CN109350738B - Pseudorabies live vaccine diluent and preparation method and application thereof - Google Patents
Pseudorabies live vaccine diluent and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a pseudorabies live vaccine diluent, which comprises microcrystalline cellulose and astragalus polysaccharide; also disclosed is a method of preparing the diluent comprising the steps of: adding microcrystalline cellulose into a reaction vessel containing water for injection, stirring, controlling temperature, standing, adding Astragalus polysaccharides, stirring, mixing, and sterilizing. The invention has the advantages that: the nasal drop immune effect of the pseudorabies vaccine can be improved, the occupying effect of the pseudorabies virus in the cranial nerves and the lymphatic system is enhanced, the intramuscular injection immunity can generate antibodies in advance, the high-level antibody maintenance time is prolonged, and the immune effect of the pig is further improved. The latent infection of the pseudorabies wild virus is effectively prevented, and the guarantee is provided for preventing and eradicating the pseudorabies. The preparation method of the diluent has the characteristics of easy production, low cost, convenient use and the like.
Description
Technical Field
The invention belongs to the technical field of vaccine processing, and particularly relates to a pseudorabies live vaccine diluent as well as a preparation method and application thereof.
Background
China is the first breeding industry of the world and about 10 hundred million pigs, cattle and sheep are bred every year. In recent years, animal infectious diseases cause death of livestock and poultry in a large amount, and the health development of the breeding industry is seriously harmed. After the large-scale breeding degree is improved, epidemic diseases are easy to spread, and the task of preventing and controlling animal epidemic diseases is very arduous. However, in recent years, immunosuppressive diseases such as porcine circovirus disease and porcine reproductive and respiratory syndrome are ubiquitous in farmers in various places, so that the difficulty in preventing and controlling animal epidemic diseases is increased, the phenomenon of vaccine immune failure in the breeding industry occurs occasionally, the antibody protection level of the animal cannot reach a sufficient level after immune injection of the vaccine, outbreak of diseases cannot be prevented, and great economic loss is brought to the farmers. Therefore, the vaccine has important significance for improving the immune effect of the vaccine and effectively preventing infectious diseases in the breeding industry.
The addition of an adjuvant to an animal vaccine is one of the measures for improving the immune effect of the vaccine. The vaccines injected into animals at present mainly comprise live vaccines and inactivated vaccines or subunit vaccines. The method for improving the immune effect of the vaccine by adding the adjuvant is mainly found in inactivated vaccines, such as porcine circovirus disease oil adjuvant vaccines and the like. The freeze-dried attenuated vaccine is widely used in the breeding industry due to convenient use and economy, such as porcine pseudorabies live vaccine, swine fever live vaccine, swine erysipelas live vaccine and the like. Such vaccines are used after dilution with a diluent prior to immunization of animals. Common vaccine diluents include aluminum hydroxide-aluminum gel physiological saline diluent, physiological saline or PBS buffer diluent, and the like. The disadvantage of the aluminium gel diluent is that the aluminium gel diluent only has the function of improving cellular immune reaction, has large local stimulation reaction at the injection part and is easy to damage the stability of the aluminium gel by freezing preservation, and the aluminium gel diluent loses the function of adjuvant; the physiological saline diluent and the PBS buffer diluent have no immune enhancement effect.
Pseudorabies virus (PRV) belongs to the herpes virus (Herpesviridae) and causes Pseudorabies in various domestic and wild animals. The pig is not only a storage of the pseudorabies virus, but also an infection source of the pseudorabies virus, and the control of the pseudorabies virus of the pig has very important significance not only for the pig raising industry, but also for the smooth development of other animal breeding. Adult pigs often show recessive infections and respiratory symptoms, abortions in sows, stillbirths and mummy, and boars show orchitis. Piglets exhibit fever, coma and neurological symptoms with a high mortality rate. Pseudorabies is therefore an important infectious disease of reproductive disorders in the pig industry. In developed countries such as Europe and America, a large amount of material resources and manpower are invested to start and finish the plan of purifying and eradicating the porcine pseudorabies. Facilitates the application of PRV gene deletion live seedlings and corresponding identification methods, and the disease is controlled or eradicated. However, in developing countries, the disease still frequently occurs, and the healthy development of the pig industry in the countries and the regions is severely restricted.
Since 2010, the incidence of porcine pseudorabies in China has increased significantly. The latent infection of the swinery is particularly worth attention by analyzing the epidemic characteristics of the swinery. Latent infection is a special state of viral presence and is one of the common characteristics of members of the herpesviridae family. After the virus infection, the pseudorabies virus can be planted in the nervous system of the pig, so that the latent infection of the pig is caused and the pig carries the virus for a lifetime, and when the pig is in a stress condition, the pseudorabies virus of the pig which is latent infection is activated, the virus is massively replicated, and the disease of the pig is caused. After vaccination, the latently infected pigs do not show clinical symptoms, but can still expel toxin to the outside, so that other pigs are infected, and the virus is difficult to remove in a pig farm. Latent infection is one of the main reasons why porcine pseudorabies is difficult to purify. Vaccine immunization can only prevent the onset and clinical symptoms of the animal, but cannot control the establishment and reactivation of latent PRV infection. If all PRV latently infected pigs are eliminated, huge economic and social costs are needed, and the current national situation of China is not met. Therefore, the problem of latent infection by PRV is a major obstacle to current prevention and eradication programs for pseudorabies in our country. In addition, certain stresses such as high body temperature rise are generated by vaccine immunization, which also affect virus replication and antigen recognition, thereby affecting the immune effect. How to enhance the occupying effect of the vaccine in the nervous system and the lymphatic system is the key for purifying the pseudorabies.
According to swine pathology, 9 th edition, the use of maternal antibody protection and gene-deletion vaccines increases the viral load required for PRV infection in the body, but does not effectively prevent capsid shedding and latent infection of PRV wild virus in swine. Therefore, the first step of purifying the porcine pseudorabies is the most important step, namely preventing the wild virus latent infection, and the nasal drop immunization is one of the best means for preventing the wild virus latent infection. The pseudorabies attenuated vaccine is dripped into the nose of the piglet within 3 days after birth (absorbed through respiratory mucosa), and the mucosal immunity of the piglet can be established within two hours to resist the invasion of wild viruses. Meanwhile, the vaccine virus can occupy space in the nervous system (such as trigeminal ganglion) of the pig, and the vaccine virus can prevent the wild virus from invading the nervous system after infection, thereby achieving the purpose of preventing latent virus infection. The nasal drop immunity is hardly interfered by maternal antibodies, the nasal drop immunity stimulates a mucosal immune system of an organism to generate protective force, the maternal antibodies are mainly dissociated in interstitial fluid serum lymph, only a small amount of secretory antibodies IgA exist on the surface of a mucosa, and the antibodies can not neutralize vaccine viruses and cannot influence the vaccine to play a role.
According to the research on the immune mechanism of the pseudorabies virus, the immunization can stimulate the body to generate humoral immunity and cellular immunity, so that related antibodies including neutralizing antibodies and secretory antibodies are generated, however, the antibodies cannot completely prevent the invasion of wild viruses, particularly the invasion of high-toxicity wild viruses, but specific antibodies can limit the replication of the viruses to a certain extent. The nasal drop immunity of the newborn piglets mainly considers the early occupation effect mechanism of the pseudorabies virus, if the nasal drop vaccine occupies the natural target position of the pseudorabies virus in the body in advance, on one hand, the invasion of the wild virus can be prevented, on the other hand, the nasal drop immunity can stimulate the body to generate mucosal immune response, and meanwhile, a large number of lymphatic systems are contained around the trachea and the lung bronchus to generate specific immune response, so that the infection of the body by the wild virus through a respiratory system is further prevented.
Mengeling et al (1992) reported that the most effective mode of vaccination was intranasal vaccination with live vaccines, which have the advantage of local replication of the virus and the establishment of mucosal immunity. Because PRV is transmitted primarily through the nose and nose of pigs, directly or indirectly, following oronasal infection, PRV initially replicates in the epithelial cells of the upper respiratory tract, subsequently infecting the tonsils and lungs, causing the virus to spread in vivo either freely or by a route that infects leukocytes. In addition, the virus can directly enter the sensory nerve endings of the nasopharynx, and thus enter the olfactory nerve and trigeminal nerve. While the colonization of the trigeminal nerve by PRV has a certain link with reinfection, studies have shown that neurons harboring PRV are unlikely to receive reinfection from other strains.
In the aspect of whether the nasal drip immunity can be interfered by maternal antibodies, the experiment of Weixiu and the like on a 800-multitudes breeding sow farm with the positive rate of the pseudorabies wild virus antibodies reaching 64 percent shows that the nasal drip immunity is not interfered by the maternal antibodies, and the pseudorabies wild virus infection can be effectively blocked by the nasal drip immunity of newborn piglets by using the pseudorabies gene deletion vaccine. And the Wangxiang et al also carried out pseudorabies attenuated vaccine nasal drop immunization test on piglets in 3 pig farms (high positive, weak positive and negative farms) in Zhejiang province, and adjusted the immunization program according to the gB and gE antibody detection results. The result shows that after immunization by a nasal drip and intramuscular injection program in a positive pig farm, the gE positive rate is respectively reduced from 69.57 percent, 0 percent and 100.00 percent to 41.70 percent, 29.20 percent and 20.80 percent at the ages of 90 days, 120 days and 175 days; negative and weakly positive swine farms had insignificant changes in the rate of gE antibody positivity for both immunization programs during the experimental period.
Although the nasal drop immunization of newborn piglets partially limits wild virus replication by occupying natural target sites, the effective maternal protective antibodies of pseudorabies can be maintained at about 60 days old, so that the piglet groups must obtain high-level protective antibodies by intramuscular injection immunization.
Research results show that the immunization strategy of nasal drip and intramuscular injection is superior to single intramuscular injection immunization and single nasal drip immunization, and the nasal drip immunization adopted in a PRV positive pig farm can block the early infection of PRV and protect piglets from being damaged by PRV wild viruses. The swine herd can be protected from being invaded by PRV wild virus by adopting intramuscular injection immunization at the age of 40-60 days.
The vaccine immunization is the best means for preventing and controlling infectious diseases, and in order to prevent and control porcine pseudorabies, China has developed various porcine pseudorabies vaccines, but the research on the vaccine diluent is very little, the immune effect of the existing vaccine diluent is not ideal enough, and the vaccine protection period is short.
Vaccine strains used in the current market are mainly classified into 2 major groups, one is a weak-toxicity live vaccine, and the other is an inactivated vaccine or subunit vaccine. Wherein the porcine pseudorabies live vaccine enters an animal body after being immunized and can infect host cells to be continuously replicated. In clinical practice, passive immunity can be obtained from colostrum after the piglets are born, and under the condition of passive immunity, the maternal antibody obtained from the piglets interferes with the replication of the weak live vaccine in vivo, so that the immunity fails. In the population, a balance point is difficult to find due to individual difference and interference of immunosuppressive diseases, so that a large dose or multiple immunizations are needed to achieve higher immune coverage, thereby reducing the cases of immune failure. In addition, the porcine pseudorabies live vaccine usually uses PBS buffer solution or normal saline as diluent, the antibody level begins to rise 7 days after the vaccine immunization, the antibody level can provide 60% of immune protection after about 28 days, the peak 70% is reached 35 days, and the high-level immunity can be maintained for about 6-8 weeks. The neonatal piglet nasal drip immunity mainly has an early occupying effect to prevent the invasion of wild virus, although the neonatal piglet nasal drip immunity is not interfered by maternal antibodies, the detention time of PBS buffer solution or normal saline as diluent in nasal cavities and respiratory tracts is too short, the occupied vaccine virus quantity is insufficient, and the nasal drip immunity effect is also influenced. The patent technology is provided for solving the problems of small quantity of occupied vaccine viruses, immunosuppressive disease interference, low antibody titer after immunization, long production time, short antibody maintenance time and the like.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a dilution of a live pseudorabies vaccine that can improve the immune effect.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a pseudorabies live vaccine diluent comprises microcrystalline cellulose and Astragalus polysaccharides.
Microcrystalline Cellulose (MCC) is the Cellulose with the widest application range and the largest dosage in the world at present, is a macromolecular substance, is white, odorless and tasteless, has hygroscopicity, is easily soluble in water, is insoluble in an organic solvent, and is crystalline powder consisting of porous particles. The natural cellulose is a granular powder product which is hydrolyzed in an acid medium to reduce the molecular weight to a certain range and has the size of about 10 mu m, and the main component of the product is a linear polysaccharide substance combined by beta-1, 4-glucoside bonds, the polymerization degree is about 3000-10000 glucose molecules, and the relative molecular weight is 242.16.
The dissolving method of the microcrystalline cellulose (MCC) is generally heating and boiling, the concentration is generally 0.5 to 1.0 percent, the pH of the solution is 6.5 to 7.5, and the solution is stable to heat. The aqueous solution is transparent and has certain viscosity; is stable in alkaline solution and is easy to hydrolyze when meeting acid. In the pharmaceutical industry, MCC with proper viscosity is selected as an adhesive and a disintegrating agent of a tablet, a suspending agent of a suspension, a biological adhesive tablet, a gastric retention tablet, a sustained release preparation and the like.
The microcrystalline cellulose (MCC) aqueous solution is mixed with the vaccine, and the porous particles become storage carriers, so that the vaccine virus can be dispersed and adsorbed in the porous particles, the absorption of the organism can be delayed during intramuscular injection, and the residence time on the mucosa is prolonged during respiratory tract medication so as to promote the absorption amount.
The Chinese pharmacopoeia stipulates that astragalus root is the dry root of leguminous plant astragalus mongholicus or astragalus membranaceus. The medicine is mainly distributed in northeast and north China, and has wide application in traditional medicine. The traditional Chinese medicine considers that the traditional Chinese medicine is sweet in nature and slightly warm, enters spleen and lung channels, and has the effects of tonifying qi and raising yang, benefiting defense and consolidating exterior, expelling toxin and promoting granulation, and inducing diuresis and removing edema. In recent years, researches show that astragalus contains various bioactive components, such as polysaccharide, saponin, flavone, amino acid, trace elements, riboflavin, folic acid, coumarin, organic acid, choline and the like, wherein the most main active components are polysaccharide, saponin, flavonoid, amino acid and trace elements. The research of Astragalus Polysaccharide (APS) is more, and a large number of scientific experiments prove that APS has wide influence on specific immunity and non-specific immunity of an organism. The traditional Chinese medicine composition has various biological activities of promoting immunoregulation, resisting tumors, viruses, radiation, aging, parasites and the like, can induce strong humoral immunity and cellular immunity, arouses the innate immune protection mechanism of an organism, is an excellent non-specific immunopotentiator in a plurality of traditional Chinese medicines, and has obvious promotion and enhancement effects on disease prevention and health care.
Because the viscosity of the microcrystalline cellulose is high, the content is not easy to be too high when the microcrystalline cellulose is used, and the nasal spray atomization is influenced. Preferably, the diluent contains 1-10 g of microcrystalline cellulose and 5-15 g of astragalus polysaccharide per 1000 ml.
Preferably, each 1000 ml of the diluent contains 3-7 g of microcrystalline cellulose and 8-12 g of astragalus polysaccharide.
Preferably, the dilution contains 5 g of microcrystalline cellulose and 10 g of astragalus polysaccharide per 1000 ml.
The invention also provides a method for preparing the diluent, which comprises the following steps: adding microcrystalline cellulose into a reaction vessel containing water for injection, stirring, controlling temperature, standing, adding Astragalus polysaccharides, stirring, mixing, and sterilizing.
Preferably, the stirring speed is 100-200 rpm, and the stirring time is 30-90 minutes; the temperature control is between 90 and 100 ℃; the standing time is 5-15 minutes.
Preferably, the stirring and mixing time is 5-15 minutes; the sterilization condition is sterilization at 115 ℃ for 30 minutes.
The invention also provides application of the diluent in preparing a medicament for preventing and treating diseases related to the pseudorabies virus.
The term "pseudorabies virus-related disease" as used herein refers to an acute infectious disease caused by pseudorabies virus. The suckling piglets within 2 weeks are initially febrile, vomit, diarrhea, anorexia and lassitude, some of the piglets have upward turning of eyeballs, hypopsia and dyspnea, are breathed in an abdominal manner, and then have nervous symptoms, trembling, ataxia, intermittent spasm and post-paralysis, and move forward or backward and move down the ground and limbs. It is often accompanied by epileptic seizures or lethargy, with muscle twitching on touch and eventually failing to die.
The main symptoms of the 3-4-week-old pigs are the same as those of the pigs, the disease course is slightly long, constipation is more, and the fatality rate can reach 40-60%. Pregnant sows show cough, fever, lassitude. With the occurrence of abortion, stillbirth and weak piglets, the weak piglets suffer from vomiting and diarrhea, dyskinesia, spasm and opisthotonus within 1-2 days, and die usually within 24-36 hours.
The term "prevention" as used herein refers to all actions of inhibiting the infection by pseudorabies virus or delaying the onset of the disease. The term "treatment" refers to all actions that result in the reduction or amelioration of symptoms caused by the infection with pseudorabies virus.
The invention has the following beneficial effects:
the pseudorabies live vaccine diluent prepared from microcrystalline cellulose and astragalus polysaccharide can improve the nasal drop immunity effect of the pseudorabies vaccine, enhance the occupying effect of the pseudorabies virus in cranial nerves and a lymphatic system, generate antibodies in advance by intramuscular injection immunity, prolong the high-level antibody maintenance time and further improve the immunity effect of pigs. The latent infection of the anti-fake rabies virus is effectively prevented, and the prevention and the eradication of the pseudorabies are guaranteed. The preparation method of the diluent has the characteristics of easy production, low cost, convenient use and the like.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
A method for preparing pseudorabies live vaccine diluent comprises the following steps: adding microcrystalline cellulose into a reaction vessel containing water for injection, stirring for 90 minutes at the temperature of 100 ℃ and the rotating speed of 200rpm, standing for 15 minutes, then adding astragalus polysaccharide, stirring and mixing for 15 minutes, and sterilizing for 30 minutes at the temperature of 115 ℃ to obtain the pseudorabies live vaccine diluent. Wherein, every 1000 ml of the dilution comprises 10 g of microcrystalline cellulose and 15 g of astragalus polysaccharide.
Example 2
A method for preparing pseudorabies live vaccine diluent comprises the following steps: adding microcrystalline cellulose into a reaction vessel containing water for injection, stirring for 30 minutes at the temperature of 90 ℃ and the rotating speed of 100rpm, standing for 5 minutes, then adding astragalus polysaccharide, stirring and mixing for 5 minutes, and sterilizing for 30 minutes at the temperature of 115 ℃ to obtain the pseudorabies live vaccine diluent. Wherein, every 1000 ml of the dilution comprises 1 g of microcrystalline cellulose and 5 g of astragalus polysaccharide.
Example 3
A method for preparing pseudorabies live vaccine diluent comprises the following steps: adding microcrystalline cellulose into a reaction vessel containing water for injection, stirring for 45 minutes at the temperature of 100 ℃ and the rotation speed of 150rpm, standing for 12 minutes, then adding astragalus polysaccharide, stirring and mixing for 8 minutes, and sterilizing for 30 minutes at the temperature of 115 ℃ to obtain the pseudorabies live vaccine diluent. Wherein, every 1000 ml of the dilution comprises 3 g of microcrystalline cellulose and 8 g of astragalus polysaccharide.
Example 4
A method for preparing pseudorabies live vaccine diluent comprises the following steps: adding microcrystalline cellulose into a reaction vessel containing water for injection, stirring for 75 minutes at the temperature of 90 ℃ and the rotation speed of 200rpm, standing for 12 minutes, then adding astragalus polysaccharide, stirring and mixing for 6 minutes, and sterilizing for 30 minutes at the temperature of 115 ℃ to obtain the pseudorabies live vaccine diluent. Wherein, every 1000 ml of the dilution comprises 7 g of microcrystalline cellulose and 12 g of astragalus polysaccharide.
Example 5
A method for preparing pseudorabies live vaccine diluent comprises the following steps: adding microcrystalline cellulose into a reaction vessel containing water for injection, stirring for 60 minutes at the temperature of 60 ℃ and the rotating speed of 150rpm, standing for 10 minutes, then adding astragalus polysaccharide, stirring and mixing for 10 minutes, and sterilizing for 30 minutes at the temperature of 115 ℃ to obtain the pseudorabies live vaccine diluent. Wherein, every 1000 ml of the dilution comprises 5 g of microcrystalline cellulose and 10 g of astragalus polysaccharide.
The pseudorabies live vaccine diluent obtained in the embodiment is milky uniform suspension, and has good needle penetration and good nasal spray atomization. Under a microscope, the particles are uniformly distributed by microscopic examination. After the product is stored for 2 years, no layering exists after the product is shaken at 37 ℃, and no layering exists when the product is stored at 2-8 ℃.
In order to further verify the technical effect of the pseudorabies live vaccine diluent, the following tests are specially carried out:
1. comparative test for adhesion Properties
Materials: the dilution of the pseudorabies live vaccine obtained in example 5 above; the tracheal tissue of the piglet is purchased from a farmer market; the PBS dilution and the aluminum gel physiological saline dilution were prepared by Yunnan biopharmaceutical company.
The method comprises the following steps: 0.4ml of sample liquid is sucked by a 1ml syringe and is forcibly sprayed to the inner wall of the piglet trachea, and the time for the liquid drops to flow through the piglet trachea wall with the length of 20cm is calculated.
As a result: the time for using the PBS diluent is 3.33s, the time for using the aluminum gel physiological saline diluent is 3.55s, and the time for using the pseudorabies live vaccine diluent is 11.42 s.
Experiments prove that the adhesion capacity of the pseudorabies live vaccine diluent is 3.43 times that of PBS diluent and 3.22 times that of alumina gel diluent.
2. Effect test on vaccine Virus potency
After the vaccine is diluted and placed for 1, 2, 3 and 4 hours, the titer is detected without influence.
The method comprises the following operation steps: taking out frozen ST cells from a liquid nitrogen tank, recovering, digesting with 0.25% pancreatin-EDTA solution after the ST cells grow into a monolayer, pouring out the pancreatin-EDTA solution when the temperature is moderate, adding DMEM culture solution containing 10% calf serum, blowing and dispersing with a sterilizing pipette, subpackaging the solution into small bottles, and culturing in an incubator at 37 ℃.
Determination of Pseudorabies antigen Titers TCID50The method comprises mixing the diluent with porcine pseudorabies virus, standing at room temperature (25 deg.C) for 1, 2, 3, and 4 hr, respectively, inoculating onto sensitive cell, and measuring TCID50A change in (c). Set up PBS diluent groupAnd the imported PRV vaccine diluent was the control group. Pseudorabies virus was serially diluted 10-fold in MEM medium, i.e., 10-1，10-2，10-3，10-4，10-5，10-6，10-7And the like. 100ul of different dilutions of virus were added to each well of a 96 well cell reaction plate at 8 well titers, followed by 100ul of digested and dispersed ST cells, preferably at 30-50 ten thousand/mL, or as a standard where cells grew into monolayers over 48 hours. Culturing in 37 deg.C incubator, observing daily, and continuously observing for 1 week. Recording the cytopathic wells, calculating TCID according to Reed-Muench's method50. TABLE 1 Virus TCID50The test data of (1).
TABLE 1 Virus TCID50Test data of
As can be seen from table 1, through experiments, the inventive example 5 and the PBS diluent group and the imported PRV vaccine diluent are used as control groups, and the titer is detected after the vaccine is diluted and left for 1, 2, 3, and 4 hours, without affecting the titer of the vaccine.
3. Nasal drop immunity test of weaned pigs aged 1-3 days:
3.1 for the comparison of the immune effects of examples 1-5, 10 weaned piglets 1-3 days old were subjected to nasal drop immunization, blood was collected at the first immunization, 21d, 35d, 49d, 63d, 105d, and 180d for each group, serum was separated, and gB IgG antibody of each group was detected using an IDEXX porcine pseudorabies antibody detection kit to obtain the following experimental data, which are detailed in table 2.
TABLE 2 comparison of the immunological effects of examples 1 to 5
As can be seen from Table 2, the products obtained in examples 1 to 5 all had an increased immunological effect. The examples 1,4 and 5 are the most effective, and the examples 1 and 4 are inferior to the example 5 in the effect of nasal spray and the convenience of use because of the high content of microcrystalline cellulose and the high viscosity.
3.2 to further illustrate the superiority of the invention and common diluent, the porcine pseudorabies live vaccine prepared in example 5 of the invention is diluted to 1 part per 0.5ml by the diluent for standby. Dividing 40 healthy piglets aged for 1-3 days into four groups, each group comprises 10 piglets, and each porcine pseudorabies live vaccine diluted by the diluent obtained in the A1 group nasal drip immunization example 5 is 0.5 ml/head; b1 group of porcine pseudorabies live vaccine diluted by PBS buffer diluent for nasal drip immunization is 0.5 ml/head; the nasal drip immunization of group C1 was performed with 0.5 ml/head of porcine pseudorabies live vaccine diluted with the imported PRV vaccine diluent, group D1 was performed as a blank control group, and the nasal drip immunization PBS buffer diluent was performed with 0.5 ml/head. Blood was collected at 14d, 21d, 28d, 35d, 45d, 60d, 90d, 120d, and 180d of priming immunization, serum was separated, and gB IgG antibodies of each group were detected using an IDEXX porcine pseudorabies antibody detection kit, and the measurement results are shown in table 3.
