CN106940371A - A kind of pestivirus culture and malicious quantitative detecting method living - Google Patents

A kind of pestivirus culture and malicious quantitative detecting method living Download PDF

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CN106940371A
CN106940371A CN201710239185.5A CN201710239185A CN106940371A CN 106940371 A CN106940371 A CN 106940371A CN 201710239185 A CN201710239185 A CN 201710239185A CN 106940371 A CN106940371 A CN 106940371A
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pestivirus
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范学政
丁家波
王琴
徐璐
赵启祖
蒋卉
沈青春
朱良全
彭小薇
许冠龙
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China Institute of Veterinary Drug Control
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    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
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    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus

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Abstract

The present invention relates to a kind of pestivirus culture and live virus quantitative detecting method.The pestivirus cultural method of the present invention, can significantly improve viral efficiency of infection, improve CSFV, the Yield of Antigen of bovine viral diarrhea virus；When quantitatively being detected to live virus, viral efficiency of infection can be significantly improved, immunofluorescence dyeing sensitiveness is improved, while reducing immunofluorescence background, improve immunofluorescence quality, Accurate Determining CSFV, the titre of live viruses (TCID of bovine viral diarrhea virus50)。

Description

A kind of pestivirus culture and malicious quantitative detecting method living
Technical field
The present invention relates to a kind of pestivirus culture and Viral Quantification detection method, belong to field of biological product.
Background technology
CSFV (Hog clolera virus, HCV are also known as Classical swine fever virus, CSFV), Bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) belongs to flaviviridae pestivirus together.
Swine fever (Hog cholera) is a kind of acute infectious disease as caused by CSFV, and various age pigs can be sent out Disease, the course of disease is varied from acute to chronic etc..Popular throughout the year, infectiousness is extremely strong, with height morbidity and mortality. But, CSFV is cultivated because of difficulty, because not causing cytopathy, is clinically separated and identifies all extremely difficult.Current China is still Using the comprehensive control measures based on immunoprophylaxis, the CSFV Lapinized strain used (CVCC AV1412) is international public The optimal hog cholera vaccine production strain recognized, is used, it is effectively controlled the prevalence of swine fever by many countries.
The production of hog cholera lapinised virus live vaccine antigen still uses the adherent production technology based on rolling bottle, biological respinse at present Device produce hog cholera lapinised virus live vac-cine technology although the development phase (such as：CN102453698A, suspension subculture cell And the method that hog cholera vaccine is produced using it), but it is still immature, without commercial prod listing.Due to CSFV and seedling The biological characteristics of cell, often occurs that viral antigen potency is low, differences between batches big, often occurs that vaccine quality is unstable etc. to ask Topic.Therefore, optimization CSFV antigen production method is very necessary.
In addition, China's live vaccines of hog cholera effect detection at present uses the hot method of rabbit precursor reactant.But this method is present a lot Drawback, not only needs many large ear rabbits, testing result easily by rabbit body individual difference and such environmental effects, waste time and energy and Cost is higher, it is often more important that China's large ear rabbit is without pure line cultivar, and strain is more and more miscellaneous, the large ear rabbit of separate sources Testing result is widely different, therefore is difficult that urgent need sets up new swine fever come the accurate effect for detecting live vaccine with rabbit precursor reactant heat Live vaccine effect detection method.
In terms of hog cholera lapinised virus culture and live vaccines of hog cholera effect detection method, China researcher has been carried out greatly Amount is attempted, and also achieves encouraging progress, e.g., Wu Hua (A of Chinese patent CN 101879311), Rui Pu (Baoding)) biological medicine company has Company of limit company (A of Chinese patent CN 104940922) etc. is carried out in the culture of hog cholera lapinised virus and production of vaccine technique Explore, Shandong Binzhou Bo Laiwei Bioisystech Co., Ltd (A of Chinese patent CN 103698518), Xinjiang Tian Kang herdings are biological Technical concern Co., Ltd (A of Chinese patent CN 103105493), Luoyang Pulaike Biological Engineering Co., Ltd.'s (Chinese patent The A of CN 101846683) etc. establish a series of new hog cholera lapinised virus TCID50Method, but exist corresponding not enough. At present in immunofluorescence dyeing more than use swine fever positive serum as primary antibody, the rabbit-anti pig IgG that FITC is marked is used as secondary antibody, pig Pest positive serum is prepared with swine fever virus infection pig, and potency is low, nonspecific reaction is strong, mark rabbit-anti pig plus FITC Cause non-specific dyeing strong after IgG, background depth is as a result difficult to judge, therefore, it is necessary to further optimization.
In addition, cultural method and malicious assay method living for the wild poison in swine fever field, lack corresponding research, still use Method the most traditional, the problem of separating for the wild poison in field with titer determination hardly possible, also in the urgent need to setting up sensitive culture side Method and assay method.
