阅读说明：本技术 牛传染性鼻气管炎病毒与牛支原体二联灭活疫苗及其制备方法与所用悬浮mdbk细胞 (Infectious bovine rhinotracheitis virus and mycoplasma bovis combined inactivated vaccine, preparation method thereof and suspension MDBK (multidrug-associated Virus) cells used in vaccine ) 是由 吴文学 王朋朋 格勒图 于 2020-07-31 设计创作，主要内容包括：本发明提供了一种牛传染性鼻气管炎病毒与牛支原体二联灭活疫苗及其制备方法与所用悬浮MDBK细胞。本发明提供了悬浮MDBK细胞,在此基础上建立了悬浮培养IBRV抗原病毒液的方法,采用该方法所得的IBRV抗原病毒液具有抗原含量高、抗原批量大、批次稳定等特点,而且避免了因培养基中加入的牛血清引起的副反应,在节约经济成本的同时又能生产出更加安全和高效的疫苗。同时本发明的IBRV与MB二联灭活疫苗一针可防两病,简化免疫程序,减少了动物的免疫次数,降低了动物的应激,也节省了劳动力,进一步降低了动物养殖的成本。(The invention provides a combined inactivated vaccine of infectious bovine rhinotracheitis virus and mycoplasma bovis, a preparation method thereof and a suspension MDBK cell used by the combined inactivated vaccine. The invention provides a suspension MDBK cell, and establishes a method for suspension culture of IBRV antigen virus liquid on the basis, the IBRV antigen virus liquid obtained by the method has the characteristics of high antigen content, large antigen batch, stable batch and the like, the side reaction caused by bovine serum added into a culture medium is avoided, and the safe and efficient vaccine can be produced while the economic cost is saved. Meanwhile, one needle of the IBRV and MB dual inactivated vaccine can prevent two diseases, simplifies the immunization procedure, reduces the immunization times of animals, reduces the stress of the animals, saves the labor force and further reduces the cost of animal breeding.)

1. The preparation method of the combined inactivated vaccine of the infectious bovine rhinotracheitis virus and the mycoplasma bovis is characterized by comprising the following steps of: the method comprises the following steps:

1) culturing infectious bovine rhinotracheitis virus by using suspended MDBK cells to obtain infectious bovine rhinotracheitis virus liquid, and inactivating the infectious bovine rhinotracheitis virus liquid to obtain inactivated infectious bovine rhinotracheitis virus antigen liquid; culturing mycoplasma bovis by using a liquid culture medium to obtain mycoplasma bovis body fluid, and inactivating the mycoplasma bovis body fluid to obtain inactivated mycoplasma bovis antigen fluid;

the suspended MDBK cells were prepared according to a method comprising the steps of: subculturing adherent MDBK cells by using a low serum suspension culture medium, and subculturing by using a serum-free suspension culture medium to obtain cells, namely the suspension MDBK cells;

the low serum suspension culture medium is a liquid culture medium obtained by adding fetal calf serum, penicillin and streptomycin into a ST cell full suspension culture medium of the family Yi Sheng; the volume percentage content of fetal calf serum in the low serum suspension culture medium is 3%, the penicillin content is 100U/ml, and the streptomycin content is 0.1 mg/ml;

the serum-free suspension culture medium is a liquid culture medium obtained by adding penicillin and streptomycin into a full suspension culture medium of ST cells of the family Yi Sheng; in the serum-free suspension culture medium, the content of penicillin is 100U/ml, and the content of streptomycin is 0.1 mg/ml;

2) and mixing the inactivated infectious bovine rhinotracheitis virus antigen liquid and the inactivated mycoplasma bovis antigen liquid to obtain an active component, thereby obtaining the combined inactivated vaccine of infectious bovine rhinotracheitis virus and mycoplasma bovis.

2. The method of claim 1, wherein: the subculturing by using the low-serum suspension culture medium is carried out for 7-15 generations by using the low-serum suspension culture medium, and the culturing is carried out under a shaking condition; the subculturing by using the serum-free suspension culture medium is carried out for 25-40 generations by using the serum-free suspension culture medium, and the culture is carried out under the shaking condition.

