CN110862972A - Canine adenovirus type I serum-free culture method - Google Patents
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Abstract
The invention relates to a canine adenovirus type I serum-free culture method, which adopts a culture medium consisting of a basic culture medium, a cell growth promoting factor and a virus proliferation promoting factor, wherein the basic culture medium is an MEM culture medium, the cell growth promoting factor consists of an epidermal growth factor EGF, a transfer growth factor TGF- β, L-lysine and glucose, the virus proliferation promoting factor consists of EDTA-Na2, taurine and cortisol, and the CAV cultured by the serum-free culture medium has high titer, can improve the yield, improve the production efficiency and reduce the cost.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a canine adenovirus type I serum-free culture method.
Background
Canine Adenovirus (CAV) is the most pathogenic virus of the mammalian genus adenovirus. Studies have shown that the virus has 2 serotypes: type I and type II, wherein, the serum type I adenovirus can cause canine infectious hepatitis and fox encephalitis, so the adenovirus is also called fox encephalitis and canine infectious hepatitis; type II serum causes infectious laryngotracheitis and enteritis in dogs. Canine infectious hepatitis is characterized by acute encephalitis, manifested as fever, watery nasal discharge, mild diarrhea, nystagmus. Then central nervous symptoms appear, convulsion, paralysis and syncope appear, death is usually realized within 24 hours, and the death rate can reach more than 50 percent. The canine adenovirus type I is DNA virus, the virus survives for 3-11 days at normal temperature, survives for 10-16 weeks in cell culture and survives for more than 6 months at 4 ℃, and common fat-soluble preparations have no killing effect on the canine adenovirus type I and have strong resistance to the environment.
At present, the main method for preventing and treating canine infectious hepatitis in China is vaccination. The commercial vaccines are all attenuated vaccines, namely, high-quality virus liquid is prepared in vitro by adopting a cell culture method to prepare the vaccine. In the dog quintuplet live vaccine developed by the university of liberalization of military agriculture and animal husbandry of the original Chinese people in China, the canine infectious hepatitis virus adopts MDCK (canine kidney) cell monolayer, the virus is inoculated according to 1% volume of maintenance liquid (containing 1-2% of fetal bovine serum with final concentration), the maintenance liquid is placed at 37 ℃ for culture, the liquid is changed after 24 hours, and when the cytopathic effect (CPE) reaches more than 75%, the virus is harvested. Harr in Chinese academy of agricultural sciencesIn the dog triple vaccine developed by Binhe veterinary research institute, the canine infectious hepatitis component also adopts MDCK cell monolayer, and according to 2% of cell maintenance liquid (containing 2% of fetal bovine serum), the virus is inoculated, cultured at 37 deg.C, observed for 48 hr, when the cytopathic effect is up to above 80%, the virus is harvested, and used for preparing vaccine, and the canine adenovirus titer in all the prepared vaccines is 106TCID50。
For vaccines, the titer of the virus culture is the main factor determining the cost and effectiveness of the product, and the heterologous substances in the virus culture (e.g. fetal calf serum used in the above process) are liable to induce immune rejection in the subject animal and cause side effects. Therefore, how to reduce or not add heterologous nutrients such as fetal calf serum to improve the safety of the vaccine, and how to improve the prior art to improve the titer of the canine adenovirus type I so as to greatly improve the yield of the virus and reduce the cost are problems to be solved urgently by the current canine adenovirus type I vaccine.
Disclosure of Invention
In order to solve the problems caused by serum use in the preparation process of the canine adenovirus type I vaccine and improve the safety of the vaccine, the invention aims to provide a canine adenovirus type I serum-free culture process, on one hand, the culture titer of the virus is improved, the cost is reduced, and on the other hand, the biological safety of vaccine raw materials is enhanced by using the serum-free culture process.
The purpose of the invention is realized by the following technical scheme:
a canine adenovirus type I serum-free culture medium is characterized by comprising a basic culture medium, a cell growth promoting factor and a virus proliferation promoting factor, wherein the basic culture medium is an MEM culture medium, the cell growth promoting factor comprises an epidermal growth factor EGF, a transfer growth factor TGF- β, L-lysine and glucose, and the virus proliferation promoting factor comprises EDTA-Na2Taurine and cortisol.
Preferably, the canine adenovirus type I serum-free medium takes MEM culture medium as a basic culture medium, and is added with cell growth promoting factors and virus proliferation promoting factors, wherein the cell growth promoting factors comprise epidermal growth factor EGF and transfer growth factor TGF- βL-lysine and glucose; the virus multiplication promoting factor is EDTA-Na2The serum-free medium is added with epidermal growth factor EGF with final concentration of 0.05 mu g/mL-0.4 mu g/mL, transfer growth factor TGF- β with final concentration of 0.06 mu g/mL-0.3 mu g/mL, L-lysine with final concentration of 0.1 mu g/mL-5 mu g/mL, glucose with final concentration of 2 mu g/mL-5 mu g/mL, EDTA-Na with final concentration of 0.05 mu g/mL-0.15 mu g/mL2Taurine with a final concentration of 50 mug/mL-75 mug/mL and cortisol with a final concentration of 10 mug/mL-15 mug/mL.
Preferably, the canine adenovirus type I serum-free medium takes an MEM (minimum essential medium) as a basic medium, and is added with a cell growth promoting factor and a virus proliferation promoting factor, wherein the cell growth promoting factor consists of an epidermal growth factor EGF, a transfer growth factor TGF- β, L-lysine and glucose, and the virus proliferation promoting factor consists of EDTA-Na2The serum-free medium is added with epidermal growth factor EGF with the final concentration of 0.1 mug/mL, transfer growth factor TGF- β with the final concentration of 0.15 mug/mL, L-lysine with the final concentration of 2.5 mug/mL, glucose with the final concentration of 3.5 mug/mL, and EDTA-Na with the final concentration of 0.1 mug/mL2Taurine with a final concentration of 50 mug/mL and cortisol with a final concentration of 10 mug/mL.
Preferably, the canine adenovirus type I serum-free medium takes an MEM (minimum essential medium) as a basic medium, and is added with a cell growth promoting factor and a virus proliferation promoting factor, wherein the cell growth promoting factor consists of an epidermal growth factor EGF, a transfer growth factor TGF- β, L-lysine and glucose, and the virus proliferation promoting factor consists of EDTA-Na2The serum-free medium is added with epidermal growth factor EGF with the final concentration of 0.2 mug/mL, transfer growth factor TGF- β with the final concentration of 0.3 mug/mL, L-lysine with the final concentration of 5 mug/mL, glucose with the final concentration of 5 mug/mL, and EDTA-Na with the final concentration of 0.15 mug/mL2Taurine with a final concentration of 75 mug/mL and cortisol with a final concentration of 15 mug/mL.
The invention also provides a canine adenovirus type I serum-free culture process, which is characterized by comprising the following steps:
step one, a serum-free culture acclimatization process of canine kidney cells (MDCK):
using a spinner flask culture process, the MDCK cells grown in a monolayer were digested with pancreatin to prepare 5 mL (10 mL) of suspension8The method comprises the steps of a, inoculating cells/mL) into a roller bottle, adding a commercialized MEM culture medium to culture 300 mL, adding fetal bovine serum with a final concentration of 5% according to a volume ratio, adding a transfer growth factor TGF- β with a final concentration of 0.05 mug/mL-0.2 mug/mL, adding L-lysine with a final concentration of 0.1 mug/mL-5 mug/mL, continuously culturing the glucose with a final concentration of 2 mug/mL-5 mug/mL by adopting a roller bottle, setting the culture temperature to 38.5 ℃, carrying out closed culture for 96 hours, inoculating the cells with a full monolayer according to a proportion of 1:10, carrying out continuous inoculation, wherein the serum concentration in a culture medium is reduced by 0.2% on the basis of the final serum concentration of the previous generation, the serum concentration of the serum is 0.05 mug/0.2 g/mL, the final growth factor TGF-5 mug/mL, continuously transferring the serum concentration of the serum to the final growth factor TGF-5 mug/mL, continuously culturing the serum of 0.05 mug/5-5 mL, and the serum-0.2 after continuous transfer culture of the serum-0.05 to the final growth factor TGF-0.5-5-mL, and continuously culturing the serum-0.2-5-4-g-5-4-1-100-4-g-4-5-4-g-4-subculture medium, and continuously transferring the serum-0-5-4-subcultd continuous subculture medium is adopted for continuous culture medium, and the serum-4-subculture culture medium, and the subcultum culture medium, and the subcultdepicted serum-4-2-4-.
