阅读说明：本技术 一种犬腺病毒ⅰ型灭活疫苗及其制备方法 (Canine adenovirus type I inactivated vaccine and preparation method thereof ) 是由 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 于 2019-11-07 设计创作，主要内容包括：本发明涉及一种犬腺病毒Ⅰ型灭活疫苗及其制备方法,采用无血清培养工艺制备得到犬腺病毒Ⅰ型灭活疫苗,其与含血清培养的犬腺病毒Ⅰ型的免疫原性相当,而采用本发明的无血清工艺培养的犬腺病毒Ⅰ型病毒所制备得到的疫苗相比有血清工艺制备得到的疫苗,其在免疫早期能够获得更好的中和抗体滴度。(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.)

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 ratio^TMPET 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.

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 ratio^TMPET 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-10^10.6TCID₅₀mL), while the serum-containing culture had a slight decrease in CAV (from 10)^9.2TCID₅₀Reduction in/mL to 10^8.9TCID₅₀/mL). The titer of the CAV cultured by the serum-free culture process can reach 10 after 120h of culture^10.6TCID50/mL, while CAV in control serum culture was 10^8.9TCID50/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.

Drawings

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.