阅读说明：本技术 鸭圆环病毒病、新型鸭呼肠弧病毒病、鸭病毒性肝炎三联灭活疫苗及其制备方法 (Triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and preparation method thereof ) 是由 宋扬 柴华 梁宛楠 徐刚 李应鹤 曹宝珠 刘洋 宋�莹 张影 孟福强 于 2020-12-31 设计创作，主要内容包括：本发明公开了鸭圆环病毒病、新型鸭呼肠弧病毒病、鸭病毒性肝炎三联灭活疫苗及其制备方法,所述三联灭活疫苗包括免疫上有效量的灭活抗原和免疫佐剂,所述灭活抗原包括鸭圆环病毒抗原、新型鸭呼肠弧病毒抗原和鸭肝炎病毒抗原。本发明进一步公开了制备所述三联灭活疫苗的方法。本发明鸭圆环病毒病、新型鸭呼肠弧病毒病、鸭病毒性肝炎三联灭活疫苗安全性良好,不会产生抗原成份的相互干扰或影响。免疫保护效力试验证明,本发明三联灭活疫苗能够同时预防鸭呼肠弧病毒,鸭圆环病毒和鸭肝炎病毒,可避免多次接种免疫出现的不良反应,具有制备方法简单、方便、多效、低成本、疫苗效价含量高等优点。(The invention discloses a triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and a preparation method thereof. The invention further discloses a method for preparing the triple inactivated vaccine. The triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis has good safety and can not generate mutual interference or influence of antigen components. Immune protection efficacy tests prove that the triple inactivated vaccine can prevent duck reovirus, duck circovirus and duck hepatitis virus simultaneously, can avoid adverse reactions caused by multiple times of vaccination and immunization, and has the advantages of simple and convenient preparation method, multiple effects, low cost, high vaccine titer content and the like.)

1. The triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis comprises an immunologically effective amount of inactivated antigen and an immunoadjuvant, and is characterized in that the inactivated antigen comprises a duck circovirus antigen, a novel duck reovirus antigen and a duck hepatitis virus antigen.

2. The triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis according to claim 1, wherein the duck circovirus antigen is a recombinant protein obtained by recombinant expression of a duck circovirus cap gene through a prokaryotic or eukaryotic system.

3. The triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis according to claim 2, wherein the duck circovirus antigen is a recombinant duck circovirus cap protein obtained by cloning a codon-optimized duck circovirus cap gene shown in SEQ ID N.1 to a eukaryotic expression plasmid and transfecting CHO cells for induced expression.

4. The triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis according to claim 3, wherein the method for cloning the codon-optimized cap gene of duck circovirus shown in SEQ ID N.1 to eukaryotic expression plasmid and transfecting CHO cells to induce expression comprises:

(1) cloning the codon-optimized duck circovirus Cap gene shown in SEQ ID No.1 onto a pUC-57 vector, and amplifying a DUCV-Cap gene fragment from the pUC-DUCV-Cap recombinant vector by adopting a PCR method;

(2) carrying out enzyme digestion on the pCI-GS plasmid vector and the purified PCR product by using Xho I and EcoRI, recovering the enzyme-digested pCI-GS plasmid and the DUCV-Cap gene fragment, and connecting to obtain a pCI-DUCV-Cap-GS eukaryotic expression plasmid;

(3) transfecting the pCI-DUCV-Cap-GS eukaryotic expression plasmid into a CHO cell, inducing the expression of recombinant Cap protein in the CHO cell, and collecting the expressed recombinant Cap protein.

5. The triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis according to claim 1, wherein the duck reovirus antigen is preferably a virus solution obtained by propagating a duck reovirus strain S in LMH cells; the microbial preservation number of the duck reovirus S strain is as follows: CGMCC No. 20000.

6. The triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis according to claim 1, wherein the duck hepatitis virus antigen is prepared by inoculating virus seeds for producing duck hepatitis virus types 1 and 3 to susceptible duck embryos, and harvesting the embryo solution to obtain the duck hepatitis virus antigen.

7. The triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis according to claim 1, wherein the immune adjuvant is any one or more of white oil, span-80, aluminum stearate or tween-80.

8. The triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease, and duck viral hepatitis according to claim 1, further comprising a cryoprotectant.

9. A method for preparing the triple inactivated vaccine for the duck circovirus disease, the novel duck reovirus disease and the duck viral hepatitis of any one of claims 1 to 8, which comprises the following steps:

(1) preparing an oil phase: mixing white oil with span-80, adding 2% aluminum stearate, stirring, and sterilizing;

(2) preparation of an aqueous phase: mixing the inactivated duck reovirus antigen, the duck viral hepatitis antigen and the recombinant duck circovirus cap protein to obtain an antigen mixed solution; mixing the antigen mixed solution and sterile tween-80 until tween-80 is completely dissolved;

(3) emulsification: and (3) taking the oil phase, slowly stirring in a tissue homogenizer, simultaneously adding the water phase, and stirring after the water phase is added to obtain the composition.

10. The method of claim 9, wherein in step (1) the white oil is mixed with span-80 in a ratio of 94: 6, uniformly mixing the materials, and adding 2 percent of aluminum stearate; the stirring is carried out until the color is light yellow, and the mixture is clarified and transparent, and then the high-pressure steam sterilization is carried out at the temperature of 121 ℃;

controlling the duck reovirus antigen not less than 10 in the antigen mixed solution in the step (2)^8.0TCID₅₀The content of the cap protein of the recombinant duck circovirus in each feather vaccine is not less than 5 mug, and the content of the duck hepatitis viruses type 1 and 3 is not less than 10^5.5ELD₅₀0.1 ml; the antigen mixture and sterile tween-80 were mixed according to 97.5: 2.5 until the Tween-80 is completely dissolved;

and (3) taking 3 parts of the oil phase, placing the oil phase into a tissue homogenizer, slowly stirring, simultaneously adding 2 parts of the water phase, and stirring at the rotating speed of 10000r/min for 4-5 min after the addition is finished.

Technical Field

The invention relates to a triple inactivated vaccine for preventing and treating duck virus diseases, in particular to a triple inactivated vaccine for preventing and treating duck circovirus diseases, novel duck reovirus diseases and duck viral hepatitis.

Background

The novel duck reoviridae is a disease mainly characterized by splenomegaly, hemorrhage and necrosis, and is found in duck farms in Shandong, Henan, Jiangsu and other areas since 2017. The susceptible days of the traditional Chinese medicine are mostly in the range of 5-25 d, the morbidity is generally between 10% and 70%, and the mortality is generally between 50% and 60%. The smaller the age of the day, the higher the morbidity and mortality. In cold weather, morbidity and mortality are significantly increased.

The duck circovirus disease is one of the main epidemic diseases harming the duck breeding industry, and besides causing primary infection and death of ducks, the duck circovirus disease causes more serious damage to the immune function of infected ducks, causes the body resistance to be reduced, is easy to suffer from the complication or secondary infection of other pathogens, causes the disease condition to be aggravated and causes more loss. The virus, which causes immunosuppression in the body, is often overlooked because it often appears as a subclinical infection. In recent years, with the rapid development of the duck breeding industry in China and the growing scale of breeding, the prevention and treatment of the duck circovirus disease are gradually emphasized, but the duck circovirus virus has difficulty in proliferation in various cell lines and embryoid bodies, so that the preparation of the vaccine by using the whole virus as the antigen is more difficult. The CHO expression system has accurate post-transcriptional modification function, so that the expressed protein is closest to natural protein molecules in the aspects of molecular structure, physicochemical property and biological function; the high-efficiency amplification and expression capacity of the recombinant gene; has the adherent growth characteristic, has higher shear stress resistance and osmotic pressure resistance, can be used for suspension culture, has higher expression level and the like, and is widely used for the expression of protein in recent years. The CHO expression system is utilized to successfully express the Cap protein of the duck circovirus, and a safe and effective inactivated vaccine of the duck circovirus disease is prepared.

Duck viral hepatitis is a rapidly transmitted and highly lethal infectious disease of ducklings caused by duck hepatitis viruses. It is mainly characterized by hepatomegaly, bleeding spots and neurological symptoms. The duck hepatitis virus has 3 serotypes, and the duck viral hepatitis types 1 and 3 are mainly prevalent in China. The disease mainly occurs in 4-20 days old ducklings, adult ducks have resistance, and chickens and ducks cannot naturally attack the disease. Diseased ducks and duck with poison are main infection sources and mainly infect through digestive tracts and respiratory tracts. Poor feeding management, lack of vitamins and minerals, and damp and crowded duck houses can all promote the occurrence of the disease. The disease occurs in the season of hatching the ducklings, once the disease occurs, the disease is quickly spread in ducklings groups, and the disease incidence can reach 100%.

In recent years, the duck breeding industry in China is rapidly developed and continuously developed in large scale, but the breeding level and the technology are relatively lagged behind, and various duck epidemic diseases occur successively. From the aspect of detecting the pathological materials of ducks of different provinces in China, the duck circovirus, the duck hepatitis and the novel reovirus are the common epidemic diseases of duck mixed infection, and huge economic losses are caused to the Chinese breeding industry. At present, no effective vaccine for simultaneously preventing and controlling the three epidemic diseases exists, so that the safe and effective triple inactivated vaccine for simultaneously preventing and controlling the duck circovirus disease, the novel duck reovirus disease and the duck viral hepatitis is developed as soon as possible.

Disclosure of Invention

One of the purposes of the invention is to provide a safe and effective triple inactivated vaccine for simultaneously preventing and treating novel duck reovirus viruses, duck circovirus diseases and duck viral hepatitis;

the invention also aims to provide a method for preparing the triple inactivated vaccine.

The above object of the present invention is achieved by the following technical solutions:

the invention firstly provides a triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis, which comprises an immunologically effective amount of inactivated antigen and an immunological adjuvant, wherein the inactivated antigen comprises duck circovirus antigen, novel duck reovirus antigen and duck hepatitis virus antigen.

The duck circovirus antigen is preferably recombinant protein obtained by recombinant expression of a cap gene of the duck circovirus through a prokaryotic or eukaryotic system; as a preferred embodiment of the invention, the duck circovirus cap gene optimized by the codon shown in SEQ ID N.1 is preferably cloned to eukaryotic expression plasmid and transfected into CHO cells, and the recombinant duck circovirus cap protein is obtained by induced expression.

For reference, the present invention provides a method for preparing a recombinant duck circovirus cap protein, comprising:

(1) cloning the codon-optimized duck circovirus Cap gene shown in SEQ ID No.1 onto a pUC-57 vector, and amplifying a DUCV-Cap gene fragment from the pUC-DUCV-Cap recombinant vector by adopting a PCR method;

(2) carrying out enzyme digestion on the pCI-GS plasmid vector and the purified PCR product by using Xho I and EcoRI, recovering the enzyme-digested pCI-GS plasmid and the DUCV-Cap gene fragment, and connecting to obtain a pCI-DUCV-Cap-GS eukaryotic expression plasmid;

(3) transfecting the pCI-DUCV-Cap-GS eukaryotic expression plasmid into a CHO cell, inducing the expression of recombinant Cap protein in the CHO cell, and collecting the expressed recombinant Cap protein.

The duck reovirus antigen is preferably virus liquid obtained by propagating a duck reovirus S strain (the microorganism preservation number is CGMCC No. 20000; the preservation unit is China general microbiological culture Collection center; the preservation date is 2020, 7 and 6 days; the classification name is novel duck reovirus; the preservation address is No. 3 Xilu No.1 North Chen West Chen of the sunward area of Beijing City) in LMH cells.

The duck hepatitis virus antigen can be prepared by inoculating virus seeds for producing duck hepatitis virus types 1 and 3 to susceptible duck embryos, and harvesting embryo solution to obtain the duck hepatitis virus antigen.

In the triple combined inactivated vaccine, the duck reovirus antigen is not less than 10^8.0TCID₅₀The content of DUCV-cap protein in each feather vaccine is not less than 5 mug, and the content of 1 type and 3 type duck hepatitis virus is not less than 10^5.5ELD₅₀/0.1ml。

The immunological adjuvant can be any conventional immunological adjuvant, and as a specific embodiment of the invention, the immunological adjuvant can be any one or more of white oil, span-80 (span-80), aluminum stearate or tween-80.

The inactivated vaccine part of the invention may also comprise a lyoprotectant.

The invention further provides a method for preparing the novel triple inactivated vaccine for duck reovirus, duck circovirus disease and duck viral hepatitis, which comprises the following steps:

(1) preparing an oil phase: mixing white oil with span-80, adding 2% aluminum stearate, stirring, and sterilizing;

(2) preparation of an aqueous phase: mixing the inactivated duck reovirus antigen, the duck viral hepatitis antigen and the recombinant duck circovirus cap protein to obtain an antigen mixed solution; mixing the antigen mixed solution and sterile tween-80 until tween-80 is completely dissolved;

(3) emulsification: and (3) taking the oil phase, slowly stirring in a tissue homogenizer, simultaneously adding the water phase, and stirring after the water phase is added to obtain the composition.

As a preferred embodiment of the invention, in step (1), the white oil is mixed with span-80 according to a ratio of 94: 6, uniformly mixing the materials, and adding 2 percent of aluminum stearate; the stirring is carried out until the color is light yellow, and the mixture is clarified and transparent, and then the mixture is sterilized by high-pressure steam at 121 ℃.

As a preferred embodiment of the present invention, the antigen mixture in step (2) controls the duck reovirus antigen not less than 10^8.0TCID₅₀The content of the cap protein of the recombinant duck circovirus in each feather vaccine is not less than 5 mug, and the content of the duck hepatitis viruses type 1 and 3 is not less than 10^5.5ELD₅₀0.1 ml; antigen mixture and sterile Tween-80 (Tween-80) were mixed according to 97.5: shaking and mixing the mixture according to the proportion of 2.5 until the Tween-80 is completely dissolved.

As a preferred embodiment of the invention, 3 parts of oil phase are taken and put into a tissue homogenizer to be slowly stirred, 2 parts of water phase are slowly added at the same time, and after the addition is finished, the mixture is stirred for 4min-5min at the rotating speed of 10000 r/min.

The safety test and inspection results show that the triple inactivated vaccine prepared by the invention is used for inoculating the ducklings, the ducklings are completely healthy and alive during observation, no local and systemic adverse reactions occur, and the triple inactivated vaccine has good safety.

According to the immune protection efficacy test result of the triple inactivated vaccine, duck circovirus attack is virulent 21 days after duckling immunization, DuCV nucleic acid copy number in whole blood is detected by whole blood collected 21 days later by an RT-QPCR method, and the duckling 10/10 in the immune group is protected as a result and the duckling 10/10 in the attack control group is attacked; blood is collected 3 weeks after duckling immunization, and the neutralizing antibody level of duck reovirus is determined by a neutralization test method, and the result shows that the neutralizing antibody titer of the ducklings in the immune group is not lower than 1:256 in 21 days compared with the control group. Blood is collected 3 weeks after duckling immunization, and the neutralizing antibody level of the duck hepatitis virus is determined by a neutralization test method, and the result shows that the neutralizing antibody titer of the ducklings in the immune group is not lower than 1:128 in 21 days compared with the control group.

The triple inactivated vaccine provided by the invention does not generate mutual interference or influence of antigen components, can simultaneously prevent duck reovirus, duck circovirus and duck hepatitis virus, can avoid adverse reactions caused by multiple times of vaccination, and has the advantages of simple and convenient preparation method, multiple effects, low cost, high vaccine titer content and the like.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. 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 within the scope of the invention.

Example 1 preparation of triple inactivated vaccine against Duck circovirus disease, novel Duck reoviridae, Duck viral hepatitis

Seed of poisonous creature

The novel duck reovirus strain S (preserved in China general microbiological culture Collection center, CGMCC No.20000), the type 1 duck hepatitis A virus strain JS (preserved in China general microbiological culture Collection center, with the microorganism preservation number of CGMCC No.6852) and the type 3 duck hepatitis A virus strain SD (preserved in China general microbiological culture Collection center, with the microorganism preservation number of CGMCC No.8506) for vaccine production are separated, identified, preserved and supplied by Hayao biological vaccine Limited.

Preparation method

Preparation of 1 duck circovirus CAP protein

1.1 design and Synthesis of Duck circovirus CAP Gene

The Cap gene of the duck circovirus is cloned to a pUC-57 vector after codon optimization (the optimized nucleotide sequence is shown as SEQ ID No. 1). The DUCV-Cap gene fragment was amplified from the pUC-DUCV-Cap recombinant vector by PCR.

1.2 construction of pCI-DUCV-Cap-GS eukaryotic expression vector

The pCI-GS plasmid vector and the purified PCR product were digested with Xho I and EcoRI at 37 ℃ for 3 hours, and the digested pCI-GS plasmid and DUCV-Cap gene fragment were recovered using DNA recovery kits, respectively, and T was used₄DNA ligase was ligated in a water bath at 16 ℃ overnight. mu.L of the ligation product was added to 200. mu.L of ice-thawed E.coli DH 5. alpha. competent cells, gently mixed and ice-cooled for 30min, heat-shocked at 42 ℃ for 45s, rapidly placed on ice for 2min, added with 900. mu.L of S.O.C medium, shake-cultured at 37 ℃ and 220rpm for 4h, centrifuged and concentrated to 150. mu.L of a cell suspension, spread on LB solid medium containing Amp, and cultured at 37 ℃ for 16 h.

1.3 identification of pCI-DUCV-Cap-GS recombinant plasmid

And (3) selecting single colonies on the plate, respectively inoculating the single colonies into an LB liquid culture medium, culturing for 2 hours at 37 ℃, and performing PCR amplification by using a DUCV-cap-R/DUCV-cap-F primer by using a bacterial liquid as a template. Colony PCR was performed with VP2-F and VP2-R primers. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 10min, wherein 30 cycles are set; extension at 72 ℃ for 10 min. The length of the PCR amplified fragment is estimated to be about 750bp, and the positive bacteria liquid is subjected to sequence determination.

TABLE 1PCR amplification primers

1.4 transfection of recombinant pCI-DUCV-Cap-GS plasmid into CHO cells

1.4.1 preparing cells

CHO cells in logarithmic growth phase were centrifuged at 800rpm for 5 minutes, and the supernatant was discardedResuspending the cells in about 20ml of fresh CHO-WM medium, discarding the supernatant, then counting the cells by a small amount of medium, adjusting the cell density to 1.5X 10⁷cells/ml。

1.4.2 mixing of plasmid with cells

5. mu.g of pCI-DUCV-Cap-GS recombinant plasmid was taken, and added to an EP tube containing 0.7mL of cells, and after mixing well, the mixture was allowed to stand for 15 minutes.

1.4.3 electrotransfer

Shocking at 260V for 15ms for 2 times, each time at 1min, immediately transferring cells into shake flask, performing suspension culture for 48 hr, observing cell state, changing culture solution, and growing to 0.6 × 10⁶For cells/ml, 50. mu.M MSX (L-methionine sulphoxide) was added for pressure screening.

1.5 monoclonal screening

Cells were resuspended in CHO cell serum-free protein-free medium CHO-WM cells + 50. mu.M MSX and counted. The cells were diluted to 5 cells/mL, and 20mL of the cell lysate were added to a 96-well plate at 200. mu.l/well, left at 37 ℃ and 5% CO₂And incubating for 4-6h in the cell incubator. Wells of individual cells were recorded. When the wells of the individual cells in the 96-well plate were grown up, the medium was discarded, PBS was washed once, 100. mu.l of 0.25% trypsin-EDTA was digested at room temperature for about 2min, the digestion reaction was stopped by adding CHO-WM medium (containing 10% FBS + 50. mu.M MSX), and the cells were blown off with a pipette. And transferring the cells to a 12-pore plate, taking the supernatant when the 12-pore plate is full, detecting whether the clone is positive by ELISA, continuously performing amplification culture on the positive clone for efficiently expressing the DUCV-Cap, and freezing and storing.

1.6 measurement of expression amount of target protein

The concentration of the DUCV-Cap protein was determined using a Bradford protein quantitative assay kit using a microplate reader method, and the assay was performed strictly according to the instructions.

1.7 cell Shake flask fermentation

Cells were diluted to 3.0-3.5X 10 with CHO-WM medium supplemented with 50. mu.M MSX medium⁵cells/mL inoculated 30mL culture medium in a 125mL shake flask. The cell culture bottle is placed at 37 ℃ and 5% CO₂The culture was carried out overnight in a constant temperature shaker at 100 rpm/min. Cell density was counted every 24hAnd activity, measuring glucose, and adding glucose to 4g/L when the sugar is lower than 2 g/L; samples were taken at 1mL per day and the supernatant was used to detect protein expression.

Preparation and inspection of duck reovirus stock solution

2.1 preparation of Virus solution

Transferring the LMH cell suspension to a cell bottle, inoculating the virus seed into LMH cell full of monolayer at 2% of total culture medium, and placing at 37 deg.C with 5% CO₂Culturing in incubator, adsorbing for 1 hr, adding DMEM cell culture solution with serum content of 2%, and continuing to culture at 37 deg.C with 5% CO₂Culturing in an incubator. After inoculation, the cells are cultured for 40-48 hours, and when the cytopathic effect reaches more than 80%, the cells and cell cultures are harvested. After repeated freeze thawing for 2 times, TCID is used₅₀The method detects the virus titer.

2.2 measurement of Virus content

LMH cells were prepared as single cell suspensions and plated on 96-well cell culture plates at 100. mu.l/well (cell content 4.0X 105 cells/ml) at 37 ℃ with 5% CO₂Culturing in an incubator for 24 h. Serial 10-fold dilution of virus liquid in DMEM culture medium, 10 times of virus liquid^-6、10^-7、10^-8、10^-94 dilutions were added to each of the 96-well cell culture plates cultured for 24h, and 6 wells of the 96-well cell culture plates were inoculated with each dilution in an amount of 100. mu.l per well, while 6 wells of normal cells were set as negative controls. DMEM medium containing 2% newborn calf serum was added to the wells, and 100. mu.l of the DMEM medium was added to each well. Placing at 37 ℃ with 5% CO₂Culturing in incubator for 5 days, observing and recording virus infection condition in each well every day, and calculating half cell infectious Titer (TCID) of virus according to Reed-Muench method₅₀)。

2.3 inactivation and inactivation assay

2.3.1 inactivation

Slowly adding formaldehyde solution into the harvested duck reoviridae solution to make the final concentration of the solution be 0.1%, fully mixing the solution, inactivating the solution at 37 ℃ for 24 hours, and stirring the solution once every two hours. And (4) storing the inactivated virus liquid at 2-8 ℃.

2.3.2 inactivation assay

And (3) taking the inactivated virus liquid, inoculating the virus liquid to LMH cells which form a good monolayer, culturing and observing the LMH cells at 37 ℃ for 4 days, repeatedly freezing and thawing for 2 times, conducting blind passage for 2 generations, and observing the pathological change condition of the cells.

Preparation and test of 31 type and 3 type duck hepatitis virus stock solution

3.1 preparation of Duck hepatitis Virus solution for preparing vaccine

Diluting the virus seeds for producing the type 1 and type 3 duck hepatitis virus with sterilized normal saline by 1000 times respectively, inoculating 11-day-old susceptible duck embryos in allantoic cavities respectively, sealing the embryos with 0.2ml each, sealing the pores with wax, and standing and incubating at 37 ℃. Discarding the duck embryos which die before 24 hours, taking eggs every 6 hours for 1 time, collecting the dead duck embryos for 24-120 hours, and cooling at 4 ℃ for 6-12 hours. Sterilizing air chamber on egg shell surface with iodine tincture, removing egg shell in air chamber by aseptic operation, collecting embryo solution, placing in a sterilizing container, marking to obtain antigen, storing at-20 deg.C, and sampling for virus content (ELD)₅₀) And (4) measuring. The virus content should be not less than 10^5.5ELD₅₀/0.1ml。

3.2 inactivation and testing

3.2.1 inactivation

Unfreezing the qualified antigen at the temperature of 2-8 ℃, and respectively adding 10% formaldehyde solution while stirring to make the final concentration of the antigen 0.2%. Inactivating at 37 deg.C for 16h while stirring. And (4) storing the inactivated virus liquid at 2-8 ℃ with the preservation period not exceeding 30 days.

3.2.2 inactivation assay

Inoculating the inactivated virus solution to susceptible duck embryos of 10 days, culturing at 37 ℃ for 168h, recording the survival conditions of the duck embryos, harvesting allantoic fluid of the healthy duck embryos, performing blind transfer for 1 generation, and recording the survival conditions of the duck embryos.

4 preparation of inactivated vaccine

4.1 oil phase preparation

Mixing white oil with span-80 (span-80) according to a proportion of 94: 6, then adding 2 percent of aluminum stearate, stirring until the mixture is light yellow, clear and transparent, and sterilizing by high-pressure steam at 121 ℃ for later use.

4.2 preparation of the aqueous phase

Uniformly mixing duck circular ring CAP protein, duck respiratory arc virus liquid, duck hepatitis virus liquid/1 and duck hepatitis virus liquid 3 qualified by inactivation inspection according to the proportion of 1:1:1:1, and mixing the mixed liquid with sterile Tween-80 (Tween-80) according to the proportion of 97.5: shaking and mixing the mixture according to the proportion of 2.5 until the Tween-80 is completely dissolved.

4.3 emulsification

Placing 2 parts of oil phase into tissue homogenizer, stirring slowly, adding 1 part of water phase slowly, and stirring at 10000r/min for 4-5 min.

5 inspection of the finished product

5.1 sterility test according to appendix of the existing Chinese veterinary pharmacopoeia.

Third, test results

1 Duck circovirus preparation

1.1 assay of DUCV-Cap protein

And (3) drawing a standard curve according to the measured OD values of the standard substances with different concentrations, taking the OD values as vertical coordinates and the concentrations as horizontal coordinates, and calculating the concentration of the DUCV-Cap protein to be 1.2mg/ml according to a linear regression equation.

Preparation of duck reovirus stock solution

2.1 Duck reovirus content determination

Inoculating the duck reovirus seed virus to LMH cells on one side, culturing for 44 hr, harvesting, repeatedly freezing and thawing for 2 times, and determining virus content to be 10^7.5TCID₅₀/ml。

2.2 inactivation assay

The inactivated reovirus is inoculated to LMH cells and blind transferred for 2 generations, no pathological changes are generated in the cells, and the result shows that the virus liquid is completely inactivated.

Preparation of duck hepatitis virus stock solution

3.1 Duck hepatitis Virus content determination

Respectively carrying out 10-time serial dilution on the type 1 and type 3 duck hepatitis disease liquid, inoculating 5 susceptible duck embryos with each dilution being 0.2ml, culturing and observing for 168h, and recording the death condition of the duck embryos. The virus content of the virus solution is respectively 10 according to the Reed-Muench method^6.32ELD₅₀ ^/0.2ml and 10^6.22ELD₅₀ ^/0.2ml。

3.2 inactivation assay

The inactivated duck hepatitis virus is inoculated with susceptible duck embryos of 10 days old, the susceptible duck embryos are subjected to blind transmission for 1 generation, all the duck embryos are healthy, and the result shows that the virus liquid is completely inactivated.

4 inspection of finished product

4.1 sterility test

The inactivated vaccine is subjected to aseptic examination according to the appendix of the current Chinese veterinary pharmacopoeia. The result shows that the finished product is qualified by aseptic inspection.

Test example 1 safety test of triple inactivated vaccine against Duck circovirus disease, novel Duck reovirus disease, and Duck viral hepatitis

Taking 20 healthy and susceptible ducklings of 1 day old, randomly dividing the ducklings into 2 groups, wherein one group is a test group, and injecting 1.0ml of inactivated vaccine subcutaneously at the neck part of the ducklings; the other group was a control group, and the same amount of physiological saline was injected subcutaneously into the neck of the control group. After inoculation, the animals were observed every day for mental and ingestion and for adverse reactions.

And (4) testing results: the triple inactivated vaccine is used for inoculating the ducklings, the ducklings are all healthy and alive during observation, and no local and systemic adverse reactions occur, so that the triple inactivated vaccine prepared by the method has good safety.

Test example 2 Effect test of triple inactivated vaccine against Duck circovirus disease, novel Duck reovirus disease, and Duck viral hepatitis

Test method 1

1.1 Duck Ring part

30 healthy and susceptible ducklings at the age of 1 day are randomly divided into 2 groups of 10 ducklings. Group 1, 0.5ml of vaccine was subcutaneously inoculated in each neck, group 2, 10 were challenge controls, and 0.5ml of physiological saline was subcutaneously injected in each neck. On 21 days after immunization, 1.0ml of duck circovirus was injected into the leg muscle of each group of ducklings. Collecting blood from the vein of the wing 21 days after the toxin attack. Adding an anticoagulant according to the ratio of fresh blood to anticoagulant of 6:1, and detecting the copy number of DuCV nucleic acid in whole blood by using an RT-QPCR method, wherein the specific detection method comprises the following steps:

1.1.1 detection primers

The detection was performed using the specific primers of Table 2.

TABLE 2 detection primers

1.1.2 RT-QPCR reaction

The synthesis system of RT-QPCR using 2 XSSYBR GREE Mix kit and SYBR GREE I dye method is shown in Table 3.

TABLE 3 RT-QPCR Synthesis System

The RT-QPCR reaction procedure was: pre-denaturation at 95 ℃ for 60 seconds; denaturation at 95 ℃ for 15 seconds and annealing at 60 ℃ for 60 seconds for 40 cycles.

1.1.3 determination of results

If Ct is less than or equal to 26, the result is positive, and if Ct is more than 26, the result is negative. The immune group should be protected by at least 8/10, and the challenge control group should have at least 8/10 onset of disease.

1.2 Duck reointestine arc part

20 healthy and susceptible ducklings at the age of 1 day are randomly divided into 2 groups of 10 ducklings. Group 1, each neck was vaccinated subcutaneously with 0.5ml of vaccine subcutaneously, and group 2 was not immunized and served as a blank control. Serum was isolated 21 days after inoculation and the neutralizing antibody titer of duck reovirus was determined by neutralization assay. The neutralizing antibody effect of duck reovirus is not less than 1: 256.

1.3 Duck hepatitis fraction

20 healthy and susceptible ducklings at the age of 1 day are randomly divided into 2 groups of 10 ducklings. Group 1, each neck was vaccinated subcutaneously with 0.5ml of vaccine subcutaneously, and group 2 was not immunized and served as a blank control. Serum was isolated 21 days after inoculation and the neutralizing antibody titers of type 1 and type 3 duck hepatitis virus were determined using the neutralization assay. The neutralizing antibody titer of the duck hepatitis virus type 1 and 3 is not less than 1: 128.

2 results of the test

2.1 Duck Ring part

Duck circovirus attack is virulent 21 days after duckling immunization, whole blood is collected 21 days later, DuCV nucleic acid copy number in the whole blood is detected by an RT-QPCR method, duckling 10/10 in the immunization group is protected as a result, duckling 10/10 in the attack-poison control group is attacked, and the result is shown in table 4.

TABLE 4 Duck Ring immune challenge protection results

Note: "+" indicates that Ct is less than or equal to 26, and "" Ct > 26, the product is positive, and "" Ct > 26, the product is negative.

2.2 Duck reointestine arc part

Blood is collected 3 weeks after duckling immunization, and the neutralizing antibody level of duck reovirus is determined by a neutralization test method, and the result shows that the neutralizing antibody titer of the ducklings in the immune group is not lower than 1:256 in 21 days compared with the control group. The test results are shown in Table 5.

TABLE 5 measurement results of antibody titer against Duck reovirus

2.3 Duck hepatitis fraction

Blood is collected 3 weeks after duckling immunization, and the neutralizing antibody level of the duck hepatitis virus is determined by a neutralization test method, and the result shows that the neutralizing antibody titer of the ducklings in the immune group is not lower than 1:128 in 21 days compared with the control group. The test results are shown in Table 6.

TABLE 6 Duck hepatitis Virus antibody titer assay results

SEQUENCE LISTING

<110> Harbin group biological vaccine Co., Ltd

Triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and preparation method thereof

<130> HLJ-3002-201208A

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 770

<212> DNA

<213> Artifical sequence

<400> 1

ctcgaggcac gcgaattatc aaagtcaatg tcgagtgcga gttacgtctc tatgtaattc 60

tgcgcgtcca ctcctgctgc gttgtccgat tcacgaaaac tcatgcacac gctgtcatgc 120

accatttagt attgagtgcg gtcgacccgt gcgaggtaac gtctccagag gaggagggcc 180

gcgactttgt cccatcaccg gcaccatgta tgatgggatt gggccaaaaa tatctccgct 240

gtactctaga aggctctcag gtcctctgct gcttgtcgaa ccatagggat ctatcgacca 300

ccctgtggtc gcagtattta ccatatgtcc atccttgtct ataacagtcg acccatgcgt 360

attactattt tgatatatct tatgtgccgg cctaagggtg atagccacac cattaataat 420

gtagtaatca tagggataat ggtaattcgg agttgacccc gttatgtaac tgttcgtgtc 480

accaaatata gcccatcttc atttcaaacc gcatatctta ttaccgcgcg ctggtgggtc 540

tgtgtattca gcaccctcat ctaagcttaa actctgtcat ttgccagcag ctgtcggacc 600

agtctgtagt cgaaaaagtc gcctacgatg ttccttgtgg ccctacatgt cactacggaa 660

aaccgtcgtc ggggtcttgc gattcgtagc cttcgtcttc tgaatctcct gcgtagaccc 720

cgcctcttcc tccgaccacg atatgcacgc cgataggagc gacctcgcat 770