Step two, serum-free culture process of canine adenovirus type I
And (3) carrying out passage on the obtained serum-free domesticated MDCK cell line by the same method, pouring all the culture solution in a spinner flask when the cell confluence is about 90%, adding 100mL of 0.9% (w/v) NaCl washing solution, washing the cells, pouring the washing solution, and repeatedly washing once again. Adding the canine adenovirus type I serum-free culture medium,And canine adenovirus type I diluted by 1000 times so that the virus multiplicity of infection is 1TCID50Fully and uniformly mixing; and (3) performing culture in a rotary bottle at the culture temperature of 38.5 ℃ for 48-72 hours in a closed manner, and harvesting the virus culture solution when the cytopathic effect reaches 90-100%.
Based on the technical scheme, the invention has the following advantages and beneficial effects:
on one hand, the invention adopts a serum-free culture process to culture the canine adenovirus type I, obtains higher titer compared with serum-containing culture, and leads the serum-free cultured CAV to be in a stable situation (10)10.5TCID50/mL-1010.6TCID50mL), while the serum-containing culture had a slight decrease in CAV (from 10)9.2TCID50/mL is reduced to 108.9TCID50/mL). The titer of the CAV cultured by the serum-free culture process can reach 10 after 120h of culture10.6TCID50/mL, while the CAV for the control serum culture was 108.9TCID50/mL, which are 10 times different (P)<0.05). The result shows that the CAV titer cultured by the serum-free culture medium is high, the yield can be improved, the production efficiency can be improved, and the cost can be reduced.
On the other hand, the invention optimizes the serum-free culture medium and finds that EDTA-Na2The results show that the composition and the proportion of the virus propagation promoting factor are the key points for obtaining the serum-free culture of the canine adenovirus type I virus propagation and obtaining the high-titer virus culture solution.
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FIG. 1: MDCK cell culture morphology, fig. 1-a is serum-free acclimated MDCK cell morphology; FIG. 1-B shows the morphology of MDCK cells cultured in serum.
FIG. 2: proliferation properties of serum-free and serum-containing MDCK were compared.
FIG. 3: effect of various additives on canine adenovirus type i proliferation properties in serum-free culture.
FIG. 4: immunostaining of serum-free cultured canine adenovirus type i was identified (100 ×).
Detailed Description
The present invention will be further described with reference to specific embodiments, and features and advantages of the present invention will become more apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1: serum-free culture process domestication of canine kidney cells (MDCK)
(1) The growing monolayer of canine kidney cells (MDCK) was washed 2 times with phosphate buffered saline (pH 7.4);
(2) adding 0.25% trypsin for digestion, and discarding the trypsin solution when the cells become round;
(3) adding MEM culture medium containing fetal calf serum (v/v) with final concentration of 5%, adding tylosin with final concentration of 2-10 mug/mL, shaking up, and placing at 37 ℃ for sealed culture for 24 hours;
(4) setting the culture temperature to 38.5 ℃, and continuously carrying out closed culture for 72 hours;
(5) the cells grow full of a monolayer, are inoculated according to the proportion of 1:10, are subjected to passage, and are continuously subjected to passage culture for 3 times according to the steps 1-4 to obtain seed cells;
(6) the growing monolayer of canine kidney cells (MDCK) was washed 2 times with phosphate buffered saline (pH 7.4);
(7) adding 0.25% trypsin for digestion, and discarding the trypsin solution when the cells become round;
(8) adding a commercialized MEM culture medium to culture 300 mL, adding fetal calf serum with a final concentration of 5% according to a volume ratio, adding epidermal growth factor EGF with a final concentration of 0.05 mug/mL-0.2 mug/mL, transfer growth factor TGF- β with a final concentration of 0.01 mug/mL-0.3 mug/mL, L-lysine with a final concentration of 0.1 mug/mL-5 mug/mL, and glucose with a final concentration of 2 mug/mL-5 mug/mL, adopting a rotary bottle for culture, setting a culture temperature to 38.5 ℃, and carrying out closed culture for 96 hours;
(9) the cells grow in a monolayer, inoculation is carried out according to the proportion of 1:10, continuous passage is carried out, each generation, the serum concentration in the culture medium is reduced by 0.2% on the basis of the serum concentration of the previous generation, epidermal growth factor EGF with the final concentration of 0.05 mug/mL-0.2 mug/mL, transfer growth factor TGF- β with the final concentration of 0.01 mug/mL-0.3 mug/mL, L-lysine with the final concentration of 0.1 mug/mL-5 mug/mL and glucose with the final concentration of 2 mug/mL-5 mug/mL are added in each generation, and rotary bottle culture is adopted, the culture temperature is set to be 38.5 ℃;
(10) continuously carrying out subculture to serum-free culture according to the method of the step (9), continuing the subculture, adding no fetal calf serum for each generation, adding epidermal growth factor EGF with the final concentration of 0.05 mug/mL-0.2 mug/mL, transfer growth factor TGF- β with the final concentration of 0.01 mug/mL-0.3 mug/mL, L-lysine with the final concentration of 0.1 mug/mL-5 mug/mL and glucose with the final concentration of 2 mug/mL-5 mug/mL for 50 generations of continuous subculture, and obtaining the MDCK cells after serum-free domestication;
(11) morphological observation of serum-free cultured cells: serum-free culture was continued for 50 passages to obtain serum-free acclimatized MDCK cells with good morphology (fig. 1A) and no morphological difference from serum-containing MDCK cells (fig. 1B).
(12) The cell growth characteristics before and after acclimation were determined by the MTS method ([ 3- (4, 5-diethylthiazole-2-yl) -5-salt ] (3-carboxymethyxyphenyl) -2- (4-sulfophenyl) -2H-itrazolium, inner) according to the following protocol:
12.1 the serum-free acclimated MDCK cells obtained were digested, inoculated into a 96-well plate at a density of 1000 cells/well, and co-inoculated into 15 wells, and cultured under the serum-free culture conditions in step (10).
12.2 MDCK cultured in serum (fetal bovine serum at a final concentration of 5%) was digested, inoculated into a 96-well plate at a density of 1000 cells/well, and cultured in MEM medium containing fetal bovine serum (v/v) at a final concentration of 5% in the medium, and co-inoculated into 15 wells, and the plate was placed at 37 ℃ for closed culture to obtain control cells.
12.3 after 24, 48, 72, 96 and 120 hours after the culture, 20 μ L of MTS was added to each well of cells cultured in 3 wells of 12.1 and 12.2, respectively, and the cells were cultured for 4 hours with gentle shaking. The absorbance of each well was measured at a wavelength of 490 nm.
12.4 as can be seen from fig. 2, the serum-free MDCK cells after acclimation and the serum-free MDCK cells without acclimation both grew well, and the proliferation test results showed no significant difference in growth at each time point (P > 0.05).
Example 2: effect of various additives on canine adenovirus type I proliferation characteristics in serum-free culture
(1) Test materials: the canine adenovirus type I (CAV-I) strain is a canine adenovirus type I which is isolated and identified by CAV-ZY 180711 strain in 2018 of the inventor, and particularly refers to the published paper of the inventor: yicheng, Danli, Tang Qinghai and the like, "separation and identification of a strain of canine adenovirus I", "Chinese animal quarantine inspection", 36 nd 2 nd of 2019, pages 82-87, herein, both the applicant and the inventor ensure that the biomaterial can be delivered to the public within 20 years from the application date.
(2) And (3) experimental design: based on the prior research of the inventor, the EDTA-Na is found2Taurine and cortisol respectively have certain promotion effects on the proliferation of the canine adenovirus type I on MDCK cells, so that EDTA-Na is used for better serum-free culture of the canine adenovirus type I2Taurine and cortisol are prepared into a virus proliferation promoting factor according to a certain proportion, and in order to verify the influence of each additive component in the virus proliferation promoting factor of a serum-free culture medium on the proliferation characteristics of the canine adenovirus type I, the invention provides the following test, wherein the serum-free culture media of example 2-1, example 2-2 and a control group 1-5 are respectively prepared according to the culture medium composition shown in the following table 1.
