CN102719407B - Porcine parvovirus BQ-C strain and application of porcine parvovirus BQ-C strain in preparation of inactivated porcine parvovirus vaccine - Google Patents

Abstract

The invention discloses a porcine parvovirus BQ-C strain and application of the porcine parvovirus BQ-C strain to the preparation of an inactivated porcine parvovirus vaccine, and belongs to the field of biological vaccines. The porcine parvovirus BQ-C strain has the advantages of high immunogenicity, good cultural character and the like; and porcine parvovirus (BQ-C strain) are inoculated to swine testis (ST) cells, a culture is harvested and inactivated by using binary ethylenimine (BEI), and the culture and a commercial MontanideTM ISA15AVG adjuvant of SEPPIC are mixed and emulsified to form the inactivated porcine parvovirus vaccine. The experiment proves that: diseases caused by porcine parvoviruses can be prevented by immunizing primiparous healthy female sows by using the prepared inactivated porcine parvovirus vaccine; and moreover, the invention has the advantages that the vaccine is safe, an antibody can be quickly generated, the immune period is long-lasting, and the like.

Description

Porcine parvovirus BQ-C strain and its application in the preparation of porcine parvovirus inactivated vaccine

technical field

The invention relates to a porcine parvovirus and its application in preparing porcine parvovirus inactivated vaccine, in particular to a porcine parvovirus BQ-C strain isolated by itself and an inactivated vaccine prepared from the virus strain. It belongs to the field of biological vaccines.

Background technique

Porcine parvovirus (Porcine parvovirus, PPV) is one of the main pathogens that cause reproductive disorders in pigs. PPV infects mainly pregnant pigs, especially primiparous sows are susceptible to infection before pregnancy, causing abortion, stillbirth, Fetal mummification, etc., and the mother usually lacks clinical symptoms; of course, other symptoms can also be caused. Since Mayr and Mahnel discovered and confirmed the existence and pathogenicity of PPV in 1966, scholars at home and abroad have conducted extensive and in-depth research on it. In recent years, PPV infection has been expanding and rising, which has caused huge economic losses to the global pig industry. In my country, Pan Xuezhu first isolated PPV in 1983. Since then, several strains of PPV have been isolated in various places. At present, it has been found that porcine parvovirus is often mixed with other viruses, such as porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus, resulting in multisystem syndrome in weaned piglets. Syndrome, dermatitis, respiratory syndrome etc., cause very big loss to the swine industry in the world. Although different isolates belong to the same serotype, they can cause a variety of clinical symptoms, but they all mainly cause reproductive disorders. At present, the PPV positive rate of multiparous sows and breeding boars in my country is as high as 80%.

The inventor of the present invention isolated a strain of porcine parvovirus from a pig farm in Heilongjiang Province in 2005, which was identified as a virulent strain by the laboratory, and the strain after domestication and cloning was named PPV BQ-C strain. Using the PPV BQ-C strain, the research and development of porcine parvovirus inactivated vaccine was carried out, and the virulent BQ-C strain of porcine parvovirus with good immunogenicity and stable titer was screened and bred, and the most suitable for the virus propagation was selected. The ST cells meet the inactivation agent, adjuvant and other conditions for vaccine manufacture. The production process, safety, protection rate, immunization program, minimum dose, antibody duration and storage period of the product have been tested and determined, and a large number of test data have been obtained, which proves that the vaccine is safe and effective. On this basis, the pilot production was carried out. After sampling inspection, all of them conformed to the proposed quality standards, and the safety test and efficacy test were carried out on the pilot products. The results also confirmed that this vaccine can effectively prevent porcine parvovirus. On the basis of laboratory experiments and intermediate trial production, the present invention has developed an inactivated vaccine for porcine parvovirus (BQ-C strain), and the results have also confirmed that this vaccine can effectively prevent porcine parvovirus. The reproductive barriers of sows further verified the quality standards of this vaccine.

Contents of the invention

One of the purposes of the present invention is to provide a porcine parvovirus strain with good immunogenicity and stable titer;

The second object of the present invention is to provide the application of the porcine parvovirus strain in the preparation of vaccines;

The third object of the present invention is to provide a vaccine prepared from the porcine parvovirus strain;

The fourth object of the present invention is to provide the application of the vaccine in the preparation of medicines for preventing porcine parvovirus.

