CN111349619A - Duck circovirus tissue inactivated vaccine and preparation method of yolk antibody thereof - Google Patents
Duck circovirus tissue inactivated vaccine and preparation method of yolk antibody thereof Download PDFInfo
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Abstract
The invention relates to a duck circovirus tissue inactivated vaccine and a preparation method of a yolk antibody thereof, wherein the antigen liquid of the vaccine is prepared by grinding, repeatedly freezing and thawing, centrifuging and filtering a grinding liquid containing duck circovirus liver tissue and sterile water according to the mass ratio of 1:3-6, and pollen pini polysaccharide with the concentration of 5-40mg/mL is added into the antigen liquid as an immunopotentiator and is emulsified with a white oil adjuvant; the yolk antibody is obtained by immunizing laying hens with the vaccine and then extracting and purifying the yolk of the hyperimmune egg; the provided duck circovirus tissue inactivated vaccine has the advantages of simple preparation process, low cost, good effect, stable dosage form, easy storage, huge development and utilization potential in the duck breeding industry and wide application prospect. The yolk antibody prepared by the invention has good safety, does not have any local or systemic adverse reaction caused by the yolk antibody, can effectively prevent and treat the infection of the duck circovirus, and has good commercial development prospect.
Description
Technical Field
The invention relates to the field of poultry epidemic disease immune control, in particular to a duck circovirus tissue inactivated vaccine and a preparation method of a yolk antibody thereof.
Background
Duck circovirus (DuCV) is a novel member of the genus circovirus of the family circoviridae, a membrane-free, single-stranded circular DNA construct. The virus was first reported in germany by Hanemann in 2003. In China, DuCV is detected for the first time from riemerella anatipestifer disease duck samples collected in Fujian province in 2006 by Jiangshiki et al (2007). Liu Shao Ning and the like (2008-2009) detect 343 tissue samples of 36 duck groups from 5 provinces of Shandong, Jiangsu, Sichuan, Fujian and Guangdong, and find that the DuCV detection rate is up to 81.63 percent and the duck group positive rate is up to 94.44 percent; wherein the positive rate of 18 meat duck farms in Shandong is 88.89%, and the positive rate of 12 duck farms is 75%. These data indicate that the duck circovirus is quite common in duck flock in the provincial city of duck breeding in China. From the data of 2016 + 2019 detection of diseased duck groups all over the country, the infection rate of DuCV is still extremely high, and most DuCV is infected with pathogens such as adenovirus, duck parvovirus, Tembusu virus, reovirus, influenza virus and bacteria. The treatment of the mixed duck group shows that the duck group with DuCV has poor treatment effect. DuCV infection is slow in process and does not cause obvious clinical symptoms per se, so that DuCV is very easy to ignore, and due to the fact that DuCV mainly attacks immune systems of ducks and causes resistance reduction of the ducks, the DuCV is very easy to cause complications or secondary infection of other pathogens, the treatment effect is seriously influenced, and huge economic loss is caused. Because DuCV is difficult to multiply and culture on animal cells and avian embryos at present, no commercial vaccine exists at present.
The yolk antibody is an antibody against a specific antigen extracted from an immunized egg, and is called yolk immunoglobulin IgY because the yolk contains only specific IgY antibodies. Egg yolk antibodies have many unique advantages over serum antibodies: (1) the source is wide, the yield is high, the cost is low, and the egg can be obtained by only continuously immunizing laying hens and purifying egg yolks of high-immunity eggs; (2) the yolk antibody has good stability, heat resistance and acid resistance, and long storage time at normal temperature; (3) the specificity is strong, specific antibodies are generated aiming at different antigens, and the treatment effect is exact; (4) no allergen, safety, high efficiency, no residue and environment friendship. Due to the limitation of the preparation technology of the duck circovirus vaccine, no commercial vaccine is available on the market at present, the market vacancy is huge, and the egg yolk antibody for preventing and treating early infection is not reported.
Disclosure of Invention
The invention mainly aims to overcome the defects of the prior art and provides a duck circovirus tissue inactivated vaccine and a preparation method of a yolk antibody thereof, wherein a duck circovirus antigen is obtained by infecting ducklings of 1 day age through intraperitoneal injection, carrying out intravenous injection for boosting infection once every 7 days, simultaneously injecting 250mg/kg of cyclophosphamide for each inoculation to induce the reproduction of immunosuppressive boosting virus, and collecting the liver tissues of infected ducks at 25 days; the antigen liquid required by the vaccine is prepared by grinding, repeatedly freezing and thawing, centrifuging and filtering a grinding liquid containing duck circovirus liver tissues and sterile water in a mass ratio of 1:3-6, adding pollen pini polysaccharide with the concentration of 5-40mg/mL as an immunopotentiator, and then emulsifying with a conventional white oil adjuvant; the yolk antibody is obtained by immunizing laying hens with the vaccine, extracting and purifying from the yolk of a hyperimmune egg, and is used for preventing and treating duck circovirus; the provided duck circovirus tissue inactivated vaccine has the advantages of simple preparation process, low cost, good effect, stable dosage form, easy storage, huge development and utilization potential in the duck breeding industry and wide application prospect. The yolk antibody prepared by the invention has good safety, does not have any local or systemic adverse reaction caused by the yolk antibody, can effectively prevent and treat the infection of the duck circovirus, and has good commercial development prospect.
