CN108265099A - A kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect - Google Patents
A kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect Download PDFInfo
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Abstract
The present invention provides a kind of methods for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect, include the following steps：1）Cell culture；2）Cell inoculation controls cell density；3）Porcine reproductive and respiratory syndrome virus live vaccine and Swine spleen transfer factor are splined in tissue culture plate, cultivate incubated cell；4）Cell with MTT solution is reacted, continues to be incubated, then carries out dissolution process with DMSO, finally cell plates are placed and OD values are measured in microplate reader, calculate cell activity.The method of the present invention has many advantages, such as that detection background is low, high sensitivity, specificity is strong, accuracy is high, reproducible, is particularly suitable for the detection of bioactivity and the foundation of quality standard when Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of detection Swine spleen transfer factor and pig breeding are integrated with breathing
The method for levying viral lived vaccine combined immunization effect.
Background technology
Transfer factor（Transfer Factor, TF）Animal body immune function can be adjusted, cooperates with vaccine immunity, is shortened
The vaccine immune response phase improves animal immune level, has boundless application prospect.Transfer factor is to be present in people and move
Cellular immune function possessed by donor can be transferred to receptor by the small-molecule substance of dialysing in object leucocyte, from rather than it is special
Enhance the immune function of receptor different in naturely.The ingredient of transfer factor mainly includes oligonucleotide and polypeptide, can induce body and exempts from
Epidemic disease cell activation enhances immunity of organisms, can shift and include virus, bacterium, parasite, fungi, histocompatibility antigen and swell
The cellular immunity of a variety of antigens such as tumor antigen.In addition, transfer factor has, molecular mass is small, nontoxic, no antigen, did not caused
It is quick reaction, do not create antagonism antibody and can surmount germline boundary application the advantages that.
Since transfer factor is safe to use, acts on rapid, significant effect, drug residue free, no antigen, nontoxic secondary work
With, and source is wide, meanwhile, as immune stimulating agent and auxiliary agent, it is remarkably improved the control effect to Animal diseases, city
Field degree of recognition is gradually increased.Therefore, transfer factor will discharge its application advantage, transfer factor in veterinary clinic field step by step
The exploitation of product will be of great practical significance to the development of animal husbandry with application.
Pig blue-ear disease is also known as " pig breeds and respiratory disorder syndrome ", is to be bred to cause with respiratory syndrome virus by pig
The infectious disease that is characterized with Adult Pig dysgenesia, premature labor, miscarriage and stillborn foetus and piglet adnormal respiration of one kind, it is destroyed
The immune function of pig body macrophage, causes immunosupress, easy secondary various respiratory tract disease.In the world it is many country and
Pig blue-ear disease all occurred for area, which is passed to China in mid-nineties 90 in last century, and World Organization for Animal Health is classified as method
Surely animal epidemic is reported, China is classified as two class animal epidemics.Epidemic situation invasion breeding and respiratory system, are mainly shown as that sow is numerous
Grow high mortality, three big symptom of the breathing problem of bred pigs before obstacle, weaned piglet.Height is shown as through production and first farrowing sow more
Heat, spirit are depressed, apocleisis, expiratory dyspnea, and a small number of sow ears, nipple, vulva, abdomen, tail portion are issued, the most normal with have sharp ears
See.After there are these symptoms, it is reachable to give birth to mummy, stillborn foetus and sick and weak piglet, mortinatality for a large amount of farrowing sow miscarriages or premature labor
One.Premature labor Farrowing has some setbacks, and lacks milk or without milk.Piglet particularly sucks the breast pig, and the death rate is very high, up to more than 80%.
A large amount of experimental study shows that transfer factor has association to pig breeding with respiratory disorder syndrome virus virus live vaccine
Same-action can significantly improve Cellular Immunity level, shorten the immune response phase, promote the generation of antibody, and extend anti-
Body peak period is allowed to maintain the longer time.But detection transfer factor is lived with pig breeding with respiratory disorder syndrome virus at present
Vaccine is used in combination the main of effect and is showed by detecting antibody and cell factor, and testing cost is high, the period is long and organism
Individual difference is big, and testing result is more unstable.Therefore establish it is a kind of quick, conveniently, inspection cost is low and inspection result is relatively steady
Fixed detection Swine spleen transfer factor and the method for porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect have important
Meaning.
Invention content
Present invention aims at providing, a kind of showing on cell, inspection cost is low and the metastable inspection of inspection result
The method for surveying Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect
To achieve these goals, the present invention uses following technical scheme：
A kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect, packet
Include following steps：
1）Porcine reproductive and respiratory syndrome virus live vaccine stoste is taken, with newborn bovine serum containing 1.8-2.2%, 0.8-1.2% moulds
The dual anti-complete DMEM culture mediums of element-streptomysin are diluted to containing 200 TCID50Virus liquid；
Swine spleen transfer factor is taken, with the dual anti-complete DMEM of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps
Content of peptides is diluted to 1 mg/mL by culture medium dilution；
2）Cell is trained in the dual anti-complete DMEM culture mediums of newborn bovine serum containing 8-12%, 0.8-1.2% Pen .- Streps
It supports to individual layer；
3) cell monolayer in step 2) is taken, with dual anti-complete of newborn bovine serum containing 8-12%, 0.8-1.2% Pen .- Streps
Full DMEM culture mediums dilution, adjustment cell concentration are 2 ~ 3 × 105A/mL, by the cell inoculation after dilution in tissue culture plate
In, continue to cultivate；Phosphate buffer wash cell plate is used after forming cell monolayer, removes culture medium；
4）By step 1）The Swine spleen transfer factor after virus liquid and dilution after dilution is splined on step 3）Tissue culture plate
In, and cultivate incubated cell；Applied sample amount is 100 μ L/ holes, specific as follows：
The dual anti-complete DMEM culture mediums 35-45 μ L of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
Virus liquid 20-30 μ L；
Swine spleen transfer factor 30-40 μ L；
5) by step 4）The cell of incubation is reacted with MTT solution, continues to be incubated；
6) by step 5）The cell of incubation carries out dissolution process with dimethyl sulfoxide (DMSO)；
7）By step 6）Treated, and cell plates are positioned over measurement OD values in microplate reader, measure cell activity.
Step 1）In, the porcine reproductive and respiratory syndrome virus live vaccine is CH-1R plants.
Step 2）In, the cell is pk-15 cells.
Step 3）In, the tissue culture plate is 96 porocyte culture plates.
Further, step 3）In, cell is diluted to be as follows：By the cell monolayer in step 2) no calcium, magnesium
The phosphate buffer of ion is washed 2-3 times, is then added in 1.0 mL pancreatin digestive juices, is gently shaken culture dish, make digestive juice cloth
Full all cell surfaces, place several minutes in 37 DEG C, then discard digestive juice, green with newborn bovine serum containing 8-12%, 0.8-1.2%
The dual anti-complete DMEM culture mediums of mycin-streptomysin elute cell, and the cell suspension after elution is diluted, and adjust cell
A concentration of 2 ~ 3 × 105A/mL.
Further, step 3）In, the cell culture condition after dilution is：100 μ L/ holes of cell inoculation amount, in 36.5-
37.5℃、4.5-5.5 % CO2Under the conditions of culture 18-24 H-shapeds into cell monolayer.
Preferably, step 4）In, applied sample amount is specific as follows：
The dual anti-40 μ L of complete DMEM culture mediums of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
25 μ L of virus liquid；
35 μ L of Swine spleen transfer factor（That is a concentration of 350 μ g/mL of Swine spleen transfer factor d）.
Step 4）In, the condition for cultivating incubated cell is in 36.5-37.5 DEG C, 4.5-5.5 % CO2Under the conditions of cultivate 40-
48 h 。
Step 5）In, cell continues the time being incubated as 3.5-4.5h.
Step 7）In, OD values are measured at 480-500nm wavelength.
The present invention, which combines Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine using above method, to be exempted from
Epidemic disease effect is detected and prior art difference is, the prior art is detected on entity animal（Pig）Detection, is examined into
This height, the period is long and bion difference easily leads to greatly unstable result, and the method for the present invention is showed on cell, quick,
Convenient, inspection cost is low, as a result stablizes relatively.
Description of the drawings
Fig. 1 is measurement result of the different TF concentration to pk-15 cell proliferation in vitro；
Fig. 2 is measurement result of the different volumes vaccine diluent to pk-15 cell proliferation in vitro；
When Fig. 3 is used in combination for 8 batches of TF solution with porcine reproductive and respiratory syndrome virus live vaccine, the measure knot of cell Proliferation
Fruit.
Specific embodiment
A kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect,
Include the following steps：
1）Take porcine reproductive and respiratory syndrome virus live vaccine（CH-1R plants）Stoste, with newborn bovine serum containing 1.8-2.2%, 0.8-
The dual anti-complete DMEM culture mediums of 1.2% Pen .- Strep are diluted to containing 200 TCID50Virus liquid；
Swine spleen transfer factor is taken, with the dual anti-complete DMEM of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps
Content of peptides is diluted to 1 mg/mL by culture medium dilution；
2）By pk-15 in the dual anti-complete DMEM culture mediums of newborn bovine serum containing 8-12%, 0.8-1.2% Pen .- Streps
Cell culture is to individual layer；
3) by step 2）In cell monolayer washed 2-3 times with the phosphate buffer of no calcium and magnesium ion, then add in 1.0 mL
Pancreatin digestive juice, gently shakes culture dish, and digestive juice is made to be covered with all cell surfaces, places several minutes in 37 DEG C, then discards
Digestive juice is washed cell with the dual anti-complete DMEM culture mediums of newborn bovine serum containing 8-12%, 0.8-1.2% Pen .- Streps
It is de-, the cell suspension after elution is diluted, adjustment cell concentration is 2 ~ 3 × 105A/mL, by the cell inoculation after dilution
In 96 porocyte culture plates, 100 μ L/ holes, in 36.5-37.5 DEG C, 4.5-5.5 % CO2Under the conditions of cultivate 20 h or so
（18-24 h）Cell monolayer is formed, then with phosphate buffer wash cell plate, removes culture medium；
4）By step 1）The Swine spleen transfer factor after virus liquid and dilution after dilution is splined on step 3）Tissue culture plate
In, in 36.5-37.5 DEG C, 4.5-5.5 % CO2Under the conditions of culture 40-48 h；
Wherein, applied sample amount is 100 μ L/ holes, specific as follows：
The dual anti-complete DMEM culture mediums 35-45 μ L of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
Virus liquid 20-30 μ L；
Swine spleen transfer factor 30-40 μ L（Preferably 35 μ L）；
5) in step 4）Tissue culture plate in add in MTT solution, 10 μ L/ holes, continue be incubated 3.5-4.5h, then discard
Clearly, dimethyl sulphoxide solution is added in, 100 μ L/ holes slowly shake up 10min；It is measured at 480-500nm wavelength per hole extinction
Value measures cell activity, compared with the control group, calculates cell proliferation rate.
Embodiment 1
A kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect, including
Following steps：
1）Take porcine reproductive and respiratory syndrome virus live vaccine（CH-1R plants）Stoste, with containing 2% newborn bovine serum, 1% penicillin-
The dual anti-complete DMEM culture mediums of streptomysin are diluted to containing 200 TCID50Virus liquid；
Swine spleen transfer factor is taken, being diluted with the dual anti-complete DMEM culture mediums containing 2% newborn bovine serum, 1% Pen .- Strep will
Content of peptides is diluted to 1 mg/mL；
2）Pk-15 cells are trained in the complete DMEM culture medium dual anti-containing 10% newborn bovine serum, 1.0% Pen .- Strep
It supports to individual layer；
3) by step 2）In cell monolayer washed 2 times with the phosphate buffer of no calcium and magnesium ion, then add in 1.0 mL pancreases
Enzymic digestion liquid, gently shakes culture dish, and digestive juice is made to be covered with all cell surfaces, is placed several minutes in 37 DEG C, then discards and disappear
Change liquid, cell is eluted with the dual anti-complete DMEM culture mediums containing 10% newborn bovine serum, 1.0% Pen .- Strep, will be eluted
Cell suspension afterwards is diluted, and adjustment cell concentration is 2 ~ 3 × 105A/mL, by the cell inoculation after dilution in 96 hole cells
In culture plate, 100 μ L/ holes, in 37 DEG C, 5.0 % CO2Under the conditions of cultivate 20 h or so formed cell monolayer, then use phosphoric acid
W salt buffer washes cell plates remove culture medium；
4）By step 1）The Swine spleen transfer factor after virus liquid and dilution after dilution is splined on step 3）Tissue culture plate
In, in 37 DEG C, 5.0 % CO2Under the conditions of cultivate 44 h；
Wherein, applied sample amount is 100 μ L/ holes, specific as follows：
Containing the dual anti-40 μ L of complete DMEM culture mediums of 2% newborn bovine serum, 1.0% Pen .- Strep；
25 μ L of virus liquid；
35 μ L of Swine spleen transfer factor；
5) in step 4）Tissue culture plate in add in MTT solution, 10 μ L/ holes, continue be incubated 4h, then discard supernatant, add
Enter dimethyl sulphoxide solution, 100 μ L/ holes slowly shake up 10min；It is measured at 490nm wavelength per hole light absorption value, measures cell
Activity.
