CN105543120B - Haemophilus parasuis pentavalent inactivated vaccine - Google Patents
Haemophilus parasuis pentavalent inactivated vaccine Download PDFInfo
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Abstract
The invention provides a Haemophilus parasuis strain, which is a Haemophilus parasuis strain LYD1(Haemophilus parasuis), is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 21 days 07-2015, and has the address of No. 3 Hospital No.1 of West Lu of the Korean district in Beijing city and the preservation number of CGMCC No. 11143. The strain LYD1 can be used for preparing vaccines for preventing and treating diseases caused by multiple serotypes of haemophilus parasuis, and the finally prepared vaccines have good safety and prevention and treatment capability.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the field of biology, and particularly relates to a haemophilus parasuis strain.
[ background of the invention ]
Haemophilus parasuis has once been considered as a stress-induced conditional and sporadic disease of piglets, and has become the most serious bacterial disease of piglets and growing pigs nowadays with the improvement of industrialization and intensification of pig raising.
The haemophilus parasuis mainly harms piglets and growing pigs at the age of 2-28 weeks, and weaning and nursing piglets at the age of 5-8 weeks are the most serious, so that symptoms such as multiple serositis, arthritis and the like are caused, the morbidity is up to 15-90%, and the mortality can be up to 90% at times. Initially, haemophilus parasuis was called haemophilus suis or haemophilus influenzae, and later it was shown that factor X (heme and other porphyrins) is not required for growth, and was therefore renamed to haemophilus parasuis. The haemophilus parasuis has polymorphism, strictly needs NAD or V factors during growth, has complex and various serotypes, can be divided into at least 15 serotypes according to the difference of outer membrane protein antigens of homotypic bacteria, and more than 20 percent of isolates cannot be divided into types; and the popular serotypes in different countries or regions are different, and according to reports of Cai Xuanwang and the like, the currently popular serotypes in China are considered to be mainly 4, 5, 12 and 13, but the dominant serotypes in different places are different, so that the difficulty in preventing and controlling diseases is increased. According to the research of serum epidemiology and the identification of separated strains in China, the types 4 and 5 are most popular, and the types 12 and 13 are secondly popular. In recent years, the interactive immunity among haemophilus parasuis serogroups is not strong, and the vaccinated strain serogroup is not consistent with the pathogenic serogroup of the currently and locally epidemic haemophilus parasuis, so that certain difficulty is brought to the prevention and treatment of haemophilus parasuis.
The current domestic vaccines for immunoprophylaxis of haemophilus parasuis disease include inactivated vaccine (Z-1517 strain) of haemophilus parasuis disease produced by Boringer Yiger animal health promotion (USA) Limited company, inactivated vaccine of haemophilus parasuis disease types 1 and 6 produced by Spain Haiblee biological big pharmaceutical factory, inactivated vaccine of haemophilus parasuis disease types 4 and 5 produced by Huazhong agriculture university, animal biological product Limited liability company before Wuhan department, and animal husbandry part Limited company; the application of these vaccines plays a certain role in prevention, and the epidemic strains of the disease and the infection of various serotypes make the expected immune effect of the existing vaccines difficult to achieve, so that the preparation of suitable strains for participating in the preparation of the vaccine for preventing and treating the haemophilus parasuis caused by various serotype haemophilus parasuis is an urgent problem to be solved by practitioners.
[ summary of the invention ]
The invention aims to solve the technical problem of providing a haemophilus parasuis strain LYD1(Haemophilus sparassis) which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 21.07.2015, wherein the address is No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC No. 11143.
The invention solves the technical problems through the following technical scheme:
in 2009, the applicant collects lung, joint effusion and trachea of suspected Hps-infected piglets in a new Luo pig farm in Quanzhou city, Fujian province under aseptic condition, streaks and inoculates the lung, joint effusion and trachea in a TSA culture medium respectively, places the TSA culture medium in a constant-temperature incubator at 37 ℃, carries out anaerobic culture in a candle jar for 24-48h, carries out physicochemical and molecular identification on the cultured strain, and finally determines that the strain is a strain of haemophilus parasuis.
The invention has the beneficial effects that: the Haemophilus parasuis strain LYD1(Haemophilus parasuis) is provided, the strain can be used for preparing vaccines for preventing and treating diseases caused by various serotypes of Haemophilus parasuis, and the finally prepared vaccines have good safety and prevention and treatment capacity.
[ description of the drawings ]
The invention will be further described with reference to the following examples with reference to the accompanying drawings.
FIG. 1 is a 1.0% agarose gel electrophoresis of the PCR products of examples 1-5 of the present invention.
