CN117942391A - Preparation method and application of brucella component vaccine - Google Patents
Preparation method and application of brucella component vaccine Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a preparation method and application of a brucella component vaccine, wherein the preparation method comprises the following steps: (1) Crushing brucella suspension by a physical method, and centrifuging to obtain a supernatant serving as a component A vaccine; (2) And weighing the precipitate obtained by centrifugation, adding each chemical reagent, centrifuging to obtain the precipitate, and concentrating by dialysis to obtain the E-component vaccine. The preparation method of the brucella component vaccine has simple process, and the prepared component vaccine has good vaccine protection efficacy and biological safety. The brucella component vaccine of the invention retains most antigens of brucella and fully utilizes the characteristic of high protection generated by antigen induction. The vaccine improves the protection efficacy of the traditional vaccine for treating the brucellosis and solves the safety problem of the live vaccine.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a brucella component vaccine.
Background
Brucellosis (Brucellosis, simply called brucellosis) is a zoonosis caused by facultative intracellular gram-negative bacteria, and can cause symptoms such as wavy heat, night sweat, body deficiency, general debilitation, insomnia, anorexia, headache, arthralgia and the like of human beings. The vaccine is taken as an important means for preventing and controlling the brucellosis, the currently used vaccine is an attenuated live vaccine, the most representative vaccine is the brucellosis 104M live vaccine which is the only human brucellosis vaccine approved for use in China, but the vaccine is taken as the live vaccine and has the problems of residual virulence, pathogenicity and the like, the currently studied novel vaccines such as recombinant subunit vaccine, DNA vaccine and the like solve the safety problem, but the protection effect is different from that of the attenuated live vaccine, and the development of a novel vaccine is needed to solve the problems of the safety and the protection effect of the brucellosis vaccine.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a preparation method and application of a brucella component vaccine.
The technical scheme adopted for solving the technical problems is as follows:
a method for preparing a brucella component vaccine, comprising the following steps:
(1) Crushing brucella suspension by a physical method, and centrifuging to obtain a precipitate;
(2) Weighing the precipitate obtained by centrifugation, adding each chemical reagent, centrifuging to obtain precipitate, and concentrating by dialysis to obtain E-component vaccine;
Adding a chemical reagent PbMgS solution and an SDS solution into the step (2), re-suspending and precipitating, oscillating for 30 minutes, then standing at 4 ℃ overnight, centrifuging to obtain a supernatant, adding an ammonium sulfate solution, standing at 4 ℃ overnight, centrifuging, dialyzing and concentrating to obtain an E component vaccine;
Wherein, the centrifugal parameters after PbMgS liquid and SDS solution are added are 4 ℃, 7600rpm or 10000rpm,30 minutes; the centrifugation parameters after the addition of the ammonium sulfate solution were 4℃at 7600rpm or 10000rpm for 15 minutes.
Further, the concentration of the Brucella suspension in the step (1) is 100 hundred million/ml or 500 hundred million/ml;
Ultrasonic crushing is carried out on the bacterial suspension at-20-room temperature, wherein the ultrasonic crushing treatment time is 30 minutes each time, and the total time is 3 times;
the centrifugation parameters were 4℃at 7600rpm or 10000rpm for 30 minutes.
Further, the preparation method of PbMgS liquid comprises the following steps: mixing 0.01M Na 2HPO4 and 0.01M NaH 2PO4 proportionally until pH=7 to obtain a mixture, and mixing 0.03M MgCl 2 and 0.44M sucrose with the above mixture until pH=6.4 to obtain PbMgS liquid;
The preparation method of the SDS solution comprises the following steps: 5g SDS was added per 1000ml water;
the preparation method of the ammonium sulfate solution comprises the following steps: every 800g of solid ammonium sulfate is dissolved in 1000ml of water, the solution is heated to be fully dissolved, the ammonium sulfate crystals are precipitated at the bottom after cooling, and the supernatant is the required ammonium sulfate solution.