Embodiment 2
A kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect, including
Following steps：
1）Take porcine reproductive and respiratory syndrome virus live vaccine（CH-1R plants）Stoste, with containing 1.8% newborn bovine serum, 0.8% mould
The dual anti-complete DMEM culture mediums of element-streptomysin are diluted to containing 200 TCID50Virus liquid；
Swine spleen transfer factor is taken, it is dilute with the dual anti-complete DMEM culture mediums containing 1.8% newborn bovine serum, 0.8% Pen .- Strep
It releases and content of peptides is diluted to 1 mg/mL；
2）By pk-15 cell culture in the complete DMEM culture medium dual anti-containing 8% newborn bovine serum, 0.8% Pen .- Strep
To individual layer；
3) by step 2）In cell monolayer washed 2 times with the phosphate buffer of no calcium and magnesium ion, then add in 1.0 mL pancreases
Enzymic digestion liquid, gently shakes culture dish, and digestive juice is made to be covered with all cell surfaces, is placed several minutes in 37 DEG C, then discards and disappear
Change liquid, cell is eluted with the dual anti-complete DMEM culture mediums containing 8% newborn bovine serum, 0.8% Pen .- Strep, will be eluted
Cell suspension afterwards is diluted, and adjustment cell concentration is 2 ~ 3 × 105A/mL, by the cell inoculation after dilution in 96 hole cells
In culture plate, 100 μ L/ holes, in 36.5 DEG C, 4.5 % CO2Under the conditions of cultivate 20 h or so formed cell monolayer, then use phosphorus
Phthalate buffer washs cell plates, removes culture medium；
4）By step 1）The Swine spleen transfer factor after virus liquid and dilution after dilution is splined on step 3）Tissue culture plate
In, in 36.5 DEG C, 4.5 % CO2Under the conditions of cultivate 48 h；
Wherein, applied sample amount is 100 μ L/ holes, specific as follows：
Containing the dual anti-35 μ L of complete DMEM culture mediums of 1.8% newborn bovine serum, 0.8% Pen .- Strep；
25 μ L of virus liquid；
40 μ L of Swine spleen transfer factor；
5) in step 4）Tissue culture plate in add in MTT solution, 10 μ L/ holes, continue be incubated 3.5h, then discard supernatant,
Dimethyl sulphoxide solution is added in, 100 μ L/ holes slowly shake up 10min；It is measured at 490nm wavelength per hole light absorption value, measured thin
Cytoactive.
