CN110859956B - A kind of canine parvovirus inactivated vaccine and preparation method thereof - Google Patents

Abstract

Based on the CPV strains circulating in Hengyang, Hunan, the present invention obtains a strong pathogenic strain through screening, and the inactivated vaccine prepared after inactivating it has a high protection level for animals, and It can effectively stimulate the vaccinated animals to produce an effective level of IgG antibodies, and the test shows that the CPV-HuN1703 strain inactivated vaccine can deal with the infection of different CPV strains, and the protection rate of other vaccine strains to animals is not as good as the vaccine strain of the present invention; The invented CPV-HuN1703 vaccine strain has good immunogenicity, can well stimulate the body to produce effective antibodies, and has a high level of antibody production and has the highest protection performance to animals.

Description

A kind of canine parvovirus inactivated vaccine and preparation method thereof

technical field

The invention belongs to the field of biotechnology, and in particular relates to a canine parvovirus inactivated vaccine and a preparation method thereof.

Background technique

Canine parvovirus disease is a highly contagious acute and severe infectious disease characterized by vomiting, diarrhea, leukopenia and hemorrhagic enteritis caused by canine parvovirus (CPV) infection. Since its discovery, it has been one of the most serious infectious diseases that endanger canine health, especially puppies aged 2 to 6 months. Canine parvovirus is distributed all over the world and can infect canines and mustelids, with a high incidence rate. 70%-100%, the mortality rate is as high as 50%. With the improvement of living standards in my country, the increase in the breeding of police dogs and experimental pet dogs, the canine parvovirus disease is becoming more and more serious, which brings huge losses to the dog breeding industry and also seriously threatens the development of my country's dog breeding industry.

Canine Parvovirus (CPV) belongs to the family Parvoviridae, one of the members of the genus Parvovirus. Hexagonal, without capsule. Because CPV has a very compact structure, it has strong resistance to various physical and chemical stimuli, and is still infectious after being treated at 65 °C for 30 minutes; under long-term low temperature environment, its infectivity will not change, and it can survive at room temperature. The time is about 3 months, and it can survive in the feces for more than half a year. After CPV infection, the onset is rapid and the mortality rate is high, so prevention of CPV is more effective than treatment. Vaccination is the main method to control the outbreak and epidemic of CPV. Puppies are susceptible to virus infection, even if vaccinated, because maternal antibodies in puppies interfere with the action of the vaccine and lead to immune failure, so many researchers are working to overcome the interference and study more effective vaccines.

Inactivated vaccine is to use inactivated virus to inoculate animals. The initial inactivated vaccine is to use FPV to inoculate dogs. This is because the proportion of homology between FPV and CPV is high, which can play a certain preventive effect. Later, some researchers inactivated the virulent strain of CPV and injected dogs to immunize them. Compared with the inactivated FPV vaccine, the immunization effect is more ideal, and the adverse reactions after vaccination are not obvious, which is safe and reliable. There is only one antigenic type of CPV so far, namely, Thomson and Kelly isolated CPV-2, but CPV has antigenic drift, and then different subtypes CPV-2 have changed in host range, hemagglutination and antigenicity. 2a, CPV-2b, CPV-2c(a) and CPV-2c(b). With the continuous mutation of the virus, new challenges have been brought to the treatment and prevention of this disease.

Invention patent application CN201611031801.X discloses a canine parvovirus strain CPV-YH, and the use of the canine parvovirus strain CPV-YH in the preparation of canine parvovirus vaccines and diagnostic reagents; the inventor also participated in the early years Research on CPV related work, isolated CPV-NY130615 strain (Bu Bin et al., "Isolation, Identification and Biological Characteristics of a Canine Parvovirus", Henan Agricultural Science, 2015, 44(18): 121-127 , 140). However, as pointed out in the patent application CN201611031801.X, the CPV isolates in my country also have genetic variation phenomenon, and the result is very serious when the natural host drift occurs through the virus, because the virus can be transmitted through innate immunity and non-adaptive The host population spreads widely. Due to the rapid mutation rate, the immune effects of inactivated vaccines and attenuated vaccines currently in clinical use have declined to varying degrees, and the existing vaccines cannot fully meet the needs of disease prevention and control and market consumption.