[ detailed description ] embodiments
The Haemophilus parasuis strain LYD1(Haemophilus parasuis) is a strain obtained by culturing lung and joint effusion of suspected Hps-infected piglets collected from a New Luo pig farm in Quanzhou city, Fujian province in 2009. In addition, the invention also relates to other strains, namely haemophilus parasuis strain LYH5, haemophilus parasuis strain LY02, haemophilus parasuis strain LYW1 and haemophilus parasuis strain LYC 2.
For the purpose of illustrating the present invention in detail, the applicant gives the following specific examples for illustrating the present invention in detail.
EXAMPLE 1 isolation and characterization of Strain LYD1
1. Isolation of strain LYD 1:
in 2009, the applicant collected lungs, joint effusion and trachea of suspected Hps-diseased piglets in a new luo pig farm in quanzhou, Fujian province under aseptic condition, streaked and inoculated in a TSA culture medium respectively, placed in a constant temperature incubator at 37 ℃, anaerobically cultured in a jar for 24-48h, and the obtained strain is named as strain LYD 1.
2. Identification of strain LYD 1:
preliminary identification:
the obtained strain LYD1 was subjected to morphological and biochemical determination, and the results were as follows: gram-negative, tiny bacilli, with many different morphologies, from single coccobacillus to long, elongated, filamentous, thallus; the haemolysis phenomenon is avoided, no indole is produced in the isolate, glucose, fructose and sucrose can be fermented, xylose, L-arabinose, lactose and raffinose are not fermented, the contact enzyme and urease tests are negative, the biochemical reaction of mannose is unstable, and V factors and X factors are required for growth; therefore, it was preliminarily confirmed that the strain LYD1 belongs to the genus Haemophilus parasuis.
Further identification:
extracting a template: selecting single colony of strain LYD1 for subclone culture, carefully scraping lawn with sterilized double distilled water, placing in 1.5mL centrifuge tube, centrifuging at 12000rpm for 30s, discarding supernatant, and retaining cell precipitate; adding 567 mu LTE suspension precipitate, extracting with equal volume of first extractive solution (phenol, chloroform and isoamylol at volume ratio of 25: 24: 1) to mix the two phases completely, and ice-cooling for 10 min; centrifuging at 12000rpm for 10min, collecting supernatant, and extracting with equal volume of second extractive solution (chloroform: isoamyl alcohol at volume ratio of 24: 1) for 1 time until flocculent DNA precipitate appears; transferring the DNA precipitate into 1mL of 70% ethanol for washing, and drying in a drying oven at 65 ℃ for 2 minutes; finally, 50-100 mu LTE buffer solution (containing 20 mu g/mLRNase) is used for dissolving DNA precipitate, namely the required template is obtained, and the required template is stored at 4 ℃ for later use.
And (3) PCR identification: the Shanghai Bioengineering Co., Ltd was entrusted with the synthesis of the upstream primer P1 (shown in SEQ ID NO: 1): 5'-GTGATGAGGAAGGGTGGTGT-3', respectively; downstream primer P2 (shown as SEQ ID NO: 2): 5'-GGCTTCGTCACCCTCTGT-3' are provided.
The template obtained by extraction in this example was used as a template, and the above primers (P1/P2) were used as a primer pair to perform PCR reaction, and the currently recognized Haemophilus parasuis standard strain was used as a negative control:
PCR reaction (50. mu.L): 10 × PCR Buffer (Mg)2+) mu.L, 2.5mmol/L dNTPs 4. mu.L, 5U/. mu.L TaqDNA polymerase 0.5. mu.L, H2O36.5 mu L, 10 mu mol/L upstream primer 1 mu L, 10 mu mol/L downstream primer 1 mu L and template 2 mu L;
the PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, renaturation at 72 ℃ for 1min, and repeating for 30 cycles; finally, extension is carried out for 7min at 72 ℃.
Detection of PCR products: the PCR product was identified by electrophoresis on 1% agarose gel using DNAmarker DL2000 as a standard molecular weight, at 80V for 20min in 1 XTAE buffer.
The gel electrophoresis separation test is carried out, and the result of the gel electrophoresis separation test is shown in figure 1 (in figure 1, M is DNA ladder, 1 is the strain to be tested in the embodiment, 2 is a negative control, and 3 is a double distilled water blank control), and as can be seen from figure 1, the strain to be tested in the embodiment, namely the strain LYD1 is the same as the haemophilus parasuis standard strain, and a single band is amplified at the position of 821bp, so that the strain LYD1 and the haemophilus parasuis standard strain are confirmed to belong to the same genus.
According to the above identification, it was finally confirmed that the strain LYD1 belongs to a strain of the genus Haemophilus parasuis (Haemophilus parasuis).
Serotype identification: inoculating strain LYD1 into TSA culture medium, culturing at 37 deg.C for mass proliferation, and centrifuging at 7000r/min to obtain thallus; adding 9 volume times of PBS buffer solution with pH value of 7.4 into the thallus, treating with steam at 121 ℃ for 2h, centrifuging at 7000r/min for 10min, collecting supernatant, performing agar diffusion test, and identifying Hps serotype according to the agar diffusion test method established by Keilstein and Rapp-Gabrielson, wherein the identification result is Hps 1 type.