3. The method according to claim 1 or 2, wherein the infectious bovine rhinotracheitis virus and mycoplasma bovis combined inactivated vaccine contains mycoplasma bovis antigen in an amount of mycoplasma bovis before inactivation, infectious bovine rhinotracheitis virus in an amount of infectious bovine rhinotracheitis virus before inactivation, and the infectious bovine rhinotracheitis virus antigen in the combined vaccine is not less than 0.5 × 10⁷TCID₅₀The content of mycoplasma bovis antigen in per ml is not less than 1 ×

10⁸cfu/ml。

4. A combined inactivated vaccine of infectious bovine rhinotracheitis virus and mycoplasma bovis, prepared by the method of any one of claims 1-3.

5. A method of preparing suspended MDBK cells, comprising: subculturing adherent MDBK cells by using a low-serum suspension culture medium, and subculturing by using a serum-free suspension culture medium to obtain the cells, namely the suspension MDBK cells; the low serum suspension culture medium is a liquid culture medium obtained by adding fetal calf serum, penicillin and streptomycin into a ST cell full suspension culture medium of the family Yi Sheng; the volume percentage content of fetal calf serum in the low serum suspension culture medium is 3%, the penicillin content is 100U/ml, and the streptomycin content is 0.1 mg/ml; the serum-free suspension culture medium is a liquid culture medium obtained by adding penicillin and streptomycin into a full suspension culture medium of ST cells of the family Yi Sheng; in the serum-free suspension culture medium, the content of penicillin is 100U/ml, and the content of streptomycin is 0.1 mg/ml.

6. The method of claim 5, wherein: the subculturing by using the low-serum suspension culture medium is carried out for 7-15 generations by using the low-serum suspension culture medium, and the culturing is carried out under the suspension condition; the subculturing by using the serum-free suspension culture medium is carried out for 25-40 generations by using the serum-free suspension culture medium, and the culture is carried out under the suspension condition.

7. Suspended MDBK cells prepared by the method of claim 5 or 6.

8. Any one of the following Y1-Y7:

use of Y1, the suspended MDBK cells of claim 7 for suspension culture of viral antigens;

use of Y2, the suspension MDBK cells of claim 7 in the suspension culture of an IBRV antigen;

use of Y3, the suspension MDBK cells of claim 7 for the preparation of a vaccine;

use of Y4, the suspension MDBK cells of claim 7 for the preparation of a vaccine comprising an IBRV antigen;

use of Y5, the suspension MDBK cells of claim 7 in preparation of IBRV and MB combined inactivated vaccine.

9. A method for suspension culture of IBRV virus liquid is characterized in that: the method is characterized in that the IBRV is cultured by the suspension MDBK cells in a suspension manner, the virus inoculation amount of the IBRV is MOI (molar equivalent of 0.1), and the virus is collected 72-96 hours after virus inoculation to obtain IBRV virus liquid.

10. An IBRV virus fluid prepared by the method of claim 9.

Technical Field

The invention relates to a combined inactivated vaccine of infectious bovine rhinotracheitis virus and mycoplasma bovis, a preparation method thereof and a used suspension MDBK cell in the technical field of veterinary biological products.

Background

Bovine kidney cells (MDBK) are a continuous cell line derived from the kidneys of apparently normal cattle, which usually grow adherently in a medium containing fetal bovine serum and are sensitive to a variety of viruses such as bovine infectious rhinotracheitis virus (IBRV), bovine viral diarrhea/mucosal virus (BVDV).

Infectious Bovine Rhinotracheitis Virus (IBRV), also known as bovine herpes virus type I (BHV-1), can cause symptoms such as hyperpyrexia, inappetence, cough, rhinorrhea, conjunctivitis and the like of cows and beef cattle, and cause infectious bovine rhinotracheitis.

Mycoplasma Bovis (MB) can cause respiratory related diseases in cattle, such as bronchopneumonia, laryngitis, pharyngitis, etc., while mycoplasma bovis can cause mastitis and arthritis in cattle, both of which are the major causes of bovine respiratory syndrome (BRDC) with IBRV.

At present, an effective means for preventing the infection of the infectious bovine rhinotracheitis virus and the mycoplasma bovis is an inactivated vaccine for immunizing the infectious bovine rhinotracheitis virus and the mycoplasma bovis. At present, patent documents such as CN110420323A, CN109022314A and CN107050451A relating to mycoplasma bovis vaccines are all single vaccines, and patent documents such as CN106729692B relating to IBRV vaccines do not relate to the prevention of mycoplasma bovis, and if the two pathogens need to be immunized respectively for simultaneous prevention and control, the stress of animals is increased, and the using effect of the vaccines is influenced.

