CN110841064A - Canine adenovirus type I inactivated vaccine and preparation method thereof - Google Patents

Canine adenovirus type I inactivated vaccine and preparation method thereof Download PDF

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CN110841064A
CN110841064A CN201911080832.8A CN201911080832A CN110841064A CN 110841064 A CN110841064 A CN 110841064A CN 201911080832 A CN201911080832 A CN 201911080832A CN 110841064 A CN110841064 A CN 110841064A
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唐青海
杨海
易程
王芳宇
杨灿
何丽芳
刘会敬
唐娇玉
易诚
曹丽敏
唐斯萍
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Abstract

The invention relates to a canine adenovirus type I inactivated vaccine and a preparation method thereof, wherein the canine adenovirus type I inactivated vaccine is prepared by adopting a serum-free culture process, and the immunogenicity of the canine adenovirus type I inactivated vaccine is equivalent to that of the canine adenovirus type I cultured by containing serum.

Description

Canine adenovirus type I inactivated vaccine and preparation method thereof
Technical Field
The invention relates to the technical field of biology, in particular to a canine adenovirus type I inactivated vaccine and a preparation method thereof.
Background
Canine Adenovirus (CAV) is the most pathogenic virus of the mammalian genus adenovirus. A dog, one of pets which are most closely contacted with humans and have the longest time to live with humans, is inseparable from the daily lives of humans, and the health condition thereof is often closely related to the health condition of humans themselves, and thus, attention to the health of pet dogs is of great public health significance to animal welfare and human health.
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. In the dog triple vaccine developed by Harbin veterinary research institute of Chinese agricultural academy of sciences, the canine infectious hepatitis component also adopts MDCK cell monolayer, 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 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 virus vaccine and improve the safety of the vaccine, the invention aims to provide the canine adenovirus type I inactivated vaccine and a preparation method thereof, and the biological safety of vaccine raw materials is enhanced by adopting a serum-free process for culture.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a canine adenovirus type I inactivated vaccine is characterized by comprising the following steps:
(1) subculturing the serum-free domesticated MDCK cells, pouring out all culture solution in a spinner flask when the confluence degree of the cells is about 90%, adding 100mL of 0.9% (w/v) NaCl washing solution, washing the cells, pouring out the washing solution, and repeatedly washing once again;
(2) adding serum-free culture medium for canine adenovirus type I and canine adenovirus type I diluted by 1000 times to make the virus multiplicity of infection be 1TCID50Culturing in a rotary bottle at a rotation speed of 1 circle/5 min and a culture temperature of 38.5 ℃ for 96h in a closed manner, and then harvesting the virus;
(3) repeatedly freezing and thawing for 3 times at a speed of 5000g/min, centrifuging for 5min, removing cell debris, collecting supernatant to determine virus titer, and adjusting virus titer to 107TCID50/mL；
(4) Virus inactivation: adding 2mL of formaldehyde into every 1L of virus liquid, wherein the final concentration of the formaldehyde is 0.2%; placing at 37 deg.C for 24 h at 90r/min for inactivation;
(5) neutralization of formaldehyde: placing the prepared antigen on ice (the low temperature has good effect on the antigen), stirring with a three-blade paddle stirrer (the diameter of a stirring head is 5cm), adjusting the rotating speed to 1000r/min, adding a sodium sulfite solution with the final concentration of 0.05% (1 mL of 50% sodium sulfite is added into 1L of antigen), stirring for 30min to uniformly disperse the antigen, and fully neutralizing formaldehyde;
(6) according to the formula of an antigen: adjuvant =98:2 "adjuvant (Montanide) was added slowly at specific gravity ratioTMPET GEL A), stirring with a three-blade paddle stirrer (the diameter of the stirring head is 5cm), adjusting the rotation speed to 1500r/min, and adding the adjuvant within 10 min; continuously stirring at the rotating speed of 1500r/min for 30-40 min;
(7) adding thimerosal at a final concentration of 20 μ g/mL (1L of antigen added with 1mL of 20mg/mL thimerosal storage solution), adjusting rotation speed to 800r/min, and stirring for 10 min;
(8) standing at 4 deg.C for 30min to eliminate bubbles, and emulsifying;
(9) subpackaging, capping and storing: filling the vaccine into a sterile vaccine bottle, wherein each bottle contains 40mL of vaccine, and capping; storing at 4 deg.C.
Preferably, the serum-free domesticated MDCK cell is prepared by the following method:
(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 subcultured for 3 times according to the steps (1) - (4), and seed cells are obtained;
(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) and (4) continuously subculturing to serum-free culture according to the method of the step (9), continuously subculturing, 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 each generation, and continuously subculturing for 50 generations to obtain the MDCK cells after serum-free domestication.
Preferably, the canine adenovirus type I serum-free culture medium is characterized by 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, and the virus proliferation promoting factor consists of EDTA-Na2Taurine and cortisol.
Preferably, the canine adenovirus type I serum-free medium takes MEM (minimum essential medium) as a basic medium, and cell growth promoting factors andthe cell growth promoting factor consists of EGF, 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 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 strain is CAV-ZY 180711.
The invention also claims the canine adenovirus type I inactivated vaccine prepared based on the method.
Based on the technical scheme, the invention has the following advantages and beneficial effects:
on one hand, the serum-free culture process is adopted to prepare the inactivated vaccine of the canine adenovirus type I, the immunogenicity of the inactivated vaccine is equivalent to that of the canine adenovirus type I cultured by serum, and compared with the vaccine prepared by the canine adenovirus type I cultured by the serum-free process, the vaccine prepared by the serum process can obtain better neutralizing antibody titer in the early immunization stage, and the immunization effect of the vaccine is better than that of the commercial attenuated vaccine.
In the second aspect, 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)9.2TCID50Reduction in/mL 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 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.
In a third aspect, the invention optimizes the serum-free culture medium, and discovers 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 subcultured for 3 times according to the steps (1) - (4), and seed cells are obtained;
(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).
(3-carboxymethylethoxyphenyl) -2- (4-thiophenyl) -2H- salt ] (12) the cell growth characteristics before and after acclimation were determined by MTS method ([ 3- (4, 5-diethylthiazole-2-yl) -5-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
Figure DEST_PATH_IMAGE001
(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) A serum-free culture medium for the proliferation of canine adenovirus type I virus is prepared from MEM culture medium as basic culture medium, cell growth promoting factor consisting of EGF, TGF- β, L-lysine and glucose, and virus proliferation promoting factor consisting of EDTA-Na2Taurine and cortisol, wherein the epidermal growth factor EGF with the final concentration of 0.05 mug/mL-0.4 mug/mL is added into the serum-free culture medium, and the final concentration isIs 0.06 to 0.3 mug/mL of transfer growth factor TGF- β, the final concentration of L-lysine is 0.1 to 5 mug/mL, the final concentration of glucose is 2 to 5 mug/mL, and the final concentration of EDTA-Na is 0.05 to 0.15 mug/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
Figure 815773DEST_PATH_IMAGE002
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)9.2TCID50Reduction in/mL 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 108.9TCID50A 10-fold difference between them (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 10 th generation subviral of canine adenovirus type I virus cultured in serum-free mode
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 2mL PBS, vacuum dried, added with 2mL 30% acetone-PBS, fixed at room temperature for 25 min, vacuum dried, added with 2mL (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) 2mL, incubating at 37 deg.C for 1h, washing with PBS for 3 times, adding AEC substrate developing solution 2mL, 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.
Example 5: preparation and immunogenicity research of serum-free cultured canine adenovirus type I virus inactivated vaccine
First, 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.
Secondly, preparation of inactivated vaccine of canine adenovirus type I virus
(1) Passaging the serum-free domesticated MDCK cells obtained in the example 1, pouring out all 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 out the washing solution, and repeatedly washing once again;
(2) serum-free MEM medium obtained in example 2-1 and a canine adenovirus type I diluted 1000-fold thereof were added to give a viral multiplicity of 1TCID50Culturing in a rotary bottle at a rotation speed of 1 circle/5 min and a culture temperature of 38.5 ℃ for 96h in a closed manner, and then harvesting the virus;
(3) repeatedly freezing and thawing for 3 times at a speed of 5000g/min, centrifuging for 5min, removing cell debris, collecting supernatant to determine virus titer, and adjusting virus titer to 107TCID50/mL；
(4) Virus inactivation: adding 2mL of formaldehyde into every 1L of virus liquid, wherein the final concentration of the formaldehyde is 0.2%; placing at 37 deg.C for 24 h at 90r/min for inactivation;
(5) neutralization of formaldehyde: placing the prepared antigen on ice (the low temperature has good effect on the antigen), stirring with a three-blade paddle stirrer (the diameter of a stirring head is 5cm), adjusting the rotating speed to 1000r/min, adding a sodium sulfite solution with the final concentration of 0.05% (1 mL of 50% sodium sulfite is added into 1L of antigen), stirring for 30min to uniformly disperse the antigen, and fully neutralizing formaldehyde;
(6) according to the formula of an antigen: adjuvant =98:2 "adjuvant (Montanide) was added slowly at specific gravity ratioTMPET GEL A), stirring with a three-blade paddle stirrer (the diameter of the stirring head is 5cm), adjusting the rotation speed to 1500r/min, and adding the adjuvant within 10 min; continuously stirring at the rotating speed of 1500r/min for 30-40 min;
(7) adding thimerosal at a final concentration of 20 μ g/mL (1L of antigen added with 1mL of 20mg/mL thimerosal storage solution), adjusting rotation speed to 800r/min, and stirring for 10 min;
(8) standing at 4 deg.C for 30min to eliminate bubbles, and emulsifying;
(9) subpackaging, capping and storing: filling the vaccine into a sterile vaccine bottle, wherein each bottle contains 40mL of vaccine, and capping; storing at 4 deg.C.
Third, vaccine test methods and results
3.1 character test the inactivated vaccine is clear and transparent in appearance.
3.2 sterile test the inactivated vaccine was tested according to the third appendix of the 2010 edition of the pharmacopoeia of the people's republic of China, and no colony was observed in the culture medium of T. G, G. P tube and G.A slant.
3.3 Mycoplasma test inactivated vaccine is tested according to the third appendix of the 2010 edition of the pharmacopoeia of the people's republic of China, no obvious color change of the small bottle and the small tube culture is found, and the transplanted liquid culture has no ' fried egg ' shaped mycoplasma colony on a solid culture medium.
3.4 the inactivated vaccine is tested according to the third appendix of the 2010 edition of the pharmacopoeia of the people's republic of China, no pollution of canine parvovirus, canine distemper virus, porcine parvovirus, canine coronavirus and the like, and the virus seeds are proved to be pure.
3.5 evaluation of safety: 9 Chinese puppies of 2 months old were selected and tested for CVA antigen-antibody and all were negative healthy dogs. The total weight is divided into 3 groups: groups A, B and C, 3 of the groups, 4mL (4 portions) of inactivated vaccine prepared by injecting the serum-free cultured CAV-I into neck muscles of each puppy in the group A, 4mL (4 portions) of inactivated vaccine prepared by injecting the serum-containing cultured CAV-I into neck muscles of each puppy in the group B, and no treatment is carried out on the group C. Dogs were observed daily for appetite, spirit, and body temperature. The results show that: the dogs in group A only had normal body temperature, normal appetite and slight loss of spirit on the day after vaccine injection, and all indexes recovered to be normal on day 2; the body temperature of the dogs in the group B slightly rises (39.2 ℃) on the day after vaccine injection, the appetite of the dogs is reduced within 2 days after the vaccine injection, the spirit is slightly poor, and all indexes are recovered to be normal on the 3 rd day; and the three groups of test dogs have normal appetite, spirit and body temperature, which indicates that the vaccine prepared by the CAV cultured in a serum-free way has better safety compared with the vaccine prepared by serum.
Example 6: evaluation of immunogenicity of inactivated vaccine of canine adenovirus type i virus cultured in serum-free:
12 Chinese puppies of 2 months old were tested negative for CVA antigen-antibody. Animals were divided into 5 groups: group A, group B, group C and group D, each group comprises 3 dogs, the neck of each puppy of group A is injected with inactivated vaccine prepared by serum-free cultured CAV-I intramuscularly, 1 mL/dog per time, and the immunization is carried out twice at intervals of 21D; in the group B, the neck of each puppy is injected with the inactivated vaccine prepared by CAV-I containing serum culture, 1 mL/puppy is injected at the neck, the immunization is carried out twice, and the interval is 21 d; c groups of seeds are planted with 1mL of Eupatorium fortunei per time, are immunized twice at intervals of 21 d; group D did not do anything. And respectively preparing serum with blood collected 7 days, 14 days, 21 days and 42 days after the first immunization, and measuring the titer of neutralizing antibodies of the serum by adopting a trace cell neutralization test method. The specific results are shown in Table 3 below.
TABLE 3 serum neutralizing antibody titer assay results
Figure 64351DEST_PATH_IMAGE003
Based on the results in table 3 above, the serum neutralization potency of the dogs in group a and group B can reach 1:12800 or more, which indicates that under the condition of the same vaccine strain, the immunogenicity of the serum-free cultured canine adenovirus type i virus is equivalent to the immunogenicity of the serum-containing cultured canine adenovirus type i virus, and the vaccine prepared by the serum-free process cultured canine adenovirus type i virus of the present invention has a better neutralizing antibody titer in the early stage of immunization compared with the vaccine prepared by the serum process, which may reduce the relationship between immune interference and immune stress of the components in the serum to the immunized animals by the serum-free process.
By combining the proliferation characteristics and the comprehensive evaluation of safety, the vaccine prepared from the serum-free cultured canine adenovirus type I has high immunogenicity, higher yield, lower cost and higher safety.

Claims (7)

1. A preparation method of a canine adenovirus type I inactivated vaccine is characterized by comprising the following steps:
(1) subculturing the serum-free domesticated MDCK cells, pouring out all culture solution in a spinner flask when the confluence degree of the cells is about 90%, adding 100mL of 0.9% (w/v) NaCl washing solution, washing the cells, pouring out the washing solution, and repeatedly washing once again;
(2) adding serum-free culture medium for canine adenovirus type I and canine adenovirus type I diluted by 1000 times to make the virus multiplicity of infection be 1TCID50Culturing in a rotary bottle at a rotation speed of 1 circle/5 min and a culture temperature of 38.5 ℃ for 96h in a closed manner, and then harvesting the virus;
(3) repeatedly freezing and thawing for 3 times at a speed of 5000g/min, centrifuging for 5min, removing cell debris, collecting supernatant to determine virus titer, and adjusting virus titer to 107TCID50/mL；
(4) Virus inactivation: adding 2mL of formaldehyde into every 1L of virus liquid, wherein the final concentration of the formaldehyde is 0.2%; placing at 37 deg.C for 24 h at 90r/min for inactivation;
(5) neutralization of formaldehyde: placing the prepared antigen on ice, stirring with a three-blade paddle type stirrer with a stirring head diameter of 5cm, adjusting the rotation speed to 1000r/min, adding a sodium sulfite solution with the final concentration of 0.05% into the inactivated virus solution, stirring for 30min to uniformly disperse the antigen, and fully neutralizing formaldehyde;
(6) according to the formula of an antigen: adjuvant =98:2 "adjuvant (Montanide) was added slowly at specific gravity ratioTMPET GEL A), stirring with a three-blade paddle stirrer with a stirring head diameter of 5cm, adjusting the rotation speed to 1500r/min, and adding adjuvant within 10 min; continuously stirring at the rotating speed of 1500r/min for 30-40 min;
(7) adding thimerosal at a final concentration of 20 μ g/mL at one time, adjusting the rotation speed to 800r/min, and continuously stirring for 10 min;
(8) standing at 4 deg.C for 30min to eliminate bubbles, and emulsifying;
(9) subpackaging, capping and storing: filling the vaccine into a sterile vaccine bottle, wherein each bottle contains 40mL of vaccine, and capping; storing at 4 deg.C.
2. The method according to claim 1, wherein the serum-free acclimated MDCK cells are prepared by the following method:
(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 subcultured for 3 times according to the steps (1) - (4), and seed cells are obtained;
(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) and (4) continuously subculturing to serum-free culture according to the method of the step (9), continuously subculturing, 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 each generation, and continuously subculturing for 50 generations to obtain the MDCK cells after serum-free domestication.
3. The method as claimed in claim 1, wherein the serum-free medium for canine adenovirus type I comprises a basic medium, a cell growth promoting factor and a virus proliferation promoting factor, wherein the basic medium is MEM (minimum essential medium), the cell growth promoting factor comprises EGF (epidermal growth factor), TGF- β (transfer growth factor), L-lysine and glucose, and the virus proliferation promoting factor comprises EDTA-Na (ethylene diamine tetraacetic acid-Na)2Taurine and cortisol.
4. The method as claimed in claim 3, wherein the serum-free medium for canine adenovirus type I is MEM medium as basic medium, and is supplemented with cell growth promoting factor and virus proliferation promoting factor, wherein the cell growth promoting factor comprises EGF, TGF- β, L-lysine and glucose, and the virus proliferation promoting 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 final concentration of 50 mug/mL-75 mug/mL and final concentrationCortisol with the degree of 10 mug/mL-15 mug/mL.
5. The method as claimed in claim 4, wherein the serum-free medium for canine adenovirus type I is MEM medium as basic medium, and is supplemented with cell growth promoting factor and virus proliferation promoting factor, wherein the cell growth promoting factor comprises EGF, TGF- β, L-lysine and glucose, and the virus proliferation promoting 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.
6. The method as claimed in claim 1, wherein the canine adenovirus type i strain is CAV-ZY 180711.
7. An inactivated canine adenovirus type I vaccine, prepared by the method of any one of claims 1 to 6.
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