CN117069866A - Duck hepatitis A virus type 3 recombinant immunogen and application thereof - Google Patents
Duck hepatitis A virus type 3 recombinant immunogen and application thereof Download PDFInfo
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Abstract
The invention discloses a duck hepatitis A virus type 3 (DHAV-3) recombinant immunogen and application thereof. According to the invention, protective antigen dominant regions of DHAV-3 structural proteins VP1, VP3 and VP0 are connected in series through a flexible Linker and prokaryotic expression is carried out, purified recombinant protein (rDHAV-3-VP) is taken as an immunogen, SPF duckling of 5 days old is immunized, 1000 ELD50 DHAV-3 duck embryo homogenate is challenged by 10 th d after immunization, 21 d is continuously observed, meanwhile, a yolk antibody prevention group and a PBS control group are arranged, and the result shows that the rDHAV-3-VP immunization group and the yolk antibody prevention group provide the same protection rate and are 82%; serum neutralizing antibody titer of surviving duckling after immune group challenge is 1:48; the pathological changes of liver tissues of the immune group are lower than those of the yolk antibody prevention group and the PBS control group, and the viral loads of the immune group and the yolk antibody prevention group are not different and are obviously lower than those of the PBS control group. The invention provides a new technical means for preparing the medicine for preventing DHAV-3 infection.
Description
Technical Field
The invention relates to a duck hepatitis A virus type 3 recombinant immunogen and application thereof, in particular to a recombinant immunogen for serially expressing a duck hepatitis A virus type 3 structural protein protective antigen dominant region and application thereof. The invention belongs to the field of biotechnology.
Background
Duck viral hepatitis (Duck viral hepatitis, DVH) is a highly lethal infectious disease of duckling caused by duck hepatitis virus (Duck hepatitis virus, DHV), and causes an acute and highly lethal infectious disease characterized by hepatomegaly, spotted bleeding on the surface and neurological symptoms of duckling of 1-3 weeks old, and the fatality rate of the sick duckling is as high as 80% or even 100%.
Duck hepatitis viruses include three serotypes, of which duck hepatitis A virus (Duck Hepatitis A Virus, DHAV) belongs to the family Picornaviridae, members of which are classified by genotype as A, B, C, also known as duck hepatitis A virus type 1 (DHAV-1), type 2 (DHAV-2) and type 3 (DHAV-3), with no or weak cross-protection between the respective types. The other two serotypes of duck hepatitis virus are duck astroviruses, which belong to members of the genus avian astrovirus of the family astroviridae and have no serotype cross-reaction with duck hepatitis a virus. With DHAV occurring most severely, type 1 and type 3 DHAV are mainly prevalent, and mixed infections of type 1 and type 3 DHAV often occur in production practice. VP1, VP3 and VP0 are three structural proteins of DHAV, wherein VP1 is the main protective antigen, which stimulates the body to produce protective response and neutralizing antibodies, but the degree of variation is higher compared with VP3 and VP0, and the variation at the C terminal is larger; VP3 and VP0 also have certain immunoprotection, and can stimulate organism to generate immunoprotection response. Because VP1, VP3 and VP0 structural proteins of DHAV-3 have immunogenicity, a theoretical basis is laid for the research and development of multi-epitope vaccines based on the DHAV-3 structural proteins.
At present, the duckling is protected by a mother source antibody generated by inoculating an inactivated vaccine and a weak vaccine to a female duck, and the duckling uses a yolk antibody, but the traditional vaccine has the problems of large antigen content difference among production batches of poultry embryo, difficulty in standardized production, strong virulence return, difficulty in transportation and storage, high production cost and the like. Compared with the traditional vaccine, the multi-epitope vaccine has the advantages of safety, stability, high yield, low cost, good immune effect and the like, and has good application prospect. At present, a multi-epitope vaccine based on the protective antigen dominant regions of three structural proteins of DHAV-3 is not reported, so that the protective antigen dominant regions of the three structural proteins of DHAV-3 are identified by utilizing a molecular biology and immunology method, recombinant immunogens expressed in series are constructed and subjected to immune evaluation, and the multi-epitope vaccine has important theoretical and practical significance for developing novel vaccines of DHAV-3 so as to provide effective prevention and control strategies.
Disclosure of Invention
The invention aims to provide a recombinant immunogen for serially expressing a protective antigen dominant region of a DHAV-3 structural protein and application thereof.
In order to achieve the above purpose, the invention adopts the following technical means:
The recombinant immunogen for serially expressing the protective antigen dominant region of the duck hepatitis A virus (Duck Hepatitis A Virus, DHAV) 3 type structural protein is obtained by serially connecting 90-175 aa protective antigen dominant region of structural protein VP1 of Duck hepatitis A virus strain GD (DHAV-3 GD, genbank accession number is GQ 122332) strain, 81-183 aa protective antigen dominant region of structural protein VP3 and 39-177aa amino acid sequence of VP0, wherein the 39-177aa amino acid sequence of VP0 contains 50-117 aa protective antigen dominant region of structural protein VP 0.
Preferably, the sequence of the Linker is Gly4SerGly4SerGly4Ser.
Wherein, preferably, the amino acid sequence of the recombinant immunogen is shown as SEQ ID NO. 2.
Polynucleotides encoding the recombinant immunogens of any preceding claim and prokaryotic expression vectors containing said polynucleotides are also within the scope of the present invention. Wherein, the preferable sequence of the polynucleotide is shown as SEQ ID NO. 1. Wherein, preferably, the prokaryotic expression vector is pET-30a.
Furthermore, the invention also provides application of the recombinant immunogen in preparing a vaccine for preventing duck hepatitis A virus type 3 infection. Wherein, preferably, the vaccine is a multi-epitope vaccine.
Furthermore, the invention also provides a duck hepatitis A virus type 3 multi-epitope vaccine, which contains any one of the recombinant immunogens.
Compared with the prior art, the invention has the beneficial effects that:
the invention carries out prokaryotic expression after connecting the protective antigen dominant region VP1 (90-175 aa) of the DHAV-3 structural protein VP1, the protective antigen dominant region VP3 (81-183 aa) of VP3 and the 39-177aa amino acid sequence of VP0 containing 50-117 aa protective antigen dominant region of structural protein VP0 in series through a flexible Linker to obtain a recombinant immunogen for serially expressing the protective antigen dominant region of the duck hepatitis A virus type 3 structural protein, which is named rDHAV-3-VP. Immunization of 5 day-old SPF ducklings with purified rDHAV-3-VP as immunogen, challenge 1000 ELD at 10 th d post immunization 50 The DHAV-3 duck embryo homogenate virus is continuously observed for 21 d, and meanwhile, a yolk antibody prevention group and a PBS control group are arranged, so that the result shows that the rDHAV-3-VP immunization group and the yolk antibody prevention group provide the same protection rate, and the protection rates are 82%; serum neutralizing antibody titer of surviving duckling after immune group challenge is 1:48; the liver tissue lesion degree of the rDHAV-3-VP immune group is lower than that of the yolk antibody preventive group and the PBS control group, and the viral loads of the immune group and the yolk antibody preventive group are not different and are obviously lower than those of the PBS control group. The invention provides a new technical means for preparing the medicine for preventing DHAV-3 infection.
