CN113215110A - Method for improving multiplication capacity of African swine fever virus - Google Patents
Method for improving multiplication capacity of African swine fever virus Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/12011—Asfarviridae
- C12N2710/12051—Methods of production or purification of viral material
Abstract
The invention provides a method for improving the multiplication capacity of African swine fever virus, belonging to the field of microorganism application. The method comprises the following steps: s1, inoculating the cell suspension of the primary alveolar macrophages of the pigs into a cell culture plate, and standing for 3-5 hours until the cells adhere to the wall; s2, uniformly mixing the African swine fever virus liquid and the Jielian bond diluent, incubating for 0-4 h at 24-26 ℃, and adding a culture medium to obtain a mixed liquid; replacing the culture solution in the cell culture plate with the obtained mixed solution, and culturing for 1.5-2.5 h; and S3, replacing the culture solution in the cell culture plate with a culture medium containing 10% fetal calf serum again, and culturing for 4-6 d. The method can enhance the infection efficiency of the African swine fever virus, save the preparation time and cost of primary alveolar macrophages of pigs in a laboratory, and provide basic data support for efficiently carrying out isolation, epidemic situation monitoring and vaccine research and development and production of the African swine fever virus.
Description
Technical Field
The invention belongs to the field of microorganism application and relates to a virus culture technology, in particular to a method for improving the multiplication capacity of African swine fever virus.
Background
African Swine Fever (ASF) is an acute, febrile and hemorrhagic swine infectious disease caused by African Swine Fever Virus (ASFV), and the acute infection mortality rate is 100%. The ASF is listed as one of legal animal epidemic diseases which must be reported by the world animal health Organization (OIE), and is listed as an animal epidemic disease in China. ASFV is a enveloped single-molecule linear double-stranded DNA virus, belongs to the African swine fever virus family (Asfarviridae) and the African swine fever virus genus (Asfivirus) in the virus classification, and is the only arbovirus known at present. The ASFV genome has a total length of about 170-190 kb and 151-167 Open Reading Frames (ORFs). ASF exhibits a variety of clinical phenotypes, with acute and subacute ASF often exhibiting severe vasculogenic lesions in a variety of organs, such as renal hemorrhage, diffuse bleeding of lymph nodes, pulmonary edema, congestive enlargement of the spleen, disseminated intravascular coagulation, thrombocytopenia, and the like. The main clinical symptoms are high fever, skin cyanosis, bleeding spots, vomiting, diarrhea, hematochezia and the like.
In 1921, ASF was first discovered in the kenya region of africa. In 1957, the first outbreak of ASF epidemic in european portugal was followed by some time of silence, which appeared and spread gradually in some countries in europe in 1960, and entered russia at the end of 2007. In recent years, epidemic situations occur in eastern Europe and some countries, and the international epidemic situation is very severe. In 2018, 8 and 3, the department of agricultural rural areas publishes the first ASF epidemic situation of our country in Liaoning province, so that great harm is brought to the pig raising industry of our country for two years, the production and trade round of pig products is greatly limited, and meanwhile, great economic loss is caused to relevant industries of live pigs.
Studies have shown that the ASFV infection cycle begins with viral attachment and entry into the host cell. Early studies found that ASFV invades porcine alveolar macrophages through a receptor-mediated endocytosis mechanism, and the ASFV invades porcine alveolar macrophages and is low in pH dependence and temperature dependence. At present, most ASFV strains can only replicate and proliferate in macrophages (such as alveolar macrophages) and monocytes derived from peripheral blood or various tissues, except that individual cell adapted strains, such as BA71V, Georgia2007/1 and other virus strains, can replicate in Vero cells of African green monkey. These primary cells are particularly valuable in laboratories due to their expensive preparation cost, tedious preparation process and time consuming. The characteristic is that the cost is increased for the in vitro detection of the ASFV live virus, and the difficulty of virus-related research is increased. In addition, the ASF epidemic situation monitoring mainly comprises ASFV nucleic acid detection and virus separation, and the ASF virus separation is an important basis for ASF etiology monitoring, can find the variation condition of the virus in time and plays an important role in virus vaccine development. Therefore, the improvement of the proliferation efficiency of the ASFV provides an important basis for the research of virus mechanism, the epidemiological monitoring of epidemic situation, the research and development of vaccine and the production.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for improving the multiplication capacity of the African swine fever virus. The method has low cost and easy operation.
