CN119286802A - A bovine herpes virus type 4 strain and its application - Google Patents

Abstract

The present invention discloses a bovine herpesvirus type 4 virus strain and its application, which is a bovine herpesvirus type 4 CB strain, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee, with a deposit number of CGMCC No.45255 and a deposit date of August 10, 2022. The bovine herpesvirus type 4 inactivated vaccine of the present invention is safe to use and has a good immune protection effect on bovine herpesvirus type 4 disease caused by bovine herpesvirus type 4. Immunized cattle can obtain protection for at least 4 months.

Description

Bovine herpes virus type 4 virus strain and application thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to application of bovine herpes virus type 4 (BoHV-4) and a vaccine or diagnostic product developed by using the virus.

Background

Bovine herpes virus type 4 is a virus that infects bovine, and belongs to the family of herpesviruses. The virus can cause various diseases including respiratory diseases, mastitis, genital tract infection, etc. BoHV-4 infection is widely present in cattle groups in various countries and regions worldwide, severely affecting the health of cattle and the economic benefits of the livestock industry. At present, effective prevention means are lacking, and the existing control means mainly depend on management means and supporting treatment, so that the methods cannot thoroughly eliminate viruses and cannot effectively prevent occurrence and transmission of diseases.

In view of the above background, the development of vaccines against BoHV-4 is highly desirable. The effective vaccine can prevent diseases, reduce economic loss, improve animal welfare, and simultaneously help to protect public health from animal-derived diseases, but no related vaccine is marketed in China.

In view of the deficiencies of the prior art, the present invention provides a bovine herpes virus type 4 based vaccine which is effective in eliciting an immune response in the body, thereby preventing diseases caused by BoHV-4. The invention also provides a method for preparing the vaccine by using the virus and a method for preventing diseases by using the vaccine.

Disclosure of Invention

The invention aims to provide a bovine herpes virus type 4 isolated strain and application thereof.

The bovine herpesvirus 4 type isolate strain is a bovine herpesvirus 4 type CB strain, and is classified and named as bovine herpesvirus 4 type. The culture medium is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 45255 and the preservation date of 2022, 08 and 10 months, and the preservation address of North Star, xiyu No. 1, 3 of the Korean area of Beijing, china academy of sciences, microbiological culture collection center of China general microbiological culture collection center, and post code of 100101.

The invention aims to provide a novel BoHV-4 virus strain and application thereof in vaccine preparation so as to realize effective prevention of diseases caused by BoHV-4.

(A) Characteristics of the virus strain:

the BoHV-4 virus strain related by the invention has high safety and good immunogenicity.

The strain is obtained by a specific adaptive culture method, retains the key antigen property capable of stimulating immune system reaction, and reduces pathogenicity to a host.

(B) Preparation of the vaccine:

The BoHV-4 virus strain provided by the invention is selected and propagated through a cell culture technology.

According to the requirements of disease prevention, the virus strain is selected to be inactivated and mixed with a proper adjuvant to prepare the vaccine.

The vaccine can be used for preventing diseases caused by BoHV-4 by injection or other suitable administration modes.

Compared with the prior art, the invention has the following beneficial technical effects:

The virus strain and the vaccine application thereof provided by the invention can effectively prevent diseases caused by BoHV-4, reduce the disease incidence rate of cattle and improve the economic benefit of the breeding industry.

The vaccine of the invention has high safety and good immune effect, and provides a new solution for the control of bovine herpesvirus.

Drawings

FIG. 1 shows MDBK cytopathic effects of bovine herpes virus 4 type virus CB strain of the present invention, wherein the right side is MDBK cells producing cytopathic effects, and the left side is normal MDBK cells;

FIG. 2 shows PCR detection of bovine herpesvirus 4 type CB strain of the invention, and gB gene amplification results, wherein gel electrophoresis sample adding sequence is sequentially shown in a lane 1, a negative control, lanes 2-6, different generations of bovine herpesvirus 4 type, and a M2K plus Marker, wherein the sequence is 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom.

Detailed Description

The following embodiments are merely exemplary and do not limit the scope of the present invention in any way, and modifications and substitutions may be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the present invention, but these modifications and substitutions fall within the scope of the present invention.

The method used in the present invention may be a method commonly used in the field of virus research, and is not limited to the specific description of the embodiments of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The reagents, materials, etc. used in the examples described below are commercially available unless otherwise specified.