In order to achieve the above object, the present invention adopts the following technical means:

The inventor of the present invention isolated a strain of porcine parvovirus from a pig farm in Heilongjiang Province in 2005, which was identified as a virulent strain by the laboratory, and the domesticated strain was named PPV BQ-C strain. The PPV BQ-C strain of the present invention is isolated from the weak piglets produced by pregnant sows infected with typical porcine parvovirus, so it has a higher proliferation titer on cells than other isolated strains, and is effective for pigs. good immunogenicity. The pig testis subculture cell line (ST) was used as the vaccine virus seed, and the strong virus used for the efficacy test was the 7th to 10th generation of the BQ-C strain. This strain can cause more than 90% infection of ST cells.

A kind of porcine parvovirus of the present invention is the 7th generation virus strain after passage on ST cells, named as porcine parvovirus BQ-C strain, classified and named as porcine parvovirus, and preserved in China Microorganism Culture Preservation Management Committee Common Microorganisms The center is located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

The invention also provides the application of the porcine parvovirus in the preparation of porcine parvovirus inactivated vaccine.

The inactivated porcine parvovirus vaccine of the present invention is characterized in that it contains the inactivated porcine parvovirus BQ-C strain of the present invention.

In the inactivated porcine parvovirus vaccine according to the present invention, preferably, the porcine parvovirus BQ-C strain is inactivated by the following method: 0.3% of diethyleneimine (BEI) is inactivated at 32°C After living for 96 hours, 0.02% sodium thiosulfate was added to terminate the inactivation.

In the present invention, preferably, the porcine parvovirus inactivated vaccine is composed of 15% (v/v) commercialized MontanideTM ISA15AVG adjuvant of France Sepic Company and 85% (v/v) of di Ethylene imine (BEI) inactivated porcine parvovirus (BQ-C strain) obtained after emulsification.

In the present invention, the emulsification process is to use the commercialized MontanideTMISA15AVG adjuvant of France Sepic Company, which can be directly used for vaccine emulsification preparation after sterilization; before emulsification, the inactivated virus liquid of the same batch is thawed and mixed, and used in a sterile room The homogenizer is stirred evenly at 200r/min, and then the volume ratio of the water phase antigen and the oil phase adjuvant is 8.5:1.5; the emulsification procedure is to first put the water phase into the emulsifier for stirring, and then slowly add the oil phase adjuvant Dosage, continuously stirred at 800r/min for 30 minutes; the vaccine produced is an oil-in-water dosage form, in order to stabilize the interface and eliminate air bubbles, and obtain the best emulsification effect, it needs to stand still for 30 minutes before subpackaging.

Further, the present invention also provides the use of the inactivated vaccine in the preparation of biological products for preventing diseases caused by porcine parvovirus.

In particular, said disease is sow reproductive failure in primiparous health-negative sows.

Description of drawings

Fig. 1 is the fluorescent detection result of porcine parvovirus BQ-C strain after cultivating different time on ST cell;

Fig. 1A is the fluorescence detection result of inoculation 0 hours; Fig. 1B is the fluorescence detection result of inoculation 8 hours; Fig. 1C is the fluorescence detection result of inoculation 16 hours; Fig. 1D is the fluorescence detection result of inoculation 24 hours; Fig. 1E is the inoculation 32 hours Fig. 1F is the fluorescence detection result of inoculation for 40 hours; Fig. 1G is the fluorescence detection result of inoculation 48 hours; Fig. 1H is the fluorescence detection result of inoculation 56 hours; Fig. 1I is the fluorescence detection result of normal control cells.

Fig. 2 is a one-step growth curve of ST cells infected with porcine parvovirus BQ-C strain.

Detailed ways

The present invention will be further described through experiments and examples below. It should be understood that these examples are only for the purpose of illustration, and in no way limit the protection scope of the present invention.

Example 1 Isolation and culture identification of porcine parvovirus BQ-C strain

1 Source and standard of poisonous species

According to the declaration requirements of new biological products, combined with a large amount of test data obtained by the present invention, and with reference to "Chinese Veterinary Pharmacopoeia" (2005 edition), the poisonous species used for seedling production has been identified, and the poisoned species standard of the trial procedure (draft) is now carried out as follows illustrate.