The infection characteristics and epidemic situations of duck circovirus are comprehensively considered, and the prevention and control of the pathogen are considered to be of great significance. The invention discloses an industrialized preparation method of the pathogen, which combines the pathogen to prepare a tissue inactivated vaccine, and adds an immunopotentiator to improve the immunogenicity of an antigen so as to achieve the effect of preventing the disease, thereby protecting the driving and navigating for the healthy development of the duck breeding industry.
The specific inventive concept of the invention is as follows:
the duck circovirus antigen adopted by the invention is a tissue antigen. Because the prior duck circovirus is difficult to be cultured in vitro, does not have a proper cell line, and the growth on the duck embryo is not ideal, no commercial vaccine exists all the time. The disease is easy to be overlooked clinically because the disease usually does not show typical clinical symptoms except emaciation and reduced immunity. Therefore, the development of the vaccine is always a blank in terms of clinical significance and technical difficulty. However, in recent years, the infection rate of the duck circovirus is higher and the harm is more and more serious, and the requirement of vaccine development is urgent. The key of vaccine preparation is the culture of virus or the preparation of protective antigen, and research is carried out on expressing the nucleocapsid protein of the duck circovirus by expression systems such as escherichia coli or saccharomycetes, but the method has high preparation requirement and high purification cost, the high-level structure of the expressed antigen protein is different from the natural antigen structure, the clinical protection effect is to be testified, and the problem of poor antigenicity of the whole virus antigen does not exist, but how to solve the culture of the duck circovirus is a technical problem. According to the invention, a duck circovirus is separated, cultured and continuously passaged and domesticated from pathological materials with diseases in 2019, the virus strain grows well in ducklings, 1-day-old healthy ducklings are selected to be inoculated with the virus, and an interval patch grafting technology and a cyclophosphamide artificial induction immunosuppression technology are adopted, so that the virus content in the ducks is greatly improved, and the duck circovirus can meet the requirements of vaccine preparation. According to the invention, the duck circovirus is made into the tissue inactivated vaccine for the first time, and the pine pollen polysaccharide is innovatively added into the antigen as the immunopotentiator, so that the immune effect of the antigen is further improved, and a feasible technical means is provided for prevention of the duck circovirus. The duck circovirus strain preserved by the invention is preserved for the first time in China, and the preservation number is CGMCC No. 19296.
On the basis of the above inventive concept, the inventor provides the following specific technical solutions:
the inventor firstly provides a duck circovirus, the preservation number of which is CGMCC No.19296, and on the basis of obtaining the virus, the inventor further provides a duck circovirus tissue inactivated vaccine, the effective components of which are as follows:
grinding, repeatedly freezing and thawing and filtering a grinding fluid containing duck circovirus liver tissues and sterile water according to the mass ratio of the vaccine of 1:3-6, and then compounding with pine pollen polysaccharide and emulsifying with a white oil adjuvant to obtain the vaccine; the final concentration of the pine pollen polysaccharide is 10-30 mg/mL;
the duck circovirus antigen is obtained by infecting ducklings of 1 day age by intraperitoneal injection of virus, and then performing intensive infection once every 7 days by intravenous injection, wherein the virus amount of each infection is not less than 1 × 107copies, injecting 250mg/kg cyclophosphamide to induce immunosuppression and enhance virus replication at the same time of each inoculation, collecting whole liver tissue of infected duck at 25 days old, and detecting virus content in liver by fluorescent quantitative PCR technology to be not less than 1 × 108copies/g, namely the antigen can be used as an original antigen tissue;
preparing antigen liquid for vaccine by the steps of proportioning, homogenizing, freezing and thawing, centrifuging and filtering tissues; meanwhile, pollen pini polysaccharide with the concentration of 5-40mg/mL is added into the antigen solution as an immunopotentiator;
inactivating antigen liquid by formaldehyde, and emulsifying the antigen liquid with a white oil adjuvant to prepare a vaccine;
furthermore, the optimal mass ratio of the tissue inactivated vaccine duck circovirus tissue antigen to the sterile water is 1:4-6, and the concentration of the pollen pini polysaccharide is 10-30 mg/mL;
most preferably, the optimal mass ratio of the tissue inactivated vaccine duck circovirus tissue antigen to the sterile water is 1:5, and the concentration of the pollen pini polysaccharide is 20 mg/mL;
the quality ratio is selected because the inventor finds that the duck circovirus has larger viscosity in the duck liver tissue grinding fluid, the grinding fluid is too viscous when the tissue specific gravity is too high, the extraction rate of the antigen fluid is lower, the tissue specific gravity is too low, and the antigen content can be reduced. Meanwhile, the plant polysaccharide is added into the antigen solution to serve as an immunopotentiator, so that the immune effect of the antigen is further improved, and finally the antibody titer of duck circovirus in an organism is improved, so that the cost is saved on the premise that the immune enhancement effect is fully ensured by selecting the concentration of the pollen pini polysaccharide.
The vaccine is prepared more specifically as follows:
selecting 1 day old healthy cherry valley duckling, performing intraperitoneal injection to inoculate purified duck circovirus virus seeds, performing intravenous injection and replanting once every 7 days, wherein the virus infection amount of each time is not less than 1 × 107Copies, the immune suppression is induced by injecting 250mg/kg dose of cyclophosphamide at the same time when infecting each time to facilitate the virus replication, when the duck is 25 days old, the duck liver is collected, the content of the duck circovirus is detected to be more than or equal to 1 × 108copies/g for use;
mixing duck liver with sterile water in a mass ratio of 1:5, fully grinding the duck liver by using a histiocyte wall breaking machine to break the wall and homogenate, repeatedly freezing and thawing for 3 times after the tissue homogenate, then centrifuging for 20-30min by using a rotary centrifuge with 5000 plus material rotation speed of 8000rpm, collecting supernatant, filtering by using gauze, adding pollen pini polysaccharide with the concentration of 5-40mg/mL, adding formaldehyde with the final concentration of two thousandth of volume fraction, and inactivating for 48h at 37 ℃ to obtain final antigen solution. The mass ratio is 1:1, fully emulsifying the antigen solution with a commercial white oil adjuvant to prepare the duck circovirus tissue inactivated vaccine;
wherein the mechanical emulsification method is completed by adopting a colloid mill, a homogenizer or a pulp refiner, and the characteristics of the mixture meet the above standard;
in addition, the invention further provides a preparation method for preparing the yolk antibody by using the vaccine, which comprises the following specific steps:
(1) immunizing the laying hens which are about 1 month before the laying by using the prepared inactivated vaccine for 4 times, wherein the immunization interval is 2 weeks each time; after 4 times of immunization in the early stage, the subsequent immunization is carried out every 1-2 months, and the immunization dose is 1.0-2.0 mL/mouse;
(2) collecting the immunized eggs two weeks after the 4 th immunization, and keeping the immunization once every 1-2 months in the continuous egg collecting process; separating egg yolk from egg white by using an egg yolk separator, mixing the egg yolk with purified water preheated to 30-37 ℃ according to the volume ratio of 3-5:1 to dilute egg yolk liquid, uniformly stirring to obtain egg yolk diluted liquid, and preheating for 1h at 30-37 ℃;
(3) adding 3-4% of PEG6000 into the yolk diluent, mixing with yolk, stirring, standing for 4-6 hr, and reacting;
(4) extracting the reacted supernatant, and coarsely filtering with a 100-200-mesh filter screen, wherein the supernatant is firstly filtered with 100 meshes and then filtered with 200 meshes;
(5) putting the filtered supernatant into a sterile barrel for secondary precipitation for 24-48 h;
(6) pumping out the supernatant of the secondary precipitation, coarsely filtering once by using a 200-mesh filter screen, and then filtering and sterilizing by using a 0.22-micron filter membrane; adding formaldehyde with volume fraction of one thousandth into the sterilized supernatant to inactivate unknown viruses and using the inactivated unknown viruses as a preservative, namely refined egg yolk antibody, wherein the lowest agar expansion test titer of the duck circovirus and adenovirus resisting antibody is not less than 1: 64.
(7) Sub-packaging the filtrate in sterile vaccine bottle under aseptic condition, covering with rubber plug, rolling aluminum cover, labeling, and storing at 4-8 deg.C.
Furthermore, in the step (1), after 4 immunizations in the early stage, the subsequent immunizations are performed every 1.5 months;
the dilution volume ratio in the step (2) is 3.5:1, and the temperature is 35 ℃;
the addition amount of PEG6000 in the step (3) is 3.5 percent; wherein PEG6000 can be dissolved in a small amount of warm water.
Compared with the prior art, the invention achieves the following technical effects:
1. the invention separates and preserves a live duck circovirus in the field for the first time, fills the blank in the field, provides the duck circovirus tissue inactivated vaccine by utilizing the duck circovirus, effectively solves the problem of preparation of the duck circovirus antigen, and determines the proportion of the duck circovirus antigen.
2. The duck circovirus tissue inactivated vaccine provided by the invention is a tissue inactivated vaccine, and has the advantages of simple preparation process, low equipment requirement, moderate viscosity, good needle penetration, small stress and good protection effect. Is suitable for small and medium scale production or for preparing antibodies.
3. The pine pollen polysaccharide adjuvant provided by the invention is small in addition amount and moderate in cost, and can be used for remarkably enhancing the functions of humoral immunity and cellular immunity of an organism and improving the antibody titer of two antigens.
4. The yolk antibody preparation technology provided by the invention optimizes immune procedures and extraction steps, the whole extraction process is simple and easy to operate, the method is suitable for large-scale production, no toxic reagent is used in the extraction process, concentration is not needed, and the residual yolk paste after extraction can be prepared into yolk powder for feeding for secondary use without any toxicity.
5. According to the egg yolk antibody preparation technology provided by the invention, the antibody titer of the duck circovirus is not less than 1:64, the protective effect is outstanding, and the harm caused by two viruses can be effectively prevented and treated.
In conclusion, the duck circovirus tissue inactivated vaccine and the yolk antibody thereof provided by the invention have the advantages of simple preparation process, convenience in use, good effect, safety, environmental friendliness, huge development and utilization potential in livestock and poultry breeding industry and wide application prospect.
The information on the storage of the information is stored,
preservation time: 26/2/2020
The name of the depository: china general microbiological culture Collection center
The preservation number is: CGMCC No.19296
The address of the depository: microbial research institute of western road 1 institute No. 3 of China academy of sciences, Beijing, Chaoyang
Classification naming Duck circovirus
Drawings
FIG. 1 is a bar graph of the effect of different vaccination protocols on duck circovirus content in duck liver.
Detailed Description
The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly define the scope of the invention:
EXAMPLE 1 preparation of vaccine
Separating the duck circovirus original virus seeds from the livers of the clinically-diseased ducks in 2019, determining that the diseased ducks infect the duck circovirus by using a PCR detection technology, detecting other pathogens, eliminating the possibility of virus mixed infection, homogenizing, centrifuging, and filtering and sterilizing the supernatant by using a disposable filter; the virus strain is infected by 1-day-old ducklings, the ducklings are continuously passed for 3 generations, the virus strain is used as a purified virus strain after the virus amount is detected to be qualified, the virus strain is subjected to biological preservation by the inventor, and the preservation number is as follows: CGMCC No. 19296;
selecting 1-day-old healthy cherry valley ducklings to inoculate purified duck circovirus virus seeds by intravenous injection, replanting once every 7 days, injecting 250mg/kg dose of cyclophosphamide to artificially induce immunosuppression during each virus attack so as to be beneficial to virus replication, collecting duck livers when the ducks are 25 days old, and detecting the content of the duck circovirus for later use;
this example identifies the number of replenishes and the effect of cyclophosphamide by setting up 2 cohorts (a and B), each cohort being 3 cohorts, each cohort being 5 ducklings. Three groups in the group A are respectively inoculated with duck circovirus at the age of 1 day, 1+8 days and 1+8+15 days; three groups in group B were inoculated in the same manner and simultaneously injected with cyclophosphamide at a dose of 250 mg/kg; each group was killed at 25 days old, livers were removed, and relative virus content was detected by fluorescent quantitative PCR. The result is shown in figure 1, the result proves that the virus inoculation frequency is obviously and positively correlated with the virus content, and the virus amount can be improved by more than 100 times after the virus inoculation is carried out for three times; in addition, from the results in group B, the use of cyclophosphamide induced immunosuppression significantly increased viral replication, and group B was also able to increase the virus content by 10-fold over group A under the 1+8+15 day vaccination protocol. Therefore, the invention finally determines to use a scheme of 1+8+15 days of virus inoculation and cyclophosphamide treatment to obtain high-content duck circovirus antigen tissues.
Respectively mixing the liver tissues containing the duck circovirus with sterile water in a mass ratio of 1:3, 1:4, 1:5 and 1:6, fully grinding and breaking the walls by using a tissue cell wall breaking machine, repeatedly freezing and thawing for 3 times after homogenizing the tissues, centrifuging for 20-30min by using a rotary centrifuge with a speed of 5000 plus materials of 8000rpm, collecting the supernatant, adding formaldehyde with a final concentration of two thousandth at 37 ℃ for inactivation for 48h to obtain the final antigen solution. The antigen liquid with the mass ratio of 1:1 and the commercial white oil adjuvant are fully emulsified by a homogenizer to prepare the duck circovirus tissue inactivated vaccine with different antigen contents, and the optimal antigen proportion is evaluated through the yield, the viscosity, the needle penetration and the like of the antigen liquid. The results are shown in Table 1, when the ratio of the tissue to the water is 1:5 and 1:6, the yield, the viscosity and the needle penetration of the antigen liquid meet the composite requirements, and considering that the larger the antigen amount is, the better the immune effect is, the ratio of 1:5 is preferably used as the optimal ratio for preparing the antigen liquid.
Preparing antigen solution by using the screened optimal antigen proportion, adding 10, 20 and 40mg/mL pine pollen polysaccharide or astragalus polysaccharide into the antigen solution respectively to prepare the duck circovirus tissue inactivated vaccine containing polysaccharides with different concentrations, and finally determining the optimal polysaccharide and content by comparing immune effects.
TABLE 1 Effect of different antigen contents on vaccine traits
Note: the "+" number represents the fold increase in viscosity compared to # 10 white oil.
Example 2 comparison of vaccine immunization
1. Design of experiments
1.1 test grouping:
group A, the vaccine prepared by using duck liver and water in the mass ratio of 1:5 in the invention example 1 and not adding polysaccharide is used for preparing antigen liquid.
Group B: the antigen solution prepared by using the duck liver and water in the mass ratio of 1:5 in the embodiment 1 of the invention is added with the vaccine prepared by adding the astragalus polysaccharide with the concentration of 10 mg/mL.
Group C: the antigen solution prepared by using the duck liver and water in the mass ratio of 1:5 in the embodiment 1 of the invention is added with the vaccine prepared by adding astragalus polysaccharide with the concentration of 20 mg/mL.
Group D: the antigen solution prepared by using the duck liver and water in the mass ratio of 1:5 in the embodiment 1 of the invention is added with the astragalus polysaccharide with the concentration of 40mg/mL to prepare the vaccine.
And group E, preparing antigen solution by using duck liver and water in a mass ratio of 1:5 in the embodiment 1 of the invention, and adding the vaccine prepared by 10mg/mL pine pollen polysaccharide.
And group F, preparing antigen solution by using the duck liver and water in the mass ratio of 1:5 in the embodiment 1 of the invention, and adding the vaccine prepared by pollen pini polysaccharide with the concentration of 20 mg/mL.
And group G, preparing antigen solution by using duck liver and water in a mass ratio of 1:5 in the embodiment 1 of the invention, and adding the vaccine prepared by pollen pini polysaccharide with the concentration of 40 mg/mL.
Mock negative control group: the vaccine is not immunized, and the normal saline with equal dosage is inoculated.
1.2 vaccination: 40 healthy cherry valley ducks were divided into 8 groups of 5 ducks on average. The ducklings of each group at the age of 7 days are injected with the corresponding vaccine in 1.1 through muscle, the inoculation dose is 0.5 mL/ducklings, and the ducklings are boosted for 1 time by the same dose after one week. Blood was collected 7, 14 and 21 days after the second immunization, and serum antibody titers were measured by the agar diffusion assay. All data were analyzed by Duncan's multiple comparisons using SPSS 17.0 software, with mean + -SD, and P <0.05 indicating significant differences. Duck circovirus detoxification is carried out on each group of ducks 21d after the second immunization, 0.5mL of liver homogenate supernatant containing duck circovirus is inoculated, and the positive rate of virus in the liver is detected by PCR technology 5d after infection.
2. Results and analysis
2.1 comparison of serum antibody titers
Each test group was subjected to a subpwinged venous blood collection of 1.0mL for each duck in each group at 7, 14, 21d after the secondary immunization, serum was separated, and the antibody titer was measured by the agar diffusion test, and the results were expressed as the average of the titers log 2. The results are shown in Table 2. As can be seen from Table 2, the mean values of antibody titers of the duck circovirus in the three groups at different time points are not greatly different with the increase of the content of the astragalus polysaccharides in the groups B to D, and have no significant difference compared with the group A without the astragalus polysaccharides. The result shows that the astragalus polysaccharide has no obvious enhancement effect on the immune effect of the vaccine. However, with the increase of the content of the pollen pini polysaccharide in the E-G group vaccine, the antibody titer is obviously increased at three time points, and after 21d after the second immunization, the average titer of the pollen pini polysaccharide vaccine group with the added concentration of 20mg/mL is 0.9 higher than that of the pollen pini polysaccharide group without the added polysaccharide, the average titer of the pollen pini polysaccharide vaccine group with the added concentration of 40mg/mL is 1.1 higher than that of the pollen pini polysaccharide group without the added polysaccharide, and the cost factor is comprehensively considered, so that the pollen pini polysaccharide with the concentration of 20mg/mL is preferably used as the immunopotentiator of the antigen.
TABLE 2 serum antibody agar titer test (mean, log2) for polysaccharide adjuvant-free vaccine immunization groups
2.2 challenge protection test
And (3) performing duck circovirus virus challenge according to the requirement of 1.2 at 21d after the second immunization, and detecting the positive rate of the virus in the liver by using a PCR technology at 5d after infection. The results showed that neither virus was detected in the livers of A, B, C, D, E, F and group G, whereas viral nucleic acid was detected in the Mock negative control group, indicating that the vaccine immunization group was able to effectively protect against viral infection and that the antibodies produced by the vaccine were effective. In clinical consideration, due to the existence of certain immunosuppressive factors, the higher the antibody titer generated by the vaccine is, the better the protection effect is, therefore, the invention preferably selects duck liver and water with the mass ratio of 1:5 to prepare antigen liquid, and 20mg/mL of pine pollen polysaccharide as an immunopotentiator.
Example 3 vaccination dose and safety evaluation
The vaccine was produced according to the preferred conditions found in example 2, and the vaccination dose and safety of the vaccine were evaluated. The vaccination dose is set to be 0.2, 0.5 and 1.0mL, and 5 ducklings with the ages of 1, 4 and 7 days are inoculated in each dose (only one vaccination is carried out, and immunization is not carried out). Collecting blood from the infrawing veins 28 days after inoculation, separating serum, and performing agar diffusion test to detect titer of duck circovirus antibodies, wherein the result is expressed by average value +/-SD of titer log 2; meanwhile, 0.5mL of virus liquid containing duck circovirus is injected intramuscularly for virus counteracting, and after 3d, virus positive rate in liver is detected by PCR technology. Stress conditions after inoculation were recorded for each group, and stress manifested as depressed spirit, unwilling to ambulate, reduced food intake, etc., and death in severe cases.
The results in Table 3 show that the antibody production was not greatly affected by the age of the day of inoculation, and the antibody titer was higher with the larger inoculation dose. The results in Table 4 show that 0.5mL of the strain at 1-day-old inoculation shows obvious stress reaction, 0.5mL of the strain at 4-day-old inoculation partially shows stress, and 0.5mL of the strain at 7-day-old inoculation has no stress; at the inoculation dose of 1.0mL, stress occurred at three days of age. According to the results, when 0.5mL of the virus was inoculated at the age of 7 days, not only the stress reaction was not caused, but also the antibody titer was 4.6. + -. 0.26, and the virus positive rate after challenge was 0. Therefore, the invention preferably selects 0.5mL of 7-day-old immune as a safe and effective dose, and can be applied to the immunity of clinical ducklings.
TABLE 3 Effect of different day ages and doses of inoculation on antibody titer and protection rate of duck circovirus
Note:athe left data in this column are antibody titers (mean ± SD, log2) and the right data are the virus positivity rate.
TABLE 4 Effect of different day ages of inoculation and inoculation dose on duckling stress
Note: the "/" ratio represents the total number of stress ratio occurrences.
Example 4 preparation of yolk antibody
(1) Immunizing laying hens which are 1 month before production with the vaccine used in the example 3 for 4 times, wherein the inoculation dose is 1.5 mL/egg, and the immunization interval is 2 weeks;
(2) collecting the immunized eggs two weeks after the 4 th immunization, and keeping the immunization once every 1-2 months in the continuous egg collecting process; separating yolk from egg white with yolk separator, diluting yolk liquid with purified water preheated to 35 deg.C at volume ratio of 3.5:1, stirring to obtain diluted yolk liquid, and pre-heating at 35 deg.C for 1 hr.
(3) Adding PEG6000 with final concentration of 3.5% into the yolk diluent, mixing with yolk, stirring, standing for 4 hr, and reacting.
(4) The supernatant after the reaction was extracted and filtered with a 100 mesh and then a 200 mesh filter.
(5) And putting the filtered supernatant into a sterile bucket for secondary precipitation, wherein the precipitation time is 48 hours.
(6) The supernatant of the second precipitation was extracted, first coarsely filtered with a 200 mesh filter screen, and then sterilized with a 0.22 μm filter.
(7) Adding a certain amount of preservative or formaldehyde with one thousandth of concentration into the sterilized supernatant, namely the refined egg yolk antibody.
(8) Agar diffusion test for detecting the agar diffusion test titer of the duck circovirus and adenovirus resisting antibody is more than or equal to 1: 64.
(9) and (5) subpackaging and storing. Sub-packaging the filtrate in sterile vaccine bottle under aseptic condition, covering with rubber plug, rolling aluminum cover, labeling, and storing at 4-8 deg.C.
Example 5 quality test of yolk antibody
(1) Safety inspection
After 20 healthy cherry valley ducklings of 1 day old are injected into muscles at multiple points, 2.0mL of the egg yolk antibody prepared in the embodiment 4 is injected into the ducklings at multiple points, and the ducklings are observed to be susceptible to all healthy survival for 14 days, which shows that the egg yolk antibody has good safety.
(2) Sterility testing
The results of the implementation according to the pharmacopoeia of the people's republic of China (2015 edition) show that the egg yolk antibody of the invention is free from bacterial, mycoplasma and foreign virus contamination.
(3) Efficacy test
40 healthy cherry valley ducklings of 1 day old (negative for duck circovirus antigen and antibody in serum examination) are randomly divided into A, B2 groups of 20 ducklings. Group A was intramuscularly injected with 0.5mL of the yolk antibody prepared in example 4; group B was a control group, and 0.5mL of saline was injected intramuscularly and kept separately. After 8h of injection, 0.5mL (of liver homogenate supernatant containing duck circovirus) is simultaneously injected into each duck>1×107copies). 3d after the virus attack, 10 viruses are killed, livers are collected, the positive condition of the viruses is detected by using a PCR technology, the rest 10 viruses are observed for 14 days, clinical symptoms are recorded, and then the positive condition of the viruses is detected by killing.
The results show that all the duck circovirus in the livers of all 20 ducks in the egg yolk antibody group (group A) is negative, and all the duck circovirus in the 20 ducks in the control group (group B) is positive. In addition, the A group has no clinical symptoms in the observation period, and the B group part of ducks have the symptoms of mental depression, poor appetite, unwillingness and the like. The result shows that the yolk antibody prepared in the example 4 is effective, and the ducklings of 1 day old can be injected with 0.5 mL.
Example 6 application of yolk antibody
In a suspected duck circovirus infection breeding factory, diseased ducks with obvious emaciation, inappetence and mental retardation are selected, 40 ducks (about 25 days old) diagnosed as duck circovirus infection are screened by blood collection and PCR technology, and are randomly divided into A, B2 groups, and each group comprises 20 ducks and are kept separately. The yolk antibody prepared in example 4 was injected into group A at 2.0mL, and the physiological saline was injected into group B at 2.0 mL. And observing for 14d, recording the illness and death condition of each group of ducks, killing the ducks and detecting the positive rate of the viruses in the liver by using a PCR (polymerase chain reaction) technology.
As a result: after the egg yolk antibody 2d is injected into the group A, the feed intake of the sick ducklings begins to increase, the mental state is obviously improved, and no ducklings die in 14 d. After 14 days, the ducks are killed and dissected, most of the ducks are slightly or asymptomatic in autopsy lesion, and the positive rate 2/20 of duck circovirus in the liver is detected by using a PCR technology. After the group B is injected with the physiological saline, the feed intake and the mental state of the sick ducks are not improved remarkably, the sick ducks die from the 2 nd day after the injection, and the death rate of the sick ducks within 14d is 25%. The live ducks are killed after 14 days of observation, most of the ducks have symptoms of yellowing, local swelling, bleeding spots and the like, and the positive rate of the duck circovirus in livers (including the livers of the dead ducks frozen) is detected by using a PCR (polymerase chain reaction) technology 17/20.
The test results show that the yolk antibody has good safety, good prevention effect and high cure rate, can be used for preventing and treating duck circovirus infection, and has great economic and social benefits.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (8)
1. The preservation number of the strain is CGMCC No. 19296.
2. A duck circovirus tissue inactivated vaccine is characterized in that: the effective components are as follows:
grinding, repeatedly freezing and thawing and filtering a grinding fluid containing duck circovirus liver tissues and sterile water according to the mass ratio of the vaccine of 1:3-6, and then compounding with pine pollen polysaccharide and emulsifying with a white oil adjuvant to obtain the vaccine; the final concentration of the pine pollen polysaccharide is 10-30 mg/mL;
the adopted duck circovirus has a preservation number of CGMCC No. 19296.
3. The inactivated vaccine for duck circovirus tissue according to claim 2, wherein:
the duck circovirus antigen is obtained by infecting ducklings of 1 day age by intraperitoneal injection of virus, and then performing intensive infection once every 7 days by intravenous injection, wherein the virus amount of each infection is not less than 1 × 107copies, simultaneous injection per vaccination250mg/kg cyclophosphamide induces immunosuppression to enhance virus replication, collects the whole liver tissue of the infected duck at 25 days old, and detects the virus content in the liver to be more than or equal to 1 × 10 by the fluorescent quantitative PCR technology8copies/g, namely the antigen can be used as an original antigen tissue;
preparing antigen liquid for vaccine by the steps of proportioning, homogenizing, freezing and thawing, centrifuging and filtering tissues; meanwhile, pollen pini polysaccharide with the concentration of 5-40mg/mL is added into the antigen solution as an immunopotentiator;
the antigen liquid is inactivated by formaldehyde and emulsified with white oil adjuvant to prepare the vaccine.
4. The inactivated vaccine for duck circovirus tissue according to claim 2, wherein:
the mass ratio of the tissue inactivated vaccine duck circovirus tissue antigen to the sterile water is 1: 4-6.
5. The inactivated vaccine for duck circovirus tissue according to claim 2 or 4, wherein: the tissue inactivated vaccine duck circovirus tissue antigen and sterile water have a mass ratio of 1:5, and the concentration of the pollen pini polysaccharide is 20 mg/mL.
6. The method for preparing the duck circovirus tissue inactivated vaccine as claimed in claim 2, is characterized in that: the method comprises the following specific steps:
selecting 1 day old healthy cherry valley duckling, performing intraperitoneal injection to inoculate purified duck circovirus virus seeds, performing intravenous injection and replanting once every 7 days, wherein the virus infection amount of each time is not less than 1 × 107Copies, the immune suppression is induced by injecting 250mg/kg dose of cyclophosphamide at the same time when infecting each time to facilitate the virus replication, when the duck is 25 days old, the duck liver is collected, the content of the duck circovirus is detected to be more than or equal to 1 × 108copies/g for use;
mixing duck liver with sterile water in a mass ratio of 1:5, fully grinding the duck liver by using a histiocyte wall breaking machine to break the wall and homogenate, repeatedly freezing and thawing for 3 times after the tissue homogenate, then centrifuging for 20-30min by using a rotary centrifuge with 5000 plus material rotation speed of 8000rpm, collecting supernatant, filtering by using gauze, adding pollen pini polysaccharide with the concentration of 5-40mg/mL, adding formaldehyde with the final concentration of two thousandth of volume fraction, and inactivating for 48h at 37 ℃ to obtain final antigen solution. The mass ratio is 1:1, fully emulsifying the antigen solution with a commercial white oil adjuvant to prepare the duck circovirus tissue inactivated vaccine;
wherein the mechanical emulsification method is completed by adopting a colloid mill, a homogenizer or a pulp refiner, and the characteristics of the mixture meet the above standard.
7. The method for preparing the yolk antibody by using the duck circovirus tissue inactivated vaccine as described in claim 2 is characterized in that: the method comprises the following specific steps:
(1) immunizing the laying hens which are about 1 month before the laying by using the prepared inactivated vaccine for 4 times, wherein the immunization interval is 2 weeks each time; after 4 times of immunization in the early stage, the subsequent immunization is carried out every 1-2 months, and the immunization dose is 1.0-2.0 mL/mouse;
(2) collecting the immunized eggs two weeks after the 4 th immunization, and keeping the immunization once every 1-2 months in the continuous egg collecting process; separating egg yolk from egg white by using an egg yolk separator, mixing the egg yolk with purified water preheated to 30-37 ℃ according to the volume ratio of 3-5:1 to dilute egg yolk liquid, uniformly stirring to obtain egg yolk diluted liquid, and preheating for 1h at 30-37 ℃;
(3) adding 3-4% of PEG6000 into the yolk diluent, mixing with yolk, stirring, standing for 4-6 hr, and reacting;
(4) extracting the reacted supernatant, and coarsely filtering with a 100-200-mesh filter screen, wherein the supernatant is firstly filtered with 100 meshes and then filtered with 200 meshes;
(5) putting the filtered supernatant into a sterile barrel for secondary precipitation for 24-48 h;
(6) pumping out the supernatant of the secondary precipitation, coarsely filtering once by using a 200-mesh filter screen, and then filtering and sterilizing by using a 0.22-micron filter membrane; adding formaldehyde with volume fraction of one thousandth into the sterilized supernatant to inactivate unknown viruses and using the inactivated unknown viruses as a preservative, namely refined egg yolk antibody, wherein the lowest agar expansion test titer of the duck circovirus and adenovirus resisting antibody is not less than 1: 64.
(7) Sub-packaging the filtrate in sterile vaccine bottle under aseptic condition, covering with rubber plug, rolling aluminum cover, labeling, and storing at 4-8 deg.C.
8. The method for producing an egg yolk antibody according to claim 7, wherein:
after 4 immunizations in the early stage in the step (1) are finished, carrying out subsequent immunizations once every 1.5 months;
the dilution volume ratio in the step (2) is 3.5:1, and the temperature is 35 ℃;
the addition amount of PEG6000 in the step (3) is 3.5 percent; wherein PEG6000 can be dissolved in a small amount of warm water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114209821A (en) * | 2021-12-30 | 2022-03-22 | 哈药集团生物疫苗有限公司 | Triple inactivated vaccine for preventing and treating duck circovirus disease, novel duck reovirus disease and duck adenovirus type 3 and preparation method thereof |
| WO2023142176A1 (en) * | 2022-01-30 | 2023-08-03 | 山东农业大学 | Pharmaceutical composition for intravenous injection, preparation containing same, and preparation method therefor and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114209821A (en) * | 2021-12-30 | 2022-03-22 | 哈药集团生物疫苗有限公司 | Triple inactivated vaccine for preventing and treating duck circovirus disease, novel duck reovirus disease and duck adenovirus type 3 and preparation method thereof |
| WO2023142176A1 (en) * | 2022-01-30 | 2023-08-03 | 山东农业大学 | Pharmaceutical composition for intravenous injection, preparation containing same, and preparation method therefor and use thereof |
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