The reagents and materials used in the examples below were CO2 incubator from Thermo, high-speed bench centrifuge from Shanghai Anting scientific instruments, gel imaging system Tanon R from Beijing Yuanzhihao Biotechnology Co., ltd, PCR instrument from Eppendorf, nucleic acid protein electrophoresis instrument from Beijing Liu instrument, trans 2K plus Marker, viral DNA/RNA extraction kit, 2X EasyTaq, PCR Supermix from Beijing full gold Biotechnology Co., ltd, 2X MDBK (Madin-Darby Bobine KIDNEY CELL) cells from China veterinary medicine inspection institute, DMEM medium and 0.25% Trypsin-EDTA (1X) pancreatin from Gibco Co., ltd, fetal bovine serum from Nerng Gu Jinyuan Bioengineering Co.

Example 1 isolation and identification of bovine herpes virus type 4 virus

1.1 Isolation of bovine herpesvirus type 4 virus

1.1.1 Treatment of disease samples nasal swabs of harvested cattle suspected of having bovine herpesvirus type 4 virus disease were placed in culture (DMEM with 1000 units/ml penicillin, 1000 μg/ml streptomycin) and stored overnight at 4 ℃. Then, the mixture is centrifuged for 10min at 12000 r/min, the supernatant is filtered and sterilized by a microporous filter membrane with the thickness of 0.22 mu m, and the temperature is kept below-70 ℃ for standby.

1.1.2 Virus isolation and passage the supernatant after the treatment described above was inoculated with a monolayer of MDBK cells in logarithmic growth phase, adsorbed for 2h at 37℃and then the liquid was decanted, DMEM maintenance solution (DMEM containing 200 units/ml penicillin, 200. Mu.g/ml streptomycin and 10 ml/L serum) was added and cultured in a 37℃5% CO2 incubator, and cytopathic effect (CPE) was observed daily. And (3) when 70% -80% of cells have CPE, the cells are attenuated, if the cells do not have CPE, the cells are blindly transmitted after 5 days, and when the cells have lesions, the harvested culture is repeatedly frozen and thawed for 2 times at-80 ℃, and then the harvested culture is preserved for standby at-80 ℃. The isolated virus produced a typical cytopathic effect (CPE) in MDBK, see fig. 1.

1.2 PCR identification of bovine herpes virus type 4 virus

1.2.1 Virus PCR detection

PCR was performed on the isolates using primers for amplification of the bovine herpes virus type 4 virus gB gene, the primer sequences are shown in Table 1 below, and the expected amplification products were 615bp.

TABLE 1BPIV3 PCR primer sequences and amplified fragment sizes

Sample nucleic acids were extracted according to commercial viral DNA/RNA extraction kits. The PCR reaction was performed in a 25. Mu.L system with 2X EasyTaq. Mu.L of PCR Supermix 12.5. Mu.L of each of the upstream and downstream primers, 2.0. Mu.L of template DNA and 6.8. Mu.L of ddH 2O.

The reaction conditions were 95℃for 5min, 94℃for 1min, 54℃for 1min,72℃for 3min, 35 cycles, and 72℃for 10 min. The PCR product was stored at 4 ℃. The reaction product was taken at 5. Mu.L, added with a proper amount of a loading buffer, mixed well, and electrophoresed at 120V voltage in 1% agarose gel for 30min, and the result was observed. The result shows that a specific band with the size of 615bp is amplified, the PCR product is sent to Shanghai Biotechnology Co., ltd for sequencing analysis, and the sequencing result is subjected to gene comparison with the sequence published by NCBI, so that the separated strain is bovine herpesvirus 4. See fig. 2.

EXAMPLE 2 Virus subculture and Virus valence determination

2.1 Proliferation culture of virus bovine herpes virus type 4 CB strain is inoculated with MDBK monolayer cells at an inoculum size of 1:1000 for culture, and is harvested when CPE reaches more than 80%. And (5) performing serial inspection such as sterile inspection, exogenous virus inspection, specificity inspection and the like on the harvested virus liquid, and freezing and preserving seeds after the virus liquid is qualified. The method is used for continuous passage for 15 generations, the virus harvest amount of each generation is at least 200ml, and the harvested virus is kept at-80 ℃ for standby.

2.2 Determination of the viral titers F1-F15 generation virus cultures are diluted by 10 times of cell maintenance solution respectively, and the half-cell infection amount (TCID 50) of the viruses is determined according to the method of annex of Chinese animal pharmacopoeia. The highest toxicity can reach 107.5TCID50/0.1ml.