1.1 Source of poisonous seeds

The virus species used to manufacture this product is porcine parvovirus BQ-C strain, which the inventor obtained after isolation and domestication from diseased pigs in 2005. Since this virus strain is obtained from pregnant sows infected by typical porcine parvovirus It is isolated from the weak piglets produced, and compared with other isolated strains, it has a higher proliferation titer on cells and has better immunogenicity to pigs, so it is selected for vaccine production. The pig testis subculture cell line (ST) was used as the vaccine virus seed, and the strong virus used for the efficacy test was the 7th to 10th generation of the BQ-C strain. The strain is the seventh-generation virus strain passed on ST cells. It is preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Strains. The address is at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number is: CGMCCNo.6091, and the deposit date is May 8, 2012.

1.2 Virus species (CGMCC No.6091) standard

1.2.1 Hemagglutination value of red blood cells

Take a 96-well U-shaped micro-reaction plate, add 25 μl PBS (0.01mol/L, pH 7.0-7.4) to each well, add 25 μl antigen to each well in the first column, and then serially dilute the antigen by 2 times until the 11th well Discard 25 μl. Add 25 μl of PBS to each well, then add 25 μl of 0.6% guinea pig erythrocyte suspension, mix well with a micro shaker, and let it react for 1 hour at room temperature. When judging the results, the highest dilution factor of the antigen at which 50% erythrocytes were agglutinated was used as the judging end point. Results: The agglutination value of the virus species on guinea pig erythrocytes should not be lower than 1:512.

1.2.2 Virus content

The virus seeds were serially diluted 10 times in DMEM medium, and 100 μl of each dilution was added to a 96-well cell culture plate. Each dilution was repeated 8 times, and then the digested and dispersed ST cell suspension was added, 100 μl per well ( The cell content was 3×10 ⁵ /mL), and a normal cell culture control was set, cultured in an incubator containing 5% CO ₂ at 37°C, and the cytopathic changes (CPE) were observed daily, cell pyknosis, aggregation, and increased granules. Some cells fused, shed, and there were many gaps, which were judged as CPE. After 7 days of observation, the number of wells with cytopathic changes was recorded, and the TCID ₅₀ of the virus was calculated according to the Reed-Muench method. Results: The virus liquid per milliliter was not less than 10 ^6.0 TCID ₅₀ .

1.2.3 Virulence

The virus generation is the 7th to 10th generation cytotoxicity of porcine parvovirus BQ-C strain; the challenge dose is that the virus content per ml should not be less than 10 ^6.0 TCID ₅₀ ; the procedure is 4 healthy negative primiparous sows, after 35 days of pregnancy Intramuscular injection of 2ml of porcine parvovirus BQ-C strain 7th to 10th generation cytotoxicity and 2ml of intranasal drops respectively, the virus content was 3×10 ^6.0 TCID ₅₀ respectively; the judging standard was necropsy 40 days after challenge, loss of 25% of fetal pigs Above, or 3/5 sows have reproductive disorders (stillbirth, mummified fetuses and fetal dissolution).

1.2.4 Immunogenicity

The virus seed was inoculated into ST cells synchronously for proliferation, the virus liquid was harvested, inactivated with BEI, mixed and emulsified with Montanide ^TM ISA 15AVG adjuvant to make an oil-in-water inactivated vaccine, and healthy susceptible primiparous sows (PPV HI Antibody titer not higher than 1:8) 5 pigs, intramuscularly inject 2ml of vaccine according to this procedure before mating, and 35-40 days after pregnancy, together with 5 control pigs with the same conditions, intranasal drip and intramuscular injection of porcine parvovirus 2ml of each BQ-C strain (10 ^6.0 TCID ₅₀ ), all pigs were killed 40 days after the challenge. In the control group, at least 4 animals should have symptoms of reproductive disorders such as stillbirth, mummified fetuses or fetal reabsorption, and at least 4 animals in the immunization group should be protected.

1.2.5 Purity test

The 10th to 11th generation of the original seed batch, the 12th to 21st generation of the basic seed batch and the 22nd to 25th generation of the production seed batch of the established porcine parvovirus BQ-C strain were tested for bacteria, mold and mycoplasma, and the 11th generation , 12 and 24 generations of virus seeds have carried out the inspection of exogenous virus, and the result shows that the original seed batch that the present invention sets up, basic seed batch, production seed batch all have no bacterium, mould, mycoplasma and exogenous virus pollution, all pure.

1.2.6 Specificity

Dilute the virus seed to 200TCID ₅₀ /0.1ml with DMEM solution, mix it with inactivated anti-porcine parvovirus-specific serum in equal amounts, place it at 37°C for 1 hour, and inoculate 3 bottles of well-grown ST cells simultaneously (cell content At 3×10 ⁵ /ml), virus control, normal cell control and negative serum control were set at the same time, and placed in a 37°C, 5% CO ₂ incubator for 5-7 days. Neither the neutralized virus group nor the normal cell control group should have cytopathic changes (CPE); both the virus control group and the negative serum control group should have CPE.

1.2.7 Generation of virus seeds

The originally isolated porcine parvovirus BQ-C strain was continuously passed on for 40 generations on ST cells (that is, 33 generations were continuously passed on the basis of the strain with the strain preservation number CGMCC No. 6091), and the TCID of each generation virus was determined. ₅₀ , the 15th, 20th, 25th, 30th, 35th, and 40th generations of viruses were selected to be inactivated to make vaccines, inoculated with 5-month-old back-up pigs, and challenged at 35 days of pregnancy. The results showed that the virus was passaged on ST cells, and after the 7th passage (including the 7th passage virus, that is, the virus strain with the strain preservation number CGMCC No.6091), the virus content of each passage was stable, not less than 10 ^6.0 TCID ₅₀ , and the titer of HA is above ²⁹ , which has reached the standard for making vaccines. The challenge test showed that the pigs immunized with vaccines prepared by the 15th, 20th, 25th, and 30th passages had no significant difference in protection against BQ-C strain challenge, and the fetuses all survived healthy; There were 2 pigs and 1 stillbirth in each of the challenged pigs, while the 4 pigs in the control group had different degrees of stillbirth and weak fetus. According to the above results, in order to ensure the good immunogenicity and safety of the produced virus seeds, the present invention determines that the highest number of passages of the virus is 27 generations, the original virus seeds are 7-11 generations, and the basic virus seeds are 12-21 generations. The maximum number of subcultures of virus seeds for production is limited to 3 generations (22nd to 25th generations).

1.2.8 Determination of the storage period of poisonous seeds

The wet virus of porcine parvovirus BQ-C strain was stored at -20°C and -70°C, and samples were taken at different times after storage for HA titer and TCID ₅₀ determination. The results showed that the wet virus was stored at -20°C for 18 months, The poison price began to drop significantly when stored at -70°C for 30 months; the freeze-dried poison was stored at -20°C and -70°C, and samples were taken every year after storage for HA titer and TCID ₅₀ determination. Results The titer of the virus stored at -20°C for 36 months decreased slightly, the HA titer and TCID ₅₀ value of the virus preserved for 48 months decreased significantly, and the HA titer of the virus stored at -70°C for 48 months And TCID ₅₀ has no obvious change, and it has dropped significantly after 60 months of storage. Considering other unfavorable factors in virus storage, to ensure the reliability of the virus storage period, the storage period of porcine parvovirus BQ-C strain wet virus at -20°C is set as 12 months, and the storage period at -70°C is 12 months. It is determined as 24 months; the storage time of porcine parvovirus freeze-dried virus at -20°C is set as 36 months; the storage time of freeze-dried virus at -70°C is 48 months.

2 cells for production

2.1 Source of cells

ST cells were purchased from the China (Wuhan) Type Culture Collection Center and preserved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

2.2 Selection of cells

In the culture process of the virus, the present invention selects the passage cell lines PK-15 and ST to carry out the multiplication of the virus. As a result, the lesion time of the virus on the ST cells is early, and the proliferation titer is the highest. Porcine parvovirus BQ-C strain (CGMCC No. 6091) on ST cells is shown in Figure 1, and the one-step growth curve of porcine parvovirus BQ-C strain (CGMCC No.6091) infected ST cells is shown in Figure 2. Foreign scholars have also reported that it is better to select ST cells for PPV proliferation, so ST cells are selected for virus proliferation.

2.3 Establishment of cell lines

The cell line used for making seedlings is ST cells. The subculture range is controlled at 40 to 55 generations, and the present invention establishes an original cell bank, a basic cell bank and a working cell bank. Among them, the original cell bank was divided into 55 bottles; the basic cell bank was divided into 370 bottles; the working cell bank was divided into 350 bottles. The cells of each generation are inspected according to the appendix of "Chinese Veterinary Pharmacopoeia", and there should be no pollution from bacteria, molds, mycoplasma and foreign viruses.

2.4 Identification of cell lines

According to the current "Chinese Veterinary Pharmacopoeia" attached to the relevant "production, testing cell standards" regulations, all meet the standards. For details, see "Report of Identification Test of ST Cell Lines".

The preparation of embodiment 2 inactivated vaccines

The preparation process of porcine parvovirus inactivated vaccine (BQ-C strain) is prepared according to the current mature commercial vaccine preparation process. Judging from the quality inspection results of 3 batches of vaccines prepared in the laboratory and 5 batches of intermediate trial-produced vaccines, the quality of the vaccine products prepared by this process is stable, and the efficacy inspection results fully meet the established quality standards.

1 Preparation of virus solution

In the process of virus reproduction, many factors will affect the proliferation of cells and viruses. The replication of PPV requires the DNA polymerase of the host cell, and it is most suitable for the proliferation in cells with strong proliferation ability and in the mitotic stage, so when PPV is propagated, it is inoculated with the cells at the same time; the serum concentration in the culture medium significantly affects the virus. Replication, after a series of comparative experiments, the serum concentration of the cell culture medium was selected to be 10%, and the serum concentration of the maintenance medium was 2%, which can reproduce stable and high-titer virus liquid; in addition, the pH value of the culture medium, cell concentration, virus The inoculation dose and the time of receiving the virus were compared. The results showed that the pH value of the cell maintenance solution was controlled between 7.2 and 7.4 when the virus propagated, and the cell concentration was selected from 1×10 ⁵ cells/ml to 5×10 ⁵ cells/ml when the virus was inoculated. ml, the cell inoculation dose is 1% (v/v), the seed virus with a virus content of more than 10 ^6.0 TCID ₅₀ /ml, and the best harvest time of the virus is 72-96 hours, and the virus liquid with high virus content can be obtained.

In this example, the serum concentration in the cell culture medium (trade name is 1640, purchased from Gibco Company) was selected as 10%, the pH value was controlled at 7.3, and the maintenance medium (trade name was MEM, purchased from Shanghai Hufeng Biotechnology Co., Ltd. The serum concentration in ) was 2%, the concentration of ST cells was 3×10 ⁵ cells/ml when inoculated, and the pig cells with a virus content of 10 ^6.0 TCID ₅₀ /ml were inoculated according to the cell inoculation dose of 1% (v/v). The 7th generation virus strain of the virus BQ-C strain (the strain preservation number is CGMCC No.6091), and the virus harvest time is 72 hours.

2 inactivation process

Select formaldehyde solution with final concentration of 0.1%, 0.2%, 0.3%, 0.4% and binary ethylenimine (BEI) with final concentration of 0.1%, 0.2%, 0.3%, 0.4% to carry out inactivation comparison test on porcine parvovirus , the test results show that 0.3% BEI can completely inactivate porcine parvovirus after 96 hours at 32°C, and the test results show that BEI has little damage to cells; PPV antigen added to 0.3% formaldehyde solution, after 37 Inactivation at ℃ for 48 hours can also achieve the purpose of completely inactivating the virus; however, formaldehyde is a strong irritant, and if the residual free formaldehyde in the vaccine enters the animal body, adverse reactions will occur. Therefore, it was finally determined that 0.3% BEI was used to inactivate porcine parvovirus at 32°C for 96 hours, and then 0.02% sodium thiosulfate was added to terminate the inactivation.