Drawings
FIG. 1 is a diagram showing PCR amplification of DHAV-3 VP1 and digestion identification of recombinant plasmid pEASY-Blunt simple-VP1;
wherein, A.M. Trans2000 plus II DNA marker, 1. Negative control, 2-4. DHAV-3 VP1 gene PCR amplification; B.M. Trans2000 plus II DNA marker 1. PEASY-Blunt simple-VP1EcoR I single enzyme digestion, 2. PEASY-Blunt simple-VP1EcoRl and RXhoI, double enzyme digestion;
FIG. 2 is a diagram showing PCR amplification and cleavage identification of truncated genes of DHAV-3 VP1-4 and pEASY-Blunt-VP1-4;
wherein A, M.Trans2000 1-4. PCR amplification results of VP 1-4 genes; 5. negative control, B.M.Trans2000 plus II DNA marker, 1, 3, 5, 7. PEASY-Blunt-VP 1-4 plasmid, 2, 4, 6, 8. PEASY-Blunt-VP 1-4 plasmidEcoIdentification of R I single enzyme digestion;
FIG. 3 is a graph showing the result of pET32a-DHAV-3-VP1 cleavage assay;
wherein, M.Trans2K Plus II DNA marker;1.Ecor I single enzyme cuts pET32a-DHAV-3-VP1 product, 2.EcoR I、XhoI double enzyme cutting pET32a-DHAV-3-VP1 product;
FIG. 4 is a SDS-PAGE analysis of Rosetta-pET32a-DHAV3-VP1 induced expression;
wherein, the M.PageRuler protein quality standard is 1.Rosetta-pET32a after induction; 2. before the induction of Rosetta-pET32a-DHAV-3-VP 1; 3. Rosetta-pET32a-DHAV-3-VP1 induced thalli; 4. supernatant after induction of Rosetta-pET32a-DHAV-3-VP 1; 5. post induction precipitation of Rosetta-pET32a-DHAV-3-VP 1;
FIG. 5 is a diagram showing the identification of cleavage of pET30a-VP 1-4;
wherein, A-B.M. Trans2K plus II DNA marker, 1, 4 pET30a-VP 1-4 plasmid, 2, 5.pET30a-VP 1-4 is processedEcoIdentification of R I single enzyme digestion; 3. 6 pET30a-VP 1-4 is processedEcoRl and RXhoI, double enzyme digestion identification;
FIG. 6 is a SDS-PAGE analysis of the prokaryotic expression of Rosetta-pET-30a-DHAV-3-VP 1-4;
wherein, A ~ D. Rosetta-pET-30a-DHAV-3-VP1-1~4 induces the expression product: page rule Marker; pre-rosetta-pET-30 a induction; after induction of Rosetta-pET-30 a; 3, before the induction of Rosetta-pET-30a-DHAV-3-VP 1-4; after induction of Rosetta-pET-30a-DHAV-3-VP 1-4; ultrasonic supernatant after induction of Rosetta-pET-30a-DHAV-3-VP 1-4; inducing Rosetta-pET-30a-DHAV-3-VP 1-4, and performing ultrasonic precipitation;
FIG. 7 is a graph showing the result of codon optimization of DHAV-3-VP1 gene;
note that: * Marked as modified codons;
FIG. 8 is a diagram showing the identification of optimized DHAV-3 VP1 gene clone and pET-32a-DHAV-3-optiVP1 cleavage;
wherein, cloning of optimized DHAV-3 VP1 gene: m, trans2K DNA Marker;1. optimized VP1 gene amplification product; 2. a negative control;
identification of pET-32a-DHAV-3-optiVP 1: trans2K Plus II Marker; pET-32a-DHAV-3-optiVP1 BamH I single enzyme digestion; pET-32a-DHAV-3-optiVP1BamH I +XhoI, double enzyme digestion; PCR identification of pET-32a-DHAV-3-optiVP 1; 4. negative control
FIG. 9 is a diagram showing SDS-PAGE analysis of prokaryotic expression before and after optimization of DHAV-3VP1 gene;
wherein, the A.Rosetta-pET-30a-DHAV-3-VP1 induces an expression product;
induction of expression products by Rosetta-pET-30a-DHAV-3-optiVP 1;
C.Rosetta-pET-32a-DHAV-3-optiVP1 induces the expression product;
page rule Marker; pre-rosetta-pET-30 a/32a induction; after Rosetta-pET-30a/32a induction; pre-induction of rosetta-pET-30a/32a-DHAV-3-VP 1; after induction of Rosetta-pET-30a/32a-DHAV-3-VP 1; ultrasonic supernatant after Rosetta-pET-30a/32a-DHAV-3-VP1 induction; ultrasonic precipitation after Rosetta-pET-30a/32a-DHAV-3-VP1 induction;
FIG. 10 is a SDS-PAGE analysis of recombinant DHAV-3VP1 and its truncated protein after purification;
wherein, M.Page rule Marker;1.250mmol/L imidazole-eluted r-30a-DHAV-3-VP1;2.500mmol/L imidazole-eluted r-32a-DHAV-3-optiVP1;3.250mmol/L imidazole eluted r-30a-DHAV-3-optiVP1; 4-7.500 mmol/L imidazole eluted r-DHAV-3-VP 1-4;
FIG. 11 is a Western blot identification chart of recombinant DHAV-3VP1 and its truncated protein after purification;
wherein, M.Page rule Marker;1. purified r-30a-DHAV-3-VP1;2. purified r-32a-DHAV-3-optiVP1;3. purified r30a-DHAV-3-optiVP1; 4-7, purifying the r-DHAV-3-VP 1-4;
FIG. 12 is a graph showing Western blot identification results of duck anti-DHAV-3-VP 1 polyclonal antibodies;
wherein, M.Page rule Marker;1.r-DHAV-3-VP1; 2-5. R-DHAV-3-VP 1-4; 6. an unrelated protein;
FIG. 13 is a view of a liver section after the DHAV-3-VP1 immunized duckling is detoxified;
wherein, PBS control group death duckling liver is examined and observed; b.r-30a-DHAV-3-optiVP1 immune group toxicity attack 21 d non-dead duckling liver section inspection;
FIG. 14 is a graph of survival of duckling immune recombinant DHAV-3-VP1 and truncated protein after DHAV-3 challenge;
FIG. 15 is a PCR amplification of DHAV-3VP3 and truncated gene fragment;
wherein M. Trans 2K/Trans 2K Plus II DNA Marker; DHAV-3VP3 gene amplification product; DHAV-3-VP3-1 gene amplification product; DHAV-3-VP3-2 gene amplification product; DHAV-3-VP3-3 gene amplification product; 5. a negative control;
FIG. 16 is a graph showing the results of the cleavage assay for recombinant expression plasmid pET-32a-DHAV-3-VP 3;
wherein M. Trans 2K/Trans 2K Plus II DNA Marker;1. pET-32a-DHAV-3-VP3BamH I single enzyme digestion; 2. pET-32a-DHAV-3-VP3BamH I +XhoI, double enzyme digestion;
FIG. 17 is a diagram showing the results of the cleavage assay of recombinant expression plasmid pET-32a-DHAV-3-VP 3-1-3;
wherein, m.trans 2K Plus II DNA Marker;1. 3, 5.pET-32a-DHAV-3-VP 3-1-3 BamH I single enzyme digestion; 2. 4, 6.PET-32a-DHAV-3-VP 3-1-3BamH I +XhoI, double enzyme digestion;
FIG. 18 is a SDS-PAGE analysis of prokaryotic expression Rosetta-pET-32a-DHAV-3-VP 3;
wherein, M.Page rule Marker;1. before Rosetta-pET-32a induction; 2. Rosetta-pET-32a
After induction; 3. pre-induction of Rosetta-pET32 a-DHAV-3-VP3; 4. after induction of Rosetta-pET32 a-DHAV-3-VP3; 5. ultrasonic supernatant after induction of Rosetta-pET32 a-DHAV-3-VP3; 6. ultrasonic precipitation after induction of Rosetta-pET32 a-DHAV-3-VP3;
FIG. 19 is a SDS-PAGE analysis of prokaryotic expression Rosetta-pET-32a-DHAV-3-VP 3-1-3;
wherein, A-C.Rosetta-pET 32a-DHAV-3-VP 3-1-3 prokaryotic expression results: page rule Marker;1. before Rosetta-pET-32a induction; 2. after induction of Rosetta-pET-32 a; 3. before the induction of Rosetta-pET32a-DHAV-3-VP 3-1-3; 4. after induction of Rosetta-pET32a-DHAV-3-VP 3-1-3; 5. ultrasonic supernatant after induction of Rosetta-pET32a-DHAV-3-VP 3-1-3; 6. ultrasonic precipitation after induction of Rosetta-pET32a-DHAV-3-VP 3-1-3;
FIG. 20 is a SDS-PAGE analysis of recombinant DHAV-3 VP3 and truncated protein after purification;
wherein, M.Page rule Marker; 1.500 mmol/L imidazole-eluted r-32a-DHAV-3-VP3; 2-4.500 mmol/L imidazole eluted r-32a-DHAV-3-VP 3-1-3;