Further, pbMgS liquid was added in an amount of 10 liquid ml PbMgS per 1g of precipitate;
the amount of SDS solution added was: pbMgS total volume of liquid and precipitate;
The amount of ammonium sulfate solution added was: pbMgS total volumes of liquid, precipitate and SDS solution.
Further, the method also comprises the steps of repeated freeze thawing and tissue homogenizer treatment, specifically: the bacterial suspension is repeatedly frozen and thawed at-20-room temperature, subjected to ultrasonic disruption and tissue homogenate device treatment, the repeated freezing and thawing time is at least 10 times, and the ultrasonic disruption and tissue homogenate device treatment time is 30 minutes each time, and the total time is 3 times.
Further, the method specifically comprises the following steps:
(1) Repeatedly freezing and thawing the brucella suspension at-20-room temperature for at least 10 times;
(2) Repeatedly treating with the tissue homogenizer for 30 minutes under the action of ultrasonic wave for 3 times;
(3) 7600rpm, centrifuging at 4deg.C for 30 min, and collecting supernatant to obtain component A vaccine;
(4) Adding PbMgS liquid into the sediment after weighing to prepare suspension, adding 10ml PbMgS liquid into each 1g sediment, adding 0.5% SDS solution with equal volume, oscillating for 30 minutes, and standing at 4 ℃ overnight;
(5) 7600rpm, centrifuging at 4deg.C for 30min, discarding precipitate, adding equal volume of saturated ammonium sulfate solution into supernatant, and standing overnight at 4deg.C;
(6) 7600rpm, centrifuging at 4deg.C for 15 min, removing supernatant, and dissolving precipitate with PbMgS;
(7) Dialyzing in distilled water at 4deg.C for at least 3 days using a dialysis bag;
(8) Concentrating and freeze-drying to obtain the E-component vaccine.
The vaccine obtained by the preparation method is applied to the preparation of products for preventing and/or diagnosing brucellosis.
The vaccine obtained by the preparation method is applied to the preparation of the antigen for preventing and/or diagnosing human brucellosis.
The colloidal gold test strip of the vaccine obtained by the preparation method is utilized.
The invention has the advantages and positive effects that:
1. The preparation method of the brucella component vaccine has simple process, and the prepared component vaccine has good vaccine protection efficacy and biological safety. The brucella component vaccine of the invention retains most antigens of brucella and fully utilizes the characteristic of high protection generated by antigen induction. The vaccine improves the protection efficacy of the traditional vaccine for treating the brucellosis and solves the safety problem of the live vaccine.
2. The component vaccine of the invention is prepared by a physical method and a chemical method, comprises protein, nucleic acid, polysaccharide and lipopolysaccharide, overcomes the defects of strong sensitization, residual toxicity and the like of a live vaccine, and solves the problem of insufficient protective efficacy of a recombinant protein vaccine.
3. The method adopts physical and chemical means to treat the Brucella, and the extracted component vaccine has the characteristics of low agglutinogenicity, weak allergenicity, strong immunoprotection and the like. The component vaccine prepared by the invention can induce strong immune response after immunization, can resist Brucella infection, solves the pathogenicity problem of the live attenuated Brucella vaccine, and improves the protection efficacy of the traditional recombinant protein vaccine.
Drawings
FIG. 1 is a schematic flow chart of the preparation method of the brucella component vaccine in examples 1 and 2.
Detailed Description
The invention will now be further illustrated by reference to the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.
The various experimental operations involved in the specific embodiments are conventional in the art, and are not specifically noted herein, and may be implemented by those skilled in the art with reference to various general specifications, technical literature or related specifications, manuals, etc. before the filing date of the present invention.