A method for improving the multiplication capacity of African swine fever virus comprises the following steps:
s1, inoculating the cell suspension of the primary alveolar macrophages of the pigs into a cell culture plate, and standing for 3-5 hours until the cells adhere to the wall;
s2, uniformly mixing the African swine fever virus liquid and the Jielian bond diluent, incubating for 0-4 h at 24-26 ℃, and adding a culture medium to obtain a mixed liquid; replacing the culture solution in the cell culture plate with the obtained mixed solution, and culturing for 1.5-2.5 h;
and S3, replacing the culture solution in the cell culture plate with a culture medium containing 10% Fetal Bovine Serum (FBS) again, and culturing for 4-6 d.
The cell density of the cell suspension of porcine primary alveolar macrophages described in step S1 was 107mL, the reagents used for resuspension were RPMI1640 medium containing 10% fetal bovine serum.
The time for the standing described in step S1 is preferably 4 hours.
The conditions for the standing described in step S1 are preferably 37 ℃ and 5% CO2。
TCID of African swine fever virus liquid described in step S250Is 1040.1mL, and the dilution ratio of the Jielian bond diluent is 1:2000 (namely, the Jielian bond: water is 1g:2000 mL); the volume ratio of the African swine fever virus liquid to the Jielian bond diluent to the culture medium is 57: 3: 240. the water used for the acid bond cleaning dilution is distilled water. The mixing according to the proportion can ensure that primary alveolar macrophages of pigs can grow normally, and simultaneously ensure that ASFV can replicate in cells.
The pH of the acid bond cleaning diluent described in step S2 was 2.75.
The medium described in steps S2 and S3 is preferably RPMI1640 medium.
The incubation time described in step S2 is preferably 0 h.
The time for the incubation described in step S2 is preferably 2 h.
The time period of the incubation described in step S3 is preferably 5 d.
The conditions of the cultivation described in steps S2 and S3 were 37 ℃, 5% CO2。
Compared with the prior art, the invention has the following beneficial effects:
according to the method, the ASFV proliferation condition is optimized, the optimal room-temperature incubation time is screened out, and the separation effect of the ASFV is effectively improved. By utilizing the technology, the proliferation efficiency of ASFV can be improved, the time and the cost for preparing primary alveolar macrophages of pigs in a laboratory are saved, the working efficiency and the monitoring quality of ASF monitoring are improved in the epidemiological research of ASF, and basic data are provided for the control of the epidemic situation of ASF and the preparation of vaccines.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Unless otherwise specified, the following examples were carried out using conventional laboratory equipment, and materials and reagents used therein were commercially available.
The Jielian bond is purchased from Guangdong Teng Jun biological nutrition science and technology company, and the product standard number is as follows: Q/TJ 28-2019.
The ASFV virus is a preserved strain ASFV/China/GZ201801 of 'China African swine fever regional laboratory (Guangzhou)' and is disclosed in a patent CN 110964849A.