Embodiment 3
A kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect, including
Following steps：
1）Take porcine reproductive and respiratory syndrome virus live vaccine（CH-1R plants）Stoste, with containing 2.2% newborn bovine serum, 1.2% mould
The dual anti-complete DMEM culture mediums of element-streptomysin are diluted to the virus liquid containing 200 TCID50；
Pig spleen transfer factor injection is taken, is trained with the dual anti-complete DMEM containing 2.2% newborn bovine serum, 1.2% Pen .- Strep
It supports base dilution and content of peptides is diluted to 1 mg/mL；
2）Pk-15 cells are trained in the complete DMEM culture medium dual anti-containing 12% newborn bovine serum, 1.2% Pen .- Strep
It supports to individual layer；
3) by step 2）In cell monolayer washed 2 times with the phosphate buffer of no calcium and magnesium ion, then add in 1.0 mL pancreases
Enzymic digestion liquid, gently shakes culture dish, and digestive juice is made to be covered with all cell surfaces, is placed several minutes in 37 DEG C, then discards and disappear
Change liquid, cell is eluted with the dual anti-complete DMEM culture mediums containing 12% newborn bovine serum, 1.2% Pen .- Strep, will be eluted
Cell suspension afterwards is diluted, and adjustment cell concentration is 2 ~ 3 × 105A/mL, by the cell inoculation after dilution in 96 hole cells
In culture plate, 100 μ L/ holes, in 37.5 DEG C, 5.5 % CO2Under the conditions of cultivate 20 h or so（18-24 h）Form cell monolayer,
Then with phosphate buffer wash cell plate, culture medium is removed；
4）By step 1）The pig spleen transfer factor injection after virus liquid and dilution after dilution is splined on step 3）Cell
In culture plate, in 37.5 DEG C, 5.5 % CO2Under the conditions of culture 40- h；
Wherein, applied sample amount is 100 μ L/ holes, specific as follows：
Containing the dual anti-45 μ L of complete DMEM culture mediums of 2.2% newborn bovine serum, 1.2% Pen .- Strep；
25 μ L of virus liquid；
30 μ L of pig spleen transfer factor injection；
5) in step 4）Tissue culture plate in add in MTT solution, 10 μ L/ holes, continue be incubated 4.5h, then discard supernatant,
Dimethyl sulphoxide solution is added in, 100 μ L/ holes slowly shake up 10min；It is measured at 490nm wavelength per hole light absorption value, measured thin
Cytoactive.
Embodiment 4
A kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect, tool
Body step with embodiment 1, difference lies in：
Step 4）In, applied sample amount is 100 μ L/ holes, specific as follows：
Containing the dual anti-35 μ L of complete DMEM culture mediums of 2.2% newborn bovine serum, 1.2% Pen .- Strep；
30 μ L of virus liquid；
35 μ L of Swine spleen transfer factor.
Embodiment 5
A kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect, tool
Body step with embodiment 1, difference lies in：
Step 4）In, applied sample amount is 100 μ L/ holes, specific as follows：
Containing the dual anti-40 μ L of complete DMEM culture mediums of 2.2% newborn bovine serum, 1.2% Pen .- Strep；
20 μ L of virus liquid；
40 μ L of Swine spleen transfer factor.
Embodiment 6
Swine spleen transfer factor determines the optimal concentration of cell proliferation in vitro
In the present invention, step 4）Swine spleen transfer factor in middle addition tissue culture plate is every hole 30-40 μ L, preferably 35 μ L,
I.e. Swine spleen transfer factor obtains preferred concentration as 350 μ g/mL, which is that screening determines by the following method：
1st, culture medium is prepared
It takes DMEM culture mediums, adds in that Pen .- Strep is dual anti-and newborn bovine serum, be configured to containing 1 % Pen .- Streps
Dual anti-and 10 % newborn bovine serum DMEM culture mediums (hereinafter referred to as 10 % DMEM culture mediums) and containing 1 % penicillin-strepto-
Dual anti-and 2 % newborn bovine serum the DMEM culture mediums (hereinafter referred to as 2 % DMEM culture mediums) of element, are placed in 4 DEG C of preservations.
2nd, cell culture
Eugonic pk-15 cell monolayers with the PBS of no calcium and magnesium ion are washed 2 times, add in 1.0 mL pancreatin digestive juices, gently
Jog moves culture dish, and digestive juice is made to be covered with all cell surfaces.It is placed several minutes in 37 DEG C.Digestive juice is discarded, with 10 % DMEM
Culture medium elutes cell.Cell suspension after elution is diluted with 10 % DMEM culture mediums, adjustment cell concentration for 2 ~
3×105A/mL.By the cell inoculation diluted in 96 porocyte culture plates, 100 μ L/ holes.In 37 DEG C, 5 % CO2Under the conditions of
It cultivates 20 h or so and forms cell monolayer.
3rd, measuring samples dilute
Swine spleen transfer factor is taken, content of peptides is diluted to 1 mg/mL with the DMEM culture mediums containing 2 % serum, makees measuring samples.
Take porcine reproductive and respiratory syndrome virus live vaccine（CH-1R plants）Stoste, it is dilute with the DMEM culture mediums containing 2% serum
It releases to containing 200 TCID50Virus liquid.
4th, Swine spleen transfer factor is taken（TF）The optimal concentration of cell proliferation in vitro is determined
TF concentration designs 7 levels, is respectively：200 μg/mL、250 μg/mL、300 μg/mL、350 μg/mL、400 μg/
mL、450 μg/mL、500 μg/mL.Cell proliferation test is carried out in 96 porocyte culture plates.It tests and sets 2 groups, 1
Test group (cell+TF), 1 control group (cell), each sample set 4 repeating holes.Result of the test is measured with mtt assay.It measures
OD490After value, cell proliferation rate is calculated, selects TF concentration optimum values.
It is as follows to calculate cell proliferation rate formula：Cell proliferation rate=test group OD values/control group 0D values.
5th, experimental result
TF concentration is to the measurement result of pk-15 cell proliferation in vitro rates as shown in Figure 1, determining TF to pk-15 cells in vitro by Fig. 1
The 350 μ g/mL of optimal concentration of proliferation.
Embodiment 7
Vaccine diluent（Virus liquid）The optimal volume of cell proliferation in vitro is determined
In the present invention, step 4）Vaccine diluent in middle addition tissue culture plate is per hole 20-30 μ L, preferably 25 μ L, is somebody's turn to do
Optimal volume is that screening determines by the following method：
1st, culture medium is prepared
It takes DMEM culture mediums, adds in that Pen .- Strep is dual anti-and newborn bovine serum, be configured to containing 1 % Pen .- Streps
Dual anti-and 10 % newborn bovine serum DMEM culture mediums (hereinafter referred to as 10 % DMEM culture mediums) and containing 1 % penicillin-strepto-
Dual anti-and 2 % newborn bovine serum the DMEM culture mediums (hereinafter referred to as 2 % DMEM culture mediums) of element, are placed in 4 DEG C of preservations.
2nd, cell culture
Eugonic pk-15 cell monolayers with the PBS of no calcium and magnesium ion are washed 2 times, add in 1.0 mL pancreatin digestive juices, gently
Jog moves culture dish, and digestive juice is made to be covered with all cell surfaces.It is placed several minutes in 37 DEG C.Digestive juice is discarded, with 10 % DMEM
Culture medium elutes cell.Cell suspension after elution is diluted with 10 % DMEM culture mediums, adjustment cell concentration for 2 ~
3×105A/mL.By the cell inoculation diluted in 96 porocyte culture plates, 100 μ L/ holes.In 37 DEG C, 5 % CO2Under the conditions of
It cultivates 20 h or so and forms cell monolayer.
3rd, Swine spleen transfer factor dilutes
Swine spleen transfer factor is taken, content of peptides is diluted to 1 mg/mL with the DMEM culture mediums containing 2 % serum.
Take porcine reproductive and respiratory syndrome virus live vaccine（CH-1R plants）Stoste, it is dilute with the DMEM culture mediums containing 2% serum
It releases to containing 200 TCID50Virus liquid.
4th, vaccine diluent determines the optimal volume of cell proliferation in vitro
Vaccine volume designs 9 levels, is respectively：10 μL、15 μL、20 μL、25 μL、30 μL、35 μL、40 μL、45 μ
L、50 μL.Cell proliferation test is carried out in 96 porocyte culture plates.Experiment sets 2 groups, 1 test group (cell+transfer
The factor+vaccine), 1 control group (cell+transfer factor), each sample sets 4 repeating holes.Result of the test is measured with mtt assay.
Measure OD490After value, calculate cell proliferation rate, select vaccine volume most just when.