Example 2 isolation and identification of Strain LYH5
1. Isolation of strain LYH 5:
in 2011, the applicant collects lung, joint effusion and trachea of suspected Hps-infected piglets in a certain permanently-defined pig farm in Longyan city, Fujian province under aseptic condition, streaks and inoculates the piglets in a TSA culture medium respectively, places the piglets in a constant-temperature incubator at 37 ℃, carries out anaerobic culture in a jar for 24-48h, and names the obtained strain through culture as a strain LYH 5.
2. Identification of strain LYH 5:
preliminary identification:
the obtained strain LYH5 was subjected to morphological and biochemical characterization, and the results were consistent with the preliminary identification results in example 1; therefore, it was preliminarily confirmed that the strain LYH5 also belongs to the genus Haemophilus parasuis.
Further identification:
extracting a template: the template extraction procedure of example 1 was referred to extract DNA of the strain LYH5 as a template, and the DNA was stored at 4 ℃ until use.
And (3) PCR identification: the template obtained by extraction in this example was used as a template, and the primers (P1/P2) described in example 1 were used as a primer pair to perform PCR reaction, and the currently recognized Haemophilus parasuis standard strain was used as a negative control:
PCR reaction (50. mu.L): 10 × PCR Buffer (Mg)2+) mu.L, 4. mu.L of 2.5mmol/L dNTPs, 0.5. mu.L of 5U/. mu.L of DNA polymerase, H2O36.5 mu L, 10 mu mol/L upstream primer 1 mu L, 10 mu mol/L downstream primer 1 mu L and template 2 mu L;
the PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, renaturation at 72 ℃ for 1min, and repeating for 30 cycles; finally, extension is carried out for 7min at 72 ℃.
Detection of PCR products: the PCR product was identified by electrophoresis on 1% agarose gel using DNAmarker DL2000 as a standard molecular weight, at 80V for 20min in 1 XTAE buffer. Namely, the gel electrophoresis separation test was carried out, and the result of the gel electrophoresis separation test was identical to that of example 1, that is, as shown in FIG. 1, it was confirmed that the strain LYH5 also belongs to the same genus as the standard strain of Haemophilus parasuis.
According to the above identification, it was finally confirmed that the strain LYH5 belongs to a strain of the genus Haemophilus parasuis (Haemophilus parasuis).
Serotype identification: the strain LYH5 was serotype-identified according to the serotype-identification procedure of example 1, and the identification result was Hps 4 type.
Example 3 isolation and identification of Strain LY02
1. Isolation of strain LY 02:
in 2012, the applicant collects lung, joint effusion and trachea of suspected Hps-infected piglets in a certain permanently-defined pig farm in Longyan city of Fujian province under aseptic condition, streaks the lung, joint effusion and trachea respectively, inoculates the piglets in a TSA culture medium, places the culture medium in a constant-temperature incubator at 37 ℃, carries out anaerobic culture in a jar for 24-48h, and names the obtained cultured strain as strain LY 02.
2. Identification of strain LY 02:
preliminary identification:
the obtained strain LY02 was subjected to morphological and biochemical characterization, and the results were consistent with the preliminary identification results in example 1; therefore, it was preliminarily confirmed that the strain LYH5 also belongs to the genus Haemophilus parasuis.
Further identification:
extracting a template: the template extraction procedure of example 1 was referenced to extract DNA of strain LY02 as the desired template, and stored at 4 ℃ until use.
And (3) PCR identification: the template obtained by extraction in this example was used as a template, and the primers (P1/P2) described in example 1 were used as a primer pair to perform PCR reaction, and the currently recognized Haemophilus parasuis standard strain was used as a negative control:
PCR reaction (50. mu.L): 10 × PCR Buffer (Mg)2+) mu.L, 4. mu.L of 2.5mmol/L dNTPs, 0.5. mu.L of 5U/. mu.L of DNA polymerase, H2O36.5 mu L, 10 mu mol/L upstream primer 1 mu L, 10 mu mol/L downstream primer 1 mu L and template 2 mu L;
the PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, renaturation at 72 ℃ for 1min, and repeating for 30 cycles; finally, extension is carried out for 7min at 72 ℃.
Detection of PCR products: the PCR product was identified by electrophoresis on 1% agarose gel using DNAmarker DL2000 as a standard molecular weight, at 80V for 20min in 1 XTAE buffer. That is, the gel electrophoresis separation test was carried out, and the result of the electrophoresis separation test was identical to that of example 1, that is, as shown in FIG. 1, it was confirmed that this strain LY02 also belongs to the same genus as the standard strain of Haemophilus parasuis.