Disclosure of Invention

The problem to be solved by the invention is how to prepare the vaccine with high antigen content, large antigen batch and/or stable batch; how to avoid side reactions caused by bovine serum; and/or how to simplify immunization programs, reduce the number of immunizations of animals, and reduce the stress of animals.

The first object of the invention is to provide a preparation method of the combined inactivated vaccine of infectious bovine rhinotracheitis virus and mycoplasma bovis, which comprises the following steps:

1) culturing infectious bovine rhinotracheitis virus by using suspended MDBK cells to obtain infectious bovine rhinotracheitis virus liquid, and inactivating the infectious bovine rhinotracheitis virus liquid to obtain inactivated infectious bovine rhinotracheitis virus antigen liquid; culturing mycoplasma bovis by using a liquid culture medium to obtain mycoplasma bovis body fluid, and inactivating the mycoplasma bovis body fluid to obtain inactivated mycoplasma bovis antigen fluid;

the suspended MDBK cells were prepared according to a method comprising the steps of: subculturing adherent MDBK cells by using a low-serum suspension culture medium, and subculturing by using a serum-free suspension culture medium to obtain a serum-free suspension culture MDBK cell line which is good in growth performance and stable, namely the suspension MDBK cells;

the low serum suspension culture medium is a liquid culture medium with the volume content of fetal calf serum being 3%; the serum-free suspension culture medium is a serum-free liquid culture medium;

2) and mixing the inactivated infectious bovine rhinotracheitis virus antigen liquid and the inactivated mycoplasma bovis antigen liquid to obtain an active component, thereby obtaining the combined inactivated vaccine of infectious bovine rhinotracheitis virus and mycoplasma bovis.

In the method, the combined inactivated vaccine of the infectious bovine rhinotracheitis virus and the mycoplasma bovis is obtained by mixing the inactivated infectious bovine rhinotracheitis virus antigen liquid and the inactivated mycoplasma bovis antigen liquid as active ingredients and mixing the active ingredients and an adjuvant in a certain ratio.

In the above method, the adherent MDBK cells are bovine kidney cells (MDBK) that undergo adherent growth in a medium containing 10% fetal bovine serum.

In the preparation method, the IBRV can be specifically IBRV-SZH strain; specifically, the MB may be a Mycoplasma bovis PD strain.

In the method, the low serum suspension culture medium is a liquid culture medium obtained by adding fetal calf serum, penicillin and streptomycin into a full suspension culture medium of ST cells of the family Yi Sheng; the volume percentage content of fetal calf serum in the low serum suspension culture medium is 3%, the penicillin content is 100U/ml, and the streptomycin content is 0.1 mg/ml;

the serum-free suspension culture medium is a liquid culture medium obtained by adding penicillin and streptomycin into a full suspension culture medium of ST cells of the family Yi Sheng; in the serum-free suspension culture medium, the content of penicillin is 100U/ml, and the content of streptomycin is 0.1 mg/ml.

In the above method, the subculturing with the low serum suspension medium is subculturing for 7 to 15 generations (specifically 13 generations) with the low serum suspension medium, and the culturing is performed under suspension conditions; the subculturing by using the serum-free suspension culture medium is carried out for 25 to 45 generations (specifically 40 generations) by using the serum-free suspension culture medium under the suspension condition.

The suspension conditions were 100rpm (radius of rotation 15 mm).

The suspension is shake culture in a shaking table. The shaker may be a lapel scientific instruments (Beijing) Inc. model 0S-30 shaker.

In the above method, the density of the seeding of the cells for each passage is 1 × 10⁶cells/ml at 100rpm/min, 37 ℃ and 5% CO₂Culturing under the condition, and carrying out passage at intervals of 48-72 h.

In the above method, the mycoplasma bovis is cultured in a liquid medium prepared as follows (taking a 100ml system as an example): taking 2.1g PPLO broth culture medium, 2.5g yeast extract, and adding 80ml ddH₂O, autoclaving, followed by addition of 20ml of horse serum (centrifugation at 12000rpm/min for 10min, filtration through a 0.45 μm filter), addition of 100. mu.l of Amp (1g of ampicillin in 10ml of ddH)₂O, filtering by a 0.45 mu m filter and subpackaging). In the method, in the combined inactivated vaccine of infectious bovine rhinotracheitis virus and mycoplasma bovis, the content of mycoplasma bovis antigen is calculated by mycoplasma bovis before inactivation, the content of infectious bovine rhinotracheitis virus antigen is calculated by infectious bovine rhinotracheitis virus before inactivation, and the infectious bovine rhinotracheitis in the combined inactivated vaccine of infectious bovine rhinotracheitis virus and mycoplasma bovis is calculated by infectious bovine rhinotracheitisThe content of virus antigen is not less than 0.5 × 10⁷TCID₅₀The content of antigen of mycoplasma bovis is not less than 1 × 10/ml and⁸cfu/ml。

the second purpose of the invention is to provide the combined inactivated vaccine of the infectious bovine rhinotracheitis virus and the mycoplasma bovis, which is prepared by the method.