3 emulsification process

The commercialized Montanide ^TM ISA15AVG adjuvant from Sepic Company in France is used, which can be directly used for vaccine emulsification preparation after sterilization; before emulsification, the inactivated virus liquid of the same batch is thawed and mixed, and the homogenizer is used in a sterile room at 200r/ min to stir evenly, and then according to the volume ratio of the water phase antigen and the oil phase adjuvant 8.5:1.5; the emulsification procedure is to first put the water phase into the emulsifier for stirring, and then slowly add the oil phase adjuvant at 800r/min Stir continuously for 30 minutes; the vaccine produced is an oil-in-water formulation. In order to stabilize the interface and eliminate air bubbles and obtain the best emulsification effect, it needs to stand still for 30 minutes before subpackaging.

4 semi-finished product inspection quality standard

4.1 Sterility test

Tested according to the appendix of the current "Chinese Veterinary Pharmacopoeia", it should grow aseptically.

4.2 Determination of virus content

The virus seeds were serially diluted 10 times in DMEM medium, and 100 μl of each dilution was added to a 96-well cell culture plate. Each dilution was repeated 8 times, and then the digested and dispersed ST cell suspension was added, 100 μl per well ( The cell content should be about 3×10 ⁵ /ml), and a normal cell culture control was set, cultured in a 37°C, 5% CO ₂ incubator, and the cytopathic changes (CPE), cell pyknosis, and aggregation were observed daily. The number of particles increased, some cells fused, shed, and many gaps appeared were judged as infection. The number of wells with cytopathic changes was recorded, and the TCID ₅₀ of the virus was calculated according to the Reed-Muench method. The results showed that the virus liquid per milliliter was not less than 10 ^6.0 TCID ₅₀ .

4.3 Inactivation test

Take the inactivated virus solution, inoculate it in 3 bottles of ST cells (cell content: 3×10 ⁵ /ml) according to the ratio of virus solution to growth solution 1:10, culture and observe at 37°C for 5 days, and re-blind those without lesions Passage for 2 generations, and set virus control at the same time. The results showed no cytopathic changes. The 3 batches of semi-finished products produced in the laboratory are all qualified.

5 about the finished product inspection quality standard

5.1 Safety Inspection Standards

In order to ensure the safety of the vaccine, the present invention has successively carried out "safety test to mice", "single-dose inoculation to piglets, single-dose repeated inoculation, one-time overdose (2 times dose)" to 5 batches of vaccines prepared in the laboratory. ) vaccination safety test", "single-dose vaccination, single-dose repeated vaccination, and one-time overdose (2 times dose) vaccination safety test for gilts", single-dose vaccination, single-dose repeated vaccination for pregnant sows , One-time super-dose (2 times dose) vaccination safety test". The test results showed that: after single-dose inoculation, single-dose repeated inoculation, and one-time large-dose inoculation of each immunized animal, the pigs were in good condition, and their food intake and drinking water were normal. , no abnormal reaction; after the vaccination, the reproductive function of the pregnant sow has no obvious impact. Above tests all prove that the vaccine is safe. The present invention has concluded that as long as local swelling, ulcers and erosion do not occur within 21 days after the vaccination As well as neural atrophy, reduced feed intake, etc., there will be no adverse reactions caused by vaccination in the future, and the safety of the vaccine can be proved. Therefore, it is stipulated in the trial procedure (draft): use 10-20kg healthy susceptible 2 piglets (HI antibody titer is not high 1:8), 4ml of the vaccine was injected into each deep muscle, and another 2 pigs with the same conditions were not vaccinated as a control, and were observed continuously for 21 days. During the observation period, there should be no any local or systemic adverse reactions.

5.2 Formulation of efficacy testing standards

5.2.1 Correlation test between antibody titer and immune challenge protection

A batch of porcine parvovirus inactivated vaccine 0701 that passed the safety test was immunized with healthy spare hens aged 3 to 5 months according to different doses of 0.5ml/head, 1ml/head, 2ml/head, and 4ml/head respectively. Pigs (PPV HI antibody not higher than 8) were challenged with 10 ^6.0 TCID ₅₀ /ml PPV BQ-C strain 10th passage virulent 4ml to determine the correlation between HI antibody titer and immune challenge protection. The test results show that when the serum HI antibody titer is not lower than 1:64, the survival rate of sows litters is 97.5%. One of the piglets in one group was aborted, but the virus isolation rate of piglets detected by PCR was 0, which is also 97.5%. There were no other pathogenic infections that caused abortion in sows. The above results show that under the experimental conditions, when the serum HI antibody titer is not lower than 1:64, it can effectively prevent the virus from being transmitted vertically from the mother to the fetus. Therefore, when determining the vaccine efficacy test, the test standard is that the serum HI antibody titer is not lower than 1:64 after 28 days of immunization.

5.2.2 Minimum immunization dose and minimum dose test

Three batches of porcine parvovirus inactivated vaccines 0701, 0702 and 0703 that passed the safety test were immunized for 3 to 5 months according to different doses of 0.5ml/head, 1ml/head, 1.5ml/head, and 2ml/head respectively Healthy gilts (PPV HI antibody not higher than 8) at the age of 28 were blood-collected for HI antibody detection 28 days after immunization. The results showed that the average titer of HI antibody was not lower than 1:64 after 28 days after immunizing pigs with a dose of 1ml/head, and the titer of HI antibody was not lower than 1:64 after 28 days after immunizing pigs with a dose of 1.5ml/head or more. According to the results of the correlation test between HI antibody titer and challenge protection, it is determined that when the HI antibody titer of porcine parvovirus reaches 1:64 or more 28 days after immunization, pregnant sows can be completely protected against the virulent attack of porcine parvovirus. Factors that may reduce the titer that may exist during the storage, transportation and use of the vaccine are taken into consideration. In the present invention, the minimum immunization dose of porcine parvovirus inactivated vaccine (BQ-C strain) is determined to be 1.5ml/head portion, and the usage dose is determined to be 2.0ml/head portion.

5.2.3 Correlation test of HI antibody between immunized guinea pigs and immunized pigs

Three batches of porcine parvovirus inactivated vaccines 0701, 0702 and 0703 produced in the laboratory were immunized to guinea pigs and pigs respectively, and the HI antibody titer was tested to analyze the correlation of HI antibodies between guinea pigs and pigs. The results showed that when guinea pigs were immunized with 0.5ml/pig and pigs were immunized with 2ml/pig, the serum HI antibody titer was not lower than 1:64 after 28 days, and there was an obvious parallel relationship between the two. The test results prove that the experimental animal guinea pig can be used instead of the target animal (pig) for efficacy testing.

Therefore, according to the above results, the present invention defines the effect test standard of this product as:

Five adult guinea pigs (PPV HI antibody titer not higher than 1:8) with a body weight of 350g-400g were injected intramuscularly with 0.5ml of the vaccine. After 28 days, blood was collected together with 4 control guinea pigs to determine the PPV HI antibody titer. The HI antibody titer of the control guinea pigs should not be higher than 1:8, and at least 4 of the immunized guinea pigs should have antibody responses, and the HI antibody titer should not be lower than 1:64.

Five 3-month-old healthy susceptible pigs (PPV HI antibody titer not higher than 1:8) were injected with 2ml of the vaccine in each deep muscle. After 28 days, blood was collected together with 4 control pigs to determine the PPV HI antibody titer . The HI antibody titer of the control pig should not be higher than 8, and all the injected pigs should have an antibody reaction, and the HI antibody titer should not be lower than 1:64.

6 Antibody growth and decline rules and immunity duration test

Three batches of porcine parvovirus inactivated vaccines 0701, 0702 and 0703 that passed the safety test were immunized to healthy gilts, multiparous pigs and boars, respectively. In order to further grasp the efficacy and duration of immunity, formulate a reasonable immunization program to ensure that the immunized pigs maintain a high and long-lasting antibody level, and carry out antibody detection on immunized pigs at different times to grasp the growth and decline of their antibodies.

The test results showed that primiparous sows aged 3 to 5 months were immunized with 2ml/head, and boosted once 2 to 3 weeks later, and multiparous sows were immunized with 2ml/head one month before mating. If it is lower than 1:64, the peak of the antibody after immunization is within 2 to 6 months, and then the antibody level gradually decreases, and it still reaches 1:64 at 9 months. In order to ensure the immune protection effect of the vaccine, the duration of immunity is determined to be 7 months.

7 Determination of immunization program

According to the duration of the maternal antibody of piglets, the initial immunization should be at 5-6 months of age, because the maternal antibody has dropped to a low level at this time (HI antibody titer is lower than 1:64). Moreover, the mating time of gilts is generally 6 months old, so that immunization at the age of 5 to 6 months can just prevent sows from being attacked by strong poison during pregnancy. For multiparous sows, due to the persistent existence of antibodies in their bodies, porcine parvovirus often does not pose a threat to them, so they were generally not immunized before, but in recent years due to the complexity of clinical disease, such as porcine parvovirus often aggravates pig Circovirus infection, etc., so the present invention advocates immunizing multiparous sows before pregnancy, so that pregnant sows can be protected from the virulent attack of porcine parvovirus throughout the pregnancy.

According to the mating time of gilts, the growth and decline of antibodies after immunization of gilts, and the immunization period, combined with the actual situation of porcine parvovirus infection in clinical practice, the present invention determines the efficacy of porcine parvovirus inactivated vaccine (BQ-C strain). The immunization program is as follows: Primiparous sows are immunized once at 5-6 months of age, and booster immunization is given once after 2-4 weeks; multiparous sows are immunized once 3-4 weeks before mating; boars are immunized twice a year. Each immunization was 2ml/head.

8 Shelf life of vaccines

The present invention has carried out experimental research on the shelf life of three batches of inactivated vaccines (0701, 0702, and 0703) prepared in the laboratory. The three batches of vaccines were stored at 2-8°C for 9, 12, 15, and 18 months. Samples were taken at different time periods to test its properties, safety and immune efficacy. The results showed that the properties of the three batches of vaccines did not change significantly after being stored at 2-8°C for 18 months, and the sterility test and safety met the requirements of the quality standards. In the potency test, the 0701 batch of vaccines stored for 18 months had a guinea pig HI antibody titer of 1:32 after the samples were immunized with guinea pigs; the 0701 and 0702 batches of vaccines were stored for 18 months and there was a pig HI antibody titer of 1: 32, but the average HI antibody level of immunized pigs is 1:64. Considering the titer loss caused by the vaccine during transportation and use, the present invention sets the storage period of the vaccine at 2-8°C for 15 months.

9Comparison of immune efficacy with similar domestic commercial vaccines

Three batches of vaccines 0701, 0702 and 0703 produced in the laboratory and commercially available vaccines A (batch number: 20071228) and B (batch number: 20071205) were immunized with 350g-400g healthy guinea pigs and 5-6 month-old healthy back-up pigs respectively. After 28 days, The serum HI antibody titers were not less than 1:64, which met the protection requirements of the virus attack. The average HI antibody of the vaccine prepared in the laboratory was slightly higher than that of the commercially available A vaccine, and significantly higher than that of the commercially available B vaccine.

Before immunization and 3, 6, 9, and 12 months after immunization, blood was collected from veins to separate serum, and the antibody production was detected. The results showed that: within 6 months after immunization, the HI antibody titers of the vaccines trial-produced in the laboratory and commercially available A and B vaccines were all above 1:64, which could meet the protection requirements of challenge. After 6 months, the HI antibody of pigs immunized with commercially available B vaccine decreased significantly.

The above description is only a preferred embodiment of the present invention, and it is only illustrative of the present invention, rather than restrictive; those of ordinary skill in the art understand that it can be used within the spirit and scope defined by the claims of the present invention. Many changes, modifications, and even equivalent changes can be made, but all will fall within the protection scope of the present invention.

Claims (6)

1. pig parvoviral (Porcine parvovirus), called after pig parvoviral BQ-C strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its culture presevation is numbered: CGMCC No.6091.

2. the application of the described pig parvoviral of claim 1 in preparation pig parvoviral inactivated vaccine.

3. pig parvoviral inactivated vaccine is characterized in that containing the described pig parvoviral of claim 1 after the deactivation.

4. inactivated vaccine as claimed in claim 3 is characterized in that described deactivation refers to divinyl imines deactivation under 32 ℃ of conditions of the described pig parvoviral employing 0.3% of claim 1 was added 0.02% Sulfothiorine and stops deactivation after 96 hours.

5. claim 3 or the 4 described inactivated vaccines application in the biological products of the disease that the preparation prevention is caused by pig parvoviral.

6. application as claimed in claim 5 is characterized in that described disease is the sow breeding difficulty that the healthy negative sow of primiparity is taken place.

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