Alveolar macrophages are derived from healthy piglets which are negative in antigen-antibody detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and porcine pseudorabies virus (PRV). The preparation method comprises the following steps: approximately 2L of PBS containing 2 × streptomycin per lung was pre-cooled. High-pressure steam sterilization is carried out on a plurality of 1L beakers, blades, scissors, hemostatic forceps, tweezers and gauzes. Dissecting a 4-week-old blue-ear pseudorabies double-negative pig until the trachea is exposed, carefully peeling off blood vessels and tissues around the trachea to avoid blood pollution, fixing the trachea by using two hemostatic forceps in an opposite direction, and cutting off redundant trachea above the hemostatic forceps by using a blade; the lungs were removed from the pig's chest cavity with lung integrity and quickly placed in pre-cooled PBS containing 2 × streptomycin to keep the lungs moist. And (5) switching to a biological safety cabinet for operation: slowly infusing precooled 2 Xqingstreptomycin PBS into the lung through the trachea, and gently patting and massaging until the whole lung is full and the liquid level of the lavage liquid at the trachea can not be reduced any more; transferring the lung lavage fluid into a 50mL centrifuge tube through a 70 mu M cell filter, and centrifuging for 10min at 500g and 4 ℃; the supernatant was discarded, the cells were resuspended in 5mL PBS and 9 tubes of cells were pooled and centrifuged at 500g for 10min at 4 ℃. The supernatant was discarded, the cells were resuspended in 10mL RPMI1640 medium, and finally pooled into one tube for each lung lavage; the cells of each lung were counted at (2-4). times.107The cell density of (a) was resuspended in a cell freezing medium and frozen in a freezer at-80 ℃.
Example 1
(1) The pH value was measured after diluting the acid bond to 1:2000 with distilled water.
(2) Taking out the cell freezing tube from the liquid nitrogen tank, directly immersing the cell freezing tube into a 37 ℃ constant-temperature water bath kettle, and shaking the cell freezing tube to melt the cell freezing tube as soon as possible without any time; after melting, opening a cover, transferring the cell suspension into a centrifuge tube, adding more than 10 times of RPMI1640 culture medium, and uniformly mixing; centrifuging at 800g for 5min, discarding supernatant, adding 10% FBS-containing RPMI1640 culture medium to resuspend cells, counting, and adjusting cell density to 107After one mL, the cells were seeded in 24-well cell plates and placed at 37 ℃ in 5% CO2Culturing for 4h in an incubator until the cells adhere to the wall. Respectively setting blank control group (570 μ L distilled water +30 μ L1640 culture medium), disinfectant negative control group (570 μ L concentration is 1:2000 acid bond +30 μ L RPMI1640 culture medium), disinfectant test group (570 μ L concentration is 1:2000 acid bond +30 μ L ASFV virus solution), and positive control group (570 μ L distilled water +30 μ L ASFV virus solution), mixing, and incubating at 24-26 deg.C for 0h (ASFV virus solution TCID)50＝104/0.1mL)。
(3) 2400 μ L of RPMI1640 medium was added to each test group to allow normal growth of porcine primary alveolar macrophages while ensuring that ASFV can replicate in cells. The culture medium in the 24-well cell plate was aspirated, and the mixed solution of the above four test groups was added to the corresponding wells, respectively, to inoculate 3 wells per group, 500. mu.L per well. Then placed at 37 ℃ with 5% CO2Culturing in an incubator. After 2h, the maintenance solution (RPMI 1640 medium containing 10% FBS) was changed, and 500. mu.L of the solution was added to each well. Finally, the mixture is placed at 37 ℃ and 5% CO2Culturing in an incubator.
(4) Observing the cell state every day, collecting the cells and the culture solution thereof at 5 days respectively, extracting DNA, and measuring the Ct value by using a fluorescence quantitative PCR method recommended in national standard GB/T18648-2020 African swine fever diagnosis technology of the people's republic of China.
The identification primer and the probe sequence are respectively as follows:
CADC-rPCR-F：5’-ATAGAGATACAGCTCTTCCAG-3’
CADC-rPCR-R：5’-GTATGTAAGAGCTGCAGAAC-3’
CADC-Probe：5’-FAM-TATCGATAAGATTGAT-MGB-3’
the reaction system is as follows: 20 μ L of: mu.L of Universal U + Probe Master Mix V210, 0.4. mu.L of each of the upstream and downstream primers (10. mu. mol/L), 0.2. mu.L of the Probe primer, 2. mu.L of the template, and sterile distilled water to make up to 20. mu.L.
