CN117964717A - CAstV-ORF2 recombinant adenovirus vector live vaccine and application thereof - Google Patents
CAstV-ORF2 recombinant adenovirus vector live vaccine and application thereof Download PDFInfo
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- CN117964717A CN117964717A CN202410225796.4A CN202410225796A CN117964717A CN 117964717 A CN117964717 A CN 117964717A CN 202410225796 A CN202410225796 A CN 202410225796A CN 117964717 A CN117964717 A CN 117964717A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10041—Use of virus, viral particle or viral elements as a vector
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Abstract
The invention discloses a CAstV-ORF2 recombinant adenovirus vector live vaccine and application thereof, and belongs to the technical field of vaccines. According to the invention, the CAstV-ORF2 capsid protein sequence is analyzed and screened, 388aa-455aa regions with low antigenicity in the sequence are deleted, and Linker connecting arms (aa: GGGGS) are added for connection, so that the amino acid sequence of the CAstV-ORF2 recombinant protein is obtained. The rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine prepared by utilizing the target gene for encoding the recombinant protein has increased antigenicity and stronger protectiveness. The rAd5-CastV-ORF2 recombinant adenovirus vector live vaccine prepared by the invention can effectively stimulate the immune response of organisms and has stronger protectiveness, and provides a new means for preventing and controlling chicken astrovirus infection, thereby having wide development and application prospects.
Description
Technical Field
The invention relates to the technical field of vaccines, in particular to a CAstV-ORF2 recombinant adenovirus vector live vaccine and application thereof.
Background
Chicken astrovirus (CAstV) belongs to the family picornaviridae, with a diameter of 25-30 nm, and is a non-envelope single-stranded positive strand RNA virus. CastV is one of important pathogens capable of independently causing development retardation syndrome, and the sick chickens mainly show symptoms such as diarrhea enteritis, gout and the like, and can cause diseases such as chick nephritis and the like. Early CAstV and other enterovirus infections may create an abnormal intestinal environment, causing subsequent dysbacteriosis, an imbalance in natural colonisation by bacteria and viruses, and may result in impaired barrier protection of the intestinal tract due to reduced nutrient digestibility, further affecting growth performance. As a virus that infects chicken flocks by the faecal route, CAstV not only infects the intestinal tract, but also often causes infections of organs outside the intestinal tract such as the kidneys and liver. White chicken syndrome caused by infection with CAstV can have a severe impact on chicken hatching. The hatched chicken feather is pale, weak and listlessness, and the chicken will die after hatching, and the kidney and liver of the sick chicken will be anatomically shown to have lesions. The death rate of the infected embryo is high, and the hatching rate is greatly reduced.
The CAstV genome consists of a 5 'untranslated region (UTR), three open reading frames (ORFs: ORF1a, ORF1b and ORF 2), a 3' UTR and a Poly-A (Poly A) tail, approximately 7.2kb in length. ORF1a and ORF1b are involved in encoding nonstructural proteins involved in RNA transcription and replication (nonstructural protein, NSP), and the ORF2 gene encodes the capsid protein of the astrovirus (Cap protein, capsid protein) and is involved in viral pathogenicity. Cap protein is the main structural protein of astrovirus, has higher diversity, and interacts with a host to mediate virus to invade cells, stimulate the organism to generate antibodies and prevent virus infection. In addition, this protein can bind to the relevant receptor on the surface of host cell and induce immune response, and is the main antigenic determinant protein of virus.
In recent years, infection of the CastV and related clinical and pathological characteristics are reported at home and abroad, serious influence is generated on the development of the poultry industry, and research of Lee et al shows that the CastV recombinant capsid protein can generate a certain protective effect on offspring.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a CAstV-ORF2 recombinant adenovirus vector live vaccine and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a CAstV-ORF2 recombinant protein is provided, and the amino acid sequence of the CAstV-ORF2 recombinant protein is shown as SEQ ID NO. 3.
The amino acid sequence of the CAstV-ORF2 recombinant protein is obtained by deleting 388aa-455aa regions with low antigenicity in the amino acid sequence of the CAstV-ORF2 capsid protein, and adding a Linker connecting arm (aa: GGGGS) for connection.
In a second aspect of the invention, a target gene for encoding the CAstV-ORF2 recombinant protein is provided, and the nucleotide sequence of the target gene is shown as SEQ ID NO. 4.
In a third aspect, the invention provides the use of a recombinant CAstV-ORF2 protein or a gene of interest encoding a recombinant CAstV-ORF2 protein in the manufacture of a vaccine for the prevention of chicken astrovirus.
The vaccine for preventing chicken astrovirus is recombinant adenovirus vector live vaccine.
The fourth aspect of the invention provides a rAD5-CAstV-ORF2 recombinant adenovirus vector live vaccine, which is prepared by the following steps:
(1) 3 xFlag tag is added to the 3' end of the target gene for encoding the CAstV-ORF2 recombinant protein, and the target gene is cloned to a vector pcADV-EF1-CMV-MCS inserted with fluorescent protein mNeonGreen gene to obtain recombinant pcADV-EF1-mNeonGreen-CMV-CAstV-ORF2-3 xFLAG shuttle plasmid;
(2) And (3) co-transfecting the shuttle plasmid obtained in the step (1) and the adenovirus skeleton vector into 293T cells for packaging, and recombining to obtain rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine.
The adenovirus skeleton vector is a human replication defective adenovirus type 5 vector.
The virus titer of the rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine is more than or equal to 6.32x10 10 (pfu/mL).
In a fifth aspect, the invention provides an application of the rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine in preparing a biological product for preventing chicken astrovirus infection.
The invention has the beneficial effects that:
(1) According to the invention, the CAstV-ORF2 capsid protein sequence is analyzed and screened, 388aa-455aa regions with low antigenicity in the sequence are deleted, and Linker connecting arms (aa: GGGGS) are added for connection, so that the amino acid sequence of the CAstV-ORF2 recombinant protein is obtained. The antigenicity of the CAstV-ORF2 recombinant adenovirus vector live vaccine prepared by utilizing the target gene for encoding the recombinant protein is increased, so that the protection effect on animals infected by the chicken astrovirus is better improved.
(2) When the CAstV-ORF2 recombinant adenovirus vector live vaccine is prepared, a 3 xFlag tag is added at the 3' end of a synthesized target gene for encoding the CAstV-ORF2 recombinant protein, a fluorescent protein mNeonGreen gene is cloned to a shuttle vector, and a seamless cloning technology is utilized to clone the target gene for encoding the CAstV-ORF2 recombinant protein to the shuttle vector, so that a pcADV-EF1-mNeonGreen-CMV-CAstV-ORF2-SDAU-3 xFLAG shuttle plasmid is obtained. The pcADV-EF1-mNeonGreen-CMV-CastV-ORF2-SDAU-3 xFLAG shuttle plasmid and the human replication defective adenovirus type 5 skeleton vector are co-transfected into 293T cells by utilizing AdMax systems for packaging, and the recombinant adenovirus vector live vaccine of the CastV-ORF2 is obtained by recombination, and the vaccine prepared by the method provided by the invention has high safety, can induce the production of cytokines and chemokines, and the human replication defective adenovirus type 5 vector has an adjuvant effect and does not need to be added with any adjuvant. The rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine prepared by the invention can adopt intramuscular injection or oral immunization and other immunization approaches, is convenient to use and has good market competitiveness.
Drawings
FIG. 1 is a graph of the analysis of the transmembrane region, signal peptide and hydrophilicity of the capsid protein of CAstV-ORF 2; wherein A is: and (3) a transmembrane analysis result, wherein B is a signal peptide sequence detection result, and C is a hydrophilicity analysis result.
FIG. 2 is a graph of antigenic analysis of the CAstV-ORF2 capsid protein.
FIG. 3 is a full gene map of the recombinant CAstV-ORF2 shuttle plasmid.
FIG. 4 is a diagram showing the identification of recombinant CAstV-ORF2 shuttle plasmid KPNI by single restriction enzyme. 1, plasmid, 2, KPNI single enzyme cutting, M, 10000bp Marker.
FIG. 5 is a transfection of recombinant shuttle plasmid in 293T cells (100X); a, B is a white light image of 293T cells transfected with recombinant shuttle plasmids; C. d green fluorescence image of 293T cells transfected with recombinant shuttle plasmid.
FIG. 6 is a graph of recombinant adenovirus titer at various dilutions (100X).
FIG. 7 is a graph showing the result of detecting the expression of ORF2 protein in rAD5-CastV-ORF2 by Western Blot.
FIG. 8 is a graph of body weight measurements of groups of chickens in an immunogenicity study in a clinical trial.
FIG. 9 is a graph showing the growth patterns of the antibodies of each group of chickens in the immunogenicity test in the clinical test.
FIG. 10 is a graph showing cytokine detection by each group of chickens in an immunogenicity experiment in a clinical experiment.
Figure 11 is a graph of body weight measurements of groups of chickens in an animal protection study in a clinical trial.
Fig. 12 is a view of pathological tissue sections of groups of chickens in animal protection experiments in clinical experiments.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As mentioned above, the chicken astrovirus has great harm to the growth and development of chicken, and seriously affects the development of the Chinese poultry industry. The vaccines for chicken astrovirus prevention and treatment disclosed in the prior art mainly comprise attenuated live vaccines and inactivated vaccines. The adenovirus vector vaccine has high safety and is easier to realize industrialization in terms of productivity, but no commercial adenovirus vector vaccine for chicken astrovirus prevention and treatment exists at present.
Based on the above, the invention develops and designs a rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine, firstly, the sequence analysis is carried out on the CastV-ORF2 capsid protein, the existence of a redundant segment of the capsid protein is found, the antigenicity of the amino acid sequence 388aa-455aa region is low, and in order to improve the protective effect of preparing the vaccine, firstly, the amino acid sequence of the CastV-ORF2 capsid protein is modified, the region with low antigenicity is deleted, and two sections of amino acid sequences obtained after deleting the region with low antigenicity are connected by using a Linker connecting arm (aa: GGGGS), so as to obtain the CastV-ORF2 recombinant protein. Then adding 3 xFlag label on the 3' end of target gene of coding CAstV-ORF2 recombinant protein, cloning it on carrier pcADV-EF1-CMV-MCS inserted with fluorescent protein mNeonGreen gene to obtain recombinant pcADV-EF1-mNeonGreen-CMV-CAstV-ORF2-3 xFLAG shuttle plasmid, co-transfecting the obtained shuttle plasmid and adenovirus skeleton carrier into 293T cell for packaging, and recombining to obtain rAD5-CAstV-ORF2 recombinant adenovirus carrier living vaccine.
In the process of constructing the adenovirus vector live vaccine, the inventor also verifies the immune protection effect of the adenovirus vector live vaccine constructed by inserting different target genes for encoding CAstV-ORF2 proteins on chicken flocks through experiments, sets 1-3 groups of treatment, and only replaces the target fragment inserted in the process of constructing the shuttle plasmid with the target gene for encoding CAstV-ORF2 capsid protein 1aa-387aa region in the process of preparing the rAD5-CAstV-ORF2 recombinant adenovirus vector live vaccine in the treatment 1 group. Treatment of group 2 only the target fragment inserted during the construction of the shuttle plasmid in the preparation of rAd5-CastV-ORF2 recombinant adenovirus vector live vaccine was replaced with the target gene encoding the region 456aa-739aa of the capsid protein of CastV-ORF 2. And (3) treating the 3 groups, and only replacing the target fragment inserted in the process of constructing the shuttle plasmid in the process of preparing rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine with the target gene encoding the CastV-ORF2 capsid protein (SEQ ID NO. 1). The vaccine group is rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine constructed by using a target gene encoding the CAstV-ORF2 recombinant protein.
Experiments were then performed with 403 d-aged SPF chickens, each group vaccinating 3 d-aged SPF chickens with each group of prepared rAD5-CAstV-ORF2 vaccine at a dose of 10 9 PFU/mL 100. Mu.L; 200 μL of 10 4TCID50 laboratory-stored CAstV virus solution was intraperitoneally injected into each group of SPF chickens at 7d years of age, and two weeks after inoculation was completed, the chickens were observed for onset.
The experimental results show that: the protection rate of the adenovirus vector live vaccine prepared in the treatment group 1 to the chicken flock is 41%, the protection rate of the adenovirus vector live vaccine prepared in the treatment group 2 to the chicken flock is 43%, the protection rate of the adenovirus vector live vaccine prepared in the treatment group 3 to the chicken flock is 80%, and the protection rate of the rAD5-CAstV-ORF2 recombinant adenovirus vector live vaccine prepared in the vaccine group to the chicken flock is 85%, so that the rAD5-CAstV-ORF2 recombinant adenovirus vector live vaccine constructed by the target gene for encoding the CAstV-ORF2 recombinant protein has a synergistic effect on the protection effect of the chicken flock. Treatment 3 adenovirus vector vaccines constructed using the gene of interest encoding the CAstV-ORF2 capsid protein gave lower protection rates to chicken flocks than the vaccine group, as demonstrated by the set up of treatment 3: compared with the full-length CASTV-ORF2 protein sequence serving as an immunogen, the recombinant CASTV-ORF2 protein sequence serving as the immunogen is improved, so that the immune response capability of an organism is improved on the premise of injecting an equivalent adenovirus vector live vaccine, the immune effect of the prepared adenovirus vector live vaccine is improved to a certain extent, the length of a target gene for encoding the recombinant CASTV-ORF2 protein sequence is shorter than that of a target gene for encoding a CASTV-ORF2 capsid protein, and the stability of the recombinant CASTV-ORF2 capsid protein inserted into a vector is improved.
The following describes the embodiments of the present application in further detail with reference to examples. The following detailed description is illustrative and is not intended to provide further explanation of the application, but is not intended to limit the scope of the application. The raw materials and reagents used in the following examples are commercially available unless otherwise specified.
Example 1: construction of a shuttle plasmid for the CAstV-ORF 2-target gene.
The sequence analysis was performed on the CAstV-ORF2 capsid protein (GenBank: ORF2 sequence in OR 286520) by TMHMM SERVER, SIGNALP 4.1.1 Server, protScale, respectively. The full length of the amino acid sequence of the CAstV-ORF2 capsid protein is 739aa, and the amino acid sequence is specifically shown in SEQ ID NO. 1:
MADKAGPQKKRVSRRGRGRSRSRSRSRSRSRNRVKKTVTIVETKKTPSKSILKKELENHERKDRKRFRKIEKKLNGPKIHDRMAVTTTLGVLTGNSDNNLERKMRALLNPLLLKSQNTGASASPLSLRASQYSMWKIQKCVVKFVPLVGAANVAGSVSFVSLDQDATSSQPESPDTIKAKVHAEVSIGQRFNWSIQSRYLVGPRSGWWGMDTGESPTDTVGPALDFWNLYRTVNTLQTGSTSQAYIAPLFSIEVFTVYVFSGYEPKPALATMTNSTFESQQGVTITNGSNGELLLNVPQQSGLAERLREKEVPQRGQNQAGGVGEVLWAVASGAVEGAAEALGPWGWLLRGGWWVIKKIFGQSSENANDVYVMYSSIEDANKDSRIYQTVSGTVPVQQGPLVLTQISAPNVNQAGGVVQVGVPVTTDYLPLSQAQVPLLENILYSNTGQPVTSNKSHTMRLTGFPASKMVTSTSSQWLGTADKSIQATKWLMSDYTDTGVIFGFPYSNETPGETLGNIGVIHTAKSLLKTVISRHQRRLSMTPLNSTPIPAASRGPNQMRGCFDTPYYWIRVCDNMCSNKPTNGPVTHRYDAWGIMVVSVIHNKVYVLAGYPDNQNTPSKQQMVWDTFNWDDTFPTGRIYYTTWPGVEQENEDETDADSDISSLFDPVNEVENDFHFQCSLKTSDYLKEEADFWKAKAQQLLMEKAMEKPGTNPPLVCLGEGGIEQQKQPASSRGHAE
The transmembrane analysis results show that the amino acid sequences are all outside the membrane (figure 1A), NO signal peptide sequences (figure 1B) and good hydrophilicity (figure 1C), the immunogenicity is low in 388aa-455aa region (68 amino acids total) through DNAstar software (figure 2), and the amino acid sequences with low antigenicity in the CAstV-ORF2 capsid protein are deleted, and the amino acid sequences in the deleted CAstV-ORF2 capsid protein are shown as SEQ ID NO: 2:
QTVSGTVPVQQGPLVLTQISAPNVNQAGGVVQVGVPVTTDYLPLSQAQVPLLENIL YSNTGQPVTSNK
After deletion, the amino acid sequence of the residual region in the CAstV-ORF2 capsid protein is added into a Linker connecting arm (aa: GGGGS) for connection to obtain the amino acid sequence of the CAstV-ORF2 recombinant protein, and the amino acid sequence of the obtained CAstV-ORF2 recombinant protein is specifically shown as SEQ ID NO: 3:
MADKAGPQKKRVSRRGRGRSRSRSRSRSRSRNRVKKTVTIVETKKTPSKSILKKELENHERKDRKRFRKIEKKLNGPKIHDRMAVTTTLGVLTGNSDNNLERKMRALLNPLLLKSQNTGASASPLSLRASQYSMWKIQKCVVKFVPLVGAANVAGSVSFVSLDQDATSSQPESPDTIKAKVHAEVSIGQRFNWSIQSRYLVGPRSGWWGMDTGESPTDTVGPALDFWNLYRTVNTLQTGSTSQAYIAPLFSIEVFTVYVFSGYEPKPALATMTNSTFESQQGVTITNGSNGELLLNVPQQSGLAERLREKEVPQRGQNQAGGVGEVLWAVASGAVEGAAEALGPWGWLLRGGWWVIKKIFGQSSENANDVYVMYSSIEDANKDSRIYSHTMRLTGFPASKMVTSTSSQWLGTADKSIQATKWLMSDYTDTGVIFGFPYSNETPGETLGNIGVIHTAKSLLKTVISRHQRRLSMTPLNSTPIPAASRGPNQMRGCFDTPYYWIRVCDNMCSNKPTNGPVTHRYDAWGIMVVSVIHNKVYVLAGYPDNQNTPSKQQMVWDTFNWDDTFPTGRIYYTTWPGVEQENEDETDADSDISSLFDPVNEVENDFHFQCSLKTSDYLKEEADFWKAKAQQLLMEKAMEKPGTNPPLVCLGEGGIEQQKQPASSRGHAE
the nucleotide sequence of the target gene for encoding the CAstV-ORF2 recombinant protein is 2028bp, and is specifically shown as SEQ ID NO. 4:
ATGGCCGATAAGGCTGGGCCGCAGAAAAAGAGAGTATCCAGGCGTGGACGTGGCCGTTCTCGATCAAGGTCACGCTCACGTTCTCGATCAAGAAATCGTGTCAAGAAAACAGTCACGATAGTTGAGACAAAAAAGACCCCAAGTAAATCAATACTAAAAAAGGAGTTAGAAAATCATGAGAGAAAAGATAGAAAGAGATTTAGGAAAATAGAGAAGAAACTAAATGGCCCAAAAATTCATGATCGCATGGCAGTTACAACTACTCTTGGTGTACTCACAGGAAATTCTGATAACAACCTAGAGAGGAAAATGAGGGCCTTGCTTAACCCATTGCTCTTGAAATCTCAGAACACTGGAGCTTCGGCGTCACCACTCTCCCTTAGAGCATCCCAATACTCAATGTGGAAGATACAAAAATGTGTGGTTAAATTCGTGCCCCTGGTAGGTGCTGCAAACGTTGCGGGCAGTGTGTCATTTGTGTCACTGGATCAAGACGCAACTTCATCACAGCCTGAGTCACCTGATACTATAAAGGCAAAGGTACATGCTGAGGTATCTATTGGCCAGAGGTTCAACTGGAGTATACAATCTAGATATTTGGTGGGACCGCGATCTGGATGGTