CN107299088A - B plants of infectious bovine rhinotrachetis virus IBRV and its application - Google Patents

Abstract

The invention discloses bovine infectious rhinotracheitis virus IBRV-B strain and its application. The bovine infectious rhinotracheitis virus IBRV-B strain of the present invention has the advantages of good immunogenicity and good culture characteristics. Rhinotracheitis virus (IBRV‑B strain) was inoculated into MDBK cells, the culture was harvested, inactivated with β‑propiolactone (β‑propiolactone), and mixed with commercial Montanide ^TM ISA15AVG adjuvant from Sebec, France for emulsification Prepare bovine infectious rhinotracheitis virus inactivated vaccine. Experiments have proved that using the bovine infectious rhinotracheitis virus inactivated vaccine prepared by the present invention to immunize weaned healthy negative cattle can prevent diseases caused by bovine infectious rhinotracheitis virus, and the vaccine has the advantages of safety, rapid antibody production, and long-term immunity. Durable and other advantages.

Description

Bovine Infectious Rhinotracheitis Virus IBRV-B Strain and Its Application

technical field

The invention belongs to the field of biology, more specifically, belongs to the field of biological vaccines, relates to a bovine infectious rhinotracheitis virus and its application, in particular to a bovine infectious rhinotracheitis virus IBRV-B strain isolated by itself and its The inactivated vaccine prepared by this virus strain.

Background technique

Infectious bovine rhinotracheitis virus (IBRV) can cause acute, febrile, and contact infectious diseases in cattle, manifested as inflammation of the upper respiratory tract and tracheal mucosa, dyspnea, runny nose and other symptoms, and can also cause purulent vulva Inflammation, vaginitis, balanitis, conjunctivitis, miscarriage, calf meningitis, mastitis, metritis and other symptoms. The disease was first discovered in Colorado in the United States in the early 1950s, and then other states also discovered the disease one after another. In 1956, Madm and others isolated the virus from sick cattle for the first time. Huck (1971) confirmed that IBRV belongs to herpes virus and is currently prevalent worldwide. In 1980, my country first discovered the disease in cattle imported from New Zealand, and reported the existence of the virus for the first time. The disease caused great economic losses.

Current vaccines include inactivated vaccines, attenuated vaccines, subunit vaccines and gene-deletion-marked vaccines. There are two vaccines listed in Australia, Rhinogard (V155 strain) and IBEPUR (subunit vaccine), and more than 2 million cattle have been vaccinated in Australia. Various traditional vaccines and marked vaccines (used to distinguish immunized groups from non-immunized groups) are available overseas, and Australia often uses vaccines against other pathogens in combination, that is, multivalent vaccines are used to jointly prevent bovine respiratory disease syndrome (BRD) ). At present, there is no self-developed related vaccine in the domestic market for the prevention and treatment of this disease.

The inventor of the present invention isolated a bovine infectious rhinotracheitis virus from a cattle farm in Beijing in 2015, which was identified as a virulent strain by the laboratory, and the strain after domestication and cloning was named IBRV-B strain. Using the IBRV-B strain, the research and development of the inactivated vaccine for bovine infectious rhinotracheitis virus disease was carried out, and the bovine infectious rhinotracheitis virus IBRV-B strain with good immunogenicity and stable titer was screened and bred. MDBK cells that are most suitable for the virus to proliferate and meet the inactivation agent, adjuvant and other conditions for making vaccines. 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, and after sampling inspection, all of them met the proposed quality standards, and the safety test and efficacy test were carried out on the pilot products, and the results also confirmed that this vaccine can effectively prevent bovine infectious nasal Bovine respiratory disease caused by tracheitis virus, on the basis of laboratory tests and intermediate trial production, the present invention has developed an inactivated vaccine of bovine infectious rhinotracheitis virus IBRV-B strain, and the results have also confirmed that this vaccine can effectively prevent The bovine respiratory disease caused by bovine infectious rhinotracheitis virus further verified the various quality standards of this vaccine.

Contents of the invention

One of the purposes of the present invention is to provide a bovine infectious rhinotracheitis virus strain with good immunogenicity and stable titer;

The second object of the present invention is to provide the application of the bovine infectious rhinotracheitis virus strain in the preparation of vaccines;

The third object of the present invention is to provide a vaccine prepared from the bovine infectious rhinotracheitis virus strain;

The fourth object of the present invention is to provide the application of the vaccine in the preparation of medicines for preventing bovine infectious rhinotracheitis disease.

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

The inventor of the present invention isolated a bovine infectious rhinotracheitis virus from a cattle farm in Beijing in 2015, which was identified as a virulent strain by the laboratory, and the domesticated strain was named IBRV-B strain. The IBRV-B strain of the present invention is isolated and obtained from cattle infected with typical bovine infectious rhinotracheitis virus, so compared with other strains isolated, the proliferation titer on cells is high, and the immunogenicity to cattle is high. it is good. After being passaged by MDBK cells, it is used as a vaccine virus, and the strong virus used for efficacy testing is the 7th to 10th generation of IBRV-B strain. This strain can cause more than 90% infection of MDBK cells.

A kind of bovine infectious rhinotracheitis virus of the present invention is the 7th generation virus strain after passage on MDBK cell, is named as bovine infectious rhinotracheitis virus IBRV-B strain, and is classified and named as bovine infectious rhinotracheitis virus Virus, preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, the address is at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The strain preservation number is: CGMCC No.14294, and the preservation date is 2017 June 15.

The present invention also provides the application of said bovine infectious rhinotracheitis virus in the preparation of bovine infectious rhinotracheitis virus inactivated vaccine.

The inactivated bovine infectious rhinotracheitis virus vaccine of the present invention is characterized in that it contains the inactivated bovine infectious rhinotracheitis virus IBRV-B strain of the present invention.

In the bovine infectious rhinotracheitis virus I inactivated vaccine of the present invention, preferably, the described bovine infectious rhinotracheitis virus IBRV-B strain is inactivated by the following method: 0.2% β-propane The esters were inactivated at 4°C for 24 hours and then at room temperature for another 24 hours.

In the present invention, preferably, the described feature is that the bovine infectious rhinotracheitis virus inactivated vaccine of the present invention containing the inactivated vaccine is made of 15% (v/v) of the French Sepic company. It is obtained by emulsifying commercial Montanide ^TM ISA15AVG adjuvant and 85% (v/v) bovine infectious rhinotracheitis virus IBRV-B strain inactivated with β-propiolactone.

In the present invention, the emulsification process is to use the commercialized Montanide ^TM ISA15AVG adjuvant of the French 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 the aseptic Indoor use a homogenizer to stir evenly at 200r/min, and then follow the volume ratio of the water phase antigen to 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, 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 the prevention of diseases caused by the inactivated bovine infectious rhinotracheitis virus of the present invention.

In particular, the disease is a respiratory disease occurring in healthy cattle.

Description of drawings

Figure 1 is a negative-stained electron microscope image of bovine infectious rhinotracheitis virus IBRV-B strain

Fig. 2 is the fluorescence detection result that bovine infectious rhinotracheitis virus IBRV-B strain is cultivated on MDBK cell for 48 hours;

Fig. 3 is a one-step growth curve after bovine infectious rhinotracheitis virus IBRV-B strain infects MDBK cells.

detailed description

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 bovine infectious rhinotracheitis virus IBRV-B strain

1 Source and standard of virus seeds

According to the new biological product declaration requirement, in conjunction with a large amount of test data that the present invention obtains, with reference to " Chinese Veterinary Pharmacopoeia " (2005 edition), the poisonous species used for making seedlings 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 used to manufacture this product is Bovine Infectious Rhinotracheitis Virus IBRV-B strain, which was obtained by the inventor after self-isolation and domestication from diseased cattle in 2015. Compared with other isolated strains, it has a higher proliferation titer on cells and has better immunogenicity to cattle, so it is selected for the production of vaccines. After being passaged by MDBK cells, it is used as a vaccine virus, and the strong virus used for efficacy testing is the 7th to 10th generation of IBRV-B strain. The virus strain is the seventh-generation virus strain after passage on MDBK cells. It is preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee. The address is at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The strain preservation number is: CGMCC No.14294, and the preservation date is June 15, 2017.

1.2 Poison Seed (CGMCC No.14294) Standard

1.2.1 Electron microscope morphology

The particles of IBRV-B strain are approximately spherical, with a capsule, in two forms: solid and hollow, with a diameter of about 150nm to 194nm, with an average of 170nm. The thickness of the membrane is asymmetrical, the shape and size are different, and there are villous protrusions on the surface (see Figure 1).

1.2.2 Virus content

The virus seeds were serially diluted 10 times with DMEM culture fluid, and 100 μl of each dilution was added to a 96-well cell culture plate, and each dilution was made 8 repetitions, and then MDBK cells were added, 100 μl of each well (the cell content was expressed as 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) were observed day by day. Formation of cavities in layer cells is judged as CPE. After 7 days of observation, the number of wells with cytopathic changes was recorded, and the TCID50 of the virus was calculated according to the Reed-Muench method. Results: The TCID50 per milliliter of virus liquid was not less than 10 ^11.0 .

1.2.3 Virulence

The virus generation is bovine infectious rhinotracheitis virus IBRV-B strain 7th to 10th generation cytotoxicity; the challenge dose is that the virus content per ml should not be less than 10 ^7.0 TCID50; the procedure is 4 healthy negative calves, intranasal Spray inoculated 10ml of IBRV-B strain 7th to 10th generation cytotoxicity, and the virus content was 1×10 ^8.0 TCID50; the judgment standard was that the body temperature rose from the second day, and continued to rise above 40.0°C for an average of 5 days, showing different symptoms. The clinical symptoms were severe, manifested as mental depression, thick purulent nasal fluid, red and swollen nasal mucosa with white spots, and purulent secretions from the eyes. The clinical symptoms lasted for about 9 days. Necropsy lesions were mainly concentrated in the lungs, thymus lymph nodes were enlarged, tracheal hemorrhage, and other organs had no obvious lesions visible to the naked eye. The results of the pathological section showed that the lungs were suppurative pneumonia, with fibrinous exudates, pulmonary edema, bronchiole wall destruction, the lumen was filled with inflammatory cells, lung tissue destruction, and a large number of inflammatory cells and necrotic foci were seen. Viruses were isolated from tissues in the lungs, trachea, and around the eyes.

1.2.4 Immunogenicity

The virus seed was inoculated into MDBK cells synchronously for proliferation, the virus liquid was harvested, inactivated with β-propiolactone, mixed and emulsified with Montanide ^TM ISA15 AVG adjuvant to make an oil-in-water inactivated vaccine, and used in healthy susceptible weaned calves (IBRV neutralizing antibody titer is not higher than 1:2) 5 cows, intramuscular injection of 2ml vaccine prepared according to this procedure, 5 unavoidable control cows, intranasal spray inoculation of 10ml of each IBRV-B strain (10 ^7.0 TCID50), attack All cattle were killed on the 14th day after poisoning. The control group should have at least 4 typical clinical symptoms of bovine infectious rhinotracheitis infection, and the immunization group should have at least 4 protection.

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 bovine infectious rhinotracheitis virus IBRV-B strain were tested for bacteria, mold and mycoplasma. And the 11th, 12th and 24th generations of virus seeds have been carried out the inspection of exogenous virus, the result shows, the original seed batch that the present invention establishes, basic seed batch, production seed batch all have no bacterium, mould, mycoplasma and exogenous virus pollution , are pure.

1.2.6 Specificity

Dilute the virus seed to 200TCID50/0.1ml with DMEM solution, mix it with inactivated anti-bovine infectious rhinotracheitis virus-specific serum, neutralize at 37°C for 1 hour, and simultaneously inoculate 3 bottles of well-growing MDBK cells (The cell content is 3×10 ⁵ /ml), and virus control, normal cell control and negative serum control are set at the same time, placed in a 37°C, 5% CO ₂ incubator, and cultivated for 5-7 days. Neither the neutralizing 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 bovine infectious rhinotracheitis virus IBRV-B strain that is originally isolated is continuously passed on for 40 generations on MDBK cells (that is, it is continuously passed on for 33 generations on the basis of the strain whose strain preservation number is CGMCC No.14294), and the For the TCID50 of each generation of virus, the 15th, 20th, 25th, 30th, 35th, and 40th generation viruses were selected to be inactivated respectively to make vaccines, inoculated with calves aged 2 to 4 months, and challenged 14 days later. The results showed that the virus was passaged on MDBK cells, and after the 7th generation (including the 7th generation virus, that is, the virus strain with strain preservation number CGMCC No.14294), the virus content of each generation was stable, all not less than 10 ^11.0 TCID50, And the neutralizing potency is above 32, which has reached the standard for making vaccines. The challenge test showed that the vaccines prepared by the 15th, 20th, 25th, and 30th generation viruses had no significant difference in the protection against IBRV-B strain challenge, and the calves survived healthy; the vaccines prepared by the 35th and 40th generation viruses were immunized After the challenge, 2 calves and 1 calves died respectively, while the 4 calves in the control group all had symptoms of bovine infectious rhinotracheitis virus infection to varying degrees. 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 bovine infectious rhinotracheitis virus IBRV-B strain was stored at -20°C and -70°C, and samples were taken at different times after storage for neutralization titer and TCID50 determination. The results showed that the wet virus was stored at -20°C After 18 months and 30 months of storage at -70°C, the poison price began to decrease significantly; the freeze-dried virus was stored at -20°C and -70°C, and samples were taken every year after storage for neutralization potency and TCID50 determination. Results The titer of the virus stored at -20°C for 36 months decreased slightly, the neutralizing titer and TCID50 value of the virus preserved for 48 months decreased significantly, and the neutralizing titer of the virus stored at -70°C for 48 months And TCID50 has no obvious change, and it has dropped significantly after 60 months of storage. Considering other unfavorable factors in virus storage and ensuring the reliability of the virus storage period, the storage period of wet virus of bovine infectious rhinotracheitis virus IBRV-B strain at -20°C was set at 12 months, and at -70°C The storage period at -20°C is 24 months; the storage period of the freeze-dried virus of bovine infectious rhinotracheitis virus IBRV-B strain is 36 months at -20°C; the storage period of the freeze-dried virus at -70°C is 48 months months.

2 Production cells

2.1 Source of cells

MDBK cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and preserved by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

2.2 Selection of cells

In the cultivation process of the virus, the present invention selects the passage cell line MDBK to carry out the propagation of the virus, and the virus is inoculated with the disease time on the MDBK cells as early as 0.01 according to the virus infection value (MOI), and the proliferation titer is the highest, and the MDBK cells infect the bovine infectivity 48 hours of rhinotracheitis virus IBRV-B strain is shown in Figure 2, and the one-step growth curve of bovine infectious rhinotracheitis virus IBRV-B strain after infection of MDBK cells is shown in Figure 3. Under such conditions, the highest virus titer was harvested at 48 hours.

2.3 Establishment of cell lines

MDBK cells are selected as the cell line for seedling production. 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. See "MDBK Cell Line Identification Test Report" for details.

The preparation of embodiment 2 inactivated vaccines

The preparation process of the bovine infectious rhinotracheitis virus IBRV-B 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. 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 inoculation dose 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 MDBK cells were inoculated according to the virus infection value (MOI) of 0.01, and the optimal harvest time of the virus was 48-7. After 72 hours, virus liquid with high virus content can be obtained.

In this embodiment, the serum concentration in the cell culture medium is selected to be 10%, the pH value is controlled at 7.3, and the serum concentration in the maintenance solution (the trade name is DMEM, purchased from Hyclone Company) is 2%, according to the virus infection value (MOI) Inoculate 0.01 bovine infectious rhinotracheitis virus IBRV-B strain 7th generation virus strain (bacteria preservation number is CGMCC No.14294) seed poison in MDBK cells, and the virus harvest time is 48 hours.

2 Inactivation process

Select formaldehyde solution with final concentration of 0.1%, 0.2%, 0.3%, 0.4% and β-propiolactone with final concentration of 0.1%, 0.2%, 0.3%, 0.4% to inactivate bovine infectious rhinotracheitis virus Comparative test, test result shows, 0.2% β-propiolactone can completely inactivate bovine infectious rhinotracheitis virus after 24 hours under the condition of 4 ℃, and, test result shows that β-propiolactone damages cell Smaller; bovine infectious rhinotracheitis virus can be completely inactivated by adding 0.3% formaldehyde solution and inactivating it at 37°C for 48 hours; however, formaldehyde is highly irritating. If the residual free formaldehyde in the vaccine enters the animal The body will produce adverse reactions. Therefore, it was finally determined that 0.2% β-propiolactone was used to inactivate the bovine infectious rhinotracheitis virus at 4°C for 24 hours, and then inactivated at room temperature for 24 hours to hydrolyze the residual β-propiolactone.

3 Emulsification process

The commercialized MontanideTM ISA15AVG adjuvant from Sebec, France is used, which can be directly used for vaccine emulsification preparation after sterilization; before emulsification, the same batch of inactivated virus liquid is thawed and mixed, and the homogenizer is used in a sterile room at 200r/min Stir evenly, and then follow the volume ratio of water phase antigen and oil phase adjuvant 8.5:1.5; the emulsification procedure is to first put the water phase into an emulsifier for stirring, and then slowly add the oil phase adjuvant, continuously at 800r/min Stir 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 with DMEM culture fluid, and 100 μl of each dilution was added to a 96-well cell culture plate, and each dilution was made 8 repetitions, and then MDBK cells were added, 100 μl of each well (the cell content was expressed as 3 ×10 ⁵ /ml), and a normal cell culture control was set up, cultured in a 37°C, 5% CO2 incubator, and the cytopathic changes (CPE) were observed day by day. The formation of cavities on the cells is judged as CPE. After 7 days of observation, the number of wells with cytopathic changes was recorded, and the TCID50 of the virus was calculated according to the Reed-Muench method. Results: The TCID50 per milliliter of virus liquid was not less than 10 ^11.0 .

4.3 Inactivation test

Take the inactivated virus solution, inoculate it in 3 flasks of MDBK cells at a ratio of 1:10 between the virus solution and the growth solution (the cell content is 3×10 ⁵ /ml), incubate 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 quality standard of finished product inspection

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 calves, repeated single-dose inoculation, one-time overdose (2 times) to 5 batches of vaccines prepared by the laboratory". dose) inoculation safety test", "safety test of single-dose inoculation, single-dose repeated inoculation, one-time overdose (2 times dose) inoculation for fattening cattle", single-dose inoculation, single-dose repeated inoculation for pregnant cows , Safety test of one-time super-dose (2 times dose) inoculation". The test results show that: after single-dose inoculation, single-dose repeated inoculation, and one-time large-dose inoculation of each immunized animal, the animals are in good condition, and the food intake and drinking water are normal. There is no abnormal reaction; the reproductive function of the pregnant cow is also not significantly affected after the vaccination. The above tests all prove that the vaccine is safe. The present invention has concluded that as long as there is no local redness, ulcer, erosion and Neural atrophy, reduced feed intake, etc., will not cause adverse reactions due to vaccination in the future, and can prove the safety of the vaccine. Therefore, it is stipulated in the trial procedure (draft): use 2 to 6 months old healthy and easy Infected calves (neutralizing antibody titer is not high 1:2) 2, each deeply intramuscularly injected with 4ml of vaccine, another 2 calves with the same conditions were not vaccinated as controls, and observed continuously for 14 days. During the observation period, there should be no Any local or systemic adverse reactions caused by vaccination.

5.2 Formulation of efficacy testing standards

5.2.1 Correlation test between antibody titer and immune challenge protection

One batch of bovine infectious rhinotracheitis virus inactivated vaccine 151101 that passed the safety test was immunized according to different doses of 0.5ml/head, 1ml/head, 2ml/head, and 4ml/head respectively at the age of 2 to 6 months. 5 healthy calves (neutralizing antibody not higher than 2) were immunized twice at intervals of 14 days, and 3 non-immunized control groups were immunized with 10 ^7.0 TCID50/ml bovine infectious rhinotracheitis virus IBRV-B 14 days after the second immunization Strain 10 generation virulent 4ml nasal spray inoculation to determine the correlation between neutralizing antibody titer and immune challenge protection. The test results show that when the serum neutralizing antibody titer is not lower than 1:32, the protection rate of calves challenged can be more than 80%. Therefore, when determining the vaccine efficacy test, the test standard is that the serum neutralizing antibody titer should not be lower than 1:32 14 days after the second immunization.

5.2.2 Minimum immunization dose and minimum dose test

Three batches of bovine infectious rhinotracheitis virus inactivated vaccines 151101, 151102 and 151103 that passed the safety test were immunized with different doses of 1ml/head, 2ml/head, 4ml/head, and 6ml/head respectively for 2~ 6-month-old healthy calves (IBRV neutralizing antibody not higher than 2), blood was collected 14 days after the second immunization for neutralizing antibody detection. The results showed that the average neutralizing antibody titer of calves immunized with 2ml/head dose was not lower than 1:32 after 28 days, and the neutralizing antibody titer of calves immunized with a dose of 4ml/head or more was not lower than 1:32 after 28 days. : 64. According to the results of the correlation test between neutralizing antibody titer and challenge protection, it is determined that bovine infectious rhinotracheitis virus can be completely protected when the neutralizing antibody titer of bovine infectious rhinotracheitis virus reaches 1:64 or more 28 days after immunization Poisonous attack. 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 bovine infectious rhinotracheitis virus inactivated vaccine (IBRV-B strain) is determined to be 2ml/head portion, and the usage dose is determined to be 4.0ml/head portion.

5.2.3 Correlation test between immunized guinea pigs and immunized bovine neutralizing antibodies

Three batches of bovine infectious rhinotracheitis virus inactivated vaccines 151101, 151102 and 151103 produced in the laboratory were immunized to guinea pigs and calves respectively, and the neutralizing antibody titer was tested to analyze the correlation of neutralizing antibodies between guinea pigs and calves. The results showed that when guinea pigs were immunized with 0.5ml/head and calves were immunized with 2ml/head, the serum neutralizing antibody titer was not lower than 1:32 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 (cattle) 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 with a body weight of 350g-400g (IBRV neutralizing antibody titer not higher than 1:2) were injected intramuscularly with 0.5ml of the vaccine. After 28 days, blood was collected together with 4 control guinea pigs to determine the IBRV neutralizing antibody titer. price. The HI antibody titer of the control guinea pigs should not be higher than 1:2, and at least 4 of the immunized guinea pigs should have antibody responses, and the neutralizing antibody titer should not be lower than 1:32.

Five 3-month-old healthy susceptible calves (IBRV neutralizing antibody titer not higher than 1:2) were injected with 2ml of the vaccine in each deep muscle. After 28 days, blood was collected together with 4 control calves to measure IBRV neutralization. Antibody titer. The HI antibody titer of the control cow should not be higher than 2, and all the vaccinated cows should have an antibody reaction, and the neutralizing antibody titer should not be lower than 1:32.

6 Antibody growth and decline rules and immunity duration test

Three batches of bovine infectious rhinotracheitis virus inactivated vaccines 151101, 151102 and 151103 that passed the safety test were immunized with healthy calves, pregnant cattle and adult cattle, respectively. In order to further grasp the efficacy and duration of immunity, formulate a reasonable immunization program, and ensure that the immunized cattle herd maintains a high and long-lasting antibody level, antibody detection is performed on immunized cattle at different times to grasp the law of their antibody growth and decline.

The test results show that the herd immunization is 2ml/head, and the immunization is boosted once after 2 to 3 weeks. After 28 days, the serum neutralizing antibody is not lower than 1:32. The peak of the antibody is within 4 to 6 months after immunization. The level gradually decreased and still reached 1:32 in 12 months. In order to ensure the immune protection effect of the vaccine, the duration of immunity was determined to be 10 months.

7 Determination of immunization program

According to the law of growth and decline of antibodies after herd immunization, the immunization period, and in conjunction with the actual situation of clinical bovine infectious rhinotracheitis virus infection, the present invention has determined the efficacy of bovine infectious rhinotracheitis virus inactivated vaccine (IBRV-B strain) The immunization program is: immunization twice a year. Each immunization is 2ml/head (doubled for adult cattle).

8 Shelf life of vaccines

The present invention has carried out experimental research on the shelf life of 3 batches of inactivated vaccines (151101, 151102 and 151103) prepared in the laboratory. The 3 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, 151101 batches of vaccines stored for 18 months had a guinea pig HI antibody titer of 1:16 after immunizing guinea pigs; 151102 and 151103 batches of vaccines were stored for 18 months after immunizing cattle, and there was a bovine serum neutralizing antibody titer of 1:16, but the average neutralizing antibody level of immunized cattle is 1:32. 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 moon.

9 Comparison of immune efficacy with similar domestic commercial vaccines

Three batches of vaccines 151101, 151102 and 151103 produced in the laboratory and commercially available vaccines A (batch number: 20151011) and B (batch number: 20150905) were immunized with 350g-400g healthy guinea pigs and healthy calves aged 5-6 months, respectively. After 28 days, The serum neutralizing antibody titers were not less than 1:32, which met the protection requirements of the virus challenge. The average neutralizing 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 neutralizing antibody titers of the laboratory-produced vaccine and commercially available A and B vaccines for immunizing cattle were all above 1:32, both of which could meet the challenge protection requirements. However, after 6 months, the neutralizing antibody of the commercially available B vaccine immunized cattle decreased significantly.

Adverse reactions such as abscesses and scabs did not exist at the vaccination sites of the three batches of vaccines 151211, 151212 and 151213 injected, while more than 2% of the above-mentioned adverse reactions existed at the vaccination sites of commercially available A and B vaccines.

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. A strain of bovine infectious rhinotracheitis virus, characterized in that, the bovine infectious rhinotracheitis virus is named after the bovine infectious rhinotracheitis virus IBRV-B strain, preserved in China Microbiological Cultures Preservation Committee Common Microorganisms Center, its culture preservation number is: CGMCC No.14294. 2. the application of bovine infectious rhinotracheitis virus described in claim 1 in the preparation of bovine infectious rhinotracheitis virus inactivated vaccine. 3. A bovine infectious rhinotracheitis virus inactivated vaccine, characterized in that it contains the bovine infectious rhinotracheitis virus of claim 1 after inactivation. 4. inactivated vaccine as claimed in claim 3, is characterized in that described inactivation refers to adopting 0.2% β-propiolactone at 4 ℃ of conditions with the bovine infectious rhinotracheitis virus described in claim 1 Inactivate at room temperature for 24 hours, then inactivate at room temperature for 24 hours to hydrolyze residual β-propiolactone. 5. the application of the inactivated vaccine described in claim 3 or 4 in the biological product medicine that prevents the disease caused by bovine infectious rhinotracheitis virus. 6. The application according to claim 5, characterized in that said disease is a respiratory disease in healthy negative cattle.