CN116875563B - Avian adenovirus, strain, fiber-2 protein, vaccine, subunit vaccine and application - Google Patents
Avian adenovirus, strain, fiber-2 protein, vaccine, subunit vaccine and application Download PDFInfo
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- CN116875563B CN116875563B CN202311137654.4A CN202311137654A CN116875563B CN 116875563 B CN116875563 B CN 116875563B CN 202311137654 A CN202311137654 A CN 202311137654A CN 116875563 B CN116875563 B CN 116875563B
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Abstract
The invention belongs to the field of biotechnology, and discloses an avian adenovirus, which is classified as group I4 avian adenovirus FAdV-YSHNAvian adenovirus FAdV‑YSHN) The method comprises the steps of carrying out a first treatment on the surface of the The preservation number is: CCTCC NO: V202302; preservation date: 2023, 5, 23, deposit unit: china center for type culture Collection. The virus is very immunogenic when used as a vaccine. Also discloses a strain, fiber-2 protein, vaccine, subunit vaccine and application. It is particularly emphasized that the vaccine of the invention adopts the combination of the avian adenovirus and Fiber-2 protein, and the inactivated vaccine can provide effective immune protection to the attack of the avian adenovirus (I group 4 type), and has excellent immune effect and longer immune duration.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to an avian adenovirus, a strain, fiber-2 protein, a vaccine, a subunit vaccine and application.
Background
Group I fowl adenovirus has many serotypes, the main epidemic serotypes in China are C4, D11, E8a and E8b, the harm of C4 is the most serious, the fowl has strong pathogenicity, and the fowl can be caused to have strong infection pericardial effusion symptoms and inclusion body hepatitis, and the death rate is 20-80 percent. Thus, there is an urgent need to develop an avian adenovirus disease (group i type 4) vaccine.
WO2018176837A1 discloses a vaccine composition comprising an immunizing amount of the Fiber protein of avian egg drop syndrome virus or an immunizing amount of a live vector recombinant with the Fiber protein gene of avian egg drop syndrome virus, and a veterinarily acceptable carrier.
The description is as follows: a vaccine composition against avian egg drop syndrome, wherein the vaccine composition comprises an immunizing amount of avian egg drop syndrome virus antigen and a veterinarily acceptable carrier; the avian egg drop syndrome virus antigen is an avian egg drop syndrome virus Fiber protein antigen or a living carrier recombined with the avian egg drop syndrome virus Fiber protein gene. The vaccine composition further comprises one or more of the following antigens: newcastle disease virus antigen, avian influenza virus antigen, avian infectious bronchitis virus antigen, avian infectious bursal disease virus antigen, avian adenovirus antigen, avian reovirus antigen, escherichia coli antigen, parachicken bacillus antigen, mycoplasma synoviae antigen, mycoplasma gallisepticum antigen, pasteurella multocida antigen, marek's virus antigen, avian encephalomyelitis virus antigen, avian infectious laryngotracheitis virus antigen.
The protocol is described in Table 6, wherein the fiber protein subunit vaccine is against AV127 virus (avian adenovirus group III, avian egg drop syndrome virus);
tables 16-20 of this protocol describe that the Fiber protein, when combined with other viruses (e.g., newcastle disease virus-inactivating antigen, avian influenza virus-inactivating antigen, avian infectious bronchitis virus-inactivating antigen, fiber-2 protein), exhibited 100% protection against the corresponding viruses (newcastle disease virus, avian influenza virus, avian infectious bronchitis virus, avian adenovirus).
It should be noted that although 100% protection of the avian adenovirus was achieved, the Fiber-2 protein of the proposal is derived from the avian adenovirus Fiber-2 protein described in CN 108126191B;
in CN108126191B, the fiber-2 protein had 100% protective effect against FAV-HN strain virus (Table 5) as well as against atypical serotype (serotypes 2, 3, 5, 8, 11) viruses.
It can be seen that the above protocol is capable of 100% protection against viruses because of the very strong immunological effect of the fiber-2 protein used.
However, as a pain point in the art: the persistence of immunity has been a problem with headache.
Over time, e.g., after 4 months, many vaccines or subunit vaccines exhibit increased detoxification rates and decreased protective power; this deterioration will be more pronounced after 8 months.
The technical problem to be solved by the scheme is as follows: how to improve the long-term immunity efficacy of the vaccine.
Disclosure of Invention
The aim of the invention is to provide an avian adenovirus which has very strong immunogenicity when used as a vaccine. Also discloses a strain, fiber-2 protein, vaccine, subunit vaccine and application.
It is particularly emphasized that the vaccine of the invention adopts the combination of the avian adenovirus and Fiber-2 protein, and the inactivated vaccine can provide effective immune protection to the attack of the avian adenovirus (I group 4 type), and has excellent immune effect and longer immune duration.
In order to achieve the above purpose, the present invention provides the following technical solutions: a strain of avian adenovirus is classified as group I4 avian adenovirus FAdV-YSHN @Avian adenovirus FAdV-YSHN); the preservation number is: CCTCC NO: V202302; preservation date: 2023, 5, 23, deposit unit: china center for type culture collection, with the preservation addresses: university of martial arts in chinese.
Meanwhile, the invention also discloses a recombinant escherichia coli BL21-Fiber-2 strain, which is classified as escherichia coli BL 21-Fiber-2%Escherichia coli BL 21-Fiber-2); the preservation number is: cctccc No. M2023813; preservation date: 2023, 5, 24, deposit unit: china center for type culture collection, with the preservation addresses: university of martial arts in chinese.
Meanwhile, the invention also discloses a Fiber-2 protein which is obtained by adopting the strain expression.