Therefore, in order to prevent and control canine parvovirus more effectively, it is urgent to isolate new CPV strains for the development of new canine parvovirus vaccines.

SUMMARY OF THE INVENTION

In order to solve the existing problems in CPV prevention, the present invention provides a new strain of canine parvovirus CPV-HuN1703, and prepares it into an inactivated vaccine. The canine parvovirus inactivated vaccine has good immunogenic properties, and the vaccine prepared with the canine parvovirus has good protective efficacy against canine parvovirus disease.

In order to solve the above technical problems and achieve the purpose of the present invention, the present invention adopts the following technical solutions:

An inactivated canine parvovirus vaccine, characterized in that it is prepared by inactivating the canine parvovirus CPV-HuN1703 strain, and the canine parvovirus CPV-HuN1703 strain has a microbial deposit number of CGMCC No.17583; classification Name: Canine Parvovirus (CPV); Preservation time is April 24, 2019; Preservation unit: General Microbiology Center of China Microbial Culture Collection Management Committee; Preservation Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing No., Institute of Microbiology, Chinese Academy of Sciences.

Preferably, the canine parvovirus inactivated vaccine further comprises an adjuvant.

Preferably, the adjuvant is one or more of aluminum hydroxide glue, mineral oil, Gel 01, propolis, ISA206 and ISA760VG.

Preferably, the adjuvant is ISA206.

Preferably, the preparation method of the canine parvovirus inactivated vaccine comprises the following steps:

Step a, culture cat kidney passaged cells F81 according to the conventional method: the growth medium is MEM containing 5% fetal bovine serum (100 μg/mL each of penicillin sodium and streptomycin sulfate), in a 37 °C, 5% CO ₂ incubator Culture passage, when the cells grow to 80% confluence, wash with PBS once and then change to serum-free medium;

In step b, the canine parvovirus CPV-HuN1703 strain with a content of 5×10 ⁴ TCID ₅₀ /ml was inoculated into the well-growing F81 cells prepared in step a at a volume percentage of 5%, adsorbed at 37° C. for 1 hour, and the adsorption solution was discarded. , supplemented with MEM containing 10 μg/mL trypsin as a maintenance medium to continue the culture;

Step c: After inoculation, observe 3 times a day, record the cytopathic effect (CPE), harvest when the cytopathic rate reaches more than 80%, freeze and thaw at -80°C for three times, centrifuge at 8000 rpm for 15 min, and collect the supernatant , which is the virus fluid;

In step d, the virus solution prepared in step c is concentrated by 20 times, and then inactivated by adding 0.2% formaldehyde at 37° C. for 24 hours to obtain canine parvovirus CPV-HuN1703 strain vaccine stock solution;

In step e, the canine parvovirus CPV-HuN1703 strain vaccine stock solution and the adjuvant are stirred and mixed uniformly in a sterile environment to prepare the inactivated vaccine.

Preferably, the inactivated vaccine contains canine parvovirus CPV-HuN1703 strain ≥5.5×10 ⁵ TCID ₅₀ per head, and ISA206 50% (V/V).

The present invention also claims to protect the application of the inactivated vaccine in the preparation of a drug for preventing canine parvovirus infection, where the disease caused by canine parvovirus infection is canine viral enteritis, canine infectious enteritis, or canine nonpurulent myocarditis .

Preferably, the vaccine is administered by intramuscular injection or intranasal immunization, more preferably intramuscular injection.

Based on the above technical solutions, the present invention has the following advantages and beneficial effects:

Based on the CPV strains circulating in Hengyang, Hunan, the present invention obtains a strong pathogenic strain through screening, and the inactivated vaccine prepared after inactivating it has a high protection level for animals, and It can effectively stimulate the vaccinated animals to produce an effective level of IgG antibodies, and the test shows that the CPV-HuN1703 strain inactivated vaccine can deal with the infection of different CPV strains, and the protection rate of other vaccine strains to animals is not as good as the vaccine strain of the present invention; The invented CPV-HuN1703 vaccine strain has good immunogenicity, can well stimulate the body to produce effective antibodies, and the antibody production level is high, and the protection performance to animals is the highest, and the effect obtained is not achieved by the existing strains. Yes, it was unexpected.

Description of drawings

Figure 1 : CPV-HuN1703 strain produces CPE on F81 cells: A, normal F81 cells; B, CPE produced after inoculation with CPV.

Figure 2: PCR identification of CPV-HuN1703 strain: M, Marker; lane 1, CPV-HuN1703 strain; lane 2, negative control.

Figure 3: Necropsy and pathological images of animal regression experiments: A, necropsy of canine small intestine; B, pathological section of canine small intestine.

Figure 4: Changes in serum titers in dogs after the first immunization.

Detailed ways

Example 1: Isolation and identification of canine parvovirus CPV-HuN1703 strain

1.1 Source and collection of disease materials: collected from severely ill dogs admitted to a pet hospital in Hengyang City, Hunan Province, and the disease materials after death.

It was clinically found that the dog showed depression, no appetite, severe diarrhea accompanied by vomiting, red blood clots in the stool, and shedding of the intestinal mucosa. It is difficult to prevent the deterioration of the condition by the existing drugs. Take the loose stools on the spot, and use colloidal gold test strips to detect CPV antigens, and the test results are positive.

After the dead dogs were sterilized, the heart, liver, spleen, lymph nodes, small intestine and intestinal contents of the dogs were collected and stored at -70°C.

Other virus sources:

CPV standard strain (CVCC AV298), from China Veterinary Drug Administration (HA titer is 1:1024).

CPV-NY130615 strain, isolated by the inventor at an early stage, is preserved in this laboratory.

Virus isolation and identification:

Take the preserved disease material of the small intestine and intestinal contents of the dead dog, scrape the intestinal mucosa and its intestinal content, grind the disease material and add PBS at a ratio of 1:5 (W:V), and after repeated freezing and thawing 3 times, 12000r/ min Centrifuge for 10 minutes, take the supernatant, add penicillin and streptomycin mixture (final concentration of 1000IU) and mix evenly, filter and sterilize with a 0.22 μm filter membrane, and then obtain the virus liquid sample to be inoculated, and store it at -20°C for later use. .

According to 10% of the volume of the cell culture solution, the virus solution sample to be inoculated was inoculated into well-grown F81 cells, and then placed in an incubator at 37 °C for cultivation. At the same time, blank cells were set as a control, and cytopathic conditions were observed every day. If there is no disease, harvest it after 5 days of culture, repeat freezing and thawing 3 times, and blindly pass it to the 6th generation. The virus solution was repeatedly frozen and thawed at -20°C for 3 times and then stored at -80°C for use as a virus separation solution.

It was observed that after the inoculated virus liquid sample was inoculated with F81 cells, slight CPE changes appeared, and obvious CPE changes appeared when blindly passed to the 3rd generation, which showed that the border of the adherent cells was obvious, and the cells showed a net-like shape and a shuttle. form and eventually disintegrate. On this basis, the canine parvovirus was isolated and named as CPV-HuN1703 strain after continuous inoculation and culture for 3 generations, all of which showed good CPE.

Virus identification and preservation:

(1) Cell culture and CPE: subculture the harvested third-generation virus culture medium, inoculate the virus separation medium with 5% content in well-grown F81 cells, adsorb at 37°C for 1 hour, discard the adsorption medium, and add the MEM with 10 μg/mL trypsin was used as a maintenance solution to continue the culture, and cultured at 37°C for 5 days, and the lesions were observed. For the specific lesions, see Figure 1 in the instruction manual. Typical cytopathic lesions appeared on the 3rd day after inoculation, showing cell shedding and large-scale "pulling reticular lesions" (see Figure 1 in the instructions).

(2) Nucleic acid identification: adopt the detection method that the inventor participated in the construction in the early stage (see Bu Bin et al., "Isolation, Identification and Biological Characteristics of a Canine Parvovirus", Henan Agricultural Science, 2015, 44 (18) : 121-127, 140), PCR identification was performed on the isolated third-generation virus culture medium. As can be seen from Figure 2 in the description, the third-generation virus culture solution can amplify a 682 bp band, which is consistent with the expected fragment length, while the negative control has no band. After sequencing and comparison, it was confirmed to be a CPV virus. This result indicated that the virus in the isolated virus culture medium was CPV.

(3) Blood coagulation test

Using a hemagglutination plate, add 50 μL of PBS to the first well, add 50 μL of the sample to be tested (the sixth-generation virus culture medium), mix well and transfer 50 μL to the second well for doubling dilution. Finally, discard 50 μL and repeat with one line. , the 12th well is the PBS control. Then, 50 μL of 1% porcine red blood cells (porcine red blood cells were treated with PBS) were added to each reaction well, and incubated at 4°C for 1 h. The maximum dilution of agglutinating 50% red blood cells was the HA titer of the sample. The specific results are shown in Table 1 below.

(4) Determination of virus titer:

Dilute the isolated virus (the 6th generation virus culture medium) with MEM ^10-1-10-10 times, and inoculate it on the ^F81 cell culture plate synchronously, with 6 replicates for each dilution, 100 μL/well, and place it in an incubator Static culture was performed, lesions were observed, CPE was observed on the 5th day, positive holes were recorded, and the TCID ₅₀ of the virus was calculated. The specific results are shown in Table 1 below.

Table 1 Hemagglutination titer and TCID ₅₀ control results of CPV-HuN1703 strain and control strain

Based on the above results, the hemagglutination titer of the third generation of CPV-HuN1703 strain is 1:1024, and the virus content is 10 ^8.75 TCID ₅₀ /mL. CPV standard strain and CPV-NY130615 strain isolated earlier by the inventors.

(5) Animal regression experiment:

According to the laboratory animal health requirements, 6 ordinary hybrid puppies in Hengyang City, about 40-50 days old, were selected for blood collection, blood routine testing, stool collection, PCR testing, maternal antibody ≤ 1:4, and no vaccination. The dogs were recorded clinical manifestations, observed for one week, CDV and CPV tests were negative, used for the challenge test. Four of them were in the challenge group, and each was orally administered 1 mL of the fifth-generation virus dilution (10 ^5.0 TCID ₅₀ /mL); 2 were in the control group, and each was orally administered with 1 mL of rehydration saline. The incidence after inoculation was counted, and the challenge group was necropsied to observe the pathological changes.

As a result, all 4 challenge dogs became ill on the 3rd day, with symptoms of loss of appetite, diarrhea, bloody stool, increased body temperature, and weight loss. The excretion of virus was detected from stool PCR, and the control group was normal.

All four dogs in the oral group died. The autopsy found that the main lesions were in the small intestine. The jejunum and ileum were congested and swollen, and the small intestinal mucosa was necrotic and exfoliated. There was a large amount of blood mixed in the intestine, the color was dark red, and the intestinal wall was thinned. Grey ulcers can be seen above. It can be seen from the pathological section that the lesions are mainly in the intestinal mucosa, the mucosal structure is destroyed, the intestinal glands atrophy and disappear, the superficial mucosal epithelium is focal shedding, and the lamina propria is congested.

After isolation and identification, a strain of canine parvovirus CPV-HuN1703 was obtained, whose microbial deposit number is CGMCCNo.17583; classification name: Canine Parvovirus (CPV); preservation time is April 24, 2019; : General Microbiology Center of China Microbial Culture Collection Management Committee; deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

Example 2: Canine parvovirus CPV-HuN1703 strain used for vaccine preparation

(1) Preparation of vaccines

A canine parvovirus inactivated vaccine, which is prepared by the following method:

Step a, culture cat kidney passaged cells F81 according to the conventional method: the growth medium is MEM containing 5% fetal bovine serum (100 μg/mL each of penicillin sodium and streptomycin sulfate), in a 37 °C, 5% CO ₂ incubator Culture passage, when the cells grow to 80% confluence, wash with PBS once and then change to serum-free medium;

In step b, the canine parvovirus CPV-HuN1703 strain with a content of 5×10 ⁴ TCID ₅₀ /ml was inoculated into the well-growing F81 cells prepared in step a at a volume percentage of 5%, adsorbed at 37° C. for 1 hour, and the adsorption solution was discarded. , supplemented with MEM containing 10 μg/mL trypsin as a maintenance medium to continue the culture;