A method for preparing a brucella component vaccine, comprising the following steps:
(1) Crushing brucella suspension by a physical method, and centrifuging to obtain a precipitate;
(2) Weighing the precipitate obtained by centrifugation, adding each chemical reagent, centrifuging to obtain precipitate, and concentrating by dialysis to obtain E-component vaccine;
Adding a chemical reagent PbMgS solution and an SDS solution into the step (2), re-suspending and precipitating, oscillating for 30 minutes, then standing at 4 ℃ overnight, centrifuging to obtain a supernatant, adding an ammonium sulfate solution, standing at 4 ℃ overnight, centrifuging, dialyzing and concentrating to obtain an E component vaccine;
Wherein, the centrifugal parameters after PbMgS liquid and SDS solution are added are 4 ℃, 7600rpm or 10000rpm,30 minutes; the centrifugation parameters after the addition of the ammonium sulfate solution were 4℃at 7600rpm or 10000rpm for 15 minutes.
Preferably, the concentration of the Brucella suspension in step (1) is 100 or 500 hundred million/ml;
Ultrasonic crushing is carried out on the bacterial suspension at-20-room temperature, wherein the ultrasonic crushing treatment time is 30 minutes each time, and the total time is 3 times;
the centrifugation parameters were 4℃at 7600rpm or 10000rpm for 30 minutes.
Preferably, the preparation method of PbMgS liquid comprises the following steps: mixing 0.01M Na 2HPO4 and 0.01M NaH 2PO4 proportionally until pH=7 to obtain a mixture, and mixing 0.03M MgCl 2 and 0.44M sucrose with the above mixture until pH=6.4 to obtain PbMgS liquid;
The preparation method of the SDS solution comprises the following steps: 5g SDS was added per 1000ml water;
the preparation method of the ammonium sulfate solution comprises the following steps: every 800g of solid ammonium sulfate is dissolved in 1000ml of water, the solution is heated to be fully dissolved, the ammonium sulfate crystals are precipitated at the bottom after cooling, and the supernatant is the required ammonium sulfate solution.
Preferably, pbMgS liquid is added in an amount of 10 liquid ml PbMgS per 1g of precipitate;
the amount of SDS solution added was: pbMgS total volume of liquid and precipitate;
The amount of ammonium sulfate solution added was: pbMgS total volumes of liquid, precipitate and SDS solution.
Preferably, the method further comprises the steps of repeated freezing and thawing and tissue homogenizer treatment, specifically: the bacterial suspension is repeatedly frozen and thawed at-20-room temperature, subjected to ultrasonic disruption and tissue homogenate device treatment, the repeated freezing and thawing time is at least 10 times, and the ultrasonic disruption and tissue homogenate device treatment time is 30 minutes each time, and the total time is 3 times.
Preferably, the method specifically comprises the following steps:
(1) Repeatedly freezing and thawing the brucella suspension at-20-room temperature for at least 10 times;
(2) Repeatedly treating with the tissue homogenizer for 30 minutes under the action of ultrasonic wave for 3 times;
(3) 7600rpm, centrifuging at 4deg.C for 30 min, and collecting supernatant to obtain component A vaccine;
(4) Adding PbMgS liquid into the sediment after weighing to prepare suspension, adding 10ml PbMgS liquid into each 1g sediment, adding 0.5% SDS solution with equal volume, oscillating for 30 minutes, and standing at 4 ℃ overnight;
(5) 7600rpm, centrifuging at 4deg.C for 30min, discarding precipitate, adding equal volume of saturated ammonium sulfate solution into supernatant, and standing overnight at 4deg.C;
(6) 7600rpm, centrifuging at 4deg.C for 15 min, removing supernatant, and dissolving precipitate with PbMgS;
(7) Dialyzing in distilled water at 4deg.C for at least 3 days using a dialysis bag;
(8) Concentrating and freeze-drying to obtain the E-component vaccine.
The vaccine obtained by the preparation method is applied to the preparation of products for preventing and/or diagnosing brucellosis.
The vaccine obtained by the preparation method is applied to the preparation of the antigen for preventing and/or diagnosing human brucellosis.