According to the above identification, it was finally confirmed that the strain LY02 belongs to a strain of the genus Haemophilus parasuis (Haemophilus parasuis).
Serotype identification: the serotype identification was performed on the strain LY02 in accordance with the serotype identification operation in example 1, and the identification result was Hps 5 type.
EXAMPLE 4 isolation and characterization of Strain LYW1
1. Isolation of strain LYW 1:
in 2011, the applicant collects lung, joint effusion and trachea of suspected Hps-infected piglets in a new Luo pig farm in Quanzhou city, Fujian province under aseptic condition, streaks the lung, joint effusion and trachea respectively, inoculates the piglets in a TSA culture medium, places the TSA culture medium in a constant-temperature incubator at 37 ℃, carries out anaerobic culture in a jar for 24-48h, and names the obtained strain through culture as a strain LYW 1.
2. Identification of strain LYW 1:
preliminary identification:
the obtained strain LYW1 was subjected to morphological and biochemical characterization, and the results were consistent with the preliminary identification results in example 1; therefore, it was preliminarily confirmed that the strain LYW1 also belongs to the genus Haemophilus parasuis.
Further identification:
extracting a template: the template extraction procedure of example 1 was referenced to extract DNA of the strain LYW1 as the desired template, and stored at 4 ℃ until use.
And (3) PCR identification: the template obtained by extraction in this example was used as a template, and the primers (P1/P2) described in example 1 were used as a primer pair to perform PCR reaction, and the currently recognized Haemophilus parasuis standard strain was used as a negative control:
PCR reaction (50. mu.L): 10 × PCR Buffer (Mg)2+) mu.L, 4. mu.L of 2.5mmol/L dNTPs, 0.5. mu.L of 5U/. mu.L of DNA polymerase, H2O36.5 mu L, 10 mu mol/L upstream primer 1 mu L, 10 mu mol/L downstream primer 1 mu L and template 2 mu L;
the PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, renaturation at 72 ℃ for 1min, and repeating for 30 cycles; finally, extension is carried out for 7min at 72 ℃.
Detection of PCR products: the PCR product was identified by electrophoresis on 1% agarose gel using DNAmarker DL2000 as a standard molecular weight, at 80V for 20min in 1 XTAE buffer. Namely, the gel electrophoresis separation test was carried out, and the result of the gel electrophoresis separation test was identical to that of example 1, that is, as shown in FIG. 1, it was confirmed that the strain LYW1 also belongs to the same genus as the standard strain of Haemophilus parasuis.
According to the above identification, it was finally confirmed that the strain LYW1 belongs to a strain of the genus Haemophilus parasuis (Haemophilus parasuis).
Serotype identification: the serotype identification of the strain LYW1 was carried out according to the serotype identification procedure of example 1, and the identification result was Hps 12 type.
Example 5 isolation and identification of Strain LYC2
1. Isolation of strain LYW 1:
in 2012, the applicant collects lung, joint effusion and trachea of suspected Hps-infected piglets in a new Luo pig farm in Quanzhou city, Fujian province under aseptic condition, streaks and inoculates the lung, joint effusion and trachea in TSA culture medium respectively, places the TSA culture medium in a constant-temperature incubator at 37 ℃, carries out anaerobic culture in a jar for 24-48h, and names the strain obtained by culture as a strain LYC 2.
2. Identification of strain LYC 2:
preliminary identification:
the obtained strain LYC2 was subjected to morphological and biochemical characterization, and the results were consistent with the preliminary identification results in example 1; therefore, it was preliminarily confirmed that the strain LYC2 also belongs to the genus Haemophilus parasuis.
Further identification:
extracting a template: the template extraction procedure of example 1 was referenced to extract DNA of the strain LYC2 as the desired template, and stored at 4 ℃ until use.
And (3) PCR identification: the template obtained by extraction in this example was used as a template, and the primers (P1/P2) described in example 1 were used as a primer pair to perform PCR reaction, and the currently recognized Haemophilus parasuis standard strain was used as a negative control:
PCR reaction (50. mu.L): 10 × PCR Buffer (Mg)2+) mu.L, 4. mu.L of 2.5mmol/L dNTPs, 0.5. mu.L of 5U/. mu.L of DNA polymerase, H2O36.5 mu L, 10 mu mol/L upstream primer 1 mu L, 10 mu mol/L downstream primer 1 mu L and template 2 mu L;
the PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, renaturation at 72 ℃ for 1min, and repeating for 30 cycles; finally, extension is carried out for 7min at 72 ℃.
Detection of PCR products: the PCR product was identified by electrophoresis on 1% agarose gel using DNAmarker DL2000 as a standard molecular weight, at 80V for 20min in 1 XTAE buffer. Namely, the gel electrophoresis separation test was carried out, and the result of the gel electrophoresis separation test was identical to that of example 1, that is, as shown in FIG. 1, it was confirmed that the strain LYC2 also belongs to the same genus as the standard strain of Haemophilus parasuis.
According to the above identification, it was finally confirmed that the strain LYC2 belongs to a strain of the genus Haemophilus parasuis (Haemophilus parasuis).
Serotype identification: the serotype identification of the strain LYC2 was carried out according to the serotype identification procedure of example 1, and the identification result was Hps 13 type.
Example 6 preparation of a Haemophilus parasuis pentavalent inactivated vaccine
Propagation and culture: taking the strain LYD1, strain LYH5, strain LY02, strain LYW1 and strain LYC2, streaking and inoculating on TSA agar plate with 5-10% CO2(i.e., the culture environment contains 5 to 10 mass% of CO2) Culturing at 37 ℃ for 24-48h, selecting typical colonies, inoculating the typical colonies on a TSB culture medium, and performing shake culture at 37 ℃ for 24-48h to obtain a first-stage seed solution of a strain LYD1, a first-stage seed solution of a strain LYH5, a first-stage seed solution of a strain LY02, a first-stage seed solution of a strain LYW1 and a first-stage seed solution of a strain LYC 2; respectively streaking the obtained primary seed liquid of strain LYD1, LYH5, LY02, LYW1 and LYC2 on TSA agar plate with 5-10% CO2(i.e., the culture environment contains 5 to 10 mass% of CO2) Culturing at 37 ℃ for 24-48h, selecting typical colonies, inoculating the typical colonies on a TSB culture medium, and performing shaking culture at 37 ℃ for 24-48h to obtain a secondary seed solution of a strain LYD1, a secondary seed solution of a strain LYH5, a secondary seed solution of a strain LY02, a secondary seed solution of a strain LYW1 and a secondary seed solution of a strain LYC 2; respectively inoculating the secondary seed liquid of the strain LYD1, the secondary seed liquid of the strain LYH5, the secondary seed liquid of the strain LY02, the secondary seed liquid of the strain LYW1 and the secondary seed liquid of the strain LYC2 to a fermentation culture medium, and performing fermentation at 37 deg.C,Culturing for 22-24 h at 180r/min to obtain bacterial liquid of a strain LYD1, bacterial liquid of a strain LYH5, bacterial liquid of a strain LY02, bacterial liquid of a strain LYW1 and bacterial liquid of a strain LYC 2;
wherein, the fermentation medium is a semisynthetic medium, and the preparation method of the semisynthetic medium comprises the following steps: dissolving 30g of TSB in deionized water, diluting to 1000mL, shaking up to dissolve completely, sterilizing with steam at 121 ℃ for 15min, cooling, and adding 50mL of filter sterilized newborn calf serum and 200 mu L of filter sterilized 0.02% NAD;
concentration and inactivation: concentrating the bacterial solutions obtained by propagation culture respectively, and concentrating bacterial solution of strain LYD1, bacterial solution of strain LYW1, and bacterial solution of strain LYC2 to obtain bacterial solution with antigen content of 2.5 × 109CFU/mL, bacterial liquid of strain LYH5, bacterial liquid of strain LY02, and concentrated to antigen content of 2.0 × 109CFU/mL; then inactivating the concentrated bacterial liquids respectively;
preparing a total mixed bacterial liquid: mixing the concentrated and inactivated bacterial liquid of the strain LYD1, the bacterial liquid of the strain LYW1 and the bacterial liquid of the strain LYC2 according to the volume ratio of 1:1:1 to obtain first mixed bacterial liquid; mixing the concentrated and inactivated bacterial liquid of the strain LYH5 and the bacterial liquid of the strain LY02 according to the volume ratio of 1:1 to obtain a second mixed bacterial liquid; then mixing the obtained first mixed bacterial liquid and second mixed bacterial liquid according to the volume ratio of 1:1.5 to obtain total mixed bacterial liquid;
preparing a water phase: taking sterilized tween-804 parts and 96 parts of total mixed bacterium liquid qualified by aseptic inspection according to the volume parts, introducing the sterilized tween-804 parts and the total mixed bacterium liquid into a liquid preparation tank, starting a stirring motor, and stirring at a constant speed until tween-80 is completely dissolved to obtain a water phase;
preparing an oil phase: taking 94 parts by volume of white oil for injection and 806 parts by volume of span, adding the white oil and the span into an oil phase preparation tank, starting a stirring motor, stirring at a constant speed, simultaneously starting a conduction oil switch, heating at 125 ℃ for 30min, and cooling to obtain an oil phase;
emulsification: placing 1.5 volume parts of oil phase in an emulsifying tank, starting a motor to stir at 1500-; after emulsification, sampling 10ml, centrifuging for 15min at 3000r/min, and emulsifying to obtain an emulsion which is the five-valent inactivated vaccine of the haemophilus parasuis if the solution is not layered; and if the layering phenomenon exists, repeatedly emulsifying the obtained emulsion until the emulsion is not layered, and finally obtaining the five-valent inactivated vaccine of the haemophilus parasuis.
In the invention, the preparation methods of the TSA agar plate and the TSB culture medium are as follows: taking 40g of tryptose soy agar, adding deionized water to a constant volume of 1000mL, fully shaking up and dissolving, sterilizing for 15min by high-pressure steam at 121 ℃, cooling, and adding 50mL of filter sterilized newborn calf serum and 200 mu L of filter sterilized 0.2 g/mLNAD. The fermentation medium is a semisynthetic medium, and the preparation method of the semisynthetic medium comprises the following steps: dissolving 30g of TSB in deionized water, diluting to 1000mL, shaking up to dissolve, sterilizing for 15min at 121 ℃ by high-pressure steam, cooling, and adding 50mL of filter-sterilized newborn calf serum and 200 mu L of filter-sterilized 0.02% NAD. The preparation method of the TSB culture medium comprises the following steps: dissolving 30g of Tryptose Soy Broth (TSB) in deionized water, diluting to 1000mL, shaking thoroughly to dissolve, sterilizing with 121 deg.C steam for 15min, adding 50mL of filter sterilized newborn calf serum and 200ul of filter sterilized 0.02% NAD.
Example 7 safety test of Haemophilus parasuis pentavalent inactivated vaccine
1. Safety test for one single dose vaccination of target animals with haemophilus parasuis vaccine
The five-valent inactivated vaccine of haemophilus parasuis prepared by the preparation method of the invention, namely the preparation method of example 6, is used for respectively carrying out neck intramuscular injection on 5 piglets of 10-15 days old (purchased from a certain improved pig farm in Fujian Longyan city), wherein the injection amount is 2 ml/head, and a normal saline immune control group is additionally arranged. The test pigs were observed for allergy, abnormality in spirit and diet, red swelling at the injection site and weight gain of piglets within 2 weeks after vaccination. The test results are shown in table 1 below.
TABLE 1 one-time vaccination safety test results for non-use day-old target animals
As can be seen from Table 1, the piglets after the injection of the five-valent inactivated vaccine of the haemophilus parasuis have good spirit, no red swelling phenomenon appears at the injection part after the inoculation, and the spirit and the ingestion are normal; namely the piglet state is basically consistent with that of the normal saline control group; thus showing that the haemophilus parasuis vaccine prepared by the invention is safe to one-time single-dose inoculation of target animals.
2. Safety test of one-time overdose inoculation of haemophilus parasuis vaccine to target animal
3 batches of the haemophilus parasuis pentavalent inactivated vaccine are prepared by adopting the preparation method, wherein the batches are respectively 01, 02 and 03; respectively carrying out neck intramuscular injection on healthy susceptible piglets of 10-15 days old by adopting the 3 batches of vaccines, wherein 5 injections are injected in each batch, the injection amount is 4 ml/injection, and a normal saline immune control group is arranged in each batch; and (5) observing the safety of the test pigs after the vaccines are inoculated. The results are shown in Table 2.
TABLE 2 safety test for hyperinflation of H.parasuis vaccine
As can be seen from Table 2, no redness and swelling reaction appears at the injection parts of 10-15 weeks old piglets which are respectively injected with 3 batches of the Haemophilus parasuis pentavalent inactivated vaccine, and the piglets are normal in spirit and ingestion and have no obvious difference from the piglets of a control group; thus, the prepared haemophilus parasuis pentavalent inactivated vaccine is safe for one-time overdose inoculation.
3. Single dose repeat inoculation safety test
The 01 batches of the haemophilus parasuis pentavalent inactivated vaccine prepared by the preparation method are used for vaccinating healthy susceptible piglets of 10-15 days old, the vaccination is repeated once after 14 days, and the inoculated physiological saline is used as a control group; the safety of the vaccinated pigs was observed with a single dose of repeated vaccinations of the vaccine. The results are detailed in Table 3.
Table 3 single dose repeat safety test results for experimental piglets
As shown in Table 3, the vaccine has no adverse effect on the boars, has no allergy phenomenon, has no adverse reaction such as red swelling and the like at the inoculation positions, has normal spirit and feed intake of the boars, and has no obvious difference between the weight gain of piglets and the weight gain of control groups; thus proving that the single-dose repeated inoculation of the five-valent inactivated vaccine of the haemophilus parasuis is safe.
4. Safety test of pregnant sows
In the test group, the five-valent inactivated vaccine of the haemophilus parasuis prepared by the preparation method is used for performing neck intramuscular injection on 5 pregnant sows (in a certain pig farm in Longyan city) 15 days before delivery, wherein the injection amount is 2 ml/head; a normal saline immune control group was additionally provided. The test pigs were observed for allergy, abnormality in spirit and diet, red swelling at the injection site, and normal sow feeding within 2 weeks after vaccination. The results show that the injection part of the pregnant sow does not have the red swelling phenomenon 15 days before the delivery of the test group after the inoculation, the body temperature is slightly raised on the 2 th to 3 th days after the inoculation, the body temperature is recovered to be normal on the 4 th day, the spirit and the ingestion are normal, the born piglet is normal, and the farrowing rate reaches 96.8; the control test pigs are normal, and the farrowing rate is 97.2 percent; thus, the haemophilus parasuis pentavalent inactivated vaccine is safe for the inoculation of pregnant sows.
Example 8 pharmacodynamic test of Haemophilus parasuis vaccine
The experimental group is an immunization group, the haemophilus parasuis pentavalent inactivated vaccine prepared by the preparation method is used for performing neck intramuscular injection on 25 healthy susceptible pigs of 10-15 days old, the injection amount is 2 ml/head, after 21 days, secondary immunization is performed, the dose is not changed, 14 days after the secondary immunization, the immune experimental pigs injected with the haemophilus parasuis pentavalent inactivated vaccine are randomly divided into 5 groups, and the haemophilus parasuis serum 1 type, 4 type, 5 type, 12 type and 13 type strains are used for attacking toxin respectively; meanwhile, a normal saline immune control group is set. The results are shown in Table 4.
TABLE 4 piglet challenge and protective trials
As can be seen from Table 4, the virus-attacking immunity rate of the five-valent inactivated vaccine of the haemophilus parasuis prepared by the preparation method disclosed by the invention on the haemophilus parasuis serum 1, 5 and 12 type strains reaches 100%, and the virus-attacking immunity rate on the haemophilus parasuis serum 4 and 13 type strains is 60%; therefore, the haemophilus parasuis pentavalent inactivated vaccine prepared by the preparation method disclosed by the invention can be used for simultaneously carrying out immune control on diseases caused by various serotype haemophilus parasuis, and has better immune control capability.
In conclusion, the invention provides a Haemophilus parasuis strain LYD1(Haemophilus parasuis), which can be used for preparing vaccines for preventing and treating diseases caused by various serotypes of Haemophilus parasuis, and the finally prepared vaccines have good safety and prevention and treatment capacity; in addition, the method has the characteristic of low cost.
Claims (1)
1. A haemophilus parasuis pentavalent inactivated vaccine is characterized in that: the haemophilus parasuis strain is characterized by comprising a strain model LYD1, wherein the haemophilus parasuis strain LYD1 is deposited in the common microorganism center of China Committee for culture Collection of microorganisms at 21.07.2015, the address is No. 3 of No.1 Siro-Shih of North Kyoho in the Chaoyang area in Beijing, and the deposition number is CGMCC No. 11143;
haemophilus parasuis LYH5(Haemophilus sparacuis) was deposited in the general microbiological center of China Committee for culture Collection of microorganisms at 21.07.2015, with the address of No. 3 Hospital No.1, Saint West Lu, North Kyoto area of Chaoyang, Beijing, and the deposition number of CGMCC No. 11144;
haemophilus parasuis strain LY02(Haemophilus sparacuis) was deposited in the general microbiological center of China Committee for culture Collection of microorganisms at 21.07.2015, with the address of No. 3 Hospital No.1, Saint West Lu, North Kyoto the open area, Beijing, and the deposition number of CGMCC No. 11145;
the Haemophilus parasuis strain LYW 1(Haemophilus parasuis) is preserved in the general microbiological center of China Committee for culture Collection of microorganisms at 21.07.2015, with the address of No. 3 Hospital No.1 of Xilu, North Chen of the Chaoyang area in Beijing and the preservation number of CGMCC No. 11146;
haemophilus parasuis LYC2(Haemophilus sparacuis) was deposited in the general microbiological center of China Committee for culture Collection of microorganisms at 21.07.2015, with the address of No. 3 Hospital No.1, Saint West Lu, North Kyoto area of Chaoyang, Beijing, and the deposition number of CGMCC No. 11147;
the preparation method of the haemophilus parasuis pentavalent inactivated vaccine comprises the following steps:
(1) preparing a seed solution: taking a strain LYD1, a strain LYH5, a strain LY02, a strain LYW1 and a strain LYC2, streaking and inoculating the strains on TSA agar plates respectively, and inoculating the strains on TSA agar plates containing 5-10% of CO by mass percent2Culturing at 37 ℃ for 24-48h, selecting typical colonies, inoculating the typical colonies on a TSB culture medium, and performing shake culture at 37 ℃ for 24-48h to obtain a first-stage seed solution of a strain LYD1, a first-stage seed solution of a strain LYH5, a first-stage seed solution of a strain LY02, a first-stage seed solution of a strain LYW1 and a first-stage seed solution of a strain LYC 2; respectively streaking the obtained primary seed liquid of the strain LYD1, the primary seed liquid of the strain LYH5, the primary seed liquid of the strain LY02, the primary seed liquid of the strain LYW1 and the primary seed liquid of the strain LYC2 on TSA agar plates, and placing the TSA agar plates on which the TSA agar plates contain 5-10% of CO by mass percent2Culturing at 37 ℃ for 24-48h, selecting typical colonies, inoculating the typical colonies on a TSB culture medium, and performing shaking culture at 37 ℃ for 24-48h to obtain a secondary seed solution of a strain LYD1, a secondary seed solution of a strain LYH5, a secondary seed solution of a strain LY02, a secondary seed solution of a strain LYW1 and a secondary seed solution of a strain LYC 2; respectively inoculating the secondary seed liquid of the strain LYD1, the secondary seed liquid of the strain LYH5, the secondary seed liquid of the strain LY02, the secondary seed liquid of the strain LYW1 and the secondary seed liquid of the strain LYC2 to a fermentation culture medium, and culturing at 37 ℃ and 180r/min for 22-24 hours to obtain a strain LYD1 bacterial liquid, a strain LYH5 bacterial liquid, a strain LY02 bacterial liquid, a strain LYW1 bacterial liquid and a strain LYC2 bacterial liquid;
the preparation method of the TSA agar plate comprises the following steps: taking 40g of tryptic soy agar, adding deionized water to a constant volume of 1000mL, fully shaking up and dissolving, sterilizing for 15min by high pressure steam at 121 ℃, cooling, adding 50mL of filter sterilized newborn calf serum and 200 mu L of filter sterilized 0.2g/mLNAD
The fermentation medium is a semisynthetic medium, and the preparation method comprises the following steps: dissolving TSB30g in deionized water, diluting to a constant volume of 1000mL, shaking thoroughly to dissolve, steam sterilizing at 121 deg.C for 15min, cooling, adding 50mL filter sterilized newborn calf serum, 200 μ L filter sterilized 0.02% NAD;
the preparation method of the TSB culture medium comprises the following steps: dissolving tryptose soy broth TSB30g in deionized water, diluting to 1000mL, shaking to dissolve completely, sterilizing with 121 deg.C steam for 15min, adding 50mL filter sterilized newborn calf serum and 200ul filter sterilized 0.02% NAD to obtain the final product;
(2) concentration and inactivation: concentrating the bacterial solutions obtained by propagation culture respectively, and concentrating bacterial solution of strain LYD1, bacterial solution of strain LYW1, and bacterial solution of strain LYC2 to obtain bacterial solution with antigen content of 2.5 × 109CFU/mL, bacterial liquid of strain LYH5, bacterial liquid of strain LY02, and concentrated to antigen content of 2.0 × 109CFU/mL; then inactivating the concentrated bacterial liquids respectively;
(3) preparing a total mixed bacterial liquid: mixing the concentrated and inactivated bacterial liquid of the strain LYD1, the bacterial liquid of the strain LYW1 and the bacterial liquid of the strain LYC2 according to the volume ratio of 1:1:1 to obtain first mixed bacterial liquid; mixing the concentrated and inactivated bacterial liquid of the strain LYH5 and the bacterial liquid of the strain LY02 according to the volume ratio of 1:1 to obtain a second mixed bacterial liquid; then mixing the obtained first mixed bacterial liquid and second mixed bacterial liquid according to the volume ratio of 1:1.5 to obtain total mixed bacterial liquid;
(4) preparing a water phase: taking sterilized tween-804 parts and 96 parts of total mixed bacterium liquid qualified by aseptic inspection according to the volume parts, introducing the sterilized tween-804 parts and the total mixed bacterium liquid into a liquid preparation tank, starting a stirring motor, and stirring at a constant speed until tween-80 is completely dissolved to obtain a water phase;
(5) preparing an oil phase: taking 94 parts by volume of white oil for injection and 806 parts by volume of span, adding the white oil and the span into an oil phase preparation tank, starting a stirring motor, stirring at a constant speed, simultaneously starting a conduction oil switch, heating at 125 ℃ for 30min, and cooling to obtain an oil phase;
(6) emulsification: placing 1.5 volume parts of oil phase in an emulsifying tank, starting a motor to stir at 1500-; after emulsification, sampling 10ml, and centrifuging for 15min at 3000r/min until no layering emulsification is carried out, and the obtained emulsion is the haemophilus parasuis pentavalent inactivated vaccine.
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| CN105524857A (en) | 2016-04-27 |
| CN105112342B (en) | 2019-03-15 |
| CN105112342A (en) | 2015-12-02 |
| CN105148262A (en) | 2015-12-16 |
| CN105148262B (en) | 2018-07-10 |
| CN105420141A (en) | 2016-03-23 |
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| CN105543120A (en) | 2016-05-04 |
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