Meanwhile, the invention also discloses a Fiber-2 protein, and the amino acid sequence of the Fiber-2 protein is shown as SEQ ID No. 1.
Meanwhile, the invention also discloses a culture method of the avian adenovirus, which is obtained by inoculating the avian adenovirus with LMH cells which are subjected to suspension culture in the logarithmic growth phase.
Meanwhile, the invention also discloses a vaccine which is characterized by comprising inactivated avian adenovirus as described above.
Meanwhile, the invention also discloses a subunit vaccine which contains the Fiber-2 protein.
Meanwhile, the invention also discloses a vaccine which contains inactivated avian adenovirus as described above and Fiber-2 protein as described above.
The vaccine contains inactivated virus liquid and Fiber-2 protein liquid; the volume ratio of the inactivated virus liquid to the Fiber-2 protein liquid is 1:1; the concentration of the inactivated avian adenovirus in the inactivated virus liquid is 2 multiplied by 10 8.0 TCID 50 0.1ml of the Fiber-2 protein is diluted according to the ratio of 1:16 to obtain the Fiber-2 protein solution.
Meanwhile, the invention also discloses the application of the avian adenovirus in vaccine manufacture.
Meanwhile, the invention also discloses the application of the Fiber-2 protein in vaccine manufacture.
Compared with the prior art, the invention has the beneficial effects that:
the inactivated avian adenovirus (I group 4) YSHN strain can provide protection not less than 90% within 2 months after immunization;
the Fiber-2 protein can provide protection force not lower than 90% within 2 months after immunization;
particularly excellent and unexpected is the ability to provide 100% protection for not less than 8 months when the inactivated avian adenovirus (group I type 4) YSHN strain of the invention, fiber-2 protein, is used as a vaccine.
Drawings
FIG. 1 is an electrophoresis chart of the amplification products of the Hexon gene of different avian adenovirus isolates (YSHN strain, YSC4 strain and YSSD strain) according to the example 1 of the present invention; m is a DNA marker;1 is an ADV negative control; 2 to 4 are ADV products of YSHN strain, YSC4 strain and YSSD strain respectively; 5 is H3H4 negative control; 6 to 8 are H3H4 products of YSHN strain, YSC4 strain and YSSD strain respectively; 9 is H1H2 negative control; 10 to 12 are H1H2 products of YSHN strain, YSC4 strain and YSSD strain respectively;
FIG. 2 is a genetic evolutionary tree of different avian adenoviruses (group I, type 4) virus isolates (YSHN, YSC4 and YSSD strains) according to example 1 of the present invention;
FIG. 3 is a photograph showing the construction of an avian adenovirus (group I, type 4) YSHN strain of example 1 according to the present invention cultured with adherent LMH cells for 56h to receive toxic lesion cells and normal cells;
the left panel is a photograph of normal cell culture for 72 h; the right panel is a photograph of 72h after inoculation of LMH cells with virus (YSHN strain);
FIG. 4 is an electrophoresis chart of amplification products of the Fiber-2 gene amplified by the bacterial liquid of the invention, M is DNA Marker DL2000; 1. is a negative control with double distilled water as a template; 2 is YSHN strain-Fiber-2 gene amplification product; 3 is a YSC4 strain Fiber-2 gene amplification product; 4 is a YSSD strain Fiber-2 gene amplification product;
FIG. 5 is a diagram of PCR amplification products of Fiber-2 gene of recombinant E.coli BL21-Fiber-2 strain constructed by different isolates (YSHN strain, YSC4 strain and YSSD strain) in example 3 of the present invention; m, protein markers; 1. vector control; 2. the recombinant plasmid does not induce bacteria; 3. recombinant plasmid-induced strain (YSHN strain); 4. recombinant plasmid-induced strain (YSC 4 strain) 5. Recombinant plasmid-induced strain (YSSD strain);
FIGS. 6A-6C are SDS-PAGE patterns of Fiber-2 proteins of recombinant E.coli BL21-Fiber-2 constructed from different isolates (YSHN strain, YSC4 strain and YSSD strain) of example 3 of the present invention;
wherein, FIG. 6A is a Fiber-2 protein SDS-PAGE diagram of recombinant escherichia coli BL21-Fiber-2 constructed by YSHN strain;
FIG. 6B is a SDS-PAGE diagram of Fiber-2 protein of recombinant E.coli BL21-Fiber-2 constructed by YSC4 strain;
FIG. 6C is a SDS-PAGE diagram of Fiber-2 protein of recombinant E.coli BL21-Fiber-2 constructed by YSSD strain;
m is a protein Marker; FIG. 6A-6C are each shown at 1 for YSHN-Fiber-2, YSC4-Fiber-2 and YSDS-Fiber-2, respectively.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 isolation and identification of epidemic strains of avian adenovirus (group 4)
In autumn and winter of 2013-2019, chickens suffering from three provinces of Guangdong, henan and Shandong develop mental depression, rough feathers, pale skin, meat crowns and faces and poor growth. The yellow hydrops appear in the pericardium of the sick chicken, the liver is swelled and turned yellow, and bleeding spots or kidney are swelled occasionally, the sample is collected, virus separation and DNA extraction are carried out, and the detection is positive to the group I avian adenovirus.