The colloidal gold test strip of the vaccine obtained by the preparation method is utilized.
Specifically, the related preparation and detection are as follows:
Example 1
Preparation of chemical reagents required for preparation of brucella component vaccine
(1) PbMgS liquid: mixing 0.01M Na 2HPO4 and 0.01M NaH 2PO4 proportionally until PH=7, mixing 0.03M MgCl 2 and 0.44M sucrose until PH=6.4 to obtain PbMgS liquid;
(2) SDS solution: 1000ml of water plus 5g of SDS;
(3) Ammonium sulfate solution: 800g of solid ammonium sulfate is dissolved in 1000ml of water, heated to be fully dissolved, cooled and then ammonium sulfate crystals are precipitated at the bottom, and the supernatant is the required ammonium sulfate solution.
(II) preparation of vaccine strain of Brucella 104M (which is a certain type of Brucella, brucella 104M is currently known vaccine.) and vaccine component of Brucella sheep strain 16M standard strain
As shown in FIG. 1, the cultured Brucella is prepared into bacterial suspension according to 500 hundred million/ml, the bacterial suspension is repeatedly frozen and thawed for at least 10 times at-20 ℃ to room temperature, the bacterial suspension is treated for 3 times by an ultrasonic breaker and a tissue homogenizer, 30min each time, 80-100 ml/time is treated by a temperature of 4 ℃ and a temperature of 10000 rpm for centrifugation, 30min, the supernatant is the A component vaccine, the weight of the precipitate is weighed, pbMgS liquid (10 ml PbMgS liquid is added to each 1g of the precipitate), 0.5% SDS solution with equal volume (i.e. PbMgS liquid and total volume of the precipitate) is added for mixing, shaking is carried out for 30min, overnight at 4 ℃,10000 rpm is centrifuged for 30min, the precipitate is discarded, the supernatant is added with equal volume (i.e. PbMgS liquid, total volume of the precipitate and SDS solution) ammonium sulfate solution, overnight at 4 ℃,10000 rpm is centrifuged for 15min, the precipitate is discarded, the precipitate is dissolved in a small amount of PbMgS liquid, and the E component vaccine is obtained after drying by using a dialysis bag.
(III) animal experiments to assess vaccine efficacy
Selecting 250g-350g Hartley healthy guinea pigs, setting a normal saline control group, a brucella 104M live vaccine group, a brucella 104M dead vaccine group, a brucella 104M A component vaccine group, a brucella 104M E component vaccine group, a brucella sheep species 16M standard strain A component vaccine group and a brucella sheep species 16M standard strain E component vaccine group, wherein the immune dose is as follows: 1ml of physiological saline group; the live vaccine group of brucella 104M and the dead vaccine group of brucella 104M are 1000 ten thousand/ml respectively, and the immunity is 1ml; the Brucella 104M A component vaccine group, the Brucella 104M E component vaccine group, the Brucella sheep species 16M standard strain A component vaccine group and the Brucella sheep species 16M standard strain E component vaccine group are 5mg/ml, and the immunity is 1ml. The immunization mode is subcutaneous mouse part immunization, and each experimental group adopts one immunization.
Selecting about 20g of Kunming mice, wherein the experimental group is the same as the guinea pig immunization experimental group, and the immunization dose is as follows: 1ml of physiological saline group; the live vaccine group of brucella 104M and the dead vaccine group of brucella 104M are 500-1000 ten thousand/ml respectively, and the immunity is 1ml; the brucella 104M A component vaccine group, the brucella 104M E component vaccine group, the brucella sheep species 16M standard strain A component vaccine group and the brucella sheep species 16M standard strain E component vaccine group are all 0.2-5mg/ml, and the immunity is 1ml. The immunization mode is subcutaneous mouse part immunization, and each experimental group adopts one immunization.