Step c: After inoculation, observe 3 times a day, record the cytopathic effect (CPE), harvest when the cytopathic rate reaches more than 80%, freeze and thaw at -80°C for three times, centrifuge at 8000 rpm for 15 min, and collect the supernatant , which is the virus fluid;

In step d, the virus solution prepared in step c is concentrated by 20 times, and then inactivated by adding 0.2% formaldehyde at 37° C. for 24 hours to obtain canine parvovirus CPV-HuN1703 strain vaccine stock solution;

In step e, the canine parvovirus CPV-HuN1703 strain vaccine stock solution and the adjuvant are stirred and mixed uniformly in a sterile environment to prepare the inactivated vaccine.

The adjuvant was ISA206.

The inactivated vaccine contains canine parvovirus CPV-HuN1703 strain ≥5.5×10 ⁵ TCID ₅₀ per head, ISA20650% (V/V).

(2) Inspection of vaccines

Two batches of vaccines were prepared according to the above method, and the batch numbers were 20180501 and 20180601 respectively.

Routine inspection: The appearance of the two batches of vaccines is pink latex, and the sterility test and mycoplasma test are all negative. Common virus tests are common. In addition to CPV, common canine viruses such as CDV, CCV, CRV, CAV-1 Wait, the tests are all negative.

Safety test: 10 common hybrid puppies aged 30-40 days who were negative for CPV pathogens and antibodies were randomly divided into 2 groups, 5 in each group, and 10 vaccines were intramuscularly injected into each animal, and one was clinically observed. month, all were 100% healthy, and no adverse reaction occurred, indicating that the two batches of vaccines of the present invention are safe.

Example 3: Vaccine Efficacy Detection Test

3.1 Materials and methods

As a control, CPV standard strain and CPV-NY130615 strain were respectively used to prepare a batch of products according to the preparation method of the inactivated vaccine in Example 2. The commercialized vaccines use Cubwei® Uminkang (mainly immune to canine distemper, canine paroxysmal, hepatitis and canine parainfluenza, batch number: A505A01)

Twenty-five 30-40-day-old crossbred puppies who were both negative for pathogen detection and antibody detection were randomly divided into 5 groups with 5 dogs in each group, named as control group, CPV-HuN1703 group, CPV standard group, CPV- NY130615 group and commercial vaccine group, the control group was injected with 50% ISA206 adjuvant (50% ISA206+50% normal saline), and the CPV-HuN1703 group, CPV standard group, and CPV-NY130615 group were injected with the 20180601 batches prepared in Example 2, respectively. Secondary vaccine, vaccine prepared based on CPV standard strain and CPV-NY130615 strain. The injection volume of each dog was 1 serving (1 mL/serving, the virus content was 5.5×10 ⁵ TCID ₅₀ /serving). The commercial vaccine group was injected with 1 commercial vaccine (used according to the instructions).

On the 7th, 14th, 21st and 28th days after injection, and 7 days after the second immunization, the antibody level in the test dogs was detected by ELISA detection method (see the inventor's previous patent: CN201410830855.7). The test results are shown in Figure 4.

The second immunization was carried out on the 28th day, and 7 days after the second immunization, CPV was used to challenge the virus. 1 mL of each culture medium, and the virus content was ≥1×10 ⁵ TCID ₅₀ /mL. The morbidity and survival of each group after challenge were observed, and the specific results are shown in Table 2.

Table 2 Survival after challenge

Based on the results in Table 2 above, it can be seen that the inactivated vaccine prepared by the CPV-HuN1703 obtained by the present invention has the best protective effect, and it has achieved full protection to the experimental animals, with a survival rate of 100%, while the mortality rate of the control group is 100%. High, after 14 days of challenge, all experimental dogs died, while the protection rates of CPV standard group, CPV-NY130615 group and commercial vaccine group were 4/5, 3/5 and 4/5, respectively. The vaccine strain of the invention can deal with the infection of different CPV strains, and the protection rate of other vaccine strains to animals is not as good as that of the vaccine strain of the invention.

In addition, based on Figure 4 of the accompanying drawings of the present invention, it can be seen that the inactivated vaccine prepared by CPV-HuN1703 of the present invention showed the highest serum titer in each group on the 7th day and the 14th day after inoculation, and the 21st day At the end of the day, all the vaccinated animals reached the highest antibody titer after the first immunization, and the serum titers of the CPV-HuN1703 group, CPV-NY130615 group and commercial vaccine group were all 1:6400; while the serum titer of the CPV standard group was slightly The IgG antibody showed a downward trend. On the 7th day after the second immunization on the 28th day, the titer of the inactivated vaccine prepared by the CPV-HuN1703 of the present invention was the highest at 1:12800, and all other groups were 1 :6400. It can be seen that the CPV-HuN1703 vaccine strain of the present invention has good immunogenicity, can better stimulate the body to produce effective antibodies, and has a high level of antibody production, and has the highest protection performance for animals. What cannot be achieved is unexpected.

Claims (8)

1. a canine parvovirus inactivated vaccine, it is characterized in that, it is prepared by inactivation by canine parvovirus CPV-HuN1703 strain, described canine parvovirus CPV-HuN1703 strain, its microbial deposit number is CGMCC No.17583 ; Classification name: Canine Parvovirus (CPV); Preservation time is April 24, 2019; Preservation unit: General Microbiology Center of China Microbial Culture Collection Management Committee; Preservation Address: No. 1, Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences. 2 . The inactivated canine parvovirus vaccine according to claim 1 , wherein the inactivated canine parvovirus vaccine further comprises an adjuvant. 3 . 3. canine parvovirus inactivated vaccine according to claim 2, is characterized in that, described adjuvant is one or more in aluminum hydroxide glue, mineral oil, Gel 01, propolis, ISA206 and ISA760VG. 4. The canine parvovirus inactivated vaccine according to claim 2, wherein the adjuvant is ISA206. 5. canine parvovirus inactivated vaccine according to any one of claim 1-4, is characterized in that, the preparation method of described canine parvovirus inactivated vaccine comprises the steps: Step a, culture cat kidney passage cell F81 according to the conventional method: the growth medium is MEM containing 5% fetal bovine serum, wherein the growth medium contains 100 μg/mL of penicillin sodium and streptomycin sulfate, and cultured at 37°C, 5% CO ₂ . Culture and passage in the box, when the cells grow to 80% confluence, wash with PBS once and then change to serum-free medium; In step b, the canine parvovirus CPV-HuN1703 strain with a content of 5×10 ⁴ TCID ₅₀ /ml was inoculated into the well-growing F81 cells prepared in step a at a volume percentage of 5%, adsorbed at 37° C. for 1 hour, and the adsorption solution was discarded. , supplemented with MEM containing 10 μg/mL trypsin as a maintenance medium to continue the culture; Step c: After inoculation, observe 3 times a day, record the cytopathic condition, harvest when the cytopathic rate reaches more than 80%, freeze and thaw at -80°C for three times, centrifuge at 8000 rpm for 15 min, and collect the supernatant, which is virus fluid; In step d, the virus solution prepared in step c is concentrated by 20 times, and then inactivated by adding 0.2% formaldehyde at 37° C. for 24 hours to obtain canine parvovirus CPV-HuN1703 strain vaccine stock solution; In step e, the canine parvovirus CPV-HuN1703 strain vaccine stock solution and the adjuvant are stirred and mixed uniformly in a sterile environment to prepare the inactivated vaccine. 6. The canine parvovirus inactivated vaccine according to claim 5, which contains canine parvovirus CPV-HuN1703 strain ≥ 5.5×10 ⁵ TCID ₅₀ /head, and ISA206 50% V/V. 7. the application of the canine parvovirus inactivated vaccine according to claim 1 or 6 in the preparation of a medicine for preventing canine parvovirus infection, the disease caused by the canine parvovirus infection is canine viral enteritis, canine infectious enteritis Or canine nonpurulent myocarditis. 8. The application according to claim 7, wherein the using mode of the vaccine is intramuscular injection or intranasal immunization.

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