The third purpose of the invention is to provide a method for preparing suspension MDBK cells, which comprises the steps of subculturing adherent MDBK cells by using a low serum suspension culture medium, and subculturing by using a serum-free suspension culture medium to obtain suspension MDBK cells; the low serum suspension culture medium is a liquid culture medium with the volume content of fetal calf serum being 3%; the serum-free suspension culture medium is a serum-free liquid culture medium.

In the method, the low serum suspension culture medium is a liquid culture medium obtained by adding fetal calf serum, penicillin and streptomycin into a full suspension culture medium of ST cells of the family Yi Sheng; the volume percentage content of fetal calf serum in the low serum suspension culture medium is 3%, the penicillin content is 100U/ml, and the streptomycin content is 0.1 mg/ml;

the serum-free suspension culture medium is a liquid culture medium obtained by adding penicillin and streptomycin into a full suspension culture medium of ST cells of the family Yi Sheng; in the serum-free suspension culture medium, the content of penicillin is 100U/ml, and the content of streptomycin is 0.1 mg/ml.

In the above method, the subculturing with the low serum suspension medium is subculturing for 7 to 15 generations (specifically 13 generations) with the low serum suspension medium, and the culturing is performed under suspension conditions; the subculturing by using the serum-free suspension culture medium is carried out for 25 to 45 generations (specifically 40 generations) by using the serum-free suspension culture medium under the suspension condition.

The suspension conditions were 100rpm (radius of rotation 15 mm).

The suspension is shake culture in a shaking table. The shaker may be a lapel scientific instruments (Beijing) Inc. model 0S-30 shaker.

In the above method, the density of the seeding of the cells for each passage is 1 × 10⁶cells/ml at 100rpm/min, 37 ℃ and 5% CO₂Culturing under the condition, and carrying out passage at intervals of 48-72 h.

It is a fourth object of the present invention to provide suspended MDBK cells prepared by the above method.

It is a fifth object of the present invention to provide any one of the following uses of Y1-Y7:

y1, use of the suspension MDBK cells in suspension culture of viral antigens;

y2, and the application of the suspension MDBK cells in the suspension culture of IBRV antigen;

y3, use of the suspension MDBK cells in the preparation of a vaccine;

y4, the use of the suspended MDBK cells in the preparation of a vaccine containing an IBRV antigen;

y5 and the application of the suspension MDBK cells in the preparation of IBRV and MB combined inactivated vaccines.

The invention also provides a method for suspension culture of the IBRV virus solution, which is a method for suspension culture of the IBRV by using the suspension MDBK cells, wherein the virus inoculation amount of the IBRV is MOI (equal to 0.1), and the IBRV virus solution is obtained after virus inoculation for 72-96 h (namely when the cell death rate reaches about 60%).

The invention also provides the IBRV virus liquid prepared by the method.

The invention discloses a method for domesticating bovine kidney cells (MDBK) to grow in a serum-free suspension state from adherent growth, can ensure the stability of vaccines in different batches, saves economic cost and can produce high-quality biological products at the same time, the invention screens five culture mediums including the culture medium related to patent CN108570454A, selects a serum-free suspension culture medium more suitable for MDBK cell suspension growth, and domesticated suspension MDBK cells are 0.3 × 10⁶～1×10⁶Subculturing at initial density of cells/ml, wherein the density of MDBK cells can reach 6 × 10 after culturing for 48-72 h⁶～8×10⁶cell/ml cell density of 7 × 10 was achieved by optimizing culture conditions⁶～2.5 ×10⁷cells/ml, has more obvious advantages. Compared with the patents CN108570454A and CN107201334A, the domesticated compound of the inventionThe suspension MDBK cells also keep high sensitivity to pathogens such as IBRV and the like, and can be used for preparing vaccine virus antigens. On the basis, the invention innovatively invents a preparation method of the dual inactivated vaccine capable of preventing the infection of two pathogens, namely infectious bovine rhinotracheitis virus and mycoplasma bovis, and is used for preventing and controlling the two diseases clinically.

