased from Seppic Shanghai; glucose and the like are domestic analytical pure reagents.

Example 1

Preparation of inactivated vaccine

(1) Preparation of antigen liquid

1) The preparation method of the culture medium comprises the following steps: respectively dissolving 2g of glucose and 2g of sodium pyruvate in 25ml of water, and filtering and sterilizing by using a 0.22 mu m microporous filter membrane for later use; dissolving 21g of PPLO broth in 650ml of deionized water, adding 100ml of 25% yeast extract and 2.5ml of 1% phenol red, uniformly mixing, carrying out autoclaving at 121 ℃ for 20min, cooling, adding 200ml of sterile healthy horse serum and 25ml of the filtered and sterilized 8% glucose, 25ml of 8% sodium pyruvate solution and 1ml of 10 ten thousand IU/ml penicillin, adjusting the pH value to 7.4-7.6 by using sterilized 1mol/L NaOH, carrying out sterile subpackaging, and standing at 4 ℃ for later use, thus obtaining the improved MTB culture medium.

2) Preparation of mycoplasma antigen:

recovering a mycoplasma capricolum pneumonia subspecies M1801 strain, inoculating to 4.5ml of an improved MTB culture medium, culturing at 37 ℃ for 24h, performing 3 times of continuous amplification culture according to 10% of the total amount of the culture medium when the pH value of the culture is reduced to 6.5-6.8, harvesting 1000ml of mycoplasma bacterial liquid when the pH value of the culture is reduced to 6.5-6.8, centrifuging at 4 ℃ at 12000rpm for 30min, collecting bacterial precipitation, and washing with a PBS buffer solution (0.01M, pH 7.2-7.4) for 3 times; the concentration of mycoprotein was determined using BCA protein quantification kit, and the concentration of mycoplasma antigen was adjusted to 300. mu.g/ml using PBS buffer (0.01M, pH7.2-7.4).

(2) Inactivation of antigen: adding sterile saponin filtered by 0.22 μm microporous membrane into mycoplasma antigen, inactivating at 4 deg.C for 24 hr while shaking every 2 hr. After inactivation is finished, performing inactivation inspection, sucking 0.2ml of inactivated M1801 bacterial liquid and 0.1ml of inoculated modified MTB culture medium, diluting by 10-fold gradient to 10^-6While simultaneously performing control on the modified MTB medium, culturing at 37 deg.C for 7d, inoculating 0.1ml modified MTA solid medium (phenol red removed in modified MTB medium, and agar powder 1.2 g/L), and placing at 37 deg.C with 5% CO₂The culture box is cultured for 7d, and the growth of mycoplasma is observed. No mycoplasma growth occurred.

(3) Preparation of vaccines

The inactivated vaccine of the water-in-oil-in-water emulsion is prepared by adopting a one-step emulsification method.

Liquid phase components: 20ml of inactivated antigen M1801 of mycoplasma capricolum and pneumonia subspecies of goat is added into 0.2ml of 1% thimerosal solution according to 1% of the total amount of the antigen, and the total amount is 20.2ml and accounts for 48% of the total amount.

Oil phase components: ISA201 VG adjuvant 22.22ml, accounting for 52% of the total amount.

The emulsification method comprises the following steps: adding ISA201 VG adjuvant into a sterilization bottle, adding liquid phase components, adjusting the speed to 1 (5000rpm) by a small-sized emulsifying machine Fluko for 12min, and emulsifying into a slightly viscous water-in-oil-in-water (w/o/w) emulsion inactivated vaccine.

Vaccine testing:

and (3) physical property inspection: the vaccine is milky white and slightly viscous emulsion in appearance, and is water-in-oil-in-water (w/o/w). The vaccine was stable and not stratified by centrifugation at 3000rpm for 15 min.

And (4) sterile inspection: the bacteria-free growth is carried out according to the inspection of the appendix of the current Chinese veterinary pharmacopoeia.

Example 2

Safety test of inactivated vaccine

The mycoplasma capricolum goat pneumonia subspecies inactivated vaccine is inoculated to healthy goats (Mccp antibody and antigen negative) with the age of 8-9 months, each goat is injected with 2ml of vaccine in neck muscles, a control group is injected with PBS with the same amount, and the continuous observation is carried out for 14 days. As a result, the goats of the vaccine group and the goat of the control group are healthy and alive, the goats have no symptoms such as cough and sneeze, the ingestion and drinking of the goats are abnormal, the injection part has no adverse reactions such as obvious lumps, and the vaccine is safe.

Example 3

Potency assay for inactivated vaccines

Dividing healthy goats (Mccp antibody and antigen negative) with the age of 8-9 months into a vaccine group and a control group, wherein each group comprises 5 goats, 2ml of vaccine is subcutaneously injected into the neck of each goat in the immune group, 2ml is injected again after two weeks, PBS is injected into the control group in the same way and dosage, after the first immunization, 35d, the goats in the immune group and the control group are subjected to mycoplasma capricolum goat subspecies challenge, M1801 bacterial liquid is used, and the challenge dosage is 10⁹CCU/mL, the mode of counteracting toxic substance adopts nasal cavity spraying 2mL for two consecutive days, and tracheal injection 6mL after 2d interval. After toxin counteracting, clinical symptoms and temperature measurement are observed, serum is collected, serum antibody detection is carried out, and an indirect ELISA method is adopted for Mccp antibody level detection. Performing autopsy on dead animals after toxin attack, and observing pathological changes; and (4) after attacking for 32d, other non-dead animals are subjected to autopsy for observing lesions, and lesion tissue detection and pathological histological observation are collected.

The indirect ELISA method for detecting the Mccp antibody level comprises the following steps: coating 1.6 mu g/mL of M1801 mycoprotein on an ELISA plate, 0.05M of carbonate coating solution with pH of 9.6, 100 mu L/hole, 1h at 37 ℃ and overnight at 4 ℃; PBST lotion, 300 u L/hole, room temperature wash 3 times; 5% skimmed milk powder 100 μ L/hole, sealing at 37 deg.C for 2 hr, and washing for 3 times; incubating primary antibody: sealing the serum to be testedDiluting with cloaca 1/200, setting up negative and positive controls, repeating each sample at least two times, 100 μ L/well, washing 4 times after 1h at 37 deg.C; diluting rabbit anti-sheep secondary antibody with a sealing solution 1/5000, washing and shooting for 4 times at 37 ℃ after 1h, wherein the concentration of the blocking solution is 100 mu L/hole; adding TMB 100 μ L/well under dark condition, and developing at 37 deg.C for 10 min; 2M H was added₂SO₄The solution turned from blue to yellow at 100. mu.L/well stop; and reading the OD value of 450nm wavelength in a microplate reader, wherein the critical value is 0.401, and judging the result.

As a result: the Mcp antibody level is increased after the vaccine group is immunized, 80% (4/5) goat serum antibody is converted to positive after the immunization 7d, and all antibodies are converted to positive (5/5) after the immunization 14d (figure 1, wherein 077, 079, 081, 093 and 318 are animal numbers), which indicates that the vaccine induces the body to generate the antibodies at a early time and the antibody level is high. The vaccine immunity can obviously relieve the symptoms of body temperature rise, cough, sneeze, mental depression and the like, and improve the survival rate of infected goats; the vaccine of the invention can obviously reduce the lung tissue dissection lesion and pathological damage (figure 2 and figure 3).

Example 4

Comparative testing of vaccines

Dividing healthy goats (Mccp antibody and antigen negative) with the age of about 12 months into an M1601 vaccine group, an M1801 vaccine group and a control group, wherein each vaccine is used for immunizing 5 goats respectively, the control group is used for immunizing 4 goats, 2ml of vaccine is injected subcutaneously into the neck of each goat in the immunization group, PBS is injected into the control group in the same way and dosage, 21d after immunization, the immunization group and the control group are used for performing virus attack on mycoplasma capricolumni subsp pneumoniae tissues, and 10d is performed⁹Copy/copy, the mode of counteracting toxic substance adopts nasal cavity spray 2ml for two consecutive days, trachea injection 1ml after 2d interval. Observing clinical symptoms and measuring body temperature, performing autopsy on dead animals after virus attack, and observing pathological changes; other non-dead animals are subjected to autopsy after 28d of virus attack to observe pathological changes and pathological tissue Mcpcp pathogen conventional PCR detection (upstream primer F (SEQ ID NO. 1): 5'-ATCATTTTTAATCCCTTCAAG-3'; downstream primer R (SEQ ID NO. 2): 5'-TACTATGAGTAATTATAATATATGCAA-3', PCR system: PCRmix 12.5 mu L, ddH₂O8.5. mu.L, 1. mu.L of each of the forward primer and the reverse primer, and 2. mu.L of tissue DNA; and (3) PCR reaction conditions: pre-denaturation at 95 deg.C for 5min, (30 s at 95 deg.C, 30s at 47 deg.C, 25s at 72 deg.C), 35 cycles, 10min at 72 deg.C, PCR product 316 bp).

As a result, the vaccine group can remarkably improve the morbidity and the mortality of goats, and relieve the fever, clinical symptoms and autopsy lesions of goats, compared with the M1601 vaccine group, the M1801 vaccine group has better effects of improving the fever and the clinical symptoms, relieving tissue lesions of lung, pleural effusion and the like, and reducing the detection of lung pathogens, so that the immune protection effect of the M1801 vaccine group is better than that of the M1601 vaccine of mycoplasma capriae goat pneumonia subspecies.

Example 5

Comparison of virulence of strains

The healthy goats (Mccp antibody and antigen negative) with the age of about 10 months are divided into M1801, C87001 toxin attacking groups and blank control groups, and the C87001 strain is prepared and provided by Chinese veterinary medicine supervision (China veterinary microbial strain preservation management center, the strain preservation number: CVCC87001, the strain classification name is the filiform mycoplasma goat pneumonia subspecies; see China veterinary medicine supervision institute, China veterinary microbial strain preservation management center, China veterinary bacterial catalogue (second edition), China agricultural science and technology publishing house, 2002, p89), and is preserved by the Lanzhou veterinary research institute of China academy of agricultural sciences. The goat in the toxin attacking group carries out the goat mycoplasma goat subspecies attacking with the dose of attacking being 10⁹And copying/copy, adopting nasal cavity spraying 2ml for two consecutive days in a virus counteracting mode, and performing tracheal injection 6ml after 2d interval. As a result, M1801 group fever, more severe clinical symptoms such as cough, sneeze and nasal discharge and more obvious weight loss, the IDEXX Mcpcp detection kit detects serum antibodies, the serum antibodies of M1801 group completely turn positive for 10 days of the first challenge, the serum antibodies of C87001 group completely turn positive for 21 days of the first challenge, and the caesarean change of M1801 group is severe (lung consolidation or adhesion, pleural effusion and fibrous exudation). The results show that M1801 is more virulent.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. Application of Mycoplasma capricolum subsp. caprepneumoniae (Mycoplasma capricoloum subsp. caprpneumoniae) Mcp 1801 in preparing goat infectious pleuropneumonia vaccine.

2. The use of claim 1, wherein the type of vaccine comprises an inactivated vaccine.

3. A goat infectious pleuropneumonia vaccine is characterized in that the vaccine comprises an inactivated antigen of mycoplasma capricolum subspecies pneumoniae Mcpcp 1801.

4. The vaccine of claim 3, further comprising saponin and ISA201 VG double adjuvant.

5. The vaccine of claim 3, wherein the inactivated antigen is prepared by a method comprising: (1) inoculating the recovered strain of mycoplasma capricolum subspecies of pneumonia Mcpcp 1801 in an improved MTB culture medium, and performing activation culture and amplification culture to obtain mycoplasma bacterial liquid; the modified MTB medium comprises the following components in concentration: 2g/L of glucose, 2g/L, PPLO g/L of sodium pyruvate and 21g/L of broth, 100ml/L of yeast extract with the mass percentage of 25%, 2.5ml/L of phenol red with the mass percentage of 1%, 200ml/L of horse serum and 10 ten thousand IU of penicillin, and the pH value is 7.4-7.6;

6. The vaccine of claim 5, wherein the activation culture and expansion culture of step (1) comprises: culturing at 37 ℃ for 12-24 h, carrying out continuous amplification culture for 3-6 times by using the inoculum size of 5-10% when the pH value of the culture is reduced to 6.5-6.8, and harvesting the mycoplasma bacterial liquid when the pH value of the culture is reduced to 6.5-6.8.

7. The vaccine according to claim 5, wherein the centrifugation in the step (2) is performed at 4 ℃, the rotation speed of the centrifugation is 12000rpm, and the centrifugation time is 20-40 min;

the collection of the pellet also included 3 washes with PBS buffer.

8. The vaccine according to claim 5, wherein the mass-to-volume ratio of the saponin to the mycoplasma antigen solution in step (3) is 1-4 mg/ml.

9. A method for preparing a vaccine according to any one of claims 3 to 8, comprising the steps of: 1) mixing the inactivated antigen with a thimerosal solution with the mass percentage of 1% to obtain a liquid phase component;

2) mixing the ISA201 VG adjuvant with the liquid phase component, and emulsifying for 10-30 min to obtain a water-in-oil-in-water type emulsified inactivated vaccine; the volume ratio of the ISA201 VG adjuvant to the liquid phase component is (50-55): (45-50).

10. The method of claim 9, wherein the volume of the thimerosal solution of step 1) is 1% of the volume of the inactivated antigen.