The reaction conditions are as follows: 2min at 37 ℃; 5min at 95 ℃; 10s at 95 ℃, 30s at 60 ℃ and 45 cycles; extension at 72 ℃ for 5 min.
In step 1, the acid bond is prepared at a concentration of 1:2000, and the pH value is 2.75.
In step 4, the Ct values of the test groups measured by the fluorescence quantitative PCR method are: ct value of blank control: 37.20; ct value of negative control group of disinfectant: 36.52; ct value for disinfectant test group: 18.53; positive control Ct value: 26.13. the result shows that compared with the positive control group, the multiplication capacity of the virus can be obviously improved when the virus and the acid bond are incubated for 0h at room temperature.
Example 2
(1) The pH value was measured after diluting the acid bond to 1:2000 with distilled water.
(2). Taking out the cell freezing tube from the liquid nitrogen tank, directly immersing the cell freezing tube into a 37 ℃ constant-temperature water bath kettle, and shaking the cell freezing tube to melt the cell freezing tube as soon as possible without any time; after melting, opening a cover, transferring the cell suspension into a centrifuge tube, adding more than 10 times of RPMI1640 culture medium, and uniformly mixing; centrifuging at 800g for 5min, discarding supernatant, adding 10% FBS-containing RPMI1640 culture medium to resuspend cells, counting, and adjusting cell density to 107After one mL, the cells were seeded in 24-well cell plates and placed at 37 ℃ in 5% CO2Culturing for 4h in an incubator until the cells adhere to the wall. Respectively setting blank control group (570 μ L distilled water +30 μ L1640 culture medium), disinfectant negative control group (570 μ L concentration is 1:2000 acid bond +30 μ L RPMI1640 culture medium), disinfectant test group (570 μ L concentration is 1:2000 acid bond +30 μ L ASFV virus solution), and positive control group (570 μ L distilled water +30 μ L ASFV virus solution), mixing, and incubating at 24-26 deg.C for 1h (ASFV virus solution TCID)50＝104/0.1mL)。
(3) 2400 μ L of RPMI1640 medium was added to each test group to allow normal growth of porcine primary alveolar macrophages while ensuring that ASFV can replicate in cells. The culture medium in the 24-well cell plate was aspirated, and the mixed solution of the above four test groups was added to the corresponding wells, respectively, to inoculate 3 wells of 500. mu.L per well. Then placed at 37 ℃ with 5% CO2Culturing in an incubator. After 2h, the maintenance solution (1640 medium containing 10% FBS) was changed, and 500. mu.L of the solution was added to each well. Finally, the mixture is placed at 37 ℃ and 5% CO2Culturing in an incubator.
(4) Observing the cell state every day, collecting the cells and the culture solution thereof at 5 days respectively, extracting DNA, and measuring the Ct value by using a fluorescence quantitative PCR method recommended in national standard GB/T18648-2020 African swine fever diagnosis technology of the people's republic of China.
In step 1, the acid bond is prepared at a concentration of 1:2000, and the pH value is 2.75.
In step 4, the Ct values of the test groups measured by the fluorescence quantitative PCR method are: ct value of blank control: 36.89; ct value of negative control group of disinfectant: 37.11; ct value for disinfectant test group: 21.02; positive control Ct value: 25.93. the result shows that compared with the positive control group, the multiplication capacity of the virus can be obviously improved when the virus and the acid bond are incubated for 1h at room temperature.
Example 3
(1) The pH value was measured after diluting the acid bond to 1:2000 with distilled water.
(2) Taking out the cell freezing tube from the liquid nitrogen tank, directly immersing the cell freezing tube into a 37 ℃ constant-temperature water bath kettle, and shaking the cell freezing tube to melt the cell freezing tube as soon as possible without any time; after melting, opening a cover, transferring the cell suspension into a centrifuge tube, adding more than 10 times of RPMI1640 culture medium, and uniformly mixing; centrifuging at 800g for 5min, discarding supernatant, adding 10% FBS-containing RPMI1640 culture medium to resuspend cells, counting, and adjusting cell density to 107After one mL, the cells were seeded in 24-well cell plates and placed at 37 ℃ in 5% CO2Culturing for 4h in an incubator until the cells adhere to the wall. Respectively setting blank control group (570 μ L distilled water +30 μ L1640 culture medium), disinfectant negative control group (570 μ L concentration is 1:2000 acid bond +30 μ L RPMI1640 culture medium), disinfectant test group (570 μ L concentration is 1:2000 acid bond +30 μ L ASFV virus solution), and positive control group (570 μ L distilled water +30 μ L ASFV virus solution), mixing, incubating at 24-26 deg.C for 2 hr, and adding the mixture to obtain the final product (ASFV virus solution TCID)50＝104/0.1mL)。
(3) 2400 μ L of RPMI1640 medium was added to each test group to allow normal growth of porcine primary alveolar macrophages while ensuring that ASFV can replicate in cells. The culture medium in the 24-well cell plate was aspirated, and the mixed solution of the above four test groups was added to the corresponding wells, respectively, to inoculate 3 wells of 500. mu.L per well. Then placed at 37 ℃ with 5% CO2Culturing in an incubator. After 2h, the maintenance solution (1640 medium containing 10% FBS) was changed, and 500. mu.L of the solution was added to each well. Finally, the mixture is placed at 37 ℃ and 5% CO2Culturing in an incubator.
(4) Observing the cell state every day, collecting the cells and the culture solution thereof at 5 days respectively, extracting DNA, and measuring the Ct value by using a fluorescence quantitative PCR method recommended in national standard GB/T18648-2020 African swine fever diagnosis technology of the people's republic of China.
In step 1, the acid bond is prepared at a concentration of 1:2000, and the pH value is 2.75.
In step 4, the Ct values of the test groups measured by the fluorescence quantitative PCR method are: ct value of blank control: 38.85; ct value of negative control group of disinfectant: 36.20; ct value for disinfectant test group: 20.49 of the total weight of the mixture; positive control Ct value: 25.10. the result shows that compared with the positive control group, the multiplication capacity of the virus can be obviously improved when the virus and the acid bond are incubated for 2 hours at room temperature.
Example 4
(1) The pH value was measured after diluting the acid bond to 1:2000 with distilled water.
(2) Taking out the cell freezing tube from the liquid nitrogen tank, directly immersing the cell freezing tube into a 37 ℃ constant-temperature water bath kettle, and shaking the cell freezing tube to melt the cell freezing tube as soon as possible without any time; after melting, opening a cover, transferring the cell suspension into a centrifuge tube, adding more than 10 times of RPMI1640 culture medium, and uniformly mixing; centrifuging at 800g for 5min, discarding supernatant, adding 10% FBS-containing RPMI1640 culture medium to resuspend cells, counting, and adjusting cell density to 107After one mL, the cells were seeded in 24-well cell plates and placed at 37 ℃ in 5% CO2Culturing for 4h in an incubator until the cells adhere to the wall. Respectively setting blank control group (570 μ L distilled water +30 μ L1640 culture medium), disinfectant negative control group (570 μ L concentration is 1:2000 acid bond +30 μ L RPMI1640 culture medium), disinfectant test group (570 μ L concentration is 1:2000 acid bond +30 μ L ASFV virus solution), and positive control group (570 μ L distilled water +30 μ L ASFV virus solution), mixing, and incubating at 24-26 deg.C for 4h (ASFV virus solution TCID)50＝104/0.1mL)。
(3) 2400 μ L of RPMI1640 medium was added to each test group to allow normal growth of porcine primary alveolar macrophages while ensuring that ASFV can replicate in cells. The culture medium in the 24-well cell plate was aspirated, and the four test groups were mixedThe pool solutions were added to the respective wells, and 3 wells were inoculated, 500. mu.L each. Then placed at 37 ℃ with 5% CO2Culturing in an incubator. After 2h, the maintenance solution (1640 medium containing 10% FBS) was changed, and 500. mu.L of the solution was added to each well. Finally, the mixture is placed at 37 ℃ and 5% CO2Culturing in an incubator.
(4) Observing the cell state every day, collecting the cells and the culture solution thereof at 5 days respectively, extracting DNA, and measuring the Ct value by using a fluorescence quantitative PCR method recommended in national standard GB/T18648-2020 African swine fever diagnosis technology of the people's republic of China.
In step 1, the acid bond is prepared at a concentration of 1:2000, and the pH value is 2.75.
In step 4, the Ct values of the test groups measured by the fluorescence quantitative PCR method are: ct value of blank control: 32.83; ct value of negative control group of disinfectant: 32.48, respectively; ct value for disinfectant test group: 19.01, respectively; positive control Ct value: 24.66. the result shows that compared with the positive control group, the multiplication capacity of the virus can be obviously improved when the virus and the acid bond are incubated for 4 hours at room temperature.
The experimental statistics for examples 1-4 are shown in table 1 below:
TABLE 1 statistical results of the experiments of examples 1-4
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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Claims (10)
1. A method for improving the multiplication capacity of African swine fever virus is characterized in that: the method comprises the following steps:
s1, inoculating the cell suspension of the primary alveolar macrophages of the pigs into a cell culture plate, and standing for 3-5 hours until the cells adhere to the wall;
s2, uniformly mixing the African swine fever virus liquid and the Jielian bond diluent, incubating for 0-4 h at 24-26 ℃, and adding a culture medium to obtain a mixed liquid; replacing the culture solution in the cell culture plate with the obtained mixed solution, and culturing for 1.5-2.5 h;
and S3, replacing the culture solution in the cell culture plate with a culture medium containing 10% fetal calf serum again, and culturing for 4-6 d.
2. The method for increasing the proliferation potency of African swine fever virus according to claim 1, wherein:
TCID of African swine fever virus liquid described in step S250Is 1040.1mL, wherein the dilution ratio of the clean acid bond diluent is 1: 2000; the volume ratio of the African swine fever virus liquid to the Jielian bond diluent to the culture medium is 57: 3: 240.
3. the method for increasing the proliferation potency of African swine fever virus according to claim 1, wherein:
the pH of the acid bond cleaning diluent described in step S2 was 2.75.
4. The method for increasing the proliferation potency of African swine fever virus according to claim 1, wherein:
the medium described in steps S2 and S3 was RPMI1640 medium.
5. The method for increasing the proliferation potency of African swine fever virus according to claim 1, wherein:
the cell density of the cell suspension of porcine primary alveolar macrophages described in step S1 was 107mL, the reagents used for resuspension were RPMI1640 medium containing 10% fetal bovine serum.
6. The method for increasing the proliferation potency of African swine fever virus according to claim 1, wherein:
the standing time in step S1 was 4 h.
7. The method for increasing the proliferation potency of African swine fever virus according to claim 1, wherein:
the incubation time described in step S2 was 0 h.
8. The method for increasing the proliferation potency of African swine fever virus according to claim 1, wherein:
the incubation time described in step S2 was 2 h.
9. The method for increasing the proliferation potency of African swine fever virus according to claim 1, wherein:
the incubation period described in step S3 was 5 days.
10. The method for increasing the proliferation potency of African swine fever virus according to claim 1, wherein:
the standing condition described in step S1 was 37 ℃ with 5% CO2；
The conditions of the cultivation described in steps S2 and S3 were 37 ℃, 5% CO2。
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