The invention provides a combined inactivated vaccine of infectious bovine rhinotracheitis virus and mycoplasma bovis, a preparation method thereof and a suspension MDBK cell used by the combined inactivated vaccine. The invention obtains the suspended MDBK cells through serum-free suspension domestication, and establishes a method for serum-free suspension culture of the IBRV antigen virus liquid on the basis, the IBRV antigen virus liquid obtained by the method has the characteristics of high antigen content, large antigen batch, stable batch and the like, the side reaction caused by bovine serum added into a culture medium is avoided, and the safer and more efficient vaccine can be produced while the economic cost is saved. Meanwhile, one needle of the IBRV and MB dual inactivated vaccine can prevent two diseases, simplifies the immunization procedure, reduces the immunization times of animals, reduces the stress of the animals, saves the labor force and further reduces the cost of animal breeding.

Drawings

FIG. 1 is a graph showing the results of screening serum-free suspension medium for adherent MDBK cells in example 1 of the present invention. Wherein A is treatment with ST cell suspension culture medium of Yi Sheng Ke; d is the treatment with CD MDBK 249 suspension medium and E is the treatment with OPM-MDBK SFM1 DPM suspension medium.

FIG. 2 is a line graph of cell density and mortality for adherent MDBK cells serially passaged in medium containing 3% FBS in example 1 of the present invention.

FIG. 3 is a line graph of cell density and mortality for serial passages of adherent MDBK cells in FBS-free medium in example 1 of the invention.

FIG. 4 is a photograph showing a comparison of the growth state of MDBK cells in the state of adherent MDBK cells and MDBK527 cells in the state of serum-free suspension culture (i.e., suspended MDBK cells) in the state of adherent MDBK cells in the state of adherent culture under an optical microscope in example 1 of the present invention.

FIG. 5 is a plot of the cell growth of MDBK527 in example 1 of the invention.

FIG. 6 is a photograph showing the results of the MDBK527 tumorigenic test mouse in example 1 of the present invention.

FIG. 7 is a growth curve of the suspension-cultured IBRV-SZH strain in example 2 of the present invention.

FIG. 8 is a differential significance analysis of the IBRV neutralizing antibody titers in guinea pig sera in example 2 of the present invention.

FIG. 9 is a graph showing the analysis of the difference in the antibody titer against MB in the serum of guinea pigs in example 2 of the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

1 molecular biological reagent

FBS (fetal bovine serum) (04-001-1ACS) in the following examples is a product of Biological Industries.

The formulation of the BEI solutions in the following examples is as follows: BEA (B8910, product of Solebao Biotechnology Co., Ltd.) was sufficiently dissolved in 0.175mol/L NaOH to prepare a BEA having a concentration of 0.1mol/L, and the BEA solution was placed in a 37 ℃ water bath, and when the temperature of the BEA solution was equilibrated to 37 ℃, the solution was shaken every 20min for 1 hour to obtain a 0.1mol/L BEI (diethylene imine) solution, which was then sterilized by filtration through a 0.2 μm filter. The preparation method of the 0.175mol/L NaOH comprises the following steps: 0.1g of sodium hydroxide was dissolved in 14.3ml of ddH₂O, preparing into 0.175mol/l sodium hydroxide solution.

The TN buffers in the following examples were prepared as follows: 0.12g Tris was dissolved in 96ml sterile deionized water, and 0.9g NaCl was added and dissolved, 0 was used.The pH value of the 1mol/L hydrochloric acid solution is adjusted to 7.2 +/-0.1. The preparation method of the 0.1mol/L hydrochloric acid solution comprises the following steps: 3.75ml of concentrated hydrochloric acid was dissolved in 41.25ml of ddH₂And O, preparing a 0.1mol/L hydrochloric acid solution.

Adjuvant 201 (referred to as MONTANIDE ISA 201VG throughout) (cat # 36075M) in the following examples is product of SEPPIC corporation.

PS (mixed liquid of penicillin and streptomycin, product number P140) is a product of Solebao biotechnology, Inc.

2 culture Medium

The ST cell full suspension culture medium (L10701) and the general-purpose full suspension culture medium (L11501) of the department of Yi Sheng in the following examples are all products of the company Aushenke (Shenzhen) Limited; the MDBK cell serum-free medium (FG0102503) is a product of Shanghai Kyoto Biotech, Inc.; CD MDBK 249(10502-249) is a product of Jianshun Biotechnology, Inc.; OPM-MDBK SFM1 DPM (V002201) is a product of Olymphami Biotech, Inc. of Shanghai; DMEM basal medium (C11995500BT) is a product of Gibco company; PPLO broth (LA7080) is a product of Solebao Biotechnology Ltd.

The low serum suspension medium in the following examples is a liquid medium obtained by adding fetal calf serum, penicillin and streptomycin to a whole suspension medium of ST cells of the family Yi Sheng; the volume percentage content of fetal calf serum in the low serum suspension culture medium is 3%, the penicillin content is 100U/ml, and the streptomycin content is 0.1 mg/ml. Penicillin and streptomycin were added in the form of PS (penicillin streptomycin mixture).

The serum-free suspension medium in the following examples is a liquid medium obtained by adding penicillin and streptomycin to a full suspension medium of ST cells of the family Yi Sheng; in the serum-free suspension culture medium, the content of penicillin is 100U/ml, and the content of streptomycin is 0.1 mg/ml. Penicillin and streptomycin were added in the form of PS (penicillin streptomycin mixture).

The adherent cell complete medium in the following examples is a medium obtained by adding FBS, penicillin and streptomycin to a DMEM basal medium; in the complete culture medium of the adherent cells, the content of FBS is 10 percent, the content of penicillin is 100U/ml, and the content of streptomycin is 0.1 mg/ml.

The adherent cell maintenance solution in the following examples is a liquid medium obtained by adding FBS to a DMEM basal medium. The content of FBS in the adherent cell maintenance liquid is 2%.

In the following examples, a mycoplasma bovis liquid medium was prepared as follows (taking a 100ml system as an example): taking 2.1g PPLO broth culture medium, 2.5g yeast extract, and adding 80ml ddH₂O, autoclaving, followed by addition of 20ml of horse serum (Brand: Congbo, centrifugation at 12000rpm/min for 10min, filtration through a 0.45 μm filter) and 100. mu.l of Amp solution (1g of ampicillin in 10ml of ddH)₂O, filtering by a 0.45 mu m filter and subpackaging).

In the following examples, mycoplasma bovis solid medium was prepared as follows (taking 100ml system as an example): taking 2.1g PPLO broth culture medium, 2.5g yeast extract, 1g agar, adding 80ml ddH₂O, autoclaving, then adding 100. mu.l Amp solution (1g ampicillin in 10ml ddH)₂O, filtering with a 0.45-micron filter and subpackaging), adding 20ml of horse serum when cooling to about 60-70 ℃, and pouring out a plate which is overheated or cooled to be turbid.

3 cell line

Adherent MDBK cells in the following examples are bovine kidney cells (MDBK) which are adherently grown in a medium containing fetal bovine serum, and the cell line is described in non-patent document "construction of a lipaca. mycoplasma bovis expression vector [ D ]. university of chinese agriculture, 2015", which is publicly available from the university of chinese agriculture to repeat the experiments of the present application and is not usable for other purposes.

The HeLa cells (numbered CL16) in the following examples are products of the China center for veterinary culture Collection.

The ST cells (No. CL27) in the following examples are products of the chinese veterinary microbial culture collection center.

The mycoplasma bovis PD strain in the following examples is described in non-patent document "liu meng yao, zuki, wu literature, li jin xiang, dual real-time fluorescent quantitative PCR detection method for BVDV and IBRV" china veterinary journal, 2019(55)11, 28-36 ", publicly available from the university of agriculture in china to repeat the experiments of this application, and cannot be used for other purposes.

4 Virus strains

The bovine infectious rhinotracheitis virus SZH strain (hereinafter referred to as IBRV-SZH strain) in the following examples is described in non-patent literature "Liu Meng Yao, Chun nan, Wu Wen, Li jin Xiang, BVDV and IBRV double real-time fluorescence quantitative PCR detection method establishment, China veterinary journal, 2019(55)11, 28-36", publicly available from China agricultural university to repeat the experiments of the application, and can not be used for other purposes.

5 Experimental animals

The athymic mice in the following examples are standard strains and are products of experimental animal technology ltd of viton.

In the following examples, healthy susceptible cattle are Holstein hybrid cattle purchased from Erhua livestock farming farmer's professional cooperative, Zhenhua 361699, Jilin province.

Healthy susceptible female guinea pigs were standard strains purchased from the Xinglong laboratory animal breeders in Hakko area, Beijing, in the following examples.

The shaker used in the examples described below was a model 0S-30 shaker from Leptott scientific instruments (Beijing) Inc.