EXAMPLE 3 preparation of bovine herpesvirus 4 type inactivated vaccine and immunization duration

3.1 Propagation of the virus the bovine herpes virus type 4 CB strain basic seed is diluted 1:10 with DMEM cell maintenance solution, then the diluted virus solution is inoculated into well-grown MDBK single-layer cells according to 0.1 MOI, and the cells are cultured in a 37 ℃ cell incubator containing 5% CO2 for 5 days to more than 80% to generate typical CPE harvest, and the virus content is 107.5TCID50/0.1ml, so that the bovine herpes virus type 4 CB strain basic seed can be used as an antigen for preparing vaccines.

3.2 Bovine herpesvirus type 4 virus inactivation the virus solution was added to a 10% formaldehyde solution (37% formaldehyde solution was 100%) to a final formaldehyde concentration of 0.2% and stirred while thoroughly mixing, at 37 ℃ and stirred for 24 hours for inactivation. After the inactivation is finished, sampling is respectively carried out for sterile inspection and inactivation inspection, and the sample is used for vaccine preparation after the inspection is qualified.

3.3 Preparation of vaccine, mixing the bovine herpesvirus 4 type virus inactivated solution which is qualified in inspection with 605 adjuvant according to the proportion of 1:1, and fully and uniformly mixing at 100-300 r/min.

3.4 Safety of bovine herpesvirus 4 type inactivated vaccine the vaccine neck muscle is injected with 5 healthy calves of 1-3 months of age, each head is 4.0ml, and continuous observation is carried out for 15 days, so that local and systemic adverse reactions caused by vaccine injection do not occur.

3.5 Potency and immunization duration of bovine herpesvirus 4 type inactivated vaccine

As shown in table 2, 10 healthy calves with 2-6 months of age antibody are adopted, wherein 2ml of bovine herpesvirus 4 type inactivated vaccine is respectively injected into the neck muscle of 5 calves, the same dose is used for boosting once in 21 days after the immunization, the neutralizing antibody titer is measured by blood sampling for 14 days, 2 months, 4 months, 6 months and 8 months together with 5 control calves after the immunization, blood sampling is respectively carried out, serum is separated, and the serum neutralizing antibody titer is detected.

Table 2 efficacy study of bovine herpes virus 4-strain CB inactivated vaccine against bovine immune group

The results of the immunization experiments are shown in Table 3 below, and bovine herpes virus type 4 CB strain induces high levels of antibodies with immunization durations of up to 6 months.

TABLE 3 results of potency studies on bovine herpesvirus 4-type CB-strain inactivated vaccine

The foregoing description describes several preferred embodiments of the invention, but as noted above, it is to be understood that the invention is not limited to the forms disclosed herein and is not to be considered as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept described herein, either by the foregoing teachings or by a person of ordinary skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.

Claims (9)

1. A bovine herpesvirus type 4 virus strain, characterized in that it is a bovine herpesvirus type 4 CB strain, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 45255. 2. The bovine herpesvirus type 4 strain according to claim 1, characterized in that the gB structural protein gene sequence of the bovine herpesvirus type 4 strain is the sequence shown in SEQ ID NO.1. 3. The bovine herpesvirus type 4 strain according to claim 1 is characterized in that: the bovine herpesvirus type 4 strain can proliferate well on MDBK cells, and the virus titer can reach up to 108.5TCID50/ml. 4. The use of the bovine herpesvirus type 4 strain according to any one of claims 1 to 3, characterized in that it is used to prepare a bovine herpesvirus type 4 inactivated vaccine preparation. 5. An inactivated bovine herpesvirus type 4 vaccine preparation, characterized in that it comprises the inactivated bovine herpesvirus type 4 virus strain of claim 1. 6. The method for preparing the inactivated bovine herpesvirus type 4 vaccine preparation according to claim 5, characterized in that it comprises mixing an inactivated bovine herpesvirus type 4 virus liquid and a vaccine adjuvant. 7. Use of the inactivated bovine herpesvirus type 4 vaccine preparation according to claim 5 for immunizing cattle. 8. The use according to claim 7, characterized in that each dose of the inactivated bovine herpesvirus type 4 vaccine preparation contains at least 107.5TCID50 of bovine herpesvirus type 4 strain. 9. The use according to claim 8, characterized in that the inactivated bovine herpesvirus type 4 vaccine preparation is injected intramuscularly into cattle, and the immunization dose is 2 ml/head.