The mice are subjected to intraperitoneal attack by virulent bacteria of a brucella sheep strain 16M standard strain one month after immunization, and the attack amount is 5-12.5LD 50;
Subcutaneous injection attack with brucella sheep strain 16M standard strain virulent strain 1 month, 3 months and 6.5 months after guinea pig immunization, dissection 30 days after attack, organ isolation culture, serum identification of growing colony with A, B factors, and organ infection rate calculation;
0.1ml of brucine was injected into the flank of a guinea pig at various times after immunization of the guinea pig, and the results were recorded by observation at 24, 48 and 72 hours after injection, and one of the longitudinal and transverse diameters was judged to be positive at 10 mm or more.
The guinea pig anal temperature was measured within 5 days after immunization and before feeding every morning, and the mean value of each group of body temperature was calculated, and the results are shown in table 1: the body temperature response of the brucella component vaccine group is not more than that of the brucella 104M live vaccine group, the body temperature response is slightly higher than that of the normal group by the third day after inoculation, and the reaction is slight.
Animals (4-5) were taken 1,3, and 6 months after immunization for histopathological examination of liver, spleen, heart, kidney, lung, and the results are shown in Table 1: when the animals are immunized for 1 month, 2 animals are in the Brucella 104M live vaccine group, 1 animal is in the Brucella sheep species 16M standard strain A component vaccine group, and no necrosis focus exists in the kidney tissues except for 1 animal in the Brucella 104M live vaccine group. When immunized for 3 months, the reactions of each group are similar, and all groups have no necrotic foci. At 6 months of immunization, spleen proliferation was enhanced in each group.
As can be seen from table 2, A, E component vaccines (brucella 104M A component vaccine group, brucella 104M E component vaccine group, brucella sheep species 16M standard strain a component vaccine group, brucella sheep species 16M standard strain E component vaccine group) extracted from brucella 104M and brucella sheep species 16M standard strain have a protection rate of Yu Bulu live vaccine group and brucella 104M dead vaccine group, which indicates that the prepared brucella component vaccine can provide high-efficiency protection for immunized mice.
As can be seen from tables 3-6, the Brucella component vaccine can induce the organism to generate effective immune response, and the analysis of the number of the spleen bacterial-yielding organs of the guinea pigs shows that the bacterial-yielding strains can block the colonization of the body, wherein the Brucella sheep strain 16M standard strain A component vaccine group has a higher protection rate than the Brucella 104M live vaccine group, the protection rate of the other groups is not as high as that of the Brucella 104M live vaccine group, but the protection duration is longer, and the long-acting protection can be provided for the immunized guinea pigs.
As can be seen from Table 7, the brucella 104M live vaccine group showed similar skin allergy to the brucella sheep strain 16M standard strain A component vaccine, with the remaining groups being negative. Compared with the 104M live vaccine, the prepared component vaccine can alleviate adverse reaction of the live vaccine, improves safety, and has the characteristics of weak allergenicity and high safety.
Table 8-Table 11 shows that serological tests showed that the response of the Brucella sheep strain 16M standard strain A-component vaccine group after immunization was similar to that induced by Brucella 104M live vaccine group, and that the protective force of the other groups after immunization was almost negative, although not as good as that of Brucella 104M live vaccine.
To sum up:
1. In the immune mice toxicity test, the protection rate of the Brucella sheep strain 16M standard strain A component vaccine is 70%, and the Brucella 104M live vaccine is 69.6%; in the toxicity test after 1,3 and 6.5 months of guinea pig immunization, the protection rates of the A-component vaccine of the 16M standard strain of the Brucella sheep are 71.2%, 86.7% and 94.3% respectively, and the 104M live vaccine of the Brucella is 57.5%, 90.5% and 94.5% respectively. Serological and skin allergy examination results show that the sheep 16M standard strain A component vaccine group is capable of producing a similar response to Brucella 104M live vaccine after immunization. Therefore, the prepared brucella sheep strain 16M standard strain A component vaccine has similar protective power as that of a brucella 104M live vaccine, but the brucella sheep strain 16M standard strain A component vaccine does not have the toxicity of the 104M live vaccine, and the safety is higher than that of the 104M live vaccine.
2. In an immune mouse toxicity test, the brucella 104M A, an E component vaccine and an E component vaccine group of the goat species 16M standard strain are high in protection rate, namely a A component vaccine group and a 104M live vaccine group of the Yu Bulu goat species 16M standard strain; in the toxicity attack tests after 1, 3 and 6.5 months of guinea pig immunization, the protection rate is not as high as that of the A component vaccine group and the 104M live vaccine group of the 16M standard strain of the Brucella sheep, but the protection rate still has obvious long-acting protection effect, and the serum and skin test reactions are almost negative, so that the method is safer and more reliable and can bring advantages to possible application in actual work in future.
3. The reaction caused by the component vaccine prepared by us is not great from the body temperature reaction and the analysis of the organ histopathological examination result, and is safe, in addition, other used Brucella vaccines (most recombinant protein vaccines) are used for immunizing animals, the animals are mostly used with the adjuvant, the animals are subjected to repeated injection for immunization, the observation period is not more than 3 months, the vaccine prepared by us is not used with the adjuvant, the subcutaneous inoculation is carried out for 1 time, and the observation period is 6.5 months at most, so that the immunogenicity of the vaccine prepared by the real reaction can be realized.
Example 2
The brucellosis 104M component vaccine was extracted in this test example by the same method as that of the brucellosis component vaccine prepared in example 1.
The experimental animals are selected from mice with the weight of 18-20g, a normal saline control group, a brucella 104M live vaccine group, a brucella 104M A component vaccine group and a brucella 104M E component vaccine group are arranged, 10 animals and females are half-male in each group, the immune dose is 0.5ml of the normal saline control group, 7 x 10 6 bacteria of the brucella 104M live vaccine group, 4mg/ml of each of the brucella 104M A component vaccine group and the brucella 104M E component vaccine group, 0.5ml of each of the brucella 104M E component vaccine group is injected, and the immune modes are subcutaneous immunization of the mouse parts, and the immunization is one-time, and no adjuvant is added. The method comprises the steps of selecting guinea pigs with the weight of 250-300g, wherein the experimental group is the same as the mice immunization experimental group, 20 mice in each group have the immunization dose of 1ml of physiological saline control group, 10 7 bacteria in brucella 104M live vaccine group, 5mg/ml each in brucella 104M A component vaccine group and 5mg/ml each in brucella 104M E component vaccine group, injecting 1ml, and the immunization modes are subcutaneous immunization of the mouse parts, wherein the immunization modes are all one-time immunization without adding any adjuvant. The guinea pigs were injected laterally with 0.1ml brucine at various times after immunization, and the results were recorded 24 and 48 hours later. Mice were challenged with 3 x 10 9 bacteria in a 16M suspension of sheep at 1ml dose for 30 and 90 days post immunization and animals were scored for mortality 15 days post injection.
As can be seen from Table 12, no skin allergy occurred after immunization of guinea pigs with the two component vaccine. Compared with 104M live vaccine, the A, E component vaccine prepared can alleviate adverse reaction after live vaccine inoculation, has improved safety, and has the characteristics of weak allergenicity and high safety.
As can be seen from Table 13, the protective rate of the Brucella A, E component vaccine is statistically treated by chi-square test compared with that of the 104M live vaccine, and no significant difference exists, which shows that the Brucella A, E component vaccine can achieve the similar protective effect as the existing attenuated live vaccine.
Chemical composition analysis of brucella 104M A, E-component vaccine: protein is measured by a Folin phenol method, and bovine serum albumin is used as a standard substance; measuring total sugar by anthrone method, and taking glucose as a standard substance; measuring DNA by a diphenylamine method, and taking standard DNA as a standard substance; RNA was measured by the dihydroxymethyl method using standard RNA as a standard.