FIG. 21 is a Western blot identification chart after purification of recombinant DHAV-3 VP3 protein;
wherein, M.Page rule Marker; 1.500 mmol/L imidazole-eluted r-32a-DHAV-3-VP3;
FIG. 22 is a Western blot identification chart of recombinant DHAV-3 VP3-1-3 proteins after purification;
wherein, M.Page rule Marker; 1-3.500 mmol/L imidazole eluted r-32a-DHAV-3-VP 3-1-3;
FIG. 23 is a Western blot identification chart of duck anti-DHAV-3 VP3 protein polyclonal antibodies;
wherein M. Page Ruler Marker;1. r-32a-DHAV-3-VP3; 2-4. R-32a-DHAV-3-VP 3-1-3; 5. purified pET-32a tag protein; 6. an unrelated protein;
FIG. 24 is a view of a liver section taken after the DHAV-3 VP3 immunized duckling has been detoxified;
wherein, PBS control group death duckling liver is examined and observed; b.r-32a-DHAV-3-VP3 immune group toxicity attack 21 d non-dead duckling liver section inspection observation
FIG. 25 is a graph of survival of duckling immune recombinant DHAV-3-VP3 and truncated protein after DHAV-3 challenge;
FIG. 26 is a PCR amplification map of the DHAV-3-VP0 gene and its truncated gene;
wherein M. Trans 2K/Trans 2K Plus II DNA Marker; DHAV-3-VP0 gene amplification product; DHAV-3-VP0-1 gene amplification product; DHAV-3-VP0-2 gene amplification product; DHAV-3-VP0-3 gene amplification product; 5. DHAV-3-VP0-4 gene amplification product;
FIG. 27 is a graph showing the identification of recombinant expression plasmid pET-30a-DHAV-3-VP 0;
wherein, M.Trans 2K Plus II Marker; pET-30a-DHAV-3-VP0EcoR I single enzyme digestion; pET-30a-DHAV-3-VP0EcoR I +XhoI, double enzyme digestion; PCR identification of pET-30a-DHAV-3-VP 0; 4. a negative control;
FIG. 28 is a graph showing the results of the identification of recombinant expression plasmid pET-32a-DHAV-3-VP 0;
wherein, M.Trans 2K Plus II Marker; pET-32a-DHAV-3-VP0 1 plasmidEcoR I single enzyme digestion; pET-32a-DHAV-3-VP0 2 plasmidEcoR I +XhoI, double enzyme digestion;
FIG. 29 is a graph showing the results of identifying recombinant expression plasmids pET-32a-DHAV-3-VP 0-1-4;
wherein, M.Trans 2K Plus II Marker;1. 3, 5, 7.pET-32a-DHAV-3-VP 0-1-4EcoR I single enzyme digestion; 2. 4, 6, 8.PET-32a-DHAV-3-VP 0-1-4EcoR I +XhoI, double enzyme digestion;
FIG. 30 is a SDS-PAGE analysis of prokaryotic expression of DHAV-3-VP0 gene;
wherein, the A.Rosetta-pET-30a-DHAV-3-VP0 induces the expression result; induction of expression results by Rosetta-pET-32a-DHAV-3-VP 0; 1.M.Page Ruler Marker; pre-rosetta-pET-30 a/32a induction; after Rosetta-pET-30a/32a induction; pre-induction of rosetta-pET-30a/32a-DHAV-3-VP 0; after induction of Rosetta-pET-30a/32a-DHAV-3-VP 0; ultrasonic supernatant after Rosetta-pET-30a/32a-DHAV-3-VP0 induction; ultrasonic precipitation after Rosetta-pET-30a/32a-DHAV-3-VP0 induction;
FIG. 31 is a SDS-PAGE analysis of prokaryotic expression of DHAV-3-VP 0-1-4 gene fragments;
wherein, A ~ D. Rosetta-pET-32a-DHAV-3-VP0-1~4 induces the expression result: page rule Marker; pre-rosetta-pET-32 a induction; after induction of Rosetta-pET-32 a; 3, before the induction of Rosetta-pET-32a-DHAV-3-VP 0-1-4; after induction of Rosetta-pET-32a-DHAV-3-VP 0-1-4; ultrasonic supernatant after induction of Rosetta-pET-32a-DHAV-3-VP 0-1-4; ultrasonic precipitation after induction of Rosetta-pET-32a-DHAV-3-VP 0-1-4;
FIG. 32 is a SDS-PAGE analysis of recombinant DHAV-3-VP0 and truncated protein after purification;
wherein, M.Page rule Marker; 1-2.250 mmol/L imidazole eluted r-30a/32a-DHAV-3-VP0;3.250mmol/L imidazole-eluted r-32a-DHAV-3-VP0-1; 4-5.500 mmol/L imidazole eluted r-32a-DHAV-3-VP0-2/-3;6.250mmol/L imidazole-eluted r-32a-DHAV-3-VP0-4;
FIG. 33 is a Western blot identification of recombinant DHAV-3-VP0 and truncated protein after purification;
wherein, M.Page rule Marker; 1-2. R-30a/32a-DHAV-3-VP0 after purification; 3-6, purifying r-32a-DHAV-3-VP 0-1-4;
FIG. 34 is a Western blot identification chart of duck anti-DHAV-3-VP 0 protein polyclonal antibody;
wherein, M.Page rule Marker;1. r-32a-DHAV-3-VP0; 2-5. R-32a-DHAV-3-VP 0-1-4; 6. an unrelated protein;
FIG. 35 is a view of a liver section after toxicity challenge of a DHAV-3-VP0 immunized duckling;
wherein, PBS control group death duckling liver is examined and observed; B.r-DHAV-3-VP0 immune group toxin-attacking 21 d non-dead duckling liver section inspection;
FIG. 36 is a graph of survival of duckling immune r-DHAV-3-VP0 and truncated protein after DHAV-3 challenge;
FIG. 37 is a schematic diagram of DHAV-3-VP gene tandem;
FIG. 38 is a graph showing the identification of recombinant expression plasmid pET-30 a-DHAV-3-VP;
wherein, M.Trans 2K Plus II Marker; pET-30a-DHAV-3-VPXhoI, single enzyme digestion; pET-30a-DHAV-3-VPBamH I+XhoI, double enzyme digestion; 3. a negative control;
FIG. 39 is a SDS-PAGE analysis of prokaryotic expression of DHAV-3-VP gene;
wherein, M.Page rule Marker; pre-rosetta-pET-30 a induction; after induction of Rosetta-pET-30 a; rosetta-pET-30a-DHAV-3-VP before induction; after induction of Rosetta-pET-30 a-DHAV-3-VP; ultrasonic supernatant after Rosetta-pET-30a-DHAV-3-VP induction; ultrasonic precipitation after induction of Rosetta-pET-30 a-DHAV-3-VP;
FIG. 40 is a SDS-PAGE analysis of recombinant DHAV-3-VP and truncated protein after purification;
wherein, M.Page rule Marker;1.500mmol/L imidazole-eluted r-DHAV-3-VP;2.500mmol/L imidazole-eluted r-DHAV-3-VP1-3;3.250mmol/L imidazole-eluted r-DHAV-3-VP3-2;4.500mmol/L imidazole-eluted r-DHAV-3-VP0-2;
FIG. 41 is a Western blot identification chart of His mab after purification of recombinant DHAV-3-VP and its truncated protein;
wherein, M.Page rule Marker;1. purified r-DHAV-3-VP;2. purified r-DHAV-3-VP1-3;3. purified r-DHAV-3-VP3-2;4. purified r-DHAV-3-VP0-2;
FIG. 42 is a graph of Western blot identification of duck anti-DHAV-3 positive serum purified from recombinant DHAV-3-VP and its truncated protein;
wherein, M.Page rule Marker;1.r-DHAV-3-VP;2.r-DHAV-3-VP1-3;3.r-DHAV-3-VP3-2;4.r-DHAV-3-VP0-2;5. an unrelated protein;
FIG. 43 is a view of a liver section after toxicity challenge of a DHAV-3-VP immune duckling;
wherein, PBS control group death duckling liver is examined and observed; B.r-DHAV-3-VP immune group virus-fighting 21 d liver section observation of a duckling which does not die;
FIG. 44 is a graph of survival of DHAV-3 challenge after immunization of duckling with DHAV-3-VP protein;
FIG. 45 is a view of liver histology of a duckling of a DHAV-3-VP immunized duckling after challenge (400X);
wherein, PBS control group; B. a yolk antibody-use group; C.r-DHAV-3-VP immunization group;
FIG. 46 is a graph of DHAV-3 viral load detection in liver tissue of a duckling after challenge;
FIG. 47 is a graph of DHAV-3 viral load detection in spleen tissue of a duckling after challenge;
Fig. 48 is a graph of DHAV-3 viral load detection in a duckling cloaca swab after challenge.
Detailed Description
The invention is further described below in connection with specific examples which are given solely for illustration of the invention and are not intended to limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
EXAMPLE 1 identification of the protective antigen dominant region of DHAV-3VP1
1.1 Prokaryotic expression of DHAV-3VP1 gene and truncated gene thereof
1.1.1 Cloning of DHAV-3VP1 Gene and its truncated Gene
cDNA is synthesized by taking extracted DHAV-3 GD strain infected duck embryo allantoic fluid RNA as Sub>A template and taking Sub>A specific downstream primer (VP 1 ORF-A) of VP1 Open Reading Frame (ORF) as Sub>A reverse transcription primer, wherein Sub>A reverse transcription system is carried out according to Sub>A reverse transcription kit instruction. PCR amplification was performed using the obtained cDNA as a template and the primer VP1 ORF-S/A, the primer sequences of which are shown in Table 1.
Note that:GAATTCis thatEcoA R I cleavage site;CTCGAGis thatXhoI, enzyme cutting site; TAA is a stop codon. The primers are synthesized by Beijing Liuhua big Gene science and technology Co.
The fragment obtained by PCR amplification is connected with a cloning vector pEASY-blue simple, and the obtained recombinant plasmid is subjected to the following steps ofEcoThe RIP is singulated, as shown in FIG. 1,a band of about 4520 and bp appears,Ecorl and RXhoA726 bp destination gene band and a pEASY-Blunt simple vector band of about 3900 bp appear after I double cleavage. The monoclonal bacterial liquid which is identified as positive is sent to Beijing Liuhua large gene technology Co.Ltd for sequencing, and the recombinant plasmid with correct sequencing is named pEASY-Blunt-simple-VP1.
1.1.1.1 Cloning of the truncated Gene of DHAV-3 VP1-1-4
The antigenicity of the DHAV-3 VP1 protein was analyzed by DNA star software, and VP1 was divided into VP1-1 (1-85 aa), VP1-2 (30-110 aa), VP1-3 (90-175 aa) and VP1-4 (155-240 aa) 4 segments. Designing a specific amplification primer according to the VP1-4 truncated gene sequence, introducing a protective base and an EcoRI restriction site at the 5 'end of the upstream primer, and introducing a protective base, an XhoI restriction site and a stop codon at the 5' end of the downstream primer, wherein the primer sequences are shown in Table 2.
Note that:GAATTCis thatEcoA R I cleavage site;CTCGAGis thatXhoI, enzyme cutting site; TAA is a stop codon. The primers are synthesized by Beijing Liuhua big Gene science and technology Co.
Using pEASY-Blunt-simple-VP1 plasmid as a template, and carrying out PCR amplification by using the specific primer pair; 1% agarose gel electrophoresis analysis, as shown in FIG. 2, A, a band consistent with the expected results appeared at around 250 bp; and (3) connecting the purified PCR product into a pEASY-Blunt Simple vector, transforming TG1 competence, extracting plasmid enzyme digestion identification results as shown in a graph of B, and delivering the identified positive monoclonal bacterial liquid to Beijing Liuhua big gene technology Co., ltd for sequencing, wherein the recombinant plasmid with correct sequencing is named as pEASY-Blunt-VP 1-4.
1.1.2 Prokaryotic expression of DHAV-3 VP1 gene and truncated gene thereof
1.1.2.1 Prokaryotic expression of DHAV-3 VP1 Gene
The pEASY-Blunt-simple-VP1 and pET-32a vector are respectively subjected to double digestion by EcoR I and Xho I, the target gene is recovered and connected with the pET32a vector, TG1 competent cells are transformed, plasmids are extracted, and digestion identification is carried out. As shown in FIG. 3, the analysis and restriction enzyme analysis result of 1% agarose gel electrophoresis shows that pET32a-DHAV-3-VP1 is cut by EcoR I to obtain a fragment of about 6620 bp, and by EcoR I and Xho I to obtain two fragments of about 5900 bp and 720 bp, the construction of recombinant expression plasmid pET32a-DHAV-3-VP1 is successful.
Positive recombinant plasmid pET-32a-DHAV-3-VP1 was transformed into Rosetta TM (DE 3) pLysS competent cells, single colonies on LB solid plates were picked up and inoculated into LB liquid medium containing 10. Mu.g/mL Amp and 30. Mu.g/mL Kan, and shake cultured at 37℃for 12 h. The cultured bacterial liquid was inoculated into 5 mL liquid LB medium containing 10. Mu.g/mL of Amp and 30. Mu.g/mL of Kan, shake-cultured at 37℃for 2 h, and then cultured with IPTG at a final concentration of 1.0 mmol/L to continue 4 h. The cultured bacterial liquid is subjected to ultrasonic treatment to break bacterial cells, and after ultrasonic treatment, the bacterial liquid is centrifuged for 10 min at 12000 g at 4 ℃, and the supernatant and the sediment are collected. Finally, the induced expression and expression pattern of the recombinant protein were analyzed by SDS-PAGE. Recombinant protein is expressed in the form of inclusion bodies, and collected thalli are subjected to ultrasonic crushing and then are centrifuged for 15 min at 4 ℃ at 5000 r/min. The expressed and sonicated product was subjected to 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and the result was shown in FIG. 4 to be about 44 kDa in Rosetta-pET32a-DHAV3-VP1 protein.
1.1.2.2 Prokaryotic expression of DHAV-3 VP1-1-4 truncated genes
The pEASY-Blunt-VP 1-4 is subjected to double digestion, target fragments are recovered, subcloned into pET-30a, and the result is shown in figure 5 after EcoRI single digestion and EcoRI double digestion of EcoRI and XhoI are carried out on 4 recombinant plasmids, the obtained fragment size accords with the expected result, and the successful construction of recombinant expression plasmids pET30a-VP 1-4 is proved.
The positive plasmid pET-30a-DHAV-3-VP1-4 is transformed into Rosetta (DE 3) pLysS competent cells, and the induction expression of the target protein is carried out according to a literature method. The specific operation is the same as 1.1.2.1, and the results are shown in FIG. 6A and FIG. 6B, the recombinant proteins expressed by Rosetta-pET-30a-DHAV-3-VP1-1 and Rosetta-pET-30a-DHAV-3-VP1-2 are respectively 18.4 kDa and 17.9 kDa in size, and are expressed in natural form; as shown in FIGS. 6C and 6D, the recombinant proteins expressed by Rosetta-pET-30a-DHAV-3-VP1-3 and Rosetta-pET-30a-DHAV-3-VP1-4 were 18.2 kDa and 19.1 kDa in size, respectively, and were expressed as inclusion bodies, the protein sizes were consistent with expectations.
1.1.3 Optimization and expression of DHAV-3-VP1 gene
1.1.3.1 DHAV-3-VP1 gene glycosylation site mutation and codon optimization
Since VP1 gene expression level is extremely low and expression is unstable, the gene is optimized. The glycosylation site of the VP1 gene of the DHAV-3 VP1 strain (GQ 122332.1) is predicted by using the http:// services, heathtech, dtu, dk/services/SignalP-4.1/output, php at the wire network station, and the 84 th, 99 th and 196 th amino acids asparagine N at the N end of the sequence are mutated into glutamine Q; the preference and rare codons of the VP1 gene were analyzed using the site at the wire mesh site http:// peple. Mbi. Ucla. Edu/sumchan/calor. Html and http:// www.kazusa.or.jp/codon/countcon. Html, and it was found that 16 rare codons were present in the VP1 sequence and dispersed in the VP1 sequence. On the premise of not changing the coding amino acid of the VP1 gene, the rare codon and the low-frequency codon in the VP1 gene are replaced by the escherichia coli preference codon, and the optimized VP1 gene is named optiVP1. The VP1 gene and the optiVP1 gene sequences are shown in FIG. 7, wherein VP1 has 240 codons, and total optimized 157 codons account for 65.42% of the number of codons. Meanwhile, a BamH I restriction enzyme site is introduced at the 5 'end of the optiVP1 gene, and an Xho I restriction enzyme site is introduced at the 3' end. The optiVP1 gene is synthesized by Beijing Liuhua big gene technology Co., ltd, and subcloned into a prokaryotic expression vector pET-30a to obtain a recombinant expression plasmid pET-30a-DHAV-3-optiVP1.
1.1.3.2 Prokaryotic expression of DHAV-3-VP1 optimized gene
The PCR product was recovered after double digestion with BamH I and Xho I using pET-30a-DHAV-3-optiVP1 as a template, and ligated with pET-32a plasmid, and the recombinant plasmid was identified by double digestion with Xho I and BamH I, and the result was shown in FIG. 8, and a fragment conforming to the expected size was obtained. And (3) sending the monoclonal bacterial liquid which is identified as positive to Beijing Liuhua large gene technology Co.Ltd for sequencing, and the recombinant plasmid which is sequenced correctly is named as pET-32a-DHAV-3-optiVP1.
Recombinant plasmids pET-30a/32a-DHAV-3-optiVP1 and pET-30a-DHAV-3-VP1 carrying optimized genes are transformed into Rosetta (DE 3) pLysS competent cells, and induced expression of target proteins is carried out according to a literature method. The specific operation was the same as 1.1.2.1, and the results are shown in fig. 9A and 9B. The results show that the recombinant proteins expressed by the Rosetta-pET-30a-DHAV-3-VP1 and the Rosetta-pET-30 a-DHAV-3-optVP 1 are 34.4 kDa in size and are expressed in a natural form, and the expression quantity of the Rosetta-pET-30 a-DHAV-3-optVP 1 which is optimized by codons is obviously increased; as shown in FIG. 9C, the recombinant protein expressed by Rosetta-pET-32 a-DHAV-3-optVP 1 had a size of 44.4 kDa, was expressed as inclusion bodies, and the protein sizes were all consistent with expectations.
1.1.3.3 Purification and identification of DHAV-3VP1 and truncated proteins
The Rosetta-pET-30a-DHAV-3-VP1, rosetta-pET-30a-DHAV-3-optiVP1 and Rosetta-pET-30a-DHAV-3-VP1-1/-2 are expressed in natural forms, so that the purification is carried out under non-denaturing conditions. Rosetta-pET-32a-DHAV-3-optiVP1 and Rosetta-pET-30a-DHAV-3-VP0-3/-4 were expressed as inclusion bodies, and thus purified under denaturing conditions. Combining the supernatant after centrifugal treatment with an NI2+ -NTA agarose gel column, and then washing the mixed protein by using a washing (de) buffer solution containing 0-500 mmol/L imidazole or 8M urea and containing different imidazole concentrations, and eluting the target protein. The imidazole concentration was 250 mmol/L or 500 mmol/L, and the bands of interest of 34.4 kDa, 44.4 kDa, 34.4 kDa and 18.4 kDa, 17.9 kDa, 18.2 kDa and 19.1 kDa were eluted, respectively, and the detected bands were single and were sized to match the expected results, indicating that the purified products of the recombinant proteins described above were obtained, as shown in FIG. 10, by SDS-PAGE analysis of r-30a-DHAV-3-VP1, r-32a-DHAV-3-opti VP1, r-30a-DHAV-3-opti VP1 and r-DHAV-3-VP 1/-2/-3/-4.
And taking the purified r-32a-DHAV-3-optiVP1 protein and r-30a-DHAV-3-VP 1-4 protein as antigens, carrying out Western blot identification, wherein the primary antibody is a 1:5000 diluted mouse anti-His-tag monoclonal antibody, the secondary antibody is a 1:5000 diluted HRP-marked goat anti-mouse IgG (H+L), and the ECL is developed. The results are shown in FIG. 11, indicating that the bands of interest appear at 34.4 kDa, 44.4 kDa, 34.4 kDa and 18.4 kDa, 17.9 kDa, 18.2 kDa, 19.1 kDa, consistent with the expected results.
1.2 preparation and identification of Duck anti-DHAV-3 VP1 polyclonal antibody
After the purified r-30a-DHAV-3-opti VP1 protein and Freund's adjuvant are emulsified, chest muscle is immunized with SPF duck, the immunization dose is 1 mg/duck, freund's complete adjuvant is adopted as one of the immunity doses, freund's incomplete adjuvant is adopted as two and three of the immunity doses, the immunization interval is 14 d, 7 d vein blood collection is carried out after one and two of the immunity doses, 5, 10, 15 d vein blood collection and 20 d heart blood collection are carried out after three of the immunity doses, and sub-packaged serum is stored at-70 ℃.
And (3) taking purified r30 a-DHAV-3-optidVP 1 protein and r-30a-DHAV-3-VP1-4 protein as antigens, taking 1:800 diluted duck anti-DHAV-3-VP 1 polyclonal antibody as primary antibody, and 1:5000 diluted goat anti-duck IgG (H+L) marked by HRP as secondary antibody, and carrying out Western blot identification. The results are shown in FIG. 12, which shows that the preparation of the duck anti-DHAV-3-VP 1 polyclonal antibody can recognize r-30a-DHAV-3-VP1-4 four-segment truncated protein.
1.3 Immunoprotection assay of DHAV-3VP1 protein and truncated proteins
The 40 SPF ducks of 5 days old were randomly divided into 8 groups of PBS control group, adjuvant control group, r-30a-DHAV-3-VP1 group, r-30a-DHAV-3-optiVP1 group, r-30a-DHAV-3-VP1-2 group, r-30a-DHAV-3-VP1-3 group and r-30a-DHAV-3-VP1-4 group, 5 animals of each group, the immune dose was 0.2 mg recombinant protein, and the PBS group was injected with equal volumes of sterilized PBS. Duckling was immunized at 5 days of age, 10 d was immunized, and then detoxified (using 1000 ELD50 of DHAV-3 duck embryo homogenate) and 21 d duckling morbidity and mortality were continuously recorded, thereby evaluating the immunoprotection of DHAV-3-VP1 and truncated protein.
After 1-2 d of toxin is attacked, duckling in the PBS control group and the adjuvant control group show typical clinical symptoms of DHAV-3 infection, and the duckling mainly shows slow response, anorexia, white and thin feces discharge, and after 3 d of toxin is attacked, the duckling starts to die successively, and after 6 d of toxin is attacked, all ducklings die. The r-30a-DHAV-3-VP1 immune group and the r-30a-DHAV-3-optiVP1 immune group have good appetite and mental state, and death phenomenon begins to appear at 16 d after the toxicity attack, and the death rate of 21 d after the toxicity attack is 40%. The r-30a-DHAV-3-VP1-1 immune group and the r-30a-DHAV-3-VP1-4 immune group have the advantages that the appetite of the duckling is reduced and the duckling is not happy after the part of 6 d is attacked, the death phenomenon starts to appear at 11 d after the part of duckling is attacked, and the death rate is 100% after 13 d and 14 d respectively after the part of duckling is attacked; the disease incidence speed of the r-30a-DHAV-3-VP1-2 immune group is lower than that of the r-30a-DHAV-3-VP1-1 immune group and the r-DHAV-3-VP1-4 immune group, the clinical symptoms are almost the same, the death phenomenon begins to appear at 13 d after the toxicity attack, and the death rate at 16 d after the toxicity attack is 100%; the disease incidence speed of the r-30a-DHAV-3-VP1-3 immune group is lower than that of the r-30a-DHAV-3-VP1-2 immune group, the duckling at 13 and d parts has neurological symptoms after the virus attack, 14 and d begin to have death phenomenon after the virus attack, and the death rate of 21 and d after the virus attack is 60%; as shown in FIG. 13, the duckling in the PBS control group was examined by a dissecting method, and the liver was swollen and bleeding, and the liver was fragile, whereas the liver of the duckling in the r-30a-DHAV-3-optiVP1 immune group was not significantly diseased. The survival rates of the duckling in the different groups are shown in fig. 14 and table 3. In conclusion, the r-30a-DHAV-3-VP1 immune group and the r-30a-DHAV-3-optiVP1 immune group provide the strongest immunoprotection, the protection rate is 60%, and the r-30a-DHAV-3-VP1-3 immune group has the protection rate of 40% and other truncated proteins have no immunoprotection, but the death time can be delayed compared with the control group. The above results demonstrate that DHAV-3-VP1, DHAV-3-opti VP1 and the truncated protein DHAV-3-VP1-3 stimulate the duckling to produce neutralizing antibodies after immunization of the duckling to provide immunoprotection against lethal virus challenge. DHAV-3-VP1-3 is the protective antigen presenting region of DHAV-3-VP 1.
Example 2 identification of the protective antigen dominant region of DHAV-3VP3
2.1 Prokaryotic expression of DHAV-3VP3 and truncated genes
2.1.1 Cloning of DHAV-3VP3 Gene and its truncated Gene
The antigenicity of DHAV-3VP3 protein was analyzed by DNA star software, and VP3 was divided into VP3-1 (1-89 aa), VP3-2 (81-183 aa), and VP3-3 (174-231 aa) 3 segments. Extracting RNA of duck embryo allantoic fluid infected by DHAV-3GD strain, synthesizing cDNA by using oligo dT as a reverse transcription primer, and performing a reverse transcription system according to a reverse transcription kit instruction. The DHAV-3-VP3 gene and its truncated gene were amplified by PCR using the obtained cDNA as a template and the primers shown in Table 4.
Note that:GGATTCis thatBamH I cleavage site;CTCGAGis thatXhoI, enzyme cutting site; ATG is the start codon and TTA is the stop codon. The primers are synthesized by Beijing Liuhua big Gene science and technology Co.
The fragments obtained by PCR amplification were subjected to 1% agarose gel electrophoresis, and as shown in FIG. 15, the bands of interest were visible at 711bp, 285 bp, 327bp and 192 bp, which were consistent with the expected results.
2.1.2 Construction of prokaryotic expression vector of DHAV-3VP3 and truncated gene
The purified PCR product and pET-32a are digested with BamH I and Xho I, target fragment is recovered, DH5 alpha competent cells are transformed after connection, and the obtained recombinant plasmid is identified by single and double digestion with BamH I and Xho I. The results of the single BamHI cleavage and the double BamHI and Xho I cleavage of pET-32a-DHAV-3-VP3 and pET-32a-DHAV-3-VP 3-1-3 are shown in FIGS. 16 and 17, and the target bands corresponding to the expected sizes are obtained. And (3) sending the monoclonal bacterial liquid identified as positive to Beijing Liuhua large gene technology Co.Ltd for sequencing, wherein the sequencing result shows that the target gene sequence is consistent with the original sequence.
2.1.3 Prokaryotic expression and purification of DHAV-3VP3 gene and truncated gene
2.1.3.1 Prokaryotic expression of DHAV-3VP3 gene and truncated gene
pET-32a-DHAV-3-VP3 and pET-32a-DHAV-3-VP 3-1-3 are respectively transformed into RosettaTM (DE 3) pLysS competence, and induction expression of target proteins is carried out according to a literature method, wherein the target proteins are all expressed in an inclusion body form. The specific procedure was the same as 1.1.2.1, and the results are shown in FIGS. 18 and 19, indicating that the target proteins of 44kDa, 28kDa, 30kDa and 25kDa, which correspond to the expected sizes, were obtained, respectively.
2.1.3.2 Purification and identification of prokaryotic expression product of DHAV-3VP3 gene and truncated gene
The four recombinant proteins are all expressed by inclusion bodies, so that the recombinant proteins are purified by utilizing NI2+ -NTA column affinity chromatography under the denaturation condition, and the result is shown in figure 20, and the destination bands of 44kDa, 28kDa, 29kDa and 25kDa can be obtained when the imidazole concentration is 500 mmol/L, which is consistent with the expected result.
The four purified proteins are used as antigens for Western blot identification, the specific operation is the same as 1.1.3.4, the results are shown in figures 21 and 22, and target bands appear at 44kDa, 28kDa, 29kDa and 25kDa, and the target bands are consistent with the expected results.
2.2 preparation and identification of Duck anti-DHAV-3 VP3 polyclonal antibody
2 SPF female ducks with the age of 6 months are purchased, blood is collected by vein before immunization, and serum is separated to serve as a negative control of the experiment. The immunization route and the immunization dose are equal to 1.2, the immunization interval is 10d, the heart is sampled 2 weeks after the 3 rd immunization, and the split charging serum is stored at-70 ℃.
And taking purified r-DHAV-3-VP3 protein and r-DHAV-3-VP 3-1-3 protein as antigens, taking 1:5000 diluted duck anti-DHAV-3-VP 3 polyclonal antibody as primary antibody, and 1:2000 diluted goat anti-duck IgG (H+L) marked by HRP as secondary antibody, and carrying out Western blot identification. The results are shown in FIG. 23, which shows that the preparation of the duck anti-DHAV-3-VP 3 polyclonal antibody can recognize three-segment truncated proteins of r-30a-DHAV-3-VP 3-1-3.
2.3 Identification of the protective antigen dominant region of the DHAV-3VP3 protein
25 SPF ducks of 5 days old were randomly divided into 5 groups of PBS control group, r-32a-DHAV-3-VP3-1 group, r-32a-DHAV-3-VP3-2 group, r-32a-DHAV-3-VP3-3 group, 5 groups each, the same immunization program as 1.3, and the immunoprotection effect of DHAV-3VP3 and truncated protein was evaluated.
After 1-2 d of toxin is attacked, typical clinical symptoms of DHAV-3 infection appear in the duckling in the PBS control group, and the duckling mainly appears as leg weakness, aversion to lying down, appetite loss, 4 d after toxin attack begin to die successively, and 5 d all die. The r-32a-DHAV-3-VP3 immune group has good appetite and mental state of most duckling, the death phenomenon starts to appear at 11 d after the toxicity attack, and the death rate at 21 d after the toxicity attack is 50%. The appetite of 7 d parts of duckling in the r-32a-DHAV-3-VP3-1/-2/-3 immune group is reduced after the virus is attacked, the duckling is coiled in corners to be tremble, death occurs in 10d after the virus is attacked, and the death rate of 12 d after the virus is attacked in the r-32a-DHAV-3-VP3-3 immune group is 100%; 14 d mortality after challenge of the r-32a-DHAV-3-VP3-1 immunogroup is 100%; 21 d mortality after challenge of the r-32a-DHAV-3-VP3-2 immunogroup is 66.7%; as shown in FIG. 24, the ducklings of the PBS control group were examined for punctate bleeding of the liver, fragile, and no lesions were found in the liver of the ducklings of the r-DHAV-3-VP3 immunized group. The survival rates of duckling in the different groups are shown in fig. 25 and table 5. In conclusion, the r-DHAV-3-VP3 immune group provides the highest immunoprotection, the protection rate is 50%, and the r-DHAV-3-VP3-2 immune group has the protection rate of 33.3%, and other truncated proteins have no immunoprotection, but can delay the death time compared with the control group. The above results demonstrate that DHAV-3-VP3 and the truncated protein DHAV-3-VP3-2 (81-183 aa) stimulate the duckling to produce neutralizing antibodies after immunization of the duckling to provide immunoprotection against lethal virus challenge.
EXAMPLE 3 identification of the protective antigen dominant region of DHAV-3VP0
3.1 Prokaryotic expression of DHAV-3-VP0 gene and truncated gene thereof
3.1.1 Cloning of DHAV-3VP0 Gene and its truncated Gene
The antigenicity of DHAV-3VP0 was analyzed by DNA star software, and VP0 was divided into VP0-1 (1-63 aa), VP0-2 (50-117 aa), VP0-3 (107-173 aa), and VP0-4 (158-256 aa) 4 segments. Extracting RNA of DHAV-3 GD strain infected duck embryo allantoic fluid, synthesizing cDNA by using oligo dT as a reverse transcription primer, and performing a reverse transcription system according to a reverse transcription kit instruction. The DHAV-3-VP0 gene and its truncated gene were cloned using the primers shown in Table 6, using the obtained cDNA as a template.
Note that:GAATTCis thatEcoR I cleavage site;CTCGAGis thatXhoI, enzyme cutting site; ATG is the start codon and TAA is the stop codon. The primers are synthesized by Beijing Liuhua big Gene science and technology Co.
The fragments obtained by PCR amplification were subjected to 1% agarose gel electrophoresis, and as shown in FIG. 26, the bands of interest were visible at 788bp, 198 bp, 223bp and 222 bp, which were consistent with the expected results.
3.1.2 Construction of prokaryotic expression vector of DHAV-3VP0 and truncated Gene
The purified PCR products were digested with EcoR I and Xho I to collect the target fragment, and DH 5. Alpha. Competent cells were transformed after ligation to construct pET-30a-DHAV-3-VP0 and pET-32a-DHAV-3-VP0-1, pET-32a-DHAV-3-VP0-2, pET-32a-DHAV-3-VP0-3 and pET-32a-DHAV-3-VP0-4 recombinant plasmids, respectively, and the obtained recombinant plasmids were identified by single and double digestion with EcoR I and Xho I, as shown in FIGS. 27, 28 and 29, and the digestion results were consistent with the expected results. And (3) sending the monoclonal bacterial liquid identified as positive to Beijing Liuhua large gene technology Co.Ltd for sequencing, wherein the sequencing result shows that the target gene sequence is consistent with the original sequence.
3.1.3 Prokaryotic expression and identification of DHAV-3VP0 gene and truncated gene thereof
3.1.3.1 Prokaryotic expression of DHAV-3VP0 Gene
The recombinant protein expressed by Rosetta-pET-30A/32a-DHAV-3-VP0 is expressed in a natural form by IPTG induction expression 4 h and by 12% SDS-PAGE analysis, and the size of the recombinant protein expressed by Rosetta-pET-30A-DHAV-3-VP0 is 39 kDa as shown in FIG. 30A; the recombinant protein expressed by pET-32a-DHAV-3-VP0 is expressed in the form of inclusion bodies. As shown in FIG. 30B, the recombinant protein expressed by pET-32a-DHAV-3-VP0 was 49 kDa in size, which was consistent with the expectations.
The Rosetta-pET-32a-DHAV-3-VP 0-1-4 is induced to express for 4 h by IPTG, 12% SDS-PAGE analysis shows that truncated genes of the DHAV-3-VP 0-1-4 segments are all expressed, as shown in a graph 31A, B, C, D, the recombinant protein sizes are 25 kDa, 35 kDa, 24 kDa and 29 kDa respectively, and the recombinant protein sizes are consistent with the expected sizes, wherein r-32a-DHAV-3-VP 0-1/-3 are all expressed in the form of inclusion bodies; the r-32a-DHAV-3-VP0-2/-4 is expressed in natural form.
3.1.3.2 Purification and identification of DHAV-3VP0 and truncated proteins
According to the expression form of the protein, the recombinant protein is purified by utilizing NI2+ -NTA column affinity chromatography in a natural form or an inclusion body form, and the recombinant r-30a/32a-DHAV-3-VP0 can be eluted respectively when the imidazole concentration is 250 mmol/L, so that target bands of 39 kDa and 49 kDa are obtained, and the target bands are consistent with the expected result; as shown in FIG. 32, recombinant proteins r-32a-DHAV-3-VP 0-1-4 can be eluted at an imidazole concentration of 250 mmol/L or 500 mmol/L, respectively, to obtain target bands of 25 kDa, 35 kDa, 24 kDa and 29 kDa, which are consistent with the expected results.
And performing Western blot identification by taking the purified DHAV-3-VP0 and truncated protein as antigens. The specific procedure is shown in FIG. 33, which shows that bands appear at the destination bands of 39 kDa, 49 kDa, 25 kDa, 35 kDa, 24 kDa and 29 kDa, the detected bands being single and of a size consistent with the expected results.
3.2 preparation and identification of Duck anti-DHAV-3-VP 0 polyclonal antibody
Two SPF ducks of 6 months old were immunized with the purified r-30a-DHAV-3-VP0 protein emulsified with Freund's adjuvant via chest muscle, collected and serum was isolated as negative serum before first-time. Immunization program is same as 1.2, after three-free 9, 12, 15 d vein blood sampling, heart blood sampling, split charging serum storing at-70 ℃.
And taking purified r-32a-DHAV-3-VP0 and r-32a-DHAV-3-VP 0-1-4 proteins as detection antigens, taking a 1:1000 diluted duck anti-DHAV-3-VP 0 polyclonal antibody as a primary antibody, and taking a 1:2000 diluted goat anti-duck IgG (H+L) marked by HRP as a secondary antibody, and carrying out Western blot identification. The results are shown in FIG. 34, which shows that the preparation of the duck anti-DHAV-3-VP 0 polyclonal antibody can recognize r-32a-DHAV-3-VP 3-1-4 four-segment truncated protein.
3.3 Immunoprotection assay of DHAV-3-VP0 protein and truncated protein
30 SPF ducks with the age of 5 days are randomly divided into 6 groups such as PBS control group, adjuvant control group, r-30a-DHAV-3-VP0 group, r-32a-DHAV-3-VP01-1 group, r-32a-DHAV-3-VP0-2 group, r-32a-DHAV-3-VP0-3 group, r-32a-DHAV-3-VP0-4 group and the like, wherein 5 immune programs are 1.3, and the immune protection effect of DHAV-3-VP0 and truncated protein is evaluated.
After 1-2 d of toxin is attacked, typical clinical symptoms of DHAV-3 infection appear in the duckling in the PBS control group, the duckling is mainly characterized by leg weakness, aversion to lying down, appetite loss, 4 d begins to die successively after toxin is attacked, and 5 d all die. The r-30a-DHAV-3-VP0 immune group has good appetite and mental state of most duckling, the death phenomenon starts to appear at 11 d after the toxicity attack, and the death rate at 21 d after the toxicity attack is 50%. The r-32a-DHAV-3-VP0-4 immune group has the advantages that after the virus is attacked, the appetite of part of duckling 5 d is reduced, the duckling is not happy, death phenomenon begins to appear at 7 d after the virus is attacked, and 11 d all dies after the virus is attacked; the disease incidence speed of the r-32a-DHAV-3-VP0-3 immune group is lower than that of the r-DHAV-3-VP0-4 immune group, the death speed is higher than that of the r-DHAV-3-VP0-4 immune group, the clinical symptoms are almost the same, death begins to occur at 8 d after the virus attack, and all the 10 d deaths after the virus attack; the disease incidence speed of the r-32a-DHAV-3-VP0-1 and the r-32a-DHAV-3-VP0-2 immune group is the same, duckling in 8 d parts after the virus attack acts slowly, white thin feces are discharged, death begins to occur in 10 d parts after the virus attack, 13 d parts after the r-32a-DHAV-3-VP0-1 immune group attacks the virus all die, and the 21 d death rate after the r-DHAV-3-VP0-2 immune group attacks the virus is 66.7%; as shown in FIG. 35, ducklings of the PBS control group were examined by dissection, and the liver of the r-DHAV-3-VP0 immunized group was found to have bleeding points, whereas the liver of the ducklings was found to be free of lesions. The survival rates of different groups of ducklings are shown in fig. 36 and table 7. In conclusion, the r-30a-DHAV-3-VP0 immune group provides the highest immunoprotection, the protection rate is 50%, the r-32a-DHAV-3-VP0-2 immune group provides the protection rate of 33.3%, and other truncated proteins have no immunoprotection, but can delay the death time compared with the control group. The above results demonstrate that DHAV-3-VP0 and the truncated protein DHAV-3-VP0-2 stimulate duckling to produce neutralizing antibodies after immunization of duckling to provide immunoprotection against lethal amounts of virus. DHAV-3-VP0-2 is the protective antigen presenting region of DHAV-3-VP 0.
EXAMPLE 4 preparation of the protective antigen-dominant region tandem antigen of DHAV-3 structural protein and immunoprotection evaluation
4.1 Tandem expression and identification of the protective antigen-dominant region of the structural protein DHAV-3
4.1.1 DHAV-3-VP codon optimization
The protective antigen dominant regions DHAV-3-VP1-3 (90-175 aa), DHAV-3-VP3-2 (81-183 aa) and 39-177aa (containing DHAV-3-VP0-2, 50-117 aa) of the identified DHAV-3 GD strain (GQ 122332.1) are connected in series through Linker (Gly 4 Ser) 3, the 5 '-end of the DHAV-3-VP3-2 is introduced with BamHI cleavage site, the 3' -end is introduced with Xho I cleavage site, and the serial genes are named DHAV-3-VP as shown in FIG. 37. Then optimizing the gene sequence of the encoded concatemer amino acid according to the codon preference of the escherichia coli, replacing rare codons and low-frequency codons in the concatemer gene with the escherichia coli preferred codons, and not changing the encoded amino acid of the DHAV-3-VP concatemer gene, wherein the VP gene contains 358 codons, and totally optimizes 229 codons, accounting for 63.97 percent of the number of the codons. The optimized VP gene (optiVP) sequence is shown as SEQ ID NO.1, the optiVP gene is synthesized by Beijing Liuhua big gene technology Co., ltd, the gene is subcloned into a prokaryotic expression vector pET-30a, a recombinant expression plasmid is obtained, and the plasmid is named pET-30a-DHAV-3-VP.
4.1.2 Identification of DHAV-3-VP recombinant expression plasmid
The recombinant expression plasmid pET-30a-DHAV-3-VP was identified by single and double digestion with BamH I and Xho I, as shown in FIG. 38, and the results were consistent with the expected results.
4.1.3 Prokaryotic expression of DHAV-3-VP recombinant protein
pET-30a-DHAV-3-VP converts Rosetta (DE 3) pLysS competent cells, and recombinant proteins expressed by Rosetta-DHAV-3-VP are expressed in the form of inclusion bodies by IPTG induction and SDS-PAGE analysis. As shown in FIG. 39, the sizes were all 47.4 kDa, which is consistent with the expectations.
4.1.4 purification and identification of recombinant proteins
The target band of 47.4 kDa was obtained by purifying r-30a-DHAV-3-VP using NI2+ -NTA column affinity chromatography at an imidazole concentration of 500 mmol/L. While the purified r-30a-DHAV-3-VP1-3, r-32a-DHAV-3-VP3-2, and r-32a-DHAV-3-VP0-2 were used as controls, the corresponding destination bands were seen at 18.2 kDa, 30 kDa, and 35 kDa, as shown in FIG. 40, all in agreement with the expected results. Western blot identification was performed on the purified proteins as antigens according to 1.1.3.4, and bands appeared at the target bands of 47.4 kDa, 18.2 kDa, 30 kDa and 35 kDa, as shown in FIG. 41, and the detected bands were single and the sizes were consistent with the expected results.
4.1.5 Western blot identification of immunoreactivity of DHAV-3-VP protein
Meanwhile, purified r-30a-DHAV-3-VP, r-30-DHAV-3-VP1-3, r-32a-DHAV-3-VP3-2 and r-32a-DHAV-3-VP0-2 are taken as detection antigens, and duck anti-DHAV-3 positive serum is taken as a primary antibody, so that Western blot identification is carried out, as shown in FIG. 42. The results show that all the 4 recombinant proteins can react with duck anti-DHAV-3 positive serum, wherein the reactivity of DHAV-3-VP is strongest, the reactivity of DHAV-3-VP1-3 is obviously higher than that of other 2-segment truncated proteins, the reactivity of DHAV-3-VP0-2 is weakest, the recombinant proteins which serially express 3 antigen dominant regions are more reactive than each antigen dominant region, and the reactivity of DHAV-3-VP1-3 is strongest in the 3 antigen dominant regions.
4.2 Immunoprotection evaluation of tandem antigen of protective antigen dominance region of DHAV-3 structural protein
4.2.1 Toxicity attack protection experiment of DHAV-3-VP protein on duckling
33 SPF ducks of 5 days old were randomly divided into PBS control group, r-30a-DHAV-3-VP and duck viral hepatitis refined yolk antibody (LY-20 strain) use group 3 group, wherein r-30a-DHAV-3-VP and yolk antibody use group were immunized with leg muscle with r-DHAV-3-VP of 0.2 mg and duck viral hepatitis refined yolk antibody (LY-20 strain) of 0.5 mL, PBS group was injected with an equal volume of sterilized PBS, and virus was challenged after immunization with 10 d, thereby evaluating immunoprotection of DHAV-3-VP. After 1-4 d of detoxification, typical clinical symptoms of infection of DHAV-3 appear in duckling of PBS control group, which are mainly manifested by slow reaction and anorexia, 5 d begins to die successively after detoxification, and 7 d die completely after detoxification; the r-30a-DHAV-3-VP immune group and the yolk antibody have good mental state, and death begins to occur at 16 d and 17 d after the virus attack, and the death rate at 21 d after the virus attack is 18%. As shown in FIG. 43, the duckling in the PBS control group was examined by a dissecting method, and the liver was swollen and bleeding, and the liver was fragile, whereas the liver of the duckling in the r-DHAV-3-VP immunized group was not diseased. The survival rates of different groups of ducklings are shown in fig. 44 and table 8. In summary, the immune group of r-DHAV-3-VP and the yolk antibody use group provided consistent immunoprotection with a protection rate of 82%.
4.2.2 Histopathological analysis of DHAV-3-VP protein and yolk antibody immunized duckling
PBS control group, r-DHAV-3-VP immune group and yolk antibody use group were collected respectively to death the liver tissue of duckling, and tissue sections were prepared by 10% formalin fixation, and HE staining was performed for histopathological observation. As shown in fig. 45A, the result shows that the PBS control group death duckling has the most serious liver histopathological change, mainly manifested by the loss of liver tissue basic structure, massive hepatocyte degeneration necrosis and severe hyperemia and hemorrhage; as shown in fig. 45B, the liver histopathological changes of the egg yolk antibody-used group were less compared to the PBS control group, and some cells were vacuolated; as shown in FIG. 45C, the r-DHAV-3-VP immunogroup livers had no obvious histopathological changes, the liver lobule structure was complete, and the hepatocytes were aligned.
4.2.3 RT-qPCR (reverse transcription-quantitative polymerase chain reaction) detection of liver and spleen tissues of duckling and cloaca swab viral load
And collecting liver and spleen tissues and cloaca swabs of the final dead duckling of each group, and extracting RNA according to the TRIzol reagent instruction. Reverse transcription was performed to synthesize cDNA with reference to the M-MLV reverse transcriptase instructions. qPCR was performed according to the primer concentration recommended by NovoStart SYBR qPCR Super Mix Plus instruction, and the reaction system was 20. Mu.L. According to RT-qPCR results, ct values of a DHAV-3 virus control group and an immune group can be obtained, the Ct values are substituted into a standard curve established in the laboratory, and the logarithm of the virus copy number is obtained, wherein a specific formula is y=3.0225x+35.142, and a correlation coefficient is R2= 0.9932. Statistical analysis is carried out on the data obtained by the test through Graph Pad Prism 6 software by utilizing a Two-way ANOVA method, and the difference significance expression result is as follows: nsP >0.05 is not significant, P <0.05 is significant, P <0.01 is very significant, P <0.001 is very significant, and P <0.0001 is very significant.
The virus load detection results of the liver and spleen tissues DHAV-3 of the duckling after the virus attack are shown in figures 46, 47 and 48, and the results show that the general change trend of the virus load of the liver and spleen tissues of the duckling and the cloaca swab is consistent, and the virus load of the duckling in a control group is obviously higher than that of the duckling in an immune group, and the copy number can reach 106.1 copies/mug cDNA, 104.5 copies/mug cDNA and 106.2 copies/mug cDNA; among them, the r-30a-DHAV-3-VP immunogroup and the egg yolk antibody used the group viral load were not different, but only 101.2 copies/. Mu.g cDNA, 100.5 copies/. Mu.g cDNA and 101.9 copies/. Mu.g cDNA.
4.2.4 Determination of serum neutralization titers of surviving ducklings after toxicity removal of DHAV-3-VP protein immune group
After the toxicity attack protection experiment is finished, the surviving duckling after the r-DHAV-3-VP immune group is subjected to toxicity attack is subjected to heart blood collection, serum is separated, diluted and mixed with the DHAV-3 duck embryo homogenate of 200 ELD50, and after incubation, duck embryos are inoculated, and the continuous observation record is 120 h. The death condition of each group of duck embryos is counted, the neutralization titer is calculated to be 1:48 by adopting a Reed-Muench method, namely, the serum of the surviving duckling after the DHAV-3-VP protein is immunized and detoxified is diluted by 1:48, 50% of duck embryos can be prevented from dying due to the infection of the DHAV-3, and specific data are shown in Table 9.
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Claims (10)
1. A recombinant immunogen for serially expressing a protective antigen dominance region of a duck hepatitis A virus (Duck Hepatitis A Virus, DHAV) 3 type structural protein is characterized in that the recombinant immunogen is obtained by serially connecting 90-175 aa protective antigen dominance region of structural protein VP1 of DHAV-3 GD strain, 81-183 aa protective antigen dominance region of structural protein VP3 and 39-177aa amino acid sequence of VP0 through flexible Linker, wherein the 39-177aa amino acid sequence of VP0 contains 50-117 aa protective antigen dominance region of structural protein VP 0.
2. The recombinant immunogen according to claim 1 wherein the Linker sequence is Gly4SerGly4Ser.
3. The recombinant immunogen according to claim 1, wherein the amino acid sequence of said recombinant immunogen is shown in SEQ ID No. 2.
4. A polynucleotide encoding the recombinant immunogen of any one of claims 1-3.
5. The polynucleotide of claim 4, wherein the polynucleotide has a sequence as set forth in SEQ ID No. 1.
6. A prokaryotic expression vector comprising the polynucleotide of claim 4 or 5.
7. The prokaryotic expression vector of claim 6, wherein said prokaryotic expression vector is pET-30a.
8. Use of a recombinant immunogen according to any one of claims 1-3 for the preparation of a vaccine for the prevention of duck hepatitis a virus type 3 infection.
9. The use of claim 8, wherein the vaccine is a multi-epitope vaccine.
10. A duck hepatitis a virus type 3 multi-epitope vaccine, characterized in that the vaccine comprises the recombinant immunogen of any one of claims 1-3.
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