It is as follows to calculate cell proliferation rate formula：Cell proliferation rate=test group OD values/control group 0D values.
5th, experimental result
Vaccine diluent volume is to the measurement result of pk-15 cell proliferation in vitro rates as shown in Fig. 2, determining that vaccine dilutes by Fig. 2
Liquid is 25 μ L to the optimal volume of pk-15 cell proliferation in vitro.
Embodiment 8
Swine spleen transfer factor（TF）The effect to pk-15 cells is used in combination with porcine reproductive and respiratory syndrome virus live vaccine
In TF concentration optimum value and vaccine diluent（Virus liquid）Under conditions of being determined to the optimal volume of cell proliferation in vitro,
96 orifice plates for having grown up to cell monolayer are taken, tests and is carried out according to following design：
Control group：+ 25 μ L porcine reproductive and respiratory syndrome virus liquid of 75 μ L, 2% DMEM culture mediums；
Test group：+ 35 μ L measuring samples of+25 μ L porcine reproductive and respiratory syndrome virus liquid of 40 μ L, 2% DMEM culture mediums；
Every group sets the repetition of 4 holes.It is placed in 37 DEG C, 5% CO2Under the conditions of cultivate 44 h, add in MTT solution, culture 4 is continued in 10 μ L/ holes
H is discarded supernatant, and adds in DMSO solution, 100 μ L/ holes slowly shake up 10 min.It is measured at 490 nm per hole light absorption value, meter
Calculate cell proliferation rate.
Different batches TF joint porcine reproductive and respiratory syndrome virus live vaccines are used to the exercising results of pk-15 cells such as
Shown in Fig. 3.Experiment selects the 8 batches of TF solution to carry out altogether, is examined according to statistics T, test group compared with the control group, except 2017008
Pk-15 cell proliferation rate indifferences (p when batch TF is used in combination with vaccine>0.05), remaining batch wise differences significantly (p<0.05).
When 2017011 crowdes of TF are used in combination with vaccine, pk-15 cell proliferation rates can reach 126.89%.The breeding of different batches TF and pig with
When breath syndrome virus live vaccine is used in combination, pk-15 cell proliferation rates average value is 119.71% ± 5.64%.
The present invention determines influence of the TF solution to pk-15 cell Proliferations using mtt assay, as a result, it has been found that, when TF is a concentration of
During 350 μ g/ml, the growth rate pole of test group pk-15 cells is significantly higher than control group（P<0.01）, and tried in five repetitions
The growth rate for testing middle test group pk-15 cells is stablized relatively；When TF combines with porcine reproductive and respiratory syndrome virus live vaccine
In use, it is stimulated when promoting the proliferation effect of pk-15 cells than porcine reproductive and respiratory syndrome virus live vaccine is used alone
Good (the p of pk-15 cell Proliferations effect<0.05).Illustrate that the method for the present invention can enhance porcine reproductive and respiratory syndrome virus live vaccine
Immune effect, detection method of the invention show on cell, and inspection cost is low, as a result stablize relatively.
Claims (10)
1. a kind of method for detecting Swine spleen transfer factor and porcine reproductive and respiratory syndrome virus live vaccine combined immunization effect,
It is characterized in that：It includes the following steps：
1）Porcine reproductive and respiratory syndrome virus live vaccine stoste is taken, with newborn bovine serum containing 1.8-2.2%, 0.8-1.2% moulds
The dual anti-complete DMEM culture mediums of element-streptomysin are diluted to containing 200 TCID50Virus liquid；
Swine spleen transfer factor is taken, with the dual anti-complete DMEM of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps
Content of peptides is diluted to 1 mg/mL by culture medium dilution；
2）Cell is trained in the dual anti-complete DMEM culture mediums of newborn bovine serum containing 8-12%, 0.8-1.2% Pen .- Streps
It supports to individual layer；
3) cell monolayer in step 2) is taken, with dual anti-complete of newborn bovine serum containing 8-12%, 0.8-1.2% Pen .- Streps
Full DMEM culture mediums dilution, adjustment cell concentration are 2 ~ 3 × 105A/mL, by the cell inoculation after dilution in tissue culture plate
In, continue to cultivate；Phosphate buffer wash cell plate is used after forming cell monolayer, removes culture medium；
4）By step 1）The Swine spleen transfer factor after virus liquid and dilution after dilution is splined on step 3）Tissue culture plate
In, and cultivate incubated cell；Applied sample amount is 100 μ L/ holes, specific as follows：
The dual anti-complete DMEM culture mediums 35-45 μ L of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
Virus liquid 20-30 μ L；
Swine spleen transfer factor 30-40 μ L；
5) by step 4）The cell of incubation is reacted with MTT solution, continues to be incubated；
6) by step 5）The cell of incubation carries out dissolution process with dimethyl sulfoxide (DMSO)；
7）By step 6）Treated, and cell plates are positioned over measurement OD values in microplate reader, measure cell activity.
2. a kind of detection Swine spleen transfer factor according to claim 1 and porcine reproductive and respiratory syndrome virus live vaccine connection
The method for closing immune effect, it is characterised in that：Step 1）In, the porcine reproductive and respiratory syndrome virus live vaccine is CH-1R
Strain.
3. a kind of detection Swine spleen transfer factor according to claim 1 and porcine reproductive and respiratory syndrome virus live vaccine connection
The method for closing immune effect, it is characterised in that：Step 2）In, the cell is pk-15 cells.
4. a kind of detection Swine spleen transfer factor according to claim 1 and porcine reproductive and respiratory syndrome virus live vaccine connection
The method for closing immune effect, it is characterised in that：Step 3）In, the tissue culture plate is 96 porocyte culture plates.
5. a kind of detection Swine spleen transfer factor according to claim 1 and porcine reproductive and respiratory syndrome virus live vaccine connection
The method for closing immune effect, it is characterised in that：Step 3）In, cell is diluted to be as follows：By the individual layer in step 2)
Cell is washed 2-3 times with phosphate buffer, and then adding in pancreatin digestive juice makes it be covered with all cell surfaces, and number is placed in 37 DEG C
Minute, digestive juice is then discarded, is trained with the dual anti-complete DMEM of newborn bovine serum containing 8-12%, 0.8-1.2% Pen .- Streps
It supports base to elute cell, the cell suspension after elution is diluted, adjustment cell concentration is 2 ~ 3 × 105A/mL.
6. a kind of detection Swine spleen transfer factor according to claim 1 and porcine reproductive and respiratory syndrome virus live vaccine
The method of combined immunization effect, it is characterised in that：Step 3）In, the cell culture condition after dilution is：100 μ of cell inoculation amount
L/ holes, in 36.5-37.5 DEG C, 4.5-5.5 % CO2Under the conditions of culture 18-24 H-shapeds into cell monolayer.
7. a kind of detection Swine spleen transfer factor according to claim 1 and porcine reproductive and respiratory syndrome virus live vaccine connection
The method for closing immune effect, it is characterised in that：Step 4）In, applied sample amount is specific as follows：
The dual anti-40 μ L of complete DMEM culture mediums of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
25 μ L of virus liquid；
35 μ L of Swine spleen transfer factor.
8. a kind of detection Swine spleen transfer factor according to claim 1 and porcine reproductive and respiratory syndrome virus live vaccine
The method of combined immunization effect, it is characterised in that：Step 4）In, the condition for cultivating incubated cell is in 36.5-37.5 DEG C, 4.5-
5.5 % CO2Under the conditions of culture 40-48 h.
9. a kind of detection Swine spleen transfer factor according to claim 1 and porcine reproductive and respiratory syndrome virus live vaccine connection
The method for closing immune effect, it is characterised in that：Step 5）In, cell continues the time being incubated as 3.5-4.5h.
10. a kind of detection Swine spleen transfer factor according to claim 1 and porcine reproductive and respiratory syndrome virus live vaccine
The method of combined immunization effect, it is characterised in that：Step 7）In, OD values are measured at 480-500nm wavelength.
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| CN113907045A (en) * | 2021-11-09 | 2022-01-11 | 福建省连江县刘氏兔业养殖场 | Method for breeding new strain of pigs and application thereof |
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