As can be seen from Table 14, the Brucella A, E component vaccine contains proteins, nucleic acids, polysaccharides, lipopolysaccharides and other components, retains most of the immunogenic substances of Brucella, and can induce the organism to generate strong immune response.
In conclusion, the protective rate of the prepared Brucella 104M A and E component vaccine for 30 days and 90 days is similar to that of the existing live vaccine Brucella 104M, but A, E component vaccine does not generate skin allergy after 104M immunization, does not have residual toxicity and has higher safety.
Example 3
The component A of the brucella 104M vaccine strain prepared by extraction is coated with a colloidal gold test strip, and the serum of a patient with confirmed diagnosis is taken as a sample, so that the coincidence rate reaches 75% (Table 15), and the brucella 104M vaccine strain A can be used as a candidate diagnosis antigen.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (9)
1. A method for preparing a brucella component vaccine, which is characterized by comprising the following steps: the method comprises the following steps:
(1) Crushing brucella suspension by a physical method, and centrifuging to obtain a precipitate;
(2) Weighing the precipitate obtained by centrifugation, adding each chemical reagent, centrifuging to obtain precipitate, and concentrating by dialysis to obtain E-component vaccine;
Adding a chemical reagent PbMgS solution and an SDS solution into the step (2), re-suspending and precipitating, oscillating for 30 minutes, then standing at 4 ℃ overnight, centrifuging to obtain a supernatant, adding an ammonium sulfate solution, standing at 4 ℃ overnight, centrifuging, dialyzing and concentrating to obtain an E component vaccine;
Wherein, the centrifugal parameters after PbMgS liquid and SDS solution are added are 4 ℃, 7600rpm or 10000rpm,30 minutes; the centrifugation parameters after the addition of the ammonium sulfate solution were 4℃at 7600rpm or 10000rpm for 15 minutes.
2. The method of manufacturing according to claim 1, characterized in that: the concentration of the brucella suspension in the step (1) is 100 hundred million/ml or 500 hundred million/ml;
Ultrasonic crushing is carried out on the bacterial suspension at-20-room temperature, wherein the ultrasonic crushing treatment time is 30 minutes each time, and the total time is 3 times;
the centrifugation parameters were 4℃at 7600rpm or 10000rpm for 30 minutes.
3. The method of manufacturing according to claim 1, characterized in that: the preparation method of PbMgS liquid comprises the following steps: mixing 0.01M Na 2HPO4 and 0.01M NaH 2PO4 proportionally until pH=7 to obtain a mixture, and mixing 0.03M MgCl 2 and 0.44M sucrose with the above mixture until pH=6.4 to obtain PbMgS liquid;
The preparation method of the SDS solution comprises the following steps: 5g SDS was added per 1000ml water;
the preparation method of the ammonium sulfate solution comprises the following steps: every 800g of solid ammonium sulfate is dissolved in 1000ml of water, the solution is heated to be fully dissolved, the ammonium sulfate crystals are precipitated at the bottom after cooling, and the supernatant is the required ammonium sulfate solution.
4. The method of manufacturing according to claim 1, characterized in that: the amount of PbMgS liquid added is 10ml PbMgS liquid per 1g of sediment;
the amount of SDS solution added was: pbMgS total volume of liquid and precipitate;
The amount of ammonium sulfate solution added was: pbMgS total volumes of liquid, precipitate and SDS solution.
5. The preparation method according to claim 2, characterized in that: the method also comprises the steps of repeated freezing and thawing and tissue homogenizer treatment, and specifically comprises the following steps: the bacterial suspension is repeatedly frozen and thawed at-20-room temperature, subjected to ultrasonic disruption and tissue homogenate device treatment, the repeated freezing and thawing time is at least 10 times, and the ultrasonic disruption and tissue homogenate device treatment time is 30 minutes each time, and the total time is 3 times.
