Bovine parainfluenza virus type 3 positive serum as well as preparation method and application thereof

Abstract

The invention provides bovine parainfluenza virus type 3 positive serum as well as a preparation method and application thereof, bovine parainfluenza virus type 3 inactivated vaccine and inactivated antigen liquid are sequentially inoculated to stimulate cattle to generate high-titer bovine parainfluenza virus type 3 positive serum, and the titer of the bovine parainfluenza virus type 3 positive serum is not lower than 1: 1024. Compared with a conventional scheme, the method has the advantages that the high-titer positive serum of the bovine parainfluenza virus type 3 can be obtained only through twice immunization, three or more immunization schemes are not needed, virus stock solution challenge is not needed, and high-efficiency preparation of the positive serum product of the bovine parainfluenza virus type 3 can be realized.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary biological products, and in particular to a bovine parainfluenza virus type 3 positive serum and a preparation method and application thereof. Background Art

[0002] Studies have shown that bovine infectious rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV3), bovine viral diarrhea virus (BVDV) and bovine respiratory syncytial virus (BRSV) are the main viral pathogens known to cause bovine respiratory infectious diseases. Among them, bovine parainfluenza virus type 3 is one of the main pathogens causing calf respiratory diseases. It is currently widely prevalent in countries around the world, and is constantly occurring or prevalent in countries in the Americas, Europe and Asia.

[0003] In recent years, severe calf respiratory disease syndrome has occurred in many provinces in my country, including Shandong, Heilongjiang, Liaoning and Inner Mongolia, causing great economic losses. Epidemiological investigations and etiological tests have shown that BPIV3 plays an important role in calf respiratory disease syndrome. The incubation period of cattle infected with BPIV3 is generally 2 to 5 days. Sick cattle have elevated body temperatures, which can reach above 41°C, loss of appetite, lethargy, tearing, sticky nasal discharge, and purulent conjunctivitis. BPIV3 infection can progressively cause tissue damage and immunosuppression in cattle, and can be secondary to bacterial or mycoplasma infection, thereby causing severe bronchopneumonia, leading to respiratory disease syndrome, and greatly increasing the mortality rate. Serological epidemiological surveys have shown that the serum positive rate of BPIV3 in some parts of my country is as high as 91.08%, which seriously endangers the development of my country's cattle breeding industry. The development of its diagnostic products and serum products has become a top priority. At present, there is a lack of commercialized bovine parainfluenza virus type 3 high-immune serum on the market, and there are no reports on the preparation method of bovine parainfluenza virus type 3 specific high-titer positive serum. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] The invention provides a bovine parainfluenza virus type 3 positive serum and a preparation method and application thereof, aiming to provide a high-titer bovine parainfluenza virus type 3 positive serum, which can be used for specificity testing, identification testing, hemagglutination inhibition experiment exogenous virus testing, diagnosis testing and the like of bovine parainfluenza virus type 3 vaccine.

[0006] (II) Technical solution

[0007] The first aspect of the present invention provides a method for preparing bovine parainfluenza virus type 3 positive serum, comprising: a first immunization, selecting cattle that are negative for bovine parainfluenza virus type 3 antibodies, foot-and-mouth disease virus antibodies, bovine viral diarrhea-mucosal disease virus antibodies, bovine infectious rhinotracheitis antibodies, and bovine typhoid fever antibodies, and inoculating each cattle with a bovine parainfluenza virus type 3 inactivated vaccine, wherein each cattle is inoculated with 2 ml of the vaccine; a second immunization, 21 days after the first immunization, continuously inoculating the cattle with a bovine parainfluenza virus type 3 inactivated vaccine, wherein each cattle is inoculated with 2 ml of the vaccine; and collecting bovine parainfluenza virus type 3 positive serum 14 days after the second immunization, and harvesting the bovine parainfluenza virus type 3 positive serum when the titer of the bovine parainfluenza virus type 3 positive serum is not less than 1:1024.

[0008] Furthermore, the method for preparing the inactivated vaccine in the first aspect comprises:

[0009] Step 1, preparation of seed virus for vaccine production: using MDBK cells to culture bovine parainfluenza virus type 3 strain in suspension to obtain seed virus;

[0010] Step 2, inoculating the seed virus obtained in step 1 into MDBK suspension cells at a ratio of 2‰ to 5‰ V / V of the culture medium, and culturing the suspension for 48 to 72 hours to obtain a virus solution;

[0011] Step 3, inactivating the virus solution obtained in step 2 to obtain an inactivated virus solution;

[0012] Step 4, using the inactivated virus liquid in step 3 to emulsify and prepare an inactivated bovine parainfluenza virus type 3 vaccine.

[0013] Preferably, the deposit number of the bovine parainfluenza virus type 3 strain is CGMCC No.21115.

[0014] Furthermore, in step 2 of the above preparation method, the cell density is adjusted to 2.0×10 6 / ml, the virus was inoculated at 2‰ of the culture volume, and the culture parameters were set as a rotation speed of 80 rpm, a temperature of 37°C, a dissolved oxygen content of 40%, and a pH value of 7.4.

[0015] Furthermore, in step 3 of the above preparation method, the virus liquid is centrifuged to obtain the antigen, and then β-propiolactone is added according to the volume ratio of β-propiolactone:antigen=1:4000, and the mixture is inactivated at 4°C for 28 hours. The inactivated liquid is harvested and hydrolyzed at 37°C for 2 hours and centrifuged.

[0016] Furthermore, after the centrifugal treatment of the inactivated virus liquid, an antigen concentration and purification step is also included: the inactivated virus liquid after centrifugation is concentrated to remove foreign proteins, the hemagglutination titer of the concentrated virus antigen is not less than 1:256, and then step 4 is entered.

[0017] Furthermore, the antigen and 206 adjuvant were emulsified at a mass ratio of 1:1 at a temperature of 32-34°C.

[0018] Furthermore, the harvested bovine parainfluenza virus type 3 positive serum was first pressure filtered through a 0.45 μm sterile filter membrane, then pressure filtered through a 0.22 μm sterile filter membrane, and then freeze-dried and stored.

[0019] In a second aspect, the present invention also provides a bovine parainfluenza virus type 3 positive serum prepared by the preparation method.

[0020] Furthermore, the present invention also includes the use of the bovine parainfluenza virus type 3 positive serum in the following ways: preparing reagents or kits for testing or identifying bovine parainfluenza virus; specific testing, identification testing, hemagglutination inhibition test, exogenous virus testing or diagnosis of bovine parainfluenza virus; preparing bovine parainfluenza virus serum standard products. Compared with conventional schemes, the present invention only requires two immunizations to obtain high-titer bovine parainfluenza virus type 3 positive serum, without the need to perform three or more immunization schemes, and without the need to perform virus stock solution attack, thereby realizing efficient preparation of bovine parainfluenza virus type 3 positive serum products.

[0021] (III) Beneficial effects

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a bovine parainfluenza virus type 3 positive serum and a preparation method thereof, and obtains a high-titer bovine parainfluenza virus type 3 positive serum, the positive serum titer of which is greater than 1:1024, and can be used for specific testing, identification testing, hemagglutination inhibition experiment exogenous virus testing, and diagnostic testing of bovine parainfluenza virus type 3 vaccines. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The methods used in the following examples are conventional methods unless otherwise specified. The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0025] Example 1: Preparation of inactivated vaccine

[0026] 1.1 Isolation and identification of the virus. The implementation steps are detailed in Example 1 in Parts 0027 to 0056 of the specific implementation method in the prior art (CN113322241A), which will not be repeated here. The obtained bovine parainfluenza virus type 3 strain (deposit number: CGMCC No. 21115) was used to prepare an inactivated bovine parainfluenza virus type 3 vaccine.

[0027] 1.2 Preparation of inactivated bovine parainfluenza virus type 3 vaccine

[0028] Specifically, it is prepared in the following manner:

[0029] Step 1, preparation of seed virus for vaccine production: using MDBK cells to culture bovine parainfluenza virus type 3 strain in suspension to obtain seed virus (see the record in paragraph 0059 of the prior art CN113322241A for details, which will not be repeated here);

[0030] Step 2, the seed virus obtained in step 1 is inoculated into MDBK suspension cells at a ratio of 2‰ to 5‰ V / V of the culture medium (2‰ in this embodiment), and the suspension culture is carried out for 48 to 72 hours to obtain a virus solution (see the records of paragraphs 0067 to 0071 in the prior art CN113322241A for details, which will not be repeated here. In this embodiment, only the culture parameters are adjusted, that is, the culture parameters are set to a speed of 80 rpm, a temperature of 37° C., a dissolved oxygen content of 40%, and a pH value of 7.4). The antigen culture conditions are shown in Table 1, and then the antigen is centrifuged after being frozen and thawed 3 times;

[0031] Table 1 Antigen culture

[0032]

[0033] Step 3, first, the virus liquid obtained in step 2 is inactivated, and after centrifugation, β-propiolactone is added to the antigen according to the volume ratio of β-propiolactone: antigen = 1:4000, and the mixture is placed at 4°C for inactivation for 28h. After the inactivated liquid is harvested, it is hydrolyzed at 37°C for 2h, centrifuged, and inactivated after centrifugation. The inactivated liquid is inoculated into a well-grown MDBK monolayer cell at a ratio of 10% (V / V) of the culture liquid, and a blank cell control is set at the same time. The cells are placed in an incubator at 37°C and containing 5% CO2 for 5 days, and observed daily. The cells are blindly propagated for 2 generations according to the above method to observe whether CPE is produced in each generation of the culture. The experimental results show that CPE does not appear in all 3 generations, proving that the liquid is completely inactivated (see Table 2), and an inactivated virus liquid is obtained (see the records of paragraphs 0074 to 0075 in the prior art CN113322241A for details, which will not be repeated here);

[0034] Table 2 Inactivation test results

[0035]

[0036] Then, the inactivated virus liquid was subjected to an antigen concentration and purification step, and the inactivated virus liquid was concentrated 5 times by a 500kd hollow fiber column to remove foreign proteins, and the protein content and hemagglutination titer before and after concentration were detected. As shown in Tables 3 and 4, the protein removal rate was as high as 88.8%, and the hemagglutination titer of the inactivated antigen (inactivated virus liquid) was above 80% was 1:128, and the hemagglutination titer of the concentrated antigen (inactivated virus liquid) was 1:256. After 5 times concentration, the hemagglutination titer was increased by 1 titer.

[0037] Table 3 Protein removal results

[0038]

[0039] Table 4 Hemagglutination titer results

[0040]

[0041] Step 4, using the inactivated virus liquid in step 3 for emulsification and vaccine preparation to prepare a bovine parainfluenza virus type 3 inactivated vaccine, the concentrated antigen obtained in step 3 and ISA206 adjuvant (purchased from Seppic, France) are emulsified at a mass ratio of 1:1 at a temperature of 32 to 34°C, and physical and chemical tests are performed after emulsification to obtain Table 5. All physical and chemical tests after emulsification are qualified, thereby obtaining a qualified inactivated vaccine.

[0042] Table 5 Physical and chemical test results of vaccines

[0043]

[0044] Example 2: Collection of bovine parainfluenza virus type 3 positive serum

[0045] 2.1 For the first immunization, select cattle (5 in this embodiment) that are negative for bovine parainfluenza virus type 3 antibody, foot-and-mouth disease virus antibody, bovine viral diarrhea-mucosal disease virus antibody, bovine infectious rhinotracheitis antibody, and bovine epidemic fever antibody, and vaccinate them with the bovine parainfluenza virus type 3 inactivated vaccine prepared in the above embodiment 1, and inject 2 ml into the buttocks muscle of each cattle each time;

[0046] 2.2 Second immunization: 21 days after the first immunization, cattle were vaccinated with inactivated bovine parainfluenza virus type 3 vaccine, 2 ml per cow per intramuscular injection in the buttocks;

[0047] 2.3 Collect blood on days 7, 14 and 21 after the second immunization and separate serum for neutralizing antibody testing.

[0048] 2.4 Neutralizing antibody detection

[0049] 2.4.1 Serum inactivation

[0050] Place the serum to be tested in a constant temperature water bath and inactivate at 56°C for 30 minutes. The volume of serum to be tested should not be less than 660 μl.

[0051] 2.4.2 Sample dilution

[0052] Use a pipette to draw up the cell maintenance medium (DMEM solution) and add it to a 96-well plate, 50 μl per well, discard the pipette tip, and then use a pipette to draw up 50 μl of the inactivated serum to be tested and add it to the corresponding wells of the 96-well microcell plate. After mixing evenly, take 50 μl and add it to the corresponding wells of the next dilution, and so on, make a 2-fold serial dilution to the required dilution, ensuring that the final content of each well is 50 μl, and each dilution has 4 wells.

[0053] 2.4.3 Virus dilution

[0054] The virus solution was stored in a -70°C refrigerator and the virus titer was determined as 200 TCID 50 / 0.1ml dilution.

[0055] 2.4.4 Neutralization

[0056] Add the diluted virus solution in 2.4.3 to the serum dilution plate in 2.4.2, 50 μl per well, mix well with a microvibrator, and neutralize in a 5% CO2 incubator at 37°C for 1 hour.

[0057] 2.4.5 Add cell suspension

[0058] After the serum and virus were neutralized for 1 hour, the 96-well plate was taken out, 100 μl of MDBK cell suspension was added to each well, and the plate was placed in a 5% CO2 incubator at 37°C for culture.

