阅读说明：本技术 一种三联灭活疫苗及其制备方法 (Triple inactivated vaccine and preparation method thereof ) 是由 张贺伟 杨振 董均 杨少宗 李琛 宋敏杰 田文静 郭少阳 张淼丹 程相朝 张春杰 于 2019-09-23 设计创作，主要内容包括：本发明提供了一种三联疫苗,所述三联灭活疫苗的抗原为灭活的鸡新城疫La Sota病毒株、灭活的H9亚型禽流感QF01株和灭活的I群8b型禽腺病毒Hexon蛋白,还提供其制备方法：I群8b型禽腺病毒Hexon蛋白经二乙烯亚胺灭活后,与灭活的鸡新城疫病毒浓缩液、灭活的禽流感病毒浓缩液混合乳化后得到三联灭活疫苗。本发明的三联灭活疫苗灭活的I群8b型禽腺病毒Hexon蛋白含量高,免疫原性强,甲醛和内毒素含量低,对当前流行的I群8b型禽腺病毒具有较好的保护效果,安全性能好,免疫效果确实,可以抵抗新城疫病毒、禽流感病毒以及I群8b型禽腺病毒对临床鸡群的感染,持续期长,仅使用一次0.3mL疫苗的情况下,免疫持续期能达到五个月,至少给鸡群提供70%的保护率。(The invention provides a triple vaccine, the antigen of which is inactivated newcastle disease La Sota virus strain, inactivated H9 subtype avian influenza QF01 strain and inactivated I group 8b type avian adenovirus Hexon protein, and a preparation method thereof: the group I8 b type avian adenovirus Hexon protein is inactivated by divinylimine, and then is mixed and emulsified with an inactivated newcastle disease virus concentrated solution and an inactivated avian influenza virus concentrated solution to obtain the triple inactivated vaccine. The triple inactivated vaccine inactivated I group 8b type avian adenovirus Hexon has high protein content, strong immunogenicity, low formaldehyde and endotoxin content, good protection effect on the currently prevalent I group 8b type avian adenovirus, good safety performance and reliable immune effect, can resist the infection of newcastle disease virus, avian influenza virus and I group 8b type avian adenovirus to clinical chicken flocks, has long duration, and can provide 70 percent of protection rate for the chicken flocks at least under the condition of only using 0.3mL vaccine once, wherein the duration of immunity can reach five months.)

1. A triple inactivated vaccine is characterized in that antigens of the triple inactivated vaccine are inactivated Newcastle disease LaSota virus strain, inactivated H9 subtype avian influenza QF01 strain and inactivated I group 8b type avian adenovirus Hexon protein; the amino acid sequence of the I group 8b type avian adenovirus Hexon protein is shown as SEQ ID NO. 2; the nucleotide of the group I8 b avian adenovirus Hexon protein is shown in SEQ ID NO. 1; the content of the Newcastle disease La Sota virus strain is more than or equal to 10^8.5EID₅₀0.1 mL; the content of the H9 subtype avian influenza QF01 strain is more than or equal to 10^7.5EID₅₀0.1 mL; the agar titer of the I group 8b type avian adenovirus Hexon protein content is more than or equal to 1: 32; the H9 subtype avian influenza QF01 strain belongs to influenza virus, is preserved in China general microbiological culture Collection center in 2019, 08 and 22 months, and has the strain preservation number: CGMCC No. 18338.

2. A method of preparing the triple inactivated vaccine of claim 1, wherein the method comprises:

s1, preparation of avian adenovirus group I8 b protein Hexon:

s101, preparation of pMD-Hexon recombinant plasmid:

designing primer pairs suitable for expressing a Hexon protein nucleic acid sequence by a baculovirus expression system, and respectively naming the primer pairs as FAdV-F and FAdV-R, wherein the nucleotide sequence of the FAdV-F is shown as SEQ ID NO. 3; the nucleotide sequence of the FAdV-R is shown as SEQ ID NO. 4; taking extracted I group 8b type poultry adenovirus DNA as a template, taking FAdV-F and FAdV-R as primers, carrying out PCR amplification to obtain a Hexon target fragment, detecting through agarose gel electrophoresis, recovering the Hexon target fragment, connecting the Hexon target fragment to a pMD-19T vector by using T4DNA ligase, then transforming DH5 alpha-T1 competent cells under aseptic conditions to obtain a recombinant plasmid a, culturing the recombinant plasmid a on an LB solid culture medium containing 50 mu g/mL ampicillin for 12-14 h, selecting a single colony, carrying out colony PCR identification by using an M13-F primer and an M13-R primer, carrying out sequencing verification on a positive colony, and naming the recombinant plasmid a with correct sequencing as a pMD-Hexon recombinant plasmid; the amino acid sequence of the Hexon target fragment is shown as SEQ ID NO.2, and the nucleotide is shown as SEQ ID NO. 1; the nucleotide sequence of the M13-F primer is shown as SEQ ID NO.5, and the nucleotide sequence of the M13-R primer is shown as SEQ ID NO. 6;

s102, preparation of a transfer plasmid pFastBac I-Hexon:

carrying out double enzyme digestion on the pFastBac I plasmid and the pMD-Hexon recombinant plasmid obtained in S101 respectively by using BamH I restriction endonuclease and EcoR I restriction endonuclease to obtain a pFastBac I enzyme digestion fragment and a pMD-Hexon enzyme digestion fragment, detecting by agarose gel electrophoresis, respectively recovering a pFastBac I enzyme digestion fragment and a pMD-Hexon enzyme digestion fragment, then T4DNA ligase is used for connection for 12 to 14 hours at the temperature of 4 ℃, then DH5 alpha-T1 competent cells are transformed under the aseptic condition to obtain recombinant plasmids b, the recombinant plasmids b are cultured for 12h to 14h on LB solid culture medium containing 50 mug/mL ampicillin, single colonies are selected to be subjected to colony PCR identification by using M13-F primers and M13-R primers, positive colonies are subjected to sequencing verification, and the recombinant plasmids b with correct sequencing are named as pFastBac I-Hexon transit plasmids; the nucleotide sequence of the M13-F primer is shown as SEQ ID NO.5, and the nucleotide sequence of the M13-R primer is shown as SEQ ID NO. 6;

s103, constructing a recombinant bacmid pF-rBac-Hexon:

transferring the pFastBac I-Hexon transfer plasmid obtained in S102 into an escherichia coli DH10Bac competent cell to obtain a recombinant escherichia coli DH10Bac, culturing on an LB solid culture medium containing 50 mug/mL ampicillin for 12-14 h, selecting a positive single colony, performing colony PCR identification by using an M13-F primer and an M13-R primer, detecting the colony size to be consistent with the expected size through agarose gel electrophoresis, recovering, and naming the colony as the recombinant bacmid pF-rBac-Hexon; the nucleotide sequence of the M13-F primer is shown as SEQ ID NO.5, and the nucleotide sequence of the M13-R primer is shown as SEQ ID NO. 6;

s104, transfecting sf9 cells by using recombinant bacmid:

transfecting the recombinant bacmid pF-rBac-Hexon obtained in S103 with insect cell sf9 by using a liposome transfection method, culturing for 96-144 h at the temperature of 27 ℃, collecting cell culture, centrifuging and taking supernatant to obtain a recombinant baculovirus rHexon strain F0 generation;

s105, amplification of the recombinant baculovirus:

inoculating insect cells sf9 to the recombinant baculovirus rHexon strain F0 obtained in S104 instead of the seed insect cells sf9, culturing for 96-144 h at the temperature of 27 ℃, collecting cell cultures, centrifuging and taking supernate to obtain F1-generation recombinant baculovirus;

s106, acquisition of avian adenovirus group I8 b protein:

inoculating the F1 generation recombinant baculovirus obtained in S105 into insect cells sf9, culturing for 96-144 h at the temperature of 27 ℃, collecting cell culture, centrifuging and taking supernatant fluid to obtain I group 8b type avian adenovirus Hexon protein;

s2, preparing an oil phase:

placing white oil for injection and aluminum stearate in an oil phase preparation tank, heating to 80 deg.C, adding span 80, maintaining for 30min until the temperature reaches 116 deg.C, and naturally cooling to room temperature to obtain oil phase; the mass ratio of the white oil for injection, the aluminum stearate and the span 80 is 94:1: 6;

s3, preparing a water phase:

culturing a Newcastle disease La Sota virus strain to obtain a Newcastle disease virus liquid, culturing an H9 subtype avian influenza QF01 strain to obtain an avian influenza virus liquid, respectively filtering, concentrating and inactivating the obtained Newcastle disease virus liquid and the avian influenza virus liquid with formaldehyde to respectively obtain an inactivated Newcastle disease virus concentrated liquid and an inactivated avian influenza virus concentrated liquid, and inactivating the I group 8b type avian adenovirus Hexon protein obtained in S106 with diethyleneimine to obtain an inactivated I group 8b type avian adenovirus Hexon protein; mixing the inactivated newcastle disease virus concentrated solution, the inactivated avian influenza virus concentrated solution and the inactivated I group 8b type avian adenovirus Hexon protein in equal mass to obtain mixed antigen solution, adding Tween 80 into the mixed antigen solution, and uniformly mixing to obtain a water phase; the mass ratio of the mixed antigen liquid in the water phase to the Tween 80 is 96: 4;

s4, emulsification:

and (3) putting the oil phase obtained in the step (S2) into an emulsifying tank, adding the water phase obtained in the step (S3) while stirring at the stirring speed of 150r/min, continuing stirring for 30min after the water phase is added to obtain an emulsion, and shearing the emulsion for 2 times at the shearing speed of 4000r/min at the temperature of 20-25 ℃ to obtain the triple inactivated vaccine.

3. The method of claim 2, wherein the reaction system of the PCR amplification in S101 is: 1.0 mu L of group I8 b type avian adenovirus DNA, 2.0 mu L of FAdV-F with the concentration of 10mmol/L, 2.0 mu L of FAdV-R with the concentration of 10mmol/L, 0.5 mu L, dNTPs 5 mu L of Taq DNA polymerase with the concentration of 5U/mu L, 5 mu L of buffer solution and sterile redistilled water to make up to 50 mu L; the reaction conditions of the PCR amplification are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 7 min.

4. The method of claim 2, wherein the colony PCR identified in S101, S102 and S103 is performed in a reaction system comprising: colony DNA 1.0 μ L, M13-F0.5 μ L with concentration of 10mmol/L, M13-R0.5 μ L with concentration of 10mmol/L, 2 XTaq DNA polymerase premix 12.5 μ L, sterile redistilled water to 25 μ L; the reaction conditions of colony PCR identification are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 90s, and 30 cycles; extension at 72 ℃ for 10 min.

5. The method according to claim 2, wherein the rotation speed of the centrifugation in S104, S105 and S106 is 4000rpm to 10000rpm, and the centrifugation time is 30min to 60 min.

6. The method of claim 2, wherein the multiplicity of infection of the recombinant baculovirus rHexon strain F0 generation-inoculated insect cell sf9 in S105 is 0.1-3.0; the multiplicity of infection of the F1 generation recombinant baculovirus inoculated insect cells sf9 in S106 is 0.1-3.0.

7. The method according to claim 2, wherein the concentration of the Newcastle disease virus liquid and the avian influenza virus liquid in S3 is 3-4 times.

8. The method according to claim 2, wherein the triple inactivated vaccine in S4 is stored in a sealed manner at a temperature of 2-8 ℃.

Technical Field

The invention belongs to the technical field of vaccines, and particularly relates to a triple inactivated vaccine and a preparation method thereof.

Background

Newcastle Disease (ND) is an acute, febrile, septic and highly contagious infectious disease of birds caused by newcastle disease virus. Characterized by high fever, dyspnea, diarrhea, neurological disturbance, mucosal and serosal bleeding. Has high morbidity and mortality, and is one of the most serious virulent infectious diseases harming the poultry industry all over the world. OIE classifies the animal epidemic diseases as A-type epidemic diseases, and is one of 5 types of animal epidemic diseases which are regulated to be preferentially prevented and mainly prevented in the national medium-long animal epidemic disease prevention and treatment plan. The world animal health Organization (OIE) ranks it as a legally reported animal epidemic.

Avian Influenza (AI) is a type of infectious disease caused by Avian influenza a virus (AIV) infection of birds, widely occurring around the world, and is subject to frequent variation and is highly harmful to humans and poultry industry. The avian influenza H9 subtype number and low-pathogenicity avian influenza have higher morbidity in a farm, have less obvious morbidity symptoms, but have great influence on intestinal tracts, oviducts and the like of chickens.

Since 2013, a highly pathogenic pathogen prevails in a plurality of provinces of China, leading to death of a large number of poultry, and the main pathological change is liver bleeding. The disease is identified as I group fowl gland virus serum 8b type infection. Epidemiological investigation on the disease discovers that the disease has high morbidity in chicken flocks in China, particularly has serious disease to chicks, and mainly occurs to the chicks, and the sick chicks are blocked in growth, disordered in feather and crouched. Death or progressive recovery occurred within more than 48 h. After the chicken group is sick, the death peak of 3-5 days can be highlighted. The mortality rate is low, possibly 10%, and individually can reach 30%. Sometimes, the course of disease may last for 2-3 days. Because of the current clinical non-approved avian adenovirus vaccine, the avian adenovirus epidemic brings huge losses to the breeding industry.

Newcastle disease and H9 subtype avian influenza are 2 common important diseases seriously threatening poultry; meanwhile, the infection of avian adenovirus group I8 b to chicken flocks also causes serious loss to the livestock industry. In order to reduce the cost input and avoid the injection stress to chicken flocks, a safe, efficient and conveniently-put multi-connected vaccine is urgently needed by a farm to deal with the continuously-changing epidemic disease prevention and control situation.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a triple inactivated vaccine aiming at the defects of the prior art, the vaccine has low endotoxin content, good safety performance and real immune effect, can resist the infection of newcastle disease virus, avian influenza virus and I group 8b type avian adenovirus to clinical chicken flocks, has long duration, and under the condition that 0.3mL vaccine is used only once by using the triple inactivated vaccine, the immunity duration can reach five months, at least 70 percent of protection rate is provided for the chicken flocks, the preparation method of the vaccine uses I group 8b type avian adenovirus Hexon protein in China, and is prepared by inoculating Sf9 cells with recombinant baculovirus rHexon strain F0 expressing chicken avian adenovirus (I group 8b type) Hexon protein instead of Sf9 cells, harvesting cell culture, centrifuging and taking supernatant fluid, mixing with the inactivated newcastle disease virus concentrated solution and the inactivated avian influenza virus concentrated solution, and emulsifying to obtain the triple inactivated vaccine.

In order to solve the technical problems, the invention adopts the technical scheme that: a triple inactivated vaccine, wherein the antigen of the triple inactivated vaccine is inactivated Newcastle disease La Sota virus strain, inactivated H9 subtype avian influenza QF01 strain and inactivated I group 8b type avian adenovirus Hexon protein; the amino acid sequence of the group I8 b type avian adenovirus Hexon protein is shown as SEQ ID NO. 2; the nucleotide of the group I8 b avian adenovirus Hexon protein is shown in SEQ ID NO. 1; the content of the Newcastle disease La Sota virus strain is more than or equal to 10^8.5EID₅₀0.1 mL; the content of the H9 subtype avian influenza QF01 strain is more than or equal to 10^7.5EID₅₀0.1 mL; the agar expansion titer of the group I8 b type avian adenovirus Hexon protein content is more than or equal to 1: 32; the H9 subtype avian influenza QF01 strain belongs to influenza virus, is preserved in the common microorganism center of China general microbiological culture preservation management Committee in 2019, 08 and 22 months, and has the strain preservation number as follows: CGMCC No. 18338.

The invention also provides a method for preparing the triple inactivated vaccine, which comprises the following steps:

s1, preparation of avian adenovirus group I8 b protein Hexon:

s101, preparation of pMD-Hexon recombinant plasmid:

designing primer pairs suitable for expressing a Hexon protein nucleic acid sequence by a baculovirus expression system, and respectively naming the primer pairs as FAdV-F and FAdV-R, wherein the nucleotide sequence of the FAdV-F is shown as SEQ ID NO. 3; the nucleotide sequence of the FAdV-R is shown in SEQ ID NO. 4; taking extracted I group 8b type poultry adenovirus DNA as a template, taking FAdV-F and FAdV-R as primers, carrying out PCR amplification to obtain a Hexon target fragment, detecting through agarose gel electrophoresis, recovering the Hexon target fragment, connecting the Hexon target fragment to a pMD-19T vector by using T4DNA ligase, then transforming DH5 alpha-T1 competent cells under aseptic conditions to obtain a recombinant plasmid a, culturing the recombinant plasmid a on an LB solid culture medium containing 50 mu g/mL ampicillin for 12-14 h, selecting a single colony, carrying out colony PCR identification by using an M13-F primer and an M13-R primer, carrying out sequencing verification on a positive colony, and naming the recombinant plasmid a with correct sequencing as a pMD-Hexon recombinant plasmid; the amino acid sequence of the Hexon target fragment is shown as SEQ ID NO.2, and the nucleotide is shown as SEQ ID NO. 1; the nucleotide sequence of the M13-F primer is shown as SEQ ID NO.5, and the nucleotide sequence of the M13-R primer is shown as SEQ ID NO. 6;

s102, preparation of a transfer plasmid pFastBac I-Hexon:

carrying out double enzyme digestion on the pFastBac I plasmid and the pMD-Hexon recombinant plasmid obtained in S101 respectively by using BamH I restriction endonuclease and EcoR I restriction endonuclease to obtain a pFastBac I enzyme digestion fragment and a pMD-Hexon enzyme digestion fragment, detecting by agarose gel electrophoresis, respectively recovering a pFastBac I enzyme digestion fragment and a pMD-Hexon enzyme digestion fragment, then connecting for 12-14 h by using T4DNA ligase at the temperature of 4 ℃, then transforming DH5 alpha-T1 competent cells under the aseptic condition to obtain recombinant plasmid b, culturing the recombinant plasmid b on an LB solid culture medium containing 50 mu g/mL ampicillin for 12 h-14 h, selecting a single colony, carrying out colony PCR identification by using an M13-F primer and an M13-R primer, carrying out sequencing verification on a positive colony, and naming the recombinant plasmid b with correct sequencing as a pFastBac I-Hexon transfer plasmid; the nucleotide sequence of the M13-F primer is shown as SEQ ID NO.5, and the nucleotide sequence of the M13-R primer is shown as SEQ ID NO. 6;

s103, constructing a recombinant bacmid pF-rBac-Hexon:

transferring the pFastBac I-Hexon transfer plasmid obtained in S102 into an escherichia coli DH10Bac competent cell to obtain a recombinant escherichia coli DH10Bac, culturing on an LB solid culture medium containing 50 mug/mL ampicillin for 12-14 h, selecting a positive single colony, performing colony PCR identification by using an M13-F primer and an M13-R primer, detecting the colony size to be consistent with the expected size through agarose gel electrophoresis, recovering, and naming the colony as the recombinant bacmid pF-rBac-Hexon; the nucleotide sequence of the M13-F primer is shown as SEQ ID NO.5, and the nucleotide sequence of the M13-R primer is shown as SEQ ID NO. 6;

s104, transfecting sf9 cells by using recombinant bacmid:

transfecting the recombinant bacmid pF-rBac-Hexon obtained in S103 with insect cell sf9 by using a liposome transfection method, culturing for 96-144 h at the temperature of 27 ℃, collecting cell culture, centrifuging and taking supernatant to obtain a recombinant baculovirus rHexon strain F0 generation;

s105, amplification of the recombinant baculovirus:

inoculating insect cells sf9 to the recombinant baculovirus rHexon strain F0 obtained in S104 instead of the seed insect cells sf9, culturing for 96-144 h at the temperature of 27 ℃, collecting cell cultures, centrifuging and taking supernate to obtain F1-generation recombinant baculovirus;

s106, acquisition of avian adenovirus group I8 b protein:

inoculating the F1 generation recombinant baculovirus obtained in S105 into insect cells sf9, culturing for 96-144 h at the temperature of 27 ℃, collecting cell culture, centrifuging and taking supernatant fluid to obtain I group 8b type avian adenovirus Hexon protein;

s2, preparing an oil phase:

placing white oil for injection and aluminum stearate in an oil phase preparation tank, heating to 80 deg.C, adding span 80, maintaining for 30min until the temperature reaches 116 deg.C, and naturally cooling to room temperature to obtain oil phase; the mass ratio of the white oil for injection, the aluminum stearate and the span 80 is 94:1: 6;

s3, preparing a water phase:

culturing a Newcastle disease La Sota virus strain to obtain a Newcastle disease virus liquid, culturing H9 subtype avian influenza QF01 strain to obtain an avian influenza virus liquid, respectively filtering, concentrating and inactivating the obtained Newcastle disease virus liquid and the avian influenza virus liquid with formaldehyde to respectively obtain an inactivated Newcastle disease virus concentrated liquid and an inactivated avian influenza virus concentrated liquid, and inactivating the I group 8b type avian adenovirus Hexon protein obtained in S106 with diethyleneimine to obtain an inactivated I group 8b type avian adenovirus Hexon protein; mixing the inactivated newcastle disease virus concentrated solution, the inactivated avian influenza virus concentrated solution and the inactivated I group 8b type avian adenovirus Hexon protein in equal mass to obtain mixed antigen solution, adding Tween 80 into the mixed antigen solution, and uniformly mixing to obtain a water phase; the mass ratio of the mixed antigen liquid in the water phase to the Tween 80 is 96: 4;

s4, emulsification:

and (3) putting the oil phase obtained in the step (S2) into an emulsifying tank, adding the water phase obtained in the step (S3) while stirring at a stirring speed of 150r/min, continuing stirring for 30min after the water phase is added to obtain an emulsion, and shearing the emulsion for 2 times at a shearing speed of 4000r/min at a temperature of 20-25 ℃ to obtain the triple inactivated vaccine.

Preferably, the reaction system of the PCR amplification in S101 is: 1.0 mu L of group I8 b type avian adenovirus DNA, 2.0 mu L of FAdV-F with the concentration of 10mmol/L, 2.0 mu L of FAdV-R with the concentration of 10mmol/L, 0.5 mu L, dNTPs 5 mu L of Taq DNA polymerase with the concentration of 5U/mu L, 5 mu L of buffer solution and sterile redistilled water to make up to 50 mu L; the reaction conditions of the PCR amplification are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1min, 35 cycles; extension at 72 ℃ for 7 min.

Preferably, the colony PCR identified reaction systems in S101, S102 and S103 are all: colony DNA 1.0 μ L, M13-F0.5 μ L with concentration of 10mmol/L, M13-R0.5 μ L with concentration of 10mmol/L, 2 XTaq DNA polymerase premix 12.5 μ L, sterile redistilled water to 25 μ L; the reaction conditions of colony PCR identification are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 90s, and 30 cycles; extension at 72 ℃ for 10 min.

Preferably, the rotation speed of the centrifugation in S104, S105 and S106 is 4000 rpm-10000 rpm, and the centrifugation time is 30 min-60 min.

Preferably, the infection complex number of the recombinant baculovirus rHexon strain F0 generation inoculation insect cell sf9 in S105 is 0.1-3.0; in S106, the multiplicity of infection of the F1 generation recombinant baculovirus inoculated insect cell sf9 is 0.1-3.0.

Preferably, the concentration times of the newcastle disease virus liquid and the bird flu virus liquid in S3 are both 3-4 times.

Preferably, the triple inactivated vaccine in S4 is stored in a sealed manner at a temperature of 2-8 ℃.

Compared with the prior art, the invention has the following advantages:

1. at present, most of the I group 8b avian adenoviruses are cultured mainly by avian embryo inoculation and cell culture, and the culture process is relatively complex. The I group 8b type avian adenovirus Hexon protein used by the invention is prepared by inoculating Sf9 cells with recombinant baculovirus rHexon strain F0 expressing chicken adenovirus (I group 8b type) Hexon protein, harvesting cell culture, centrifuging and taking supernatant, in the process of preparing triple inactivated vaccine, obtaining inactivated I group 8b type avian adenovirus Hexon protein after inactivation by binary ethyleneimine as antigen in the triple inactivated vaccine, mixing with inactivated newcastle disease virus concentrated solution and inactivated avian influenza virus concentrated solution to obtain mixed antigen solution, emulsifying to obtain triple inactivated vaccine, the inactivated I group 8b type avian adenovirus Hexon protein has high content, strong immunogenicity, low formaldehyde content and endotoxin content, has good protection effect on the currently popular I group 8b type avian adenovirus, and the prepared triple inactivated vaccine has low formaldehyde content and endotoxin content, the invention combines the traditional vaccine production process and genetic engineering, and lays a foundation for the improvement of the vaccine process and the guarantee of the quality.

2. The triple inactivated vaccine prepared by the invention has good safety performance, no local and systemic adverse reaction caused by the vaccine occurs after the experiment of immunization of chickens, the analysis of a potency test and an immunity duration test shows that the immune effect is reliable, the triple inactivated vaccine can resist the infection of newcastle disease virus, avian influenza virus and I group 8b type avian adenovirus to clinical chickens, the duration is long, the immunity duration can reach five months under the condition that the triple inactivated vaccine is used for only one time of 0.3mL, and at least 70 percent of protection rate is provided for the chickens, and the triple inactivated vaccine can be used as an excellent choice for preventing the newcastle disease virus, the H9 subtype avian influenza virus and the I group 8b type avian adenovirus.

The present invention will be described in further detail with reference to examples.

Detailed Description