And bovine viral diarrhoea (Bovine viral diarrhea, BVD) is drawn by bovine viral diarrhea virus (BVDV) Rise with heating, mucosal erosion ulcer, Neuroleptic Leukocytopenia, diarrhoea, cough and Pregnant cows miscarry or output malformation fetus based on Want a kind of infectious disease of feature.At present, the disease is widely present in the world.It is isolated in recent years from the multiple morbidity cattle farms of China BVDV, huge economic loss is brought to China's cattle-raising.The cow's serum sample in China part province is detected, as a result table Bright, the disease is in China in rising situation.Vaccine is one of the prevention and control sick effective measures.But, on the disease virus culture, Titer determination method etc. is used conventional methods always, is not improved for many years, needs also exist for setting up for bovine viral diarrhoea Viral sensitive cultural method and assay method.
The content of the invention
The purpose of the present invention is：Set up the cultural method and live virus assay method of new CSFV；Set up new ox Viral diarrhea virus cultural method and live virus assay method, to improve CSFV and bovine viral diarrhea virus nutrient solution Middle antigenic content；Improve the accuracy and sensitiveness of CSFV and bovine viral diarrhea virus live virus assay method.
The main technical principle of the present invention
In the presence of finite concentration high molecular polymer polyethylene glycol (PEG) etc., viral sedimentation can be made, cell membrane melts It is swollen, so as to increase the sensitiveness of cell infection virion, the probability of virus particle infection cell is greatly improved, so that significantly Improve cell Toxin producing C；In TCID50During measure, by optimizing antibody type and quality, optimize colouring method, improve immune glimmering Light dyes rate and dyeing quality, so as to improve the quantitative accuracy of live virus.
Technical scheme
1. a kind of pestivirus culture and malicious quantitative detecting method living, it is characterised in that the method for pestivirus culture includes following Step：Prepare sensitive cells；Pestivirus is inoculated with, while adding the PEG of debita spissitudo, viral infection rate is improved；Change nutrient solution, Cultivated in cell culture incubator；Harvest virus liquid.
2. a kind of pestivirus culture of the present invention and malicious quantitative detecting method living, it is characterised in that pestivirus live virus Titer determination method (TCID50) comprise the following steps：Prepare sensitive cells in 48 orifice plates；Pestivirus is dilute with nutrient solution gradient Release；Add the PEG of debita spissitudo；It is inoculated with sensitive cells, 37 DEG C, 5%CO2Cultivated in cell culture incubator；Optimize primary antibody species (profit Prepared and antibody purification with the E2 albumen of baculovirus expression) and secondary antibody species；Observe positive empty under fluorescence inverted microscope Number, calculates virus titer.
3. a kind of pestivirus culture of the present invention and malicious quantitative detecting method living, it is characterised in that the pestivirus includes CSFV and bovine viral diarrhea virus.
4. a kind of pestivirus culture of the present invention and malicious quantitative detecting method living, it is characterised in that the primary antibody species bag Include and, including：CSFV E2 protein Is gG is purified；Purify BVDV E2 protein Is gG；Secondary antibody species is：The anti-rabbit IgG of FITC marks.
5. a kind of pestivirus culture of the present invention and malicious quantitative detecting method living, it is characterised in that this method can be used for normal See virus such as Bovine Rhinotracheitis Virus (IBRV), pig breathing and breeding syndrome are viral (PRRS), Porcine epidemic diarrhea virus (PEDV), the culture such as gastroenteritis virus of swine (TGEV) and live virus are quantified.
The specific embodiment of the invention
1. pestivirus E2 protein Is gG preparation purifying (being used in the present invention as primary antibody)：
With baculovirus expression and purify E2 albumen [CSFV E2 (hog cholera antibody ELISA diagnosis reagent kit, 200810223406.0, CGMCC No.2671), BVDV E2 (expression and application of bovine viral diarrhea virus I type E2 albumen, Application number 201610518191X, CGMCC No.12544)] Japan large ear rabbit is immunized, first immunisation is helped completely with complete Freund Agent is emulsified, and the immune 200 μ g/ of subcutaneous multiple spot are only.Secondary immunity after 30 days, incomplete Freund's adjuvant adjuvant emulsion, subcutaneous multiple spot is exempted from The μ g/ of epidemic disease 200 are only.After 200 μ of E2 albumen abdominal cavity injecting immune g/ after 30 days with purifying.Blood sampling separation serum after 7 days.Will Serum Protein A antibody purification posts are purified, and use SDS-PAGE electrophoretic analysis, and protein concentration is determined with Bradford methods, plus Enter to dispense after 20% glycerine PBS and freeze.Working concentration is determined, with PBS finite concentration is diluted to using preceding.
The preparation of 2.PEG nutrient solutions：
100~140g PEG6000, plus 800ml MEM or DMEM culture mediums are weighed, is fully determined after dissolving with MEM or DMEM Amount is to 1000mL, and PEG concentration is 10%~14%.With 0.22 μm of filter membrane bacteriological filtration, 4 DEG C save backup.
3. cell growth medium is prepared：
MEM containing the 8%~10%FBS or DMEM containing 8%~10%FBS.
4. cell maintenance medium is prepared：
MEM containing the 2%FBS or DMEM containing 2%FBS.
5. Virus culture process：
(1) cell prepares：The sensitive cells for covering with Tissue Culture Flask is digested with 0.25%EDTA- pancreatin and disperseed, then Appropriate cell growth medium is added, by 1:3 ratios are dispersed to new Tissue Culture Flask.Cell length to 80%~90% it is full when use.
(2) viral dilution and inoculation：Virus liquid is suitably diluted with MEM or DMEM nutrient solutions, PEG nutrient solutions are pressed 1: 1 (v/v) ratio is added in virus liquid, is mixed.Nutrient solution is blotted, the virus liquid containing PEG is added in cell bottle, in 37 DEG C, 5% CO22~4h is incubated in cell culture incubator；After incubation terminates, virus liquid is discarded, appropriate cell maintenance medium is added, is placed in 37 DEG C, 5%CO2Cell culture incubator continues to cultivate 72~96h；
Note：For the virus of non-cell pathogenicity, such as CSFV (containing hog cholera lapinised virus vaccine strain), culture 72~ Maintaining liquid is suctioned out after 96h, the maintaining liquid more renewed puts 37 DEG C, 5%CO2Cell culture incubator continues to cultivate, every after 72~96h Change once, continuous 4 to 6 times.The maintaining liquid suctioned out every time freezes, and marks receipts time, determines the viral antigen of each receipts time Content.
6. the TCID of virus50Continuous mode：
(1) preparation of cell culture plate：The sensitive cells for covering with Tissue Culture Flask is disappeared with 0.25%EDTA- pancreatin Change and disperse, then add appropriate cell growth medium, be dispersed to 48 porocyte culture plates (by 75cm2Tissue Culture Flask scattered 4~8 The ratio of block Tissue Culture Plate is carried out).Put 37 DEG C, 5%CO2Cell culture incubator culture, cell length to 80%~90% makes when expiring With.
(2) viral dilution and inoculation：Cell length to 80%~90% it is full when, with MEM or DMEM nutrient solutions by virus liquid Gradient dilution is carried out, PEG nutrient solutions are then pressed 1:1 (v/v) ratio is added in each gradient virus liquid diluted, is mixed；Inhale Stem cell growth liquid, adds each gradient virus liquid diluted, per the μ l of hole 200, in 37 DEG C, 5%CO as early as possible2In cell culture incubator It is incubated 2~4h；After incubation terminates, virus liquid is blotted, cell maintenance medium is added as early as possible, per the μ l of hole 500, put 37 DEG C, 5%CO2Carefully Born of the same parents' incubator continues to cultivate 60~72h；
(3) Immunofluorescence test
After culture terminates, cell maintenance medium is blotted, the fixer (acetone of about 500 μ l precoolings is added per hole:Methanol=1:1) Fixed more than 20min, is exhausted into vent cabinet and dries, and 500 μ l PBS are added per hole and are rinsed one time, the purifying E2 eggs of dilution are added White IgG is (i.e.：Primary antibody) (being diluted with the PBS solution containing 3%~5% horse serum), per the μ l of hole 200,40 are incubated in 37 DEG C of incubators ~60min, is blotted, and is rinsed three times with PBS, is added 500 μ l per hole every time, is blotted, and adds the goat of the FITC marks diluted Anti-rabbit IgG antibody is (i.e.：Secondary antibody) (being diluted with the PBS solution containing 3%~5% horse serum), per the μ l of hole 200, in 37 DEG C of incubators 40~60min is incubated, is blotted, is rinsed three times with PBS, it is each that 500 μ l are added per hole, blot, the last 300 μ l of addition per hole~ 500 μ l PBS, are observed and are counted under fluorescence microscope.
7. a clearly demarcated described method can be used for common virus such as Bovine Rhinotracheitis Virus (IBRV), pig breathing is comprehensive with breeding Close syndrome virus (PRRS), Porcine epidemic diarrhea virus (PEDV), the culture such as gastroenteritis virus of swine (TGEV) and live virus are quantified.
Brief description of the drawings
In Fig. 1 CSFV proliferation tests, figure：A figures are CSFV rabbitization attenuated vaccine strain (CVCC AV1412)；B schemes For CSFV Liaoning strain (CVCC AV280).
Cell state after A. step inoculation methods 72h in cell state after Fig. 2 step inoculation methods 72h, figure；B.PEG synchronously connects Cell state after the method for kind 72h.
Fig. 3 different antibodies combine A. Antibody Combinations of the present invention in fluorescent staining result, figure：Purify CSFV E2 protein Is gG Goat anti-rabbit igg (secondary antibody) combination of (primary antibody) FITC marks；B. publication Antibody Combination：Swine fever positive serum and FITC marks Remember the combination of rabbit-anti pig IgG；C. the sheep anti-mouse igg combination of swine fever monoclonal antibody and FITC marks.
Fig. 4 bovine viral diarrhea virus proliferation tests.
The present invention relates to biomaterial resource information
1. express CSFV raq genes recombinant baculovirus VFBMEL-SE2M-HIS (CGMCC No.2671) (see：Swine fever Antibody ELISA diagnosis reagent kit 200810223406.0), have been filed on the common micro- life of China Committee for Culture Collection of Microorganisms Thing center.
Hog cholera lapinised virus vaccine strain (CVCC AV1412), CSFV Liaoning strain (CVCC AV280), CSFV Zheng State strain (CVCC AV279)；
2. express BVDV raq genes recombinant baculovirus BacVME2-His (CGMCC No.12544) (see：Bovine viral The expression and application of property diarrhea virus I type E2 albumen, application number 201610518191X), have been filed on Chinese microorganism strain preservation Administration committee's common micro-organisms center.
OregonC24v plants of bovine viral diarrhea virus (BVDV) also known as BA plants (CVCC AV69), NADL plants of BVDV (CVCC AV67), BVDV yaks strain (CVCC AV68).
The microorganism fungus kind that numbering is CVCC above is from the Chinese veterinary medicament in ZhongGuanCun south Street, Haidian District, BeiJing City 8 Supervise institute, Chinese veterinary microorganism culture presevation administrative center.Its bacterium numbering is see the websitehttp:// www.ivdc.org.cn。
The positive effect of the present invention
The present invention relates to a kind of pestivirus culture and live virus quantitative detecting method.The pestivirus cultural method of the present invention, Viral efficiency of infection can be significantly improved, CSFV, the Yield of Antigen of bovine viral diarrhea virus virus is improved；Lived to determining When Viral Quantification is detected, viral efficiency of infection can be significantly improved, immunofluorescence dyeing sensitiveness is improved, while reducing immunofluorescence Background, improves immunofluorescence quality, Accurate Determining hog cholera field virus, hog cholera lapinised virus, the work of bovine viral diarrhea virus virus Malicious titre (TCID50)。
Embodiment
Following examples are not construed as limiting to further illustrate the present invention to claimed technical scheme of the invention.
Embodiment 1
--- CSFV E2 protein Is gG preparation and purification
With baculovirus expression and the albumen (CSFV E2 (hog cholera antibody ELISA diagnosis reagent kits of purifying 200810223406.0, CGMCC No.2671) Japan large ear rabbit is immunized, first immunisation is emulsified with complete Freund's complete adjuvant, The subcutaneous immune 200 μ g/ of multiple spot are only.200 μ g/ are immunized in secondary immunity after 30 days, incomplete Freund's adjuvant adjuvant emulsion, subcutaneous multiple spot Only.After 200 μ of E2 albumen abdominal cavity injecting immune g/ after 30 days with purifying.Blood sampling separation serum after 7 days.Serum is used Protein A antibody purifications post is purified, and uses SDS-PAGE electrophoretic analysis, and protein concentration is determined with Bradford methods, adds 20% Packing freezes after glycerine PBS.Determine working concentration, using it is preceding be diluted to PBS working concentration (this measuring be 200~400 Times).
Embodiment 2
--- the culture of CSFV
1. cell prepares：Tissue Culture Flask will be covered with (with 25cm2Exemplified by Tissue Culture Flask) sensitive cells (ST, PK15) It is scattered with the digestion of 0.25%EDTA- pancreatin, 18ml cell growth mediums are then added, by 1:3 ratios are dispersed to new cell culture Bottle.Cell length to 80%~90% it is full when use.
2. the dilution and inoculation of virus：With MEM or DMEM nutrient solutions by virus liquid [with hog cholera lapinised virus vaccine strain Exemplified by (CVCC AV1412) and CSFV Liaoning strain (CVCC AV280)] dilute respectively, PEG nutrient solutions are pressed 1:1(v/v) Ratio is mixed into virus liquid；(while setting the control group for being not added with PEG, it is changed to by 1:1 (v/v) ratio adds MEM nutrient solutions, claims For without PEG groups).In 37 DEG C, 5%CO22~4h is incubated in cell culture incubator；After incubation terminates, virus liquid, every bottle of addition are blotted 6ml cell maintenance mediums, are placed in 37 DEG C, 5%CO2Cell culture incubator continues to cultivate 72~96h, suctions out maintaining liquid, the dimension more renewed Liquid is held, 37 DEG C, 5%CO are put2Cell culture incubator continues to cultivate, every being changed after 72~96h once, continuous 4 to 6 times.Inhale every time The maintaining liquid gone out freezes, and marks receipts time.Viral antigen is determined with CSFV Ag ELISA kits (IDEXX Products) Content.
As a result：Either hog cholera lapinised virus vaccine strain still CSFV Liaoning strain, is added after PEG, each to receive secondary disease Malicious antigenic content is obviously higher than without PEG groups.See Fig. 1.