[0059] 2.4.6 Control Establishment

[0060] 2.4.6.1 Negative and positive sera

[0061] The positive and negative control serum was diluted according to its titer. Four wells were set for each dilution, with 50 μl per well. 200 TCID 50 / 0.1ml virus solution 50μl for neutralization, and finally add 100μl cell suspension.

[0062] 2.4.6.2 Virus regression test

[0063] 200TCID 50 / 0.1ml virus solution as 100TCID 50 / 0.1ml, 10TCID 50 / 0.1ml, 1TCID 50 / 0.1ml, 0.1TCID 50 / 0.1ml 4 titer dilutions, 8 wells for each dilution, 100μl virus solution per well, and then add 100μl MDBK cell suspension.

[0064] 2.4.6.3 Cell Control

[0065] Set up 4 to 8 wells of normal cell control, add 100 μl of DMEM cell maintenance solution to each well, and add 100 μl of MDBK cell suspension.

[0066] 2.4.7 Result determination

[0067] 2.4.7.1 Test conditions

[0068] (1) 100TCID 50 / 0.1ml should be all holes lesion, that is, 8 / 8 holes lesion, 0.1TCID 50 / 0.1ml should have no lesions in all holes;

[0069] (2) All cell control wells should be free of pathological changes;

[0070] (3) The neutralizing antibodies in positive and negative serum should be consistent with their titers.

[0071] 2.4.7.2 Result calculation

[0072] The cytopathic effect (CPE) was observed and recorded daily. After 5 days of culture, the number of serum lesion wells to be tested was counted, and the serum neutralizing antibody titer was calculated according to the Reed-Muench method.

[0073] Referring to Table 6, the test results show that the antibody value of the serum reached its peak 14 days after the second immunization. After the test, a large amount of blood was collected at the peak to prepare bovine parainfluenza virus type 3 positive serum. The titer of the obtained bovine parainfluenza virus type 3 positive serum was not less than 1:1024.

[0074] Table 6 Positive serum antibody test results

[0075]

[0076] 2.5 Blood collection and preparation of positive serum

[0077] Blood was collected 14 days after the second immunization, and the serum was pressure-filtered through a 0.45μm sterile filter membrane and then pressure-filtered through a 0.22μm sterile filter membrane; it was then aseptically divided into 5ml vials, 2ml per vial, lyophilized by conventional methods, and stored at -70℃.

[0078] The prepared bovine parainfluenza virus type 3 positive serum can be used in the following ways: (1) preparing reagents or kits for testing or identifying bovine parainfluenza virus; (2) specific testing, identification testing, hemagglutination inhibition test, exogenous virus testing or diagnosis of bovine parainfluenza virus; (3) preparing bovine parainfluenza virus serum standard.

[0079] Comparative Example 1: Neutralizing antibody titer after one immunization

[0080] The secondary immunization scheme of steps 2.1 to 2.3 in Example 2 is replaced by a single immunization scheme, and the target serum is harvested and the neutralizing antibody titer is detected by step 2.4.

[0081] Primary immunization scheme: select cattle (4 in this comparative example, obtained from the same batch as the cattle in Example 2) that are negative for bovine parainfluenza virus type 3 antibody, foot-and-mouth disease virus antibody, bovine viral diarrhea-mucosal disease virus antibody, bovine infectious rhinotracheitis antibody, and bovine epidemic fever antibody) and vaccinate the bovine parainfluenza virus type 3 inactivated vaccine prepared in Example 1, divide the cattle into 2 groups, 2 in each group, one group is injected with 2 ml of vaccine in the buttocks muscle, and the other group is injected with 4 ml of vaccine in the buttocks muscle. Blood was collected 7, 14, and 21 days after the first immunization to detect the neutralizing antibody titer. The neutralizing antibody results are shown in Table 7, and the neutralizing antibody value in the table is the average value of the 2 cattle.

[0082] Table 7 Neutralizing antibody values ​​after immunization

[0083]

[0084] Comparative Example 2: Neutralizing antibody titer after three immunizations

[0085] The three-immunization immunization scheme is used to replace the two-immunization scheme of steps 2.1 to 2.3 in Example 2, and the target serum is harvested and the neutralizing antibody titer is detected according to step 2.4.

[0086] The cattle (two cattle in this comparative example, obtained from the same batch as the cattle in Example 2) with negative bovine parainfluenza virus type 3 antibody, foot-and-mouth disease virus antibody, bovine viral diarrhea-mucosal disease virus antibody, bovine infectious rhinotracheitis antibody, and bovine epidemic fever antibody were selected for inoculation with the bovine parainfluenza virus type 3 inactivated vaccine prepared in Example 1, and each cattle was injected with 2 ml of the buttocks muscle each time. The second immunization was performed 21 days after the first immunization, and the third immunization was performed 14 days after the second immunization. Blood was collected at 7d, 14d, and 21d after the third immunization to detect the neutralizing antibody titer. The test results are shown in Table 8.