Table 31-3 days old weaned piglets were tested for nasal drop immunization pseudorabies live vaccine immune antibody: S/N value
(positive S/N is not less than 0.6; suspicious S/N is not less than 0.6 and not more than 0.7; negative S/N is not less than 0.7).
From the above data, it can be seen that the group a1 of the present invention has the highest antibody positive rate and the longest duration, and B1 and C1 can produce antibodies but have too low positive rate and the duration can only last for more than 100 days.
To illustrate the injection effect, the porcine pseudorabies live vaccine prepared in example 5 of the present invention was diluted to 1 part/ml with a diluent for use. Dividing 30 healthy weaned piglets which are inspected for 50 days into three groups, wherein each group comprises 10 piglets, and A2 group is injected with 1.0 ml/pig pseudorabies live vaccine diluted by the diluent prepared in the embodiment 5; b2 group porcine pseudorabies live vaccine diluted by PBS liquid for injection is 1.0 ml/head; c2 group PRV vaccine diluent 0.5 ml/head; group D2 was used as a blank control group, and 1.0 ml/head of PBS buffer was injected. Blood was collected from the groups at 7d, 14d, 21d, 28d, 35d, 42d, 63d, 84d, and 105d of priming immunization, and serum was isolated, and gB IgG antibodies of each group were detected using the IDEXX porcine pseudorabies antibody detection kit, and the results of the measurements are shown in table 4.
Table 428 detection of immune pseudorabies live vaccine immune antibodies of weaned pigs at day age: and (4) S/N value.
(positive S/N is not less than 0.6; suspicious S/N is not less than 0.6 and not more than 0.7; negative S/N is not less than 0.7).
From the above data, it can be found that the group a2 of the present invention has the highest antibody positive rate and the longest duration, and B2 and C2 can produce antibodies but the positive rate duration is not as long as a2, so that the superiority of the present invention can be seen.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (5)
1. A pseudorabies live vaccine diluent is characterized in that: every 1000 ml of the diluent contains 1-10 g of microcrystalline cellulose and 5-15 g of astragalus polysaccharide;
the preparation method of the diluent comprises the following steps: adding microcrystalline cellulose into a reaction vessel containing water for injection, stirring, controlling temperature, standing, adding Astragalus polysaccharides, stirring, mixing, and sterilizing;
the stirring speed is 100-200 rpm, and the stirring time is 30-90 minutes; the temperature control is between 90 and 100 ℃; the standing time is 5-15 minutes.
2. The pseudorabies live vaccine diluent according to claim 1, wherein: every 1000 ml of the diluent contains 3-7 g of microcrystalline cellulose and 8-12 g of astragalus polysaccharide.
3. The pseudorabies live vaccine diluent according to claim 2, wherein: every 1000 ml of the dilution comprises 5 g of microcrystalline cellulose and 10 g of astragalus polysaccharide.
4. The pseudorabies live vaccine diluent according to claim 1, wherein: the stirring and mixing time is 5-15 minutes; the sterilization condition is sterilization at 115 ℃ for 30 minutes.
5. Use of the dilution according to any one of claims 1-4 in the manufacture of a medicament for the prevention and treatment of pseudorabies virus related diseases.
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| CN102247600A (en) * | 2010-05-21 | 2011-11-23 | 上海创宏生物科技有限公司 | Method for preparing diluent for pseudorabies live vaccine |
| CN103083659A (en) * | 2013-01-18 | 2013-05-08 | 北京华夏兴洋生物科技有限公司 | Preparation method and application of novel oil-free adjuvant |
| CN105997920A (en) * | 2016-05-25 | 2016-10-12 | 浙江美保龙生物技术有限公司 | Immunologic adjuvant effervescent tablet for pseudorabies live vaccine |
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| CN102247600A (en) * | 2010-05-21 | 2011-11-23 | 上海创宏生物科技有限公司 | Method for preparing diluent for pseudorabies live vaccine |
| CN103083659A (en) * | 2013-01-18 | 2013-05-08 | 北京华夏兴洋生物科技有限公司 | Preparation method and application of novel oil-free adjuvant |
| CN105997920A (en) * | 2016-05-25 | 2016-10-12 | 浙江美保龙生物技术有限公司 | Immunologic adjuvant effervescent tablet for pseudorabies live vaccine |
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