6. The method according to any one of claims 1 to 5, wherein: the method specifically comprises the following steps:
(1) Repeatedly freezing and thawing the brucella suspension at-20-room temperature for at least 10 times;
(2) Repeatedly treating with the tissue homogenizer for 30 minutes under the action of ultrasonic wave for 3 times;
(3) 7600rpm, centrifuging at 4deg.C for 30 min, and collecting supernatant to obtain component A vaccine;
(4) Adding PbMgS liquid into the sediment after weighing to prepare suspension, adding 10ml PbMgS liquid into each 1g sediment, adding 0.5% SDS solution with equal volume, oscillating for 30 minutes, and standing at 4 ℃ overnight;
(5) 7600rpm, centrifuging at 4deg.C for 30min, discarding precipitate, adding equal volume of saturated ammonium sulfate solution into supernatant, and standing overnight at 4deg.C;
(6) 7600rpm, centrifuging at 4deg.C for 15 min, removing supernatant, and dissolving precipitate with PbMgS;
(7) Dialyzing in distilled water at 4deg.C for at least 3 days using a dialysis bag;
(8) Concentrating and freeze-drying to obtain the E-component vaccine.
7. Use of a vaccine obtained by the preparation method according to any one of claims 1 to 6 for the preparation of a product for the prevention and/or diagnosis of brucellosis.
8. Use of a vaccine obtained by the preparation method according to any one of claims 1 to 6 for the preparation of a diagnostic antigen for preventing and/or diagnosing brucellosis in humans.
9. A colloidal gold test strip of a vaccine obtained by the production method according to any one of claims 1 to 6.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016541A (en) * | 2006-12-26 | 2007-08-15 | 中国农业科学院兰州兽医研究所 | Method of producing brucella vaccine antigen protein |
| WO2019056075A1 (en) * | 2017-09-21 | 2019-03-28 | Абульфат Ахмед оглы АЛЫЕВ | Method for differential diagnosis of brucellosis in animals, nutrient medium and method for preparation of same |
| CN113727999A (en) * | 2019-01-11 | 2021-11-30 | 奥默罗斯公司 | Methods and compositions for treating cancer |
| US20240044891A1 (en) * | 2022-08-02 | 2024-02-08 | Institute Of Animal Sciences, Chinese Academy Of Agricultural Sciences | Antibody detection test strip of integrating primary screening and diagnosis of sheep brucellosis |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016541A (en) * | 2006-12-26 | 2007-08-15 | 中国农业科学院兰州兽医研究所 | Method of producing brucella vaccine antigen protein |
| WO2019056075A1 (en) * | 2017-09-21 | 2019-03-28 | Абульфат Ахмед оглы АЛЫЕВ | Method for differential diagnosis of brucellosis in animals, nutrient medium and method for preparation of same |
| CN113727999A (en) * | 2019-01-11 | 2021-11-30 | 奥默罗斯公司 | Methods and compositions for treating cancer |
| US20240044891A1 (en) * | 2022-08-02 | 2024-02-08 | Institute Of Animal Sciences, Chinese Academy Of Agricultural Sciences | Antibody detection test strip of integrating primary screening and diagnosis of sheep brucellosis |
Non-Patent Citations (3)
| Title |
|---|
| 姜顺求等: "《布鲁氏菌病防治手册》", vol. 1986, 28 February 1986, 人民卫生出版社, pages: 312 - 314 * |
| 尚德秋: "提取布鲁氏菌免疫抗原接种小鼠豚鼠的探索性研究", 《流行病学杂志》, vol. 1, no. 3, 20 August 1980 (1980-08-20), pages 173 - 177 * |
| 徐震舟等: "短小棒状杆菌对布鲁氏菌组分苗免疫影响的初步试验观察", 《地方病通报》, vol. 2, no. 3, 30 August 1987 (1987-08-30), pages 50 - 53 * |
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