[0087] Table 8 Neutralizing antibody values ​​after three immunizations

[0088]

[0089] The comparison results show that the neutralizing antibody value cannot reach the peak value in one immunization, whether it is 2ml or double the dosage immunization, and the antibody level after three immunizations is consistent with the result of the second immunization, except that the duration of the antibody peak is longer than that of the two immunizations. However, the three immunizations increase the experimental time and the experimental cost. Therefore, the present invention adopts the secondary immunization shown in Example 2, and blood is collected 14 days after the second immunization to prepare high-titer bovine parainfluenza virus type 3 positive serum. Compared with the conventional scheme, the present invention only needs 2 immunizations to obtain high-titer bovine parainfluenza virus type 3 positive serum, and there is no need to perform three or more immunization schemes, and there is no need to perform virus stock solution attack, which can achieve efficient preparation of bovine parainfluenza virus type 3 positive serum products.

[0090] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing bovine parainfluenza virus type 3 positive serum, characterized in that: include: For the first immunization, select cattle that are negative for bovine parainfluenza virus type 3 antibody, foot-and-mouth disease virus antibody, bovine viral diarrhea-mucosal disease virus antibody, bovine infectious rhinotracheitis antibody, and bovine epidemic fever antibody and vaccinate them with bovine parainfluenza virus type 3 inactivated vaccine, 2 ml per cattle; The second immunization, 21 days after the first immunization, continued to vaccinate cattle with inactivated bovine parainfluenza virus type 3 vaccine, 2 ml per cattle; Fourteen days after the second immunization, the bovine parainfluenza virus type 3 positive serum was collected. When the titer of the bovine parainfluenza virus type 3 positive serum was not less than 1:1024, the bovine parainfluenza virus type 3 positive serum was harvested.

2. The preparation method according to claim 1, characterized in that: The preparation method of the inactivated vaccine comprises: Step 1, preparation of seed virus for vaccine production: using MDBK cells to culture bovine parainfluenza virus type 3 strain in suspension to obtain seed virus; Step 2, inoculating the seed virus obtained in step 1 into MDBK suspension cells at a ratio of 2‰ to 5‰ V / V of the culture medium, and culturing the suspension for 48 to 72 hours to obtain a virus solution; Step 3, inactivating the virus solution obtained in step 2 to obtain an inactivated virus solution; Step 4, using the inactivated virus liquid in step 3 to emulsify and prepare an inactivated bovine parainfluenza virus type 3 vaccine.

3. The preparation method according to claim 2, characterized in that: The deposit number of the bovine parainfluenza virus type 3 strain is CGMCC No.21115.

4. The preparation method according to claim 2, characterized in that: In step 2, the cell density was adjusted to 2.0×10 6 / ml, the virus was inoculated at 2‰ of the culture volume, and the culture parameters were set as a rotation speed of 80 rpm, a temperature of 37°C, a dissolved oxygen content of 40%, and a pH value of 7.

4.

5. The preparation method according to claim 2, characterized in that: In step 3, the virus solution is centrifuged to obtain the antigen, and then β-propiolactone is added according to the volume ratio of β-propiolactone:antigen=1:4000, and the mixture is inactivated at 4°C for 28 hours. The inactivated solution is harvested and hydrolyzed at 37°C for 2 hours and centrifuged.

6. The preparation method according to claim 5, characterized in that: After the inactivated virus liquid is centrifuged, the step of antigen concentration and purification is also included: the inactivated virus liquid after centrifugation is concentrated to remove foreign proteins, the hemagglutination titer of the concentrated virus antigen is not less than 1:256, and then step 4 is entered.

7. The preparation method according to claim 2, characterized in that: The antigen and 206 adjuvant were emulsified at a mass ratio of 1:1 at a temperature of 32-34°C.

8. The preparation method according to claim 1, characterized in that: The harvested bovine parainfluenza virus type 3 positive serum was first pressure filtered through a 0.45 μm sterile filter membrane, then pressure filtered through a 0.22 μm sterile filter membrane, and then freeze-dried and stored.

9. A bovine parainfluenza virus type 3 positive serum prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the bovine parainfluenza virus type 3 positive serum as claimed in claim 9 in the following manner: (1) preparing reagents or kits for testing or identifying bovine parainfluenza virus; (2) Specificity test, identification test, hemagglutination inhibition test, exogenous virus test or diagnosis for bovine parainfluenza virus; (3) Preparation of bovine parainfluenza virus serum standard.

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