Embodiment 3
--- plating cells and PEG dilution poison disease vaccination is synchronous and influence of the separate operations to cell
According to the embodiment of the present invention 2 --- the method culture hog cholera lapinised virus in the culture of CSFV；Meanwhile, contrast The preparation method of patent (Rui Pu, application number 201510380369.4) described live vaccines of hog cholera, by CSFV seed culture of viruses and cell Suspension synchronously in access rolling bottle, fills into cell growth medium and cultivated, and polyethylene glycol is added while CSFV is accessed (PEG), PEG concentration is 2% (m:V).Observe influence of two kinds of operating methods to ST cell growth states.
As a result：Separate operations have no significant effect to cellular growth conditions, and the synchronous inocalation method of document easily causes cell Activity decrease, later stage cell draws in the net lesion, and severe patient death comes off；Therefore, in the present invention, it is to be generated using cell is first prepared When length is to logarithmic phase, PEG dilution virus liquids are inoculated, cell maintenance medium is changed after being incubated 2~4h, sees Fig. 2.
Embodiment 4
--- the comparison of different antibodies combination
(1) preparation of cell culture plate：The sensitive cells (by taking ST cells as an example) for covering with Tissue Culture Flask is used The digestion of 0.25%EDTA- pancreatin is scattered, then adds appropriate cell growth medium, is dispersed to 48 porocyte culture plates (by 75cm2Carefully The ratio of scattered 4~8 piece of 48 porocyte culture plates of born of the same parents' blake bottle is carried out).Put 37 DEG C, 5%CO2Cell culture incubator culture, cell It is long to 80%~90% it is full when use.
(2) viral dilution and inoculation：Cell length to 80%~90% it is full when, with MEM or DMEM nutrient solutions by virus liquid (hog cholera lapinised virus vaccine strain (CVCC AV1412)) carries out 10 times of gradient dilutions, and PEG nutrient solutions then are pressed into 1:1 (v/v) compares Example is added in each gradient virus liquid diluted, is mixed；Growth-promoting media in each hole of cell plates is blotted, each ladder diluted is added as early as possible Virus liquid is spent, per the μ l of hole 200, in 37 DEG C, 5%CO22~4h is incubated in cell culture incubator；After incubation terminates, virus liquid is blotted, Cell maintenance medium is added as early as possible, per the μ l of hole 500, puts 37 DEG C, 5%CO2Cell culture incubator continues to cultivate 60~72h；
(3) Immunofluorescence test：After culture terminates, cell maintenance medium is blotted, the fixer of about 500 μ l precoolings is added per hole (acetone:Methanol=1:1) fixed more than 20min, is exhausted into vent cabinet and dries, and 500 μ l PBS are added per hole and are rinsed one time, plus Enter working concentration purifying CSFV E2 protein Is gG (primary antibody, it is dilute with the PBS solution containing 3%~5% horse serum from embodiment 1 Release), per the μ l of hole 200,40~60min is incubated in 37 DEG C of incubators, is blotted, rinsed three times with PBS, add 500 μ l per hole every time, Blot, add goat anti-rabbit IgG antibody (KPL Products 072-03-15-06) that the FITC that dilute marks (secondary antibody, with containing The PBS solution dilution of 3%~5% horse serum), per hole 200ul, 40~60min is incubated in 37 DEG C of incubators, blots, is floated with PBS Wash three times, add 500 μ l per hole every time, blot, it is last that 300~500 μ l PBS are added per hole, seen under fluorescence microscope Examine and count.
Meanwhile, swine fever positive serum and the FITC mark that contrast patent (Rui Pu, application number 201310663187.9) is used Rabbit-anti pig IgG is combined, and part sample aperture is dyed, compares the Color of Antibody Combination.
In addition, being dyed by the combination of commercially available monoclonal antibody reagent, after the fixation of 48 orifice plates is dried, 500 μ l are added per hole PBS is rinsed one time, adds swine fever monoclonal antibody (AHVLA products), per the μ l of hole 200, and 40~60min is incubated in 37 DEG C of incubators, is blotted, Rinsed three times with PBS, add 500 μ l per hole every time, blot, add the sheep anti-mouse igg antibody of the FITC marks diluted (sigma company F4018) (secondary antibody), per the μ l of hole 200, is incubated 40~60min, blots, three are rinsed with PBS in 37 DEG C of incubators Time, 500 μ l are added per hole every time, are blotted, it is last that 300~500 μ l PBS are added per hole, observed under fluorescence microscope.
As a result：As can be seen that purifying CSFV E2 protein Is gG (primary antibody, embodiment 1) FITC marks that the present invention is used Goat anti-rabbit IgG antibody (KPL Products 072-03-15-06) (secondary antibody) combination fluorescent staining works well, and background is low, cloudy Hot spot shows clearly, consistent with commercially available monoclonal antibody reagent compound staining effect；And swine fever positive serum and FITC mark rabbits Anti- pig IgG combination is dyed, and immunofluorescence background is deep, easily causes false positive results.Therefore, the Antibody Combination of the invention Immune effect is better than other published methods, sees Fig. 3.
