CN108373989A - A method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect - Google Patents
A method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect Download PDFInfo
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Abstract
The present invention provides a kind of methods of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect, include the following steps：1）Cell culture；2）Cell inoculation controls cell density；3）Live vaccines of hog cholera and Swine spleen transfer factor are splined in tissue culture plate, incubated cell is cultivated；4）Cell is reacted with MTT solution, continues to be incubated, then carries out dissolution process with DMSO, finally cell plates are placed and measure OD values 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 live vaccines of hog cholera combined immunization.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of detection Swine spleen transfer factor is combined with live vaccines of hog cholera exempts from
The method of epidemic disease effect.
Background technology
Transfer factor（Transfer Factor, TF）Animal body immune function can be adjusted, vaccine immunity is cooperateed with, 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 anisotropicly.The ingredient of transfer factor includes mainly oligonucleotide and polypeptide, can induce body and exempts from
Epidemic disease cell activation, enhance immunity of organisms, can shift 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, it is used as immune stimulating agent and auxiliary agent, 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 step by step in veterinary clinic field
The exploitation of product will be of great practical significance to the development of animal husbandry with application.
Swine fever be commonly called as " rinderpest " be it is a kind of there is highly infectious epidemic disease, be threaten pig breeding industry Infectious Diseases it
One, it is characterized in that：It is acute, change in septic, organa parenchymatosum's bleeding, necrosis and infarct；Chronic is in cellulosic gangrenosum acne intestines
Inflammation, later stage often have paratyphoid and pasteurellosis secondary.The disease is mainly by being in direct contact, or due to the medium of contact stain
And it falls ill.Alimentary canal, nasal membrane and the skin of rupture are routes of infection.Can all it occur throughout the year, with spring and summer rainy season
Section is more.
A large amount of experimental study shows that transfer factor has synergistic effect to live vaccines of hog cholera, and it is thin to significantly improve body
Born of the same parents' immune level shortens the immune response phase, promotes the generation of antibody, and extend antibody peak period, be allowed to remain longer
Time.But at present detection transfer factor and live vaccines of hog cholera be used in combination effect it is main by detect antibody and cell factor come
Show, testing cost is high, the period is long and organism individual difference is big, and testing result is more unstable.Therefore a kind of quick, side is established
Just, inspection cost is low and the side of the metastable detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect of inspection result
Method is of great significance.
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 live vaccines of hog cholera combined immunization effect
To achieve the goals above, the present invention uses following technical scheme：
A method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect comprising following steps：
1）Live vaccines of hog cholera stoste is taken, with dual anti-complete of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps
DMEM culture mediums are diluted to the virus liquid containing 100 RID；
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 single 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 holes μ L/, specific as follows：
The dual anti-complete DMEM culture mediums 25-35 μ L of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
Virus liquid 30-40 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 live vaccines of hog cholera is live vaccines of hog cholera（Rabbit source）.
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, and 1.0 mL pancreatin digestive juices are then added, gently shakes culture dish, makes 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 holes μ L/ of cell inoculation amount, in 36.5-
37.5℃、4.5-5.5 % CO2Under the conditions of culture 18-24 H-shapeds at cell monolayer.
Preferably, step 4）In, applied sample amount is specific as follows：
The dual anti-30 μ L of complete DMEM culture mediums of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
35 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）.
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 is detected Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect using above method, and existing
There is technology the difference lies in that prior art detection is on entity animal（Pig）Detection, inspection cost is high, and the period is long and biology is a
Body difference easily leads to greatly unstable result, and the method for the present invention is showed on cell, and quickly, conveniently, inspection cost is low, as a result
Stablize relatively.
Description of the drawings
Fig. 1 is measurement result of the different TF concentration to pk-15 cell proliferation in vitro；Subscript " * " indicates significant difference (P<
0.05)；Subscript " * * " indicates difference extremely significantly (P<0.01)；
Fig. 2 is measurement result of the different volumes vaccine diluent to pk-15 cell proliferation in vitro；
When Fig. 3 is that 8 batches of TF solution are used in combination with live vaccines of hog cholera, the measurement result of cell Proliferation；Subscript " * " indicates that difference is aobvious
Write (P<0.05)；Subscript " * * " indicates difference extremely significantly (P<0.01).
Specific implementation mode
A method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect include the following steps：
1）Take live vaccines of hog cholera（Rabbit source）Stoste, it is dual anti-with newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps
Complete DMEM culture mediums be diluted to the virus liquid containing 100 RID；
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 single layer；
3) by step 2）In cell monolayer washed 2-3 times with the phosphate buffer of no calcium and magnesium ion, 1.0 mL are then added
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 holes μ L/, 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, phosphate buffer wash cell plate is then used, 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 holes μ L/, specific as follows：
The dual anti-complete DMEM culture mediums 25-35 μ L of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
Virus liquid 30-40 μ L（Preferably 35 μ L）；
Swine spleen transfer factor 30-40 μ L（Preferably 35 μ L）；
5) in step 4）Tissue culture plate in be added MTT solution, 10 holes μ L/, continue be incubated 3.5-4.5h, then discard
Clearly, dimethyl sulphoxide solution is added, 100 holes μ L/ slowly shake up 10min；It is measured per hole extinction at 480-500nm wavelength
Value measures cell activity, compared with the control group, calculates cell proliferation rate.
Embodiment 1
A method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect include the following steps：
1）Take live vaccines of hog cholera（Rabbit source）Stoste is trained with the dual anti-complete DMEM containing 2% newborn bovine serum, 1% Pen .- Strep
Foster base is diluted to the virus liquid containing 100 RID；
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 single layer；
3) by step 2）In cell monolayer washed 2 times with the phosphate buffer of no calcium and magnesium ion, 1.0 mL pancreases are then added
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 holes μ L/, 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 holes μ L/, specific as follows：
Containing the dual anti-30 μ L of complete DMEM culture mediums of 2% newborn bovine serum, 1.0% Pen .- Strep；
35 μ L of virus liquid；
35 μ L of Swine spleen transfer factor；
5) in step 4）Tissue culture plate in be added MTT solution, 10 holes μ L/, continue be incubated 4h, then discard supernatant, add
Enter dimethyl sulphoxide solution, 100 holes μ L/ slowly shake up 10min；It is measured at 490nm wavelength per hole light absorption value, measures cell
Activity.
Embodiment 2
A method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect include the following steps：
1）Take live vaccines of hog cholera（Rabbit source）Stoste, with containing dual anti-complete of 1.8% newborn bovine serum, 0.8% Pen .- Strep
DMEM culture mediums are diluted to the virus liquid containing 100 RID；
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 single layer；
3) by step 2）In cell monolayer washed 2 times with the phosphate buffer of no calcium and magnesium ion, 1.0 mL pancreases are then added
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 holes μ L/, 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 holes μ L/, specific as follows：
Containing the dual anti-25 μ L of complete DMEM culture mediums of 1.8% newborn bovine serum, 0.8% Pen .- Strep；
35 μ L of virus liquid；
40 μ L of Swine spleen transfer factor；
5) in step 4）Tissue culture plate in be added MTT solution, 10 holes μ L/, continue be incubated 3.5h, then discard supernatant,
Dimethyl sulphoxide solution is added, 100 holes μ L/ slowly shake up 10min；It is measured at 490nm wavelength per hole light absorption value, is measured thin
Cytoactive.
Embodiment 3
A method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect include the following steps：
1）Take live vaccines of hog cholera（Rabbit source）Stoste, with containing dual anti-complete of 2.2% newborn bovine serum, 1.2% Pen .- Strep
DMEM culture mediums are diluted to the virus liquid containing 100 RID；
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 single layer；
3) by step 2）In cell monolayer washed 2 times with the phosphate buffer of no calcium and magnesium ion, 1.0 mL pancreases are then added
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 holes μ L/, in 37.5 DEG C, 5.5 % CO2Under the conditions of cultivate 20 h or so（18-24 h）Cell monolayer is formed,
Then phosphate buffer wash cell plate is used, 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 holes μ L/, 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；
35 μ L of virus liquid；
30 μ L of pig spleen transfer factor injection；
5) in step 4）Tissue culture plate in be added MTT solution, 10 holes μ L/, continue be incubated 4.5h, then discard supernatant,
Dimethyl sulphoxide solution is added, 100 holes μ L/ slowly shake up 10min；It is measured at 490nm wavelength per hole light absorption value, is measured thin
Cytoactive.
Embodiment 4
A method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect, specific steps are the same as embodiment 1, area
It is not：
Step 4）In, applied sample amount is 100 holes μ L/, specific as follows：
Containing the dual anti-25 μ L of complete DMEM culture mediums of 2.2% newborn bovine serum, 1.2% Pen .- Strep；
40 μ L of virus liquid；
35 μ L of Swine spleen transfer factor.
Embodiment 5
A method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect, specific steps are the same as embodiment 1, area
It is not：
Step 4）In, applied sample amount is 100 holes μ L/, specific as follows：
Containing the dual anti-30 μ L of complete DMEM culture mediums of 2.2% newborn bovine serum, 1.2% Pen .- Strep；
30 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. the preferred concentration of Swine spleen transfer factor is 350 μ g/mL, which is that screening is determining by the following method：
1, culture medium is prepared
It takes DMEM culture mediums, is added that Pen .- Strep is dual anti-and newborn bovine serum, be configured to contain 1 % Pen .- Streps
Dual anti-and 10 % newborn bovine serum DMEM culture mediums (hereinafter referred to as 10 % DMEM culture mediums) and contain 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.
2, cell culture
Eugonic pk-15 cell monolayers are washed 2 times with the PBS of no calcium and magnesium ion, 1.0 mL pancreatin digestive juices are added, 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 be 2 ~
3×105A/mL.By the cell inoculation diluted in 96 porocyte culture plates, 100 holes μ L/.In 37 DEG C, 5 % CO2Under the conditions of
It cultivates 20 h or so and forms cell monolayer.
3, 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 live vaccines of hog cholera（Rabbit source）Stoste is diluted to the virus liquid containing 100 RID with the DMEM culture mediums containing 2% serum.
4, 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.Test result 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.
5, experimental result
Experiment selects 3 batches of TF solution, determines TF to the optimal of pk-15 cell proliferation in vitro altogether under identical culture environment every time
Concentration, experiment carry out five times altogether.
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 by Fig. 1
The 350 μ g/mL of optimal concentration of in-vitro multiplication.
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 to be somebody's turn to do per hole 30-40 μ L, preferably 35 μ L
Optimal volume is that screening is determining by the following method：
1, culture medium is prepared
It takes DMEM culture mediums, is added that Pen .- Strep is dual anti-and newborn bovine serum, be configured to contain 1 % Pen .- Streps
Dual anti-and 10 % newborn bovine serum DMEM culture mediums (hereinafter referred to as 10 % DMEM culture mediums) and contain 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.
2, cell culture
Eugonic pk-15 cell monolayers are washed 2 times with the PBS of no calcium and magnesium ion, 1.0 mL pancreatin digestive juices are added, 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 be 2 ~
3×105A/mL.By the cell inoculation diluted in 96 porocyte culture plates, 100 holes μ L/.In 37 DEG C, 5 % CO2Under the conditions of
It cultivates 20 h or so and forms cell monolayer.
3, 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 live vaccines of hog cholera（Rabbit source）Stoste is diluted to the virus liquid containing 100 RID with the DMEM culture mediums containing 2% serum.
4, 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.Test result 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.
5, 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 35 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 live vaccines of hog cholera
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, experiment are taken to be carried out according to following design：
Control group：+ 35 μ L swine fever venom of 65 μ L, 2% DMEM culture mediums；
Test group：+ 35 μ L Swine spleen transfer factors of+35 μ L swine fevers venom of 30 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, MTT solution is added, culture 4 is continued in 10 holes μ L/
H is discarded supernatant, and DMSO solution is added, 100 holes μ L/ 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 live vaccines of hog cholera uses exercising result such as Fig. 3 to pk-15.Experiment selects 8 crowdes of TF molten altogether
Liquid carries out, and is examined according to statistics T, and compared with the control group, each batch TF combines live vaccines of hog cholera in use, pk-15 to test group
Proliferation rate increase, difference extremely significantly (P<0.01).2017002 crowdes of TF combine live vaccines of hog cholera in use, pk-15 proliferation rates are reachable
To 136.05%.When different batches TF is used in combination with live vaccines of hog cholera, pk-15 proliferation rate average values are 130.40% ± 5.59%.
Present invention application mtt assay determines influence of the TF solution to pk-15 cell Proliferations, as a result, it has been found that, when TF is a concentration of
When 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 and live vaccines of hog cholera are used in combination, promote pk-15
Stimulation pk-15 cell Proliferations effect is good when the proliferation effect of cell is than being used alone live vaccines of hog cholera.Illustrate that the method for the present invention can
Enhance live vaccines of hog cholera immune effect, detection method of the invention shows on cell, and inspection cost is low, as a result stablizes relatively.
Claims (10)
1. a kind of method of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect, it is characterised in that：It include with
Lower step：
1）Live vaccines of hog cholera stoste is taken, with dual anti-complete of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps
DMEM culture mediums are diluted to the virus liquid containing 100 RID；
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 single 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 holes μ L/, specific as follows：
The dual anti-complete DMEM culture mediums 25-35 μ L of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
Virus liquid 30-40 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. the method for a kind of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect according to claim 1,
It is characterized in that：Step 1）In, the live vaccines of hog cholera is rabbit source live vaccines of hog cholera.
3. the method for a kind of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect according to claim 1,
It is characterized in that：Step 2）In, the cell is pk-15 cells.
4. the method for a kind of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect according to claim 1,
It is characterized in that：Step 3）In, the tissue culture plate is 96 porocyte culture plates.
5. the method for a kind of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect according to claim 1,
It is characterized in that：Step 3）In, cell is diluted to be as follows：By the cell monolayer phosphate buffer in step 2)
It washes 2-3 times, pancreatin digestive juice, which is then added, makes it be covered with all cell surfaces, is placed several minutes in 37 DEG C, then discards digestion
Liquid is eluted cell with the dual anti-complete DMEM culture mediums of newborn bovine serum containing 8-12%, 0.8-1.2% Pen .- Streps, will
Cell suspension after elution is diluted, and adjustment cell concentration is 2 ~ 3 × 105A/mL.
6. the method for a kind of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect according to claim 1,
It is characterized in that：Step 3）In, the cell culture condition after dilution is：100 holes μ L/ of cell inoculation amount, 36.5-37.5 DEG C,
4.5-5.5 % CO2Under the conditions of culture 18-24 H-shapeds at cell monolayer.
7. the method for a kind of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect according to claim 1,
It is characterized in that：Step 4）In, applied sample amount is specific as follows：
The dual anti-30 μ L of complete DMEM culture mediums of newborn bovine serum containing 1.8-2.2%, 0.8-1.2% Pen .- Streps；
35 μ L of virus liquid；
35 μ L of Swine spleen transfer factor.
8. the method for a kind of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect according to claim 1,
It is characterized 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 cultivate
40-48 h 。
9. the method for a kind of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect according to claim 1,
It is characterized in that：Step 5）In, cell continues the time being incubated as 3.5-4.5h.
10. the method for a kind of detection Swine spleen transfer factor and live vaccines of hog cholera combined immunization effect according to claim 1,
It is characterized in that：Step 7）In, OD values are measured at 480-500nm wavelength.
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