TABLE 1 serum-free Medium composition
(3) Effect of various additives on canine adenovirus type I proliferation characteristics in serum-free culture
(a) The serum-free cell line MDCK obtained in example 1 was passaged in the same manner until the confluence degree of the cells became about 90%, the whole culture solution in the spinner flask was poured off, 100mL of 0.9% (w/v) NaCl washing solution was added, the cells were washed, the washing solution was poured off, and the washing was repeated once more.
(b) Adding serum-free MEM culture medium and diluted canine adenovirus type I with dilution multiple of 1000 times to make virus multiplicity of infection be 1TCID50(ii) a Adding each serum-free culture medium in the table 1 above for rotary bottle culture at a rotation speed of 1 circle/5 min and a culture temperature of 38.5 ℃, performing closed culture, preparing 12 repeated samples, collecting 3 repeated samples respectively at 48h, 72h, 96h and 120h after culture, and performing TCID (temperature controlled identification)50The average value is the corresponding TCID50。
(4) And (3) test results: see fig. 3. As is clear from the above tests, the present invention in example 2-1 (test group 1) and example 2-2 (test group 2) both exhibited good growth effects, and only EDTA-Na was added2The results show that the composition and the proportion of the virus proliferation promoting factor of the invention are the key points for obtaining the serum-free culture of the canine adenovirus type I virus proliferation and obtaining the high-titer virus culture solution, and the subsequent tests show that the virus proliferation effect of the invention is superior to that of the traditional serum culture process, which is not reported in the relevant literature of the canine adenovirus type I virus culture, belonging to the breakthrough effect.
(5) Serum-free medium composition for propagation of canine adenovirus type i virus: taking MEM culture medium as basic culture medium, adding cell growth promoting factor and virus proliferation promoting factor, wherein the cell growth promoting factor and virus proliferation promoting factor are addedThe promoter comprises epidermal growth factor EGF, transfer growth factor TGF- β, L-lysine and glucose, and the virus proliferation promoter comprises EDTA-Na2The serum-free medium is added with epidermal growth factor EGF with final concentration of 0.05 mu g/mL-0.4 mu g/mL, transfer growth factor TGF- β with final concentration of 0.06 mu g/mL-0.3 mu g/mL, L-lysine with final concentration of 0.1 mu g/mL-5 mu g/mL, glucose with final concentration of 2 mu g/mL-5 mu g/mL, EDTA-Na with final concentration of 0.05 mu g/mL-0.15 mu g/mL2Taurine with a final concentration of 50 mug/mL-75 mug/mL and cortisol with a final concentration of 10 mug/mL-15 mug/mL.
Example 3: serum-free culture proliferation characteristics of canine adenovirus type I
(1) The serum-free acclimated MDCK cells obtained in example 1 were passaged by the same method, and when the degree of cell confluence was about 90%, the culture solution in the spinner flask was completely removed, 100mL of 0.9% (w/v) NaCl washing solution was added, the cells were washed, the washing solution was removed, and the washing was repeated once more.
(2) Serum-free MEM medium obtained in example 2-1 and a canine adenovirus type I diluted 1000 times thereof were added to give a viral multiplicity of infection of 1TCID50The culture is carried out in a rotary bottle at a rotation speed of 1 circle/5 min and a culture temperature of 38.5 ℃ in a closed manner. 12 replicate samples were made and 3 replicate samples were collected for TCID at 48h, 72h, 96h and 120h post-incubation, respectively50The measurement of (1).
(3) Serum-containing cultures of canine adenovirus type i were set up as controls: and (3) carrying out passage on MDCK cultured by serum (fetal bovine serum with the final concentration of 5%) until the cell confluence is about 90%, pouring all the culture solution in the spinner flask, adding 100mL of 0.9% (w/v) NaCl washing solution, washing the cells, pouring the washing solution, and repeatedly washing once again. Adding 2% final fetal bovine serum (universal maintenance medium concentration) containing MEM medium; culturing in a rotary bottle at 1 ring/5 min and 38.5 deg.C under sealed condition. 12 replicate samples were made and 3 replicate samples were collected at 48h, 72h, 96h and 120h post incubation, respectively, for determination of TCID 50.
(4) The comparative experiment results are as follows: it can be seen from table 2 that the proliferation properties of CAV cultured by the serum-free culture process are significantly stronger than those of CAV cultured by the serum-containing culture process.