Group I avian adenoviruses are classified into 12 serotypes. In recent years, with the development of molecular biology technology, PCR-combined nucleotide sequence determination and alignment have been applied to the detection and typing of group I avian adenoviruses, and by sequencing and genetic evolution alignment of Hexon (Hexon) genes, different serotypes of group I avian adenoviruses can be accurately distinguished. DNA from different isolates was used to amplify the Hexon gene. 3 pairs of specific primers were designed and synthesized according to the nucleotide sequence of FAdV-I hexon gene registered in GenBank, as shown in Table 1:
TABLE 1 Synthesis of primer sequences
Sequencing result analysis shows that according to Hexon genetic evolution analysis and sequence comparison results, 3 isolated strains and avian adenovirus (I group 4) strains belong to the same branch, and the similarity of the gene sequence and the I group 4 is highest and reaches 99%. The 3 isolated strains are all avian adenovirus I group-genotype C-serotype 4;
culturing 3 isolated strains by using LMH adherent cells, wherein purity test of the obtained virus liquid meets the regulations; animal regression test results show that the 3 isolated strains have stronger toxicity. The inspection of the group I avian adenovirus is carried out according to three annexes of China animal pharmacopoeia of 2020 edition, and the result shows that 3 isolated strains are all the group I avian adenovirus; an identification test was performed with an avian adenovirus positive serum (FAdV positive serum type 4) purchased from the chinese veterinary microbiological bacterial deposit center. The results showed that all 3 isolates were positive for avian adenovirus (type 4 FAdV).
The test results show that the separated strains are identified by a molecular biological method, a serological method, a virus regression test and the like, and 3 separated strains are determined to be avian adenovirus I group-genotype C-serotype 4, and are respectively named FAdV/Chicken/HeNan/YSHN/2013 strain (YSHN strain for short), FAdV/Chicken/Guangdong/YSC4/2016 strain (YSC 4 strain for short) and FAdV/Chicken/Shandong/YSSD/2019 strain (YSSD strain for short).
Example 23 comparative immunogenicity test of isolates
(1) Preparation of full Virus single seedlings of FAdV YSHN Strain, YSC4 Strain and YSSD Strain
Antigen preparation: taking 3 bottles of cell culture bottles which grow into good LMH cell monolayers, discarding growth solution, washing 3 times with DMEM culture medium, respectively adding clinical samples, receiving the clinical samples according to 2 percent, adsorbing for 60 minutes at 37 ℃, adding 5 milliliters of cell maintenance solution into each bottle, and culturing at 37 ℃. Observing cytopathic effect twice daily, harvesting cell maintenance solution when cytopathic effect exceeds 80%, and determining TCID 50 . The virus content of the obtained fowl adenovirus (I group 4) YSHN strain virus liquid is 10 7.18 TCID 50 0.1ml; the virus content of the virus liquid of the avian adenovirus (I group 4) YSC4 strain is 10 6.68 TCID 50 0.1ml; the virus content of the virus liquid of the avian adenovirus (I group 4) YSSD strain is 10 7.0 TCID 50 /0.1ml。
Antigen inactivation: and (3) respectively adding formaldehyde solution with the final concentration of 0.1% into the 3 virus solutions, inactivating the virus solutions for 16 hours at 37 ℃, and carrying out inactivation test on the inactivated antigens.
Preparing an aqueous phase: taking inactivated fowl adenovirus (group I4) YSHN strain, YSC4 strain and YSSD strain antigen respectively, diluting with sterilized injectable water until the virus content is 10 6.50 TCID 50 0.1ml of Tween-80 was added, and the ratio of antigen to Tween-80 was 93:7 by volume, to allow Tween-80 to be dissolved sufficiently.
Preparing an oil phase: the oil phase is the mixture of injection white oil and span-80 according to the volume ratio of 95:5, and is sterilized for standby.
Preparation of an inactivated vaccine: mixing the water phase and the oil phase according to the volume ratio of 1:2, emulsifying, sub-packaging, labeling and preserving at 2-8 ℃ for standby.
The prepared vaccine is taken for carrying out the property and sterility test, and the results meet the requirements of three appendices of the current Chinese animal pharmacopoeia.
(2) Efficacy comparison of full Virus single seedlings of FAdV YSHN, YSC4 and YSSD strains
120 SPF chickens with 21-28 days of age are immunized by adopting an immune toxicity attack method, 30 SPF chickens are immunized with 3 groups of whole virus single seedlings of different isolates respectively, 0.3ml of vaccine is injected into each neck subcutaneously or intramuscularly, and 30 SPF chickens are immunized with the same group of vaccine, and the immunization groups are shown in Table 2. 21 days after immunization, all immunized chickens and control chickens, 10 in each group, each neck was subcutaneously injected with avian adenovirus (group I type 4) YSHN strain, YSC4 strain and YSSD strain cell lines at 0.2 ml/each (containing 2X 10) 6.50 TCID 50 ). Continuously observing for 14 days after the challenge, observing the morbidity and mortality of immunized chickens and control chickens, collecting oropharynx and cloacal cotton swabs of the chickens and the dead chickens which survive for 14 days after the challenge, inoculating LMH adherent cells after the cotton swabs are treated, carrying out virus separation, and carrying out blind transmission on samples which are negative to the virus separation.
Table 2 vaccine composition immune grouping situation
The test results showed that chickens immunized with single vaccine of avian adenosis (group I type 4) YSHN strain were protected from 21d with YSHN strain, YSC4 strain and YSSD strain at 0.2 ml/dose (containing 2X 10) 6.50 TCID 50 ) The dosage of the medicine is subjected to toxin expelling, the protection rate after toxin expelling is 10/10, and the toxin expelling rate is 1/10-2/10; immunizing chickens by using single seedlings of the YSC4 strain with the avian adenovirus disease (I group 4 type), wherein the later 21d is avoided, the protection rate after the virus attack is 10/10, the toxin expelling rate is 2/10, the virus attack is carried out by using the YSHN strain and the YSSD strain which are heterologous strains, the protection rate after the virus attack is 7/10 and 8/10 respectively, and the toxin expelling rate is 4/10 and 3/10; single vaccine immunization of chickens with avian adenovirus disease (group 4 type I) YSSD strain was performed for 21d after the immunization, the homologous strain YSSD strain was used for virus challenge, and the protection rate after virus challenge was 9The toxin expelling rate is 2/10, the heterologous strains YSHN and YSC4 are used for counteracting toxin, the protection rate after counteracting toxin is 8/10 and 9/10 respectively, and the toxin expelling rate is 4/10 and 3/10; the control chickens were challenged with YSHN strain, YSC4 strain and YSSD strain, the protection rate after the challenged was 0/10, 1/10 and 2/10, and the toxin expelling rate was 10/10, and the results are shown in Table 3. The test result shows that YSHN strains in the three isolated strains have the strongest toxicity; the vaccine prepared from the avian adenovirus disease (I group 4) YSHN strain can protect 100% of the vaccine by using homologous or heterologous avian adenovirus disease (I group 4) to attack the virus after immunization, has the best effect of inhibiting and expelling the virus, shows that the avian adenovirus disease (I group 4) YSHN strain has better immunogenicity and universality, and can be used as a vaccine candidate strain.
TABLE 3 post vaccine challenge protection results
Note that: 1. and judging the healthy survival as protection, wherein the protection rate is the survival rate. 2. The dead chicken is collected in time and the cotton swab of the throat and the cloaca is used for virus separation.
EXAMPLE 3 construction and identification of recombinant E.coli BL21-Fiber-2 Strain
According to the Fiber-2 gene sequence published by GenBank, designing a specific primer, extracting the virus nucleic acid of the avian adenovirus (I group 4 type) YSHN strain, amplifying the gene by using a PCR technology and identifying, subcloning the Fiber-2 gene into a prokaryotic expression vector pET-28a (+) and transforming a positive plasmid into competent cells BL21 (DE 3) for induction expression by IPTG, and carrying out SDS-PAGE and Western blot analysis on the expressed product.
Primer sequence:
Fiber-2F:5'-TATCATATGCTCCGCGCCCCTAGACGC-3' (Nde I cleavage site, SEQ ID No. 8)
Fiber-2R: 5'-TATCTCGAGCGCGAGGGAGGCAGCT-3' (Xho I cleavage site, SEQ ID No. 9)
The fragment size was about 1446bp.
PCR amplification of the target gene: DNA of YSHN-4 strain FF4 generation virus was extracted according to the operating instructions of DNeasy Blood & Tissue kit of QIAGEN company, PCR amplification was performed with the primers Fiber-2F and Fiber-2R, and a 50 mu L reaction system was prepared according to TaKaRa PrimeSTAR HS DNA polymerase specification, and the reaction conditions were: 94℃for 3min,94℃for 30s,57℃for 30s,72℃for 1min for 30s, go to 2, 35 cycles, 72℃for 10min. After the reaction, the PCR product was purified and recovered by 1% agarose gel electrophoresis.
Cloning and identification of the fragment of interest: adding an A tail into the 3' -end of the purified and recovered PCR product according to the specification of TaKaRa DNA A-Tailing Kit, connecting the purified and recovered PCR product with a pMD19-T (Simple) vector at 4 ℃ overnight, transforming the purified and recovered PCR product into DH5 alpha competent cells, culturing the obtained product at 37 ℃, picking single colony, extracting plasmid, identifying plasmid containing target fragment through PCR and enzyme digestion, sequencing the screened positive clone plasmid by Jin Weizhi biotechnology Co., ltd, and designating the positive clone as pMD-Fiber-2.
Construction and identification of recombinant expression plasmids: the pMD-Fiber-2 is digested with restriction enzymes Nde I and Xho I, the purified and recovered target gene is connected with pET-28a (+) vector digested with the same restriction enzymes at 4 ℃ overnight, transformed into DH5 alpha competent cells, single colony is selected for culture, plasmid is extracted, positive bacteria containing the target gene are screened through PCR and digestion identification, and the positive bacteria are sent to the golden-intelligence biotechnology Co for sequencing to ensure that the reading frame sequence is correct, and the obtained recombinant plasmid is named pET-Fiber-2.
Construction and identification of recombinant E.coli BL21 (DE 3) -Fiber-2: and (3) constructing a correct plasmid through sequencing inspection, and transforming the plasmid into competent BL21 (DE 3) escherichia coli to construct BL21 (DE 3) -Fiber-2 expression engineering bacteria. Single colonies were picked from LB (containing 50. Mu.g/ml kanamycin) plates, liquid amplified culture was performed to extract plasmids for restriction identification, and sequencing analysis was performed on plasmids with correct restriction identification.
The test result shows that the target gene is correctly inserted into the expression vector, the Fiber-2 protein expressed by the expression strain BL21-Fiber-2 strain is about 59kDa, and the Western blot result shows that the expressed protein has good immunoreactivity with positive serum of the avian adenovirus (I group 4 type). The construction and identification of the E.coli BL21-YSC4-Fiber-2 strain and BL21-YSSD-Fiber-2 strain were completed as described above.
EXAMPLE 4 expression of avian adenovirus (group I, type 4) Fiber-2 protein
Inoculating a sterilized fermentation medium containing 50 mug/ml kanamycin into the cultured recombinant escherichia coli BL21-Fiber-2 bacterial liquid at a ratio of 1.5%, fermenting and culturing at 37 ℃, adding IPTG with a final concentration of 0.2mmol/L to induce expression of avian adenovirus (group I4 type) Fiber-2 protein when the OD600nm of the bacterial liquid is 0.6-0.8, and culturing at 37 ℃ for 5 hours to obtain the bacterial liquid. The procedure was repeated three times to induce expression of three batches of Fiber-2 protein. The expression of YSC4-Fiber-2 protein and YSSD-Fiber-2 protein was induced as described above. The sequence of YSC4-Fiber-2 protein is shown as SEQ ID No. 10; the sequence of YSSD-Fiber-2 protein is shown as SEQ ID No. 11.
EXAMPLE 5 purification of avian adenovirus (group I type 4) Fiber-2 protein
The bacterial liquid induced to express the avian adenovirus (group 4 type I) Fiber-2 protein in example 4 is centrifuged, bacterial precipitate is harvested, bacterial cells are resuspended in 10ml physiological saline per 1g of wet bacteria, the bacterial cells are crushed by a high-pressure homogenizer at 2-8 ℃ for 4 times, and after crushing, the precipitate is removed by centrifugation, and the supernatant is collected.
106g of ammonium sulfate is added into each 1000ml of the crushed bacterial liquid supernatant, the ammonium sulfate is fully dissolved and then is placed at the temperature of 2-8 ℃ for precipitation for more than 12 hours, the low-temperature centrifugation is carried out, and the supernatant is collected after the centrifugation. Adding 143g ammonium sulfate into each 1000ml of the collected supernatant, fully dissolving, precipitating for more than 12 hours at the temperature of 2-8 ℃, centrifuging again, collecting the precipitate after centrifuging, and re-dissolving the obtained protein with physiological saline at the ratio of 1:8. The treatment was repeated 2 times.
Sampling the collected precipitated protein, re-suspending with physiological saline solution in the ratio of 1:8, adding TritonX-114 into the re-dissolved protein solution in the ratio of 1% by volume, and shaking the protein solution at the temperature of 2-8 ℃ overnight. Taking out, heating to 28 ℃, centrifuging, and collecting supernatant. The treatment was repeated 4 times. And measuring the endotoxin content of the protein antigen and the protein agar-agar titer. 1 batch of YSC4-Fiber-2 protein and YSDS-Fiber-2 protein were purified separately as described above. The test results show that the endotoxin content of the purified Fiber-2 protein is lower than 5000EU/ml, and the agar titer is 1:128, as shown in Table 4:
TABLE 4 endotoxin content and protein agar-spread potency determination of purified protein antigen
| Protein name | Endotoxin content (EU/ml) | Protein agar spread potency |
| YSHN-Fiber-2-001 | <5000 | 1:128 |
| YSHN-Fiber-2-002 | <5000 | 1:128 |
| YSHN-Fiber-2-003 | <5000 | 1:128 |
| YSC4-Fiber-2-001 | <5000 | 1:128 |
| YSSD-Fiber-2-001 | <5000 | 1:128 |
Example 6 safety and efficacy comparison tests of avian adenovirus (group 4) YSHN Strain whole Virus antigen and Fiber-2 protein compositions expressing different isolates
The vaccine was prepared by taking the whole virus antigen of YSHN strain prepared in example 2 and the avian adenovirus (group i 4) Fiber-2 protein of the different isolates prepared in example 5.
Antigen inactivation: inactivation of the whole virus liquid: adding formaldehyde solution with the final concentration of 0.1% respectively, and inactivating for 16h at 37 ℃; inactivation of Fiber-2 protein: formaldehyde solution with the final concentration of 0.1% is added respectively, and the mixture is inactivated for 48 hours at the temperature of 2-8 ℃. And (3) carrying out inactivation test on the inactivated antigen, wherein the result shows that the antigen is completely inactivated.
Preparing an aqueous phase: preparing mixed antigen according to the proportion shown in Table 5, adding Tween-80 after uniformly mixing the antigen, wherein the volume ratio of the antigen to the Tween-80 is 93:7, and fully dissolving the Tween-80.
Preparing an oil phase: the oil phase is the mixture of injection white oil and span-80 according to the volume ratio of 95:5, and is sterilized for standby.
Preparation of an inactivated vaccine: mixing the water phase and the oil phase according to the volume ratio of 1:2, packaging and labeling after emulsification, and preserving at 2-8 ℃ for standby, wherein the proportion of the vaccine is shown in table 5. The prepared vaccine is taken for carrying out the property and sterility test, and the results meet the requirements of three appendices of the current Chinese animal pharmacopoeia. The endotoxin content of the vaccine composition was determined by gel method.
Table 5 vaccine preparation ratio of various antigens and antigen titers
The composition was subjected to safety evaluation by SPF chicken
Vaccine compositions 1 to 4 were immunized with 7 to 14 day-old SPF chickens, 10 animals/group, chest intramuscular, 1 ml/animal, and observed for 14 days. The test results show that no local reaction or systemic adverse reaction caused by vaccine appears in the chickens immunized by the compositions 1-3, and slight local inflammatory reaction appears in the chickens immunized by the composition 4. Tests show that the low endotoxin vaccine composition has higher safety than the high endotoxin vaccine composition and less side effects.
The above compositions were evaluated for efficacy using SPF chickens. By usingThe immunization and toxicity counteracting method includes immunizing 50 SPF chickens of 21-28 days old with 10 SPF chickens each, immunizing 1-4 with the composition, and injecting vaccine 0.3ml subcutaneously or intramuscularly to each neck, and setting 10 control groups for the immunization group as shown in Table 6. 21 days after immunization, all immunized chickens and control chickens, each neck was subcutaneously injected with avian adenovirus (group I type 4) YSHN strain, 0.2 ml/dose (containing 2X 10) 6.50 TCID 50 ). Continuously observing for 14 days after the challenge, observing the morbidity and mortality of immunized chickens and control chickens, collecting oropharynx and cloacal cotton swabs of the chickens and the dead chickens which survive for 14 days after the challenge, inoculating LMH adherent cells after the cotton swabs are treated, carrying out virus separation, and carrying out blind transmission on samples which are negative to the virus separation.
Table 6 vaccine composition immune grouping situation
The test results showed that the chickens immunized with composition 1 were 21d post-immunization with YSHN strain, 0.2 ml/3 (containing 2X 10) 6.50 TCID 50 ) The dosage of the medicine is subjected to toxin expelling, the protection rate after toxin expelling is 10/10, and the toxin expelling rate is 0/10; after the vaccine composition 2 is free, the protection rate after toxin attack is 10/10, and the toxin expelling rate is 2/10; after the vaccine composition 3 is free, the protection rate after toxin attack is 10/10, and the toxin expelling rate is 1/10; the vaccine composition 4 was protected against challenge at a protection rate of 10/10 and a detoxification rate of 2/10, and the results are shown in Table 7. Test results show that compared with the composition of YSHN strain whole virus antigen and YSC4-Fiber-2 protein or YSSD-Fiber-2 protein, the chicken flock can be completely protected after being immunized by the composition of YSHN strain whole virus antigen and YSHN-Fiber-2, and toxin expelling can be inhibited by 100%.
TABLE 7 post vaccine challenge protection results
Note that: 1. and judging the healthy survival as protection, wherein the protection rate is the survival rate. 2. The dead chicken is collected in time and the cotton swab of the throat and the cloaca is used for virus separation.
The results of safety and efficacy comparison tests of avian adenovirus (I group 4) YSHN strain whole virus antigen and Fiber-2 protein composition expressing different isolates show that the prepared oil emulsion vaccine has good safety, small side reaction, can completely protect chicken groups after immunization, and can inhibit toxin expelling by 100 percent.
Example 7 optimal cell Density for avian adenovirus (group I type 4) YSHN strains inoculated with virus
Taking LMH cells in logarithmic growth phase suspension culture, respectively diluting to 4.0X10 6 cells/ml、5.0×10 6 cells/ml、6.0×10 6 cells/ml、7.0×10 6 Inoculating fowl adenovirus (group I4) YSHN strain at 0.1% ratio, culturing for 72 hr, harvesting virus liquid, repeating culturing for three times, and measuring virus content. The test results showed that the virus-inoculated cell density was 5.0X10 6 ～6.0×10 6 The virus content in cells/ml is not less than 10 8.50 TCID 50 0.1ml, and the results are shown in Table 8. The test results showed that the cell density was 5.0X10 6 cells/ml is the optimal cell density for virus inoculation, when the virus solution is the highest in virus content.
TABLE 8 determination of the Virus content at different cell densities
Example 8 optimal ratio of avian adenovirus (group I type 4) YSHN strains to be inoculated
Taking LMH cells in logarithmic growth phase suspension culture, and adjusting cell density to 5.0X10 6 Inoculating fowl adenovirus (group I4) YSHN strain at ratio of 0.05%, 0.1%, 0.15%, 0.20%, and 0.3%, culturing for 72 hr, collecting virus solution, repeating culturing for three times, and determining virus content. Test results show that the inoculation proportion is 0.05-0.2%, and the virus content is not lower than 10 8.50 TCID 50 0.1ml, as shown in Table 9. The experimental result shows that the inoculation proportion is 0.1% of the optimal proportion of virus inoculation, at this timeThe virus content of the virus liquid is highest.
TABLE 9 results of virus content determination for different inoculation ratios of viruses
Example 9 optimal time to harvest of avian adenovirus (group I, type 4) YSHN strain virus
Taking LMH cells in logarithmic growth phase suspension culture, and adjusting cell density to 5.0X10 6 cells/ml, avian adenovirus (group I type 4) YSHN strain was inoculated at a ratio of 0.1%, samples were taken 48h, 60h, 72h, 84h and 96h after inoculation, and the culture was repeated 3 times to determine the virus content. Test results show that the virus content is not lower than 10 after the virus inoculation culture for 60 to 72 hours 8.50 TCID 50 0.1ml, and the results are shown in Table 10. The test result shows that the culture time is the optimal harvest time when the virus content of the virus liquid is highest when the culture time is 72 hours.
TABLE 10 results of virus content determination at different culture times
EXAMPLE 10 preparation of avian adeno-associated virus (group 4) vaccine composition
Antigen inactivation: (1) Taking virus liquid (suspension cell culture) of the YSHN strain of the adenovirus disease (I group 4 type) of the poultry, adding formaldehyde solution with the final concentration of 0.1 percent, inactivating for 16 hours at 37 ℃, taking the inactivated antigen for inactivation test, and the result shows that the antigen is completely inactivated. (2) Taking purified Fiber-2 protein of the adenovirus disease (group I4 type) of the poultry, adding formaldehyde solution with the final concentration of 0.05 percent, inactivating for 48 hours at the temperature of 2-8 ℃, and carrying out inactivation test and protein agar expansion titer determination after the inactivation. The results show that the protein is completely inactivated, the endotoxin content is less than 5000EU/ml, and the agar titer is 1:128.
Preparing an aqueous phase:
aqueous phase 1: taking inactivated avian adenovirus (I group 4 type) YSHN strain antigen, diluting with sterilized water for injection until the virus content before inactivation is 10 6.50 TCID 50 0.1ml of Tween-80 was added, and the ratio of antigen to Tween-80 was 93:7 by volume, to allow Tween-80 to be dissolved sufficiently.
Aqueous phase 2: taking inactivated avian adenovirus (I group 4 type) Fiber-2 protein, diluting with sterilized water for injection until the agar expansion titer is 1:8, adding Tween-80, and making the volume ratio of antigen to Tween-80 be 93:7, so that Tween-80 is fully dissolved.
Aqueous phase 3: taking inactivated avian adenovirus (I group 4 type) YSHN strain antigen, diluting with water for injection until the virus content before inactivation is 10 6.50 TCID 50 0.1ml, taking inactivated avian adenovirus (I group 4 type) YSHN-Fiber-2 protein, diluting to 1:8 with water for injection, mixing the two according to the volume equal proportion, adding Tween-80, wherein the volume ratio of antigen to Tween-80 is 93:7, and dissolving Tween-80 to obtain water phase;
aqueous phase 4: taking inactivated avian adenovirus (I group 4 type) YSHN strain antigen, diluting with water for injection until the virus content before inactivation is 10 6.50 TCID 50 And (2) 0.1ml, taking inactivated avian adenovirus (I group 4 type) YSC4-Fiber-2 protein, diluting to 1:8 by using water for injection, mixing the two proteins according to the volume equal proportion, adding Tween-80, wherein the volume ratio of antigen to Tween-80 is 93:7, and dissolving the Tween-80 to obtain the water phase.
Aqueous phase 5: taking inactivated avian adenovirus (I group 4 type) YSHN strain antigen, diluting with water for injection until the virus content before inactivation is 10 6.50 TCID 50 And (2) 0.1ml, taking inactivated avian adenovirus (I group 4 type) YSSD-Fiber-2 protein, diluting to 1:8 by using water for injection, mixing the two proteins according to the volume equal proportion, adding Tween-80, wherein the volume ratio of antigen to Tween-80 is 93:7, and dissolving the Tween-80 to obtain the water phase.
Preparing an oil phase: the oil phase is prepared by mixing injection white oil and span-80 according to a volume ratio of 95:5, and sterilizing for later use;
preparation of an inactivated vaccine: mixing the water phase and the oil phase according to the volume ratio of 1:2, emulsifying, sub-packaging, labeling and preserving at 2-8 ℃ for standby.
Taking the prepared vaccine for carrying out character and sterility test, wherein the results meet the requirements of three appendices of the current Chinese animal pharmacopoeia, and the vaccine test result is shown as 11.
TABLE 11 characterization and sterility test of avian adeno-associated virus (group 4) vaccine compositions
EXAMPLE 13 safety testing of avian adeno-associated virus (group 4) vaccine compositions
Vaccine compositions 1 to 5 in Table 11 were immunized with SPF chickens 7 to 14 days old, 10 animals/group, chest intramuscular injection, 1 ml/animal, and observed for 14 days. The test results show that the local reaction or the systemic adverse reaction caused by the vaccine does not occur after the chicken is immunized by the composition 1 and the compositions 3 to 5, and the slight local inflammatory reaction occurs after the chicken is immunized by the composition 2.
Example 14 efficacy comparison test of avian adenosis (group 4) vaccine compositions
Vaccine compositions 1 to 5 in Table 11 were given by immunization and challenge method, and 10 animals per group were given by subcutaneous or intramuscular injection of 0.3ml vaccine to each neck, with 60 SPF chickens 21 to 28 days old, and 10 animals per control group. 21 days after immunization, all immunized and control chickens received subcutaneous injection of avian adenovirus (group I4) YSHN strain cell toxin 0.2 ml/dose (containing 2X 10) 6.50 TCID 50 ). Continuously observing for 14 days after the challenge, observing the morbidity and mortality of immunized chickens and control chickens, collecting oropharynx and cloacal cotton swabs of the chickens and the dead chickens which survive for 14 days after the challenge, inoculating LMH adherent cells after the cotton swabs are treated, carrying out virus separation, and carrying out blind transmission on samples which are negative to the virus separation.
The test result shows that the vaccine composition 1 prepared by using the fowl adenovirus (I group 4 type) YSHN strain has the protection rate of 10/10 after the immunization of chickens and the toxin expelling rate of 1/10; vaccine composition 2 prepared by YSHN-Fiber-2 protein, after immunization of chickens, the protection rate after detoxification is 10/10, and the toxin expelling rate is 2/10; vaccine composition 3 prepared from fowl adenovirus (group I4) YSHN strain and YSHN-Fiber-2 protein, wherein after immunization of chicken, the protection rate after challenge is 10/10, and the toxin expelling rate is 0/10; vaccine composition 4 prepared from fowl adenovirus (group I4) YSHN strain and YSC4-Fiber-2 protein, wherein after immunization of chicken, the protection rate after challenge is 10/10, and the toxin expelling rate is 2/10; vaccine composition 5 prepared with avian adenovirus (group I4) YSHN strain and YSSD-Fiber-2 protein, after immunization of chickens, had a protection rate of 10/10 after challenge and a detoxification rate of 1/10, and the results are shown in Table 12. The test result shows that the suspension-cultured avian adenovirus (group I4) strain YSHN strain virus liquid and fermentation-cultured BL21-Fiber-2 strain expression Fiber-2 protein are mixed and emulsified to prepare the avian adenovirus (group I4) inactivated vaccine, the protection rate is 100%, and the virus can be inhibited and discharged by 100%.
Table 12 results of challenge protection after vaccine composition against avian adenosis (group 4 type I)
Note that: "a" means survival; "B" means death; "+" indicates detoxification; "-" means not expelling toxin. 2. The protection rate is the survival rate. 3. The dead chicken is collected in time and the cotton swab of the throat and the cloaca is used for virus separation.
EXAMPLE 14 immunization duration test of avian adeno-associated virus (group 4) vaccine compositions
Vaccine compositions 1 to 5 in Table 11 were given by immunization and challenge method, and 50 SPF chickens of 21 to 28 days old were used in each group, and 50 vaccine compositions were given by subcutaneous or intramuscular injection to each neck in an amount of 0.3ml, while 50 control groups were given. 10 animals were randomly collected from the immunized group and the control group after 2 months, 4 months, 6 months and 8 months, and 0.2 ml/animal (containing 2×10) of avian adenovirus (group I4) YSHN strain cell toxin was subcutaneously injected into each neck 6.50 TCID 50 ). Continuously observing for 14 days after the challenge, observing the morbidity and mortality of immunized chickens and control chickens, collecting oropharynx and cloacal cotton swabs of the chickens and the dead chickens which survive for 14 days after the challenge, inoculating LMH adherent cells after the cotton swabs are treated, carrying out virus separation, and carrying out blind transmission on samples which are negative to the virus separation.
The test results show that the vaccine composition 1 prepared by using the fowl adenovirus disease (I group 4 type) YSHN strain has the protection rate of 9/10 until the 6 months after the prevention, the toxin expelling rate of 4/10, the protection rate of 8/10 until the 8 months after the prevention, and the toxin expelling rate of 5/10; vaccine composition 2 prepared with YSHN-Fiber-2 protein, up to 6 months post-exposure, a protection rate of 8/10, a detoxification rate of 6/10, up to 8 months post-exposure, a protection rate of 7/10, a detoxification rate of 7/10; vaccine composition 3 prepared from fowl adenovirus (group I4) YSHN strain and YSHN-Fiber-2 protein, with protection rate of 10/10, toxin expelling rate of 0/10, and toxin expelling rate of 0/10; vaccine composition 4 prepared from avian adenosis (group i 4) YSHN strain and YSC4-Fiber-2 protein, with a protection rate of 9/10, a detoxification rate of 3/10, a protection rate of 9/10, and a detoxification rate of 4/10 for 6 months to 8 months; vaccine composition 5 prepared with avian adenovirus disease (group I, type 4) YSHN strain and YSSD-Fiber-2 protein, up to 6 months post-exposure, a protection ratio of 10/10, a detoxification ratio of 3/10, up to 8 months post-exposure, a protection ratio of 9/10, a detoxification ratio of 4/10, and the results are shown in Table 13.
TABLE 13 post vaccine challenge protection results
Note that: 1. the protection rate is the survival rate; 2. the dead chicken is collected in time and the cotton swab of the throat and the cloaca is used for virus separation.
Experimental results show that the vaccine composition prepared by mixing and emulsifying the suspension-cultured avian adenovirus (group I4) strain YSHN strain virus liquid and the fermentation-cultured BL21-Fiber-2 strain expressing YSHN-Fiber-2 protein has longer protection time and is more effective in inhibiting toxin expelling compared with the vaccine composition prepared by suspending-cultured avian adenovirus (group I4) strain YSHN virus liquid and YSC 4/YSSD-Fiber-2 protein, the vaccine composition inactivated vaccine prepared by avian adenovirus (group I4) YSHN strain and the vaccine composition prepared by avian adenovirus (group I4) YSHN-2 protein.
Analysis of results:
1. compared with CN108126191B, the subunit vaccine of the Fiber-2 protein has weaker protective power;
2. the YSHN strain inactivated vaccine has similar protective capability with the subunit vaccine of Fiber-2 protein.
3. Unexpectedly, it was: when the invention adopts the combination of YSHN inactivated vaccine and YSHN-Fiber-2 protein, the invention provides unexpected ultra-long-term protection, the protection rate of the vaccine is 100% until 8 months after the protection, and the consistent toxin expelling rate is not less than 90%.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (2)
1. A vaccine, which is characterized by comprising an inactivated virus liquid and a Fiber-2 protein liquid;
the inactivated virus liquid contains inactivated avian adenovirus, and the Fiber-2 protein liquid contains Fiber-2 protein;
the fowl adenovirus is fowl adenovirusAvian adenovirus FAdV-YSHNBelongs to group I type 4; the preservation number is: CCTCC NO: V202302; preservation date: 2023, 5, 23, deposit unit: china center for type culture collection, with the preservation addresses: university of martial arts in chinese;
the amino acid sequence of the Fiber-2 protein is shown as SEQ ID No. 1;
the volume ratio of the inactivated virus liquid to the Fiber-2 protein liquid is 1:1;
the concentration of the inactivated avian adenovirus in the inactivated virus liquid is 10 6.5 TCID 50 /0.1ml；
The Fiber-2 protein liquid is obtained by diluting Fiber-2 protein liquid with a agar-agar titer of 1:128 to 1:8 with water for injection.
2. Use of an avian adenovirus and Fiber-2 protein in the manufacture of a vaccine;
the vaccine contains inactivated virus liquid and Fiber-2 protein liquid;
the inactivated virus liquid contains inactivated avian adenovirus, and the Fiber-2 protein liquid contains Fiber-2 protein;
the volume ratio of the inactivated virus liquid to the Fiber-2 protein liquid is 1:1;
the concentration of the inactivated avian adenovirus in the inactivated virus liquid is 10 6.5 TCID 50 /0.1ml；
The Fiber-2 protein liquid is obtained by diluting Fiber-2 protein liquid with a agar titer of 1:128 to 1:8 with water for injection;
the classification name of the avian adenovirus is avian adenovirusAvian adenovirus FAdV-YSHNBelongs to group I type 4; the preservation number is: CCTCC NO: V202302; preservation date: 2023, 5, 23, deposit unit: china center for type culture collection, with the preservation addresses: university of martial arts in chinese;
the amino acid sequence of the Fiber-2 protein is shown as SEQ ID No. 1.
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