Embodiment 5
--- the TCID of hog cholera lapinised virus vaccine strain50Determine
(1) preparation of cell culture plate：The sensitive cells (by taking ST cells as an example) for covering with Tissue Culture Flask is used The digestion of 0.25%EDTA- pancreatin is scattered, then adds appropriate cell growth medium, is dispersed to 48 porocyte culture plates (by 75cm2Carefully The ratio of scattered 4~8 piece of 48 porocyte culture plates of born of the same parents' blake bottle is carried out).Put 37 DEG C, 5%CO2Cell culture incubator culture, cell It is long to 80%~90% it is full when use.
(2) viral dilution and inoculation：Cell length to 80%~90% it is full when, with MEM or DMEM nutrient solutions by virus liquid (strain of CVCC AV1412 hog cholera lapinised virus vaccines) carries out 10 times of gradient dilutions, and PEG nutrient solutions then are pressed into 1:1 (v/v) ratio Add in each gradient virus liquid diluted, mix；Growth-promoting media in each hole of cell plates is blotted, each gradient diluted is added as early as possible Virus liquid, per the μ l of hole 200, in 37 DEG C, 5%CO22~4h is incubated in cell culture incubator；After incubation terminates, virus liquid is blotted, to the greatest extent It is fast to add cell maintenance medium, per the μ l of hole 500, put 37 DEG C, 5%CO2Cell culture incubator continues to cultivate 60~72h；
Meanwhile, each gradient antigen liquid of the hog cholera lapinised virus vaccine diluted is inoculated with Japan large ear rabbit, pressed《Chinese beast Pharmacopeia》Three (two 〇 First Five-Year Plans year version) methods determine antigen liquid rabbit body infective dose (RID).
(3) Immunofluorescence test：After culture terminates, cell maintenance medium is blotted, the fixer of about 500 μ l precoolings is added per hole (acetone:Methanol=1:1) fixed more than 20min, is exhausted into vent cabinet and dries, and 500 μ l PBS are added per hole and are rinsed one time, plus Enter working concentration purifying CSFV E2 protein Is gG (primary antibody, it is dilute with the PBS solution containing 3%~5% horse serum from embodiment 1 Release), per the μ l of hole 200,40~60min is incubated in 37 DEG C of incubators, is blotted, rinsed three times with PBS, add 500 μ l per hole every time, Blot, add goat anti-rabbit IgG antibody (KPL Products 072-03-15-06) that the FITC that dilute marks (secondary antibody, with containing The PBS solution dilution of 3%~5% horse serum), per the μ l of hole 200,40~60min is incubated in 37 DEG C of incubators, blots, is floated with PBS Wash three times, add 500 μ l per hole every time, blot, it is last that 300~500 μ l PBS are added per hole, seen under fluorescence microscope Examine and count.And be compared with rabbit body infective dose (RID).
As a result：As can be seen that the TCID that the present invention is created50Method with《Chinese veterinary pharmacopoeia》The defined hot method of rabbit precursor reactant is determined Result have high consistency.It is shown in Table 1.
The hot method of rabbit precursor reactant of table 1 and TCID of the present invention50Method compares
Embodiment 6
--- the TCID of classical swine fever virus isolation strain50Determine
(1) preparation of cell culture plate：The sensitive cells (by taking PK15 cells as an example) for covering with Tissue Culture Flask is used The digestion of 0.25%EDTA- pancreatin is scattered, then adds appropriate cell growth medium, is dispersed to 48 porocyte culture plates (by 75cm2Carefully The ratio of scattered 4~8 pieces of Tissue Culture Plates of born of the same parents' blake bottle is carried out).Put 37 DEG C, 5%CO2Cell culture incubator culture, cell length is extremely 80%~90% uses when full.
(2) viral dilution and inoculation：Cell length to 80~90% it is full when, with MEM or DMEM nutrient solutions by virus liquid ([by taking CSFV Zhengzhou strain (CVCC AV279) CSFV Liaoning strain (CVCC AV280) as an example] carries out gradient dilution, so PEG nutrient solutions are pressed 1 afterwards:1 (v/v) ratio is added in each gradient virus liquid diluted, is mixed；Cell growth medium is blotted, to the greatest extent Each gradient virus liquid diluted is added soon, per the μ l of hole 200, in 37 DEG C, 5%CO22~4h is incubated in cell culture incubator；It is incubated After end, virus liquid is blotted, cell maintenance medium is added as early as possible, per the μ l of hole 500, put 37 DEG C, 5%CO2Cell culture incubator continues to train Support 60~72h；
(3) Immunofluorescence test：After culture terminates, cell maintenance medium is blotted, the fixer of about 500 μ l precoolings is added per hole (acetone:Methanol=1:1) fixed more than 20min, is exhausted into vent cabinet and dries, and 500 μ l PBS are added per hole and are rinsed one time, plus Enter working concentration purifying CSFV E2 protein Is gG (primary antibody, it is dilute with the PBS solution containing 3%~5% horse serum from embodiment 1 Release), per the μ l of hole 200,40~60min is incubated in 37 DEG C of incubators, is blotted, rinsed three times with PBS, add 500 μ l per hole every time, Blot, add goat anti-rabbit IgG antibody (KPL Products 072-03-15-06) that the FITC that dilute marks (secondary antibody, with containing The PBS solution dilution of 3%~5% horse serum), per the μ l of hole 200,40~60min is incubated in 37 DEG C of incubators, blots, is floated with PBS Wash three times, add 500 μ l per hole every time, blot, it is last that the μ l PBS of 300 μ l~500 are added per hole, carried out under fluorescence microscope Observation and counting.
In addition, being dyed by the combination of commercially available monoclonal antibody reagent, after the fixation of 48 orifice plates is dried, 500 μ l are added per hole PBS is rinsed one time, adds swine fever monoclonal antibody (AHVLA products), per hole 200ul, and 40~60min is incubated in 37 DEG C of incubators, is blotted, Rinsed three times with PBS, add 500 μ l per hole every time, blot, add the sheep anti-mouse igg antibody of the FITC marks diluted (sigma company F4018), per the μ l of hole 200, is incubated 40~60min, blots in 37 DEG C of incubators, rinse three times with PBS, every time 500 μ l are added per hole, are blotted, it is last that 300~500 μ l PBS are added per hole, observed under fluorescence microscope.
As a result：As can be seen that the swine fever TCID that the present invention is created50Method, the CSFV titre of measure is apparently higher than commercially available Antibody Combination.It is shown in Table 2.
The classical swine fever virus isolation of table 2 strain TCID50Measurement result
Embodiment 7
--- BVDV E2 protein Is gG preparation and purification
With (the expression of bovine viral diarrhea virus I type E2 albumen and should of the BVDV E2 albumen of the purifying of baculovirus expression With Chinese patent, application number 201610518191X, CGMCC No.12544) Japan large ear rabbit is immunized, first immunisation is finished Full Freund's complete adjuvant emulsification, the immune 200 μ g/ of subcutaneous multiple spot are only.Secondary immunity after 30 days, incomplete Freund's adjuvant adjuvant breast Change, the immune 200 μ g/ of subcutaneous multiple spot are only.After 200 μ of E2 albumen abdominal cavity injecting immune g/ after 30 days with purifying.Adopted after 7 days Blood system is from serum.Serum Protein A antibody purification posts are purified, SDS-PAGE electrophoretic analysis is used, are surveyed with Bradford methods Determine to dispense after protein concentration, 20% glycerine PBS of addition and freeze.Working concentration is determined, with PBS working concentration is diluted to using preceding (this measuring is 200~400 times).
Embodiment 8
--- BVDV culture
(1) cell prepares：Tissue Culture Flask will be covered with (with 25cm2Exemplified by Tissue Culture Flask) sensitive cells (with MDBK Exemplified by cell) it is scattered with the digestion of 0.25%EDTA- pancreatin, appropriate cell growth medium is then added, by 1:3 ratios are dispersed to new Tissue Culture Flask.Cell length to 80%~90% it is full when use.
(2) viral dilution and inoculation：With MEM or DMEM nutrient solutions by virus liquid [with BVDVOregonC24v plants of BVDV Exemplified by OregonC24v (CVCC AV69), BVDV NADL (CVCC AV67), BVDV yaks strain (CVCC AV68)] dilution, will PEG nutrient solutions press 1:1 (v/v) ratio is mixed into virus liquid；(while setting the control group for being not added with PEG, it is changed to by 1:1(v/v) Ratio adds nutrient solution).In 37 DEG C, 5%CO22~4h is incubated in cell culture incubator；After incubation terminates, virus liquid, every bottle are blotted 6ml cell maintenance mediums are added, 37 DEG C, 5%CO are placed in2Cell culture incubator continuation 72~120h of culture, observation of cell lesion situation, Treat that cavity caused by cytopathy occurs in about 40%~60% area, suction out maintaining liquid and freeze.With BVDV Ag ELISA reagents Box (IDEXX Products) determines viral antigen content.
As a result：For each strain of bovine viral diarrhoea, add after PEG, in nutrient solution viral antigen content obviously higher than Without PEG groups.See Fig. 4.
Embodiment 9
--- BVDV TCID50Determine
(1) preparation of cell culture plate：The sensitive cells (by taking MDBK cells as an example) for covering with Tissue Culture Flask is used The digestion of 0.25%EDTA- pancreatin is scattered, then adds appropriate cell growth medium, is dispersed to 48 porocyte culture plates (by 75cm2Carefully The ratio of scattered 4~8 piece of 48 porocyte culture plates of born of the same parents' blake bottle is carried out).Put 37 DEG C, 5%CO2Cell culture incubator culture, cell It is long to 80%~90% it is full when use.
(2) viral dilution and inoculation：Cell length to 80%~90% it is full when, with MEM or DMEM nutrient solutions by virus liquid [by taking BVDV OregonC24v (CVCC AV69), BVDV NADL (CVCC AV67), BVDV yaks strain (CVCC AV68) as an example] Gradient dilution is carried out, PEG nutrient solutions are then pressed 1:1 (v/v) ratio is added in each gradient virus liquid diluted, is mixed；Inhale Stem cell growth liquid, adds each gradient virus liquid diluted, per the μ l of hole 200, in 37 DEG C, 5%CO as early as possible2In cell culture incubator It is incubated 2~4h；After incubation terminates, virus liquid is blotted, cell maintenance medium is added as early as possible, per the μ l of hole 500, put 37 DEG C, 5%CO2Carefully Born of the same parents' incubator continues to cultivate 60~72h；
(3) Immunofluorescence test：After culture terminates, cell maintenance medium is blotted, the fixer of about 500 μ l precoolings is added per hole (acetone:Methanol=1:1) fixed more than 20min, is exhausted into vent cabinet and dries, and 500 μ l PBS are added per hole and are rinsed one time, plus Enter working concentration purifying BVDV E2 protein Is gG (primary antibody come from embodiment 7, it is dilute with the PBS solution containing 3%~5% horse serum Release), per the μ l of hole 200,40~60min is incubated in 37 DEG C of incubators, is blotted, rinsed three times with PBS, add 500 μ l per hole every time, Blot, add goat anti-rabbit IgG antibody (KPL the Products) (secondary antibody, with containing 3%~5% horse blood of the FITC marks diluted Clear PBS solution dilution), per the μ l of hole 200,40~60min is incubated in 37 DEG C of incubators, is blotted, rinse three times with PBS, every time 500 μ l are added per hole, are blotted, it is last that the μ l PBS of 300 μ l~500 are added per hole, observed and counted under fluorescence microscope.
Meanwhile, dyed by commercial antibody agent combination, after the fixation of 48 orifice plates is dried, 500 μ l PBS drifts are added per hole Wash one time, add and 40~60min is incubated in BVD antibody working solution (AHVLA product RAE2020), 37 DEG C of incubators, blot, use PBS is rinsed three times, is added 500 μ l per hole every time, is blotted, and adds the sheep anti-mouse igg antibody (sigma of the FITC marks diluted Company F4018), 40~60min is incubated in 37 DEG C of incubators, is blotted, is rinsed three times with PBS, 500 μ l are added per hole every time, inhaled It is dry, it is last that 300~500 μ l PBS are added per hole, observed and counted under fluorescence microscope.
As a result：As can be seen that the bovine viral diarrhea virus TCID that the present invention is created50Method, the bovine viral diarrhoea of measure The TCID that virus titer is combined apparently higher than commercial antibody50Method.It is shown in Table 3.
The bovine viral diarrhea virus measurement result of table 3
Embodiment 10
Method of the present invention can be used for common virus such as Bovine Rhinotracheitis Virus (IBRV), and pig breathing and breeding are comprehensive Syndrome virus (PRRS), Porcine epidemic diarrhea virus (PEDV), the culture such as gastroenteritis virus of swine (TGEV) and live virus are quantified.

Claims (5)

1. a kind of pestivirus culture and malicious quantitative detecting method living, it is characterised in that the method for pestivirus culture includes following step Suddenly：Prepare sensitive cells；Pestivirus is inoculated with, while adding the PEG of debita spissitudo, viral infection rate is improved；Nutrient solution is changed, carefully Cultivated in born of the same parents' incubator；Harvest virus liquid.
2. a kind of pestivirus culture as claimed in claim 1 and malicious quantitative detecting method living, it is characterised in that pestivirus live virus Assay method (TCID50) comprise the following steps：Prepare sensitive cells in 48 orifice plates；By pestivirus nutrient solution gradient dilution； Add the PEG of debita spissitudo；It is inoculated with sensitive cells, 37 DEG C, 5%CO2Cultivated in cell culture incubator；Optimize primary antibody species；Optimization Secondary antibody species；Positive empty number is observed under fluorescence inverted microscope, virus titer is calculated.
3. a kind of pestivirus culture as claimed in claim 1 and malicious quantitative detecting method living, it is characterised in that the pestivirus bag Include CSFV and bovine viral diarrhea virus.
4. such as claim 1, a kind of pestivirus culture described in 2 and malicious quantitative detecting method living, it is characterised in that the primary antibody kind Class include and, including：CSFV E2 protein Is gG is purified；Purify BVDV E2 protein Is gG；Secondary antibody species is：The anti-rabbit of FITC marks IgG。
5. such as claim 1, a kind of pestivirus culture described in 2 and malicious quantitative detecting method living, it is characterised in that this method can use In common virus such as Bovine Rhinotracheitis Virus (IBRV), pig breathing and breeding syndrome are viral (PRRS), Porcine Epidemic Diarrhea Malicious (PEDV), the culture such as gastroenteritis virus of swine (TGEV) and live virus are quantified.
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CN103777009A (en) * 2012-10-18 2014-05-07 辽宁成大动物药业有限公司 Method using horse radish peroxidase-labeled antibody for detection of swine fever attenuated virus titer
CN104535764A (en) * 2014-12-07 2015-04-22 青岛易邦生物工程有限公司 Classical swine fever virus gene recombinant adenovirus vector vaccine virus content determination method
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