TABLE 2 comparison of the proliferation Properties of MDCK-CAV Virus in serum-free and serum-containing cultures
The results show that: both were in a growth state during 48-96h of culture, but CAV in serum-free culture was in a plateau state (10) during 96-120h10.5TCID50/mL-1010.6TCID50mL), while the serum-containing culture had a slight decrease in CAV (from 10)9.2TCID50/mL is reduced to 108.9TCID 50/mL). The titer of the CAV cultured by the serum-free culture process can reach 10 after 120h of culture10.6TCID50/mL, while CAV in control serum culture was 108.9TCID50/mL, which are 10 times different (P)<0.05). The result shows that the CAV titer cultured by the serum-free culture medium is high, the yield can be improved, the production efficiency can be improved, and the cost can be reduced.
Example 4: immunostaining identification of serum-free cultured canine adenovirus type I10 th-generation subviral
Inoculating the 10 th generation culture solution of the canine adenovirus type I cultured in the serum-free manner into MDCK cells (a culture dish with the diameter of 35 mm) with the confluency of 80%, culturing for 12 h, and then removing the culture medium; each dish of cells was washed 3 times with 2 mL PBS, vacuum dried, added with 2 mL 30% acetone-PBS, fixed at room temperature for 25 min, vacuum dried, added with 2 mL (1: 3000 fold dilution) of CAV type I monoclonal antibody, incubated at 37 ℃ for 1 h. PBS was washed 3 times. Adding enzyme-labeled secondary antibody (HRP-SPA, 1: 8000 times diluted) 2 mL, incubating at 37 deg.C for 1h, washing with PBS for 3 times, adding AEC substrate developing solution 2 mL, and incubating at 37 deg.C for 20 min. The reaction solution was discarded, washed with distilled water 2 times, and the results were observed by microscope. See in particular fig. 4.
The results show that: the CAV I type specific monoclonal antibody is specifically combined with the cultured virus in cell nucleus.
Claims (6)
1. A canine adenovirus type I serum-free culture medium is characterized by comprising a basic culture medium, a cell growth promoting factor and a virus proliferation promoting factor, wherein the basic culture medium is an MEM culture medium, the cell growth promoting factor comprises an epidermal growth factor EGF, a transfer growth factor TGF- β, L-lysine and glucose, and the virus proliferation promoting factor comprises EDTA-Na2Taurine and cortisol.
2. The serum-free medium for the canine adenovirus type I according to claim 1, wherein the serum-free medium for the canine adenovirus type I is a MEM medium as a basic medium, and is added with a cell growth promoting factor and a pro-virus propagation factor, wherein the cell growth promoting factor comprises an epidermal growth factor EGF, a transfer growth factor TGF- β, L-lysine and glucose, and the pro-virus propagation factor comprises EDTA-Na2The serum-free medium is added with epidermal growth factor EGF with final concentration of 0.05 mu g/mL-0.4 mu g/mL, transfer growth factor TGF- β with final concentration of 0.06 mu g/mL-0.3 mu g/mL, L-lysine with final concentration of 0.1 mu g/mL-5 mu g/mL, glucose with final concentration of 2 mu g/mL-5 mu g/mL, EDTA-Na with final concentration of 0.05 mu g/mL-0.15 mu g/mL2Taurine with a final concentration of 50 mug/mL-75 mug/mL and cortisol with a final concentration of 10 mug/mL-15 mug/mL.
3. The serum-free medium for the canine adenovirus type I according to claim 1, wherein the serum-free medium for the canine adenovirus type I is a MEM medium as a basic medium, and is added with a cell growth promoting factor and a pro-virus propagation factor, wherein the cell growth promoting factor comprises an epidermal growth factor EGF, a transfer growth factor TGF- β, L-lysine and glucose, and the pro-virus propagation factor comprises EDTA-Na2The serum-free medium is added with epidermal growth factor EGF with the final concentration of 0.1 mug/mL, transfer growth factor TGF- β with the final concentration of 0.15 mug/mL, L-lysine with the final concentration of 2.5 mug/mL, glucose with the final concentration of 3.5 mug/mL, and EDTA-Na with the final concentration of 0.1 mug/mL2Taurine with a final concentration of 50 mug/mL and cortisol with a final concentration of 10 mug/mL.
4. The serum-free medium for the canine adenovirus type I according to claim 1, wherein the serum-free medium for the canine adenovirus type I is a MEM medium as a basic medium, and is added with a cell growth promoting factor and a pro-virus propagation factor, wherein the cell growth promoting factor comprises an epidermal growth factor EGF, a transfer growth factor TGF- β, L-lysine and glucose, and the pro-virus propagation factor comprises EDTA-Na2The serum-free medium is added with epidermal growth factor EGF with the final concentration of 0.2 mug/mL, transfer growth factor TGF- β with the final concentration of 0.3 mug/mL, L-lysine with the final concentration of 5 mug/mL, glucose with the final concentration of 5 mug/mL, and EDTA-Na with the final concentration of 0.15 mug/mL2Taurine with a final concentration of 75 mug/mL and cortisol with a final concentration of 15 mug/mL.
5. A serum-free culture process of canine adenovirus type I is characterized by comprising the following steps:
step one, a serum-free culture acclimatization process of canine kidney cells (MDCK):
step two, a serum-free culture process of the canine adenovirus type I, wherein the serum-free culture process of the canine adenovirus type I adopts the serum-free culture medium of the canine adenovirus type I as described in any one of claims 1 to 4.
6. The serum-free culture process of canine adenovirus type I according to claim 5, comprising the following steps:
step one, a serum-free culture acclimatization process of canine kidney cells (MDCK):
using a spinner flask culture process, the MDCK cells grown in a monolayer were digested with pancreatin to prepare 5 mL (10 mL) of suspension8Individual cells/mL) is inoculated in a spinner flask, commercial MEM culture medium is added for culturing 300 mL, fetal bovine serum with final concentration of 5 percent is added according to volume ratio, epidermal growth factor EGF with final concentration of 0.05 mug/mL-0.2 mug/mL is added, and final concentration is carried outTransferring growth factor TGF- β with the degree of 0.01 mug/mL-0.3 mug/mL, L-lysine with the final concentration of 0.1 mug/mL-5 mug/mL, glucose with the final concentration of 2 mug/mL-5 mug/mL, performing continuous passage by adopting a spinner bottle culture, setting the culture temperature to 38.5 ℃, performing closed culture for 96 hours, inoculating the cells in a full monolayer according to the proportion of 1:10, performing continuous passage, adding epidermal growth factor EGF with the final concentration of 0.05 mug/mL-0.2 mug/m into a culture medium of each passage, transferring growth factor TGF- β with the final concentration of 0.05 mug/mL-0.2 mug/m, transferring growth factor TGF- β with the final concentration of 0.01 mug/mL-0.3 mug/mL, continuously performing the passage culture for 0.2-0.2 mug/mL, continuously performing the passage culture for the serum with the serum concentration of 0.01 mug/mL-0.3 mug/mL, and performing continuous passage for the serum-2K culture for the passage, and obtaining the transferred growth factor TGF- β with the final concentration of the serum-0.05 mug/mL, and the serum-0.05 mug/5 mL, and continuously performing the serum-5-medium conversion culture for the serum-free culture, and the continuous passage for the continuous passage, and the transplanting the final culture for the continuous passage for the glucose culture for the continuous passage, wherein the TGF-2-medium for the serum-0.05-0.0.05-0-0.4 continuous passage, the serum-0-0.4 continuous passage, the subcultd culture for the subcultemargination is obtained by adopting;
step two, serum-free culture process of canine adenovirus type I
And (2) carrying out passage on the obtained serum-free domesticated MDCK cell line by the same method, pouring out all culture solution in a rotary bottle when the cell confluence is about 90%, adding 100mL of 0.9% (w/v) NaCl washing solution, washing cells, pouring out the washing solution, repeatedly washing once again, adding the canine adenovirus type I serum-free culture medium and the canine adenovirus type I diluted by the same dilution multiple of 1000 to ensure that the virus infection complex number is 1TCID50Fully and uniformly mixing; and (3) performing culture in a rotary bottle at the culture temperature of 38.5 ℃ for 48-72 hours in a closed manner, and harvesting the virus culture solution when the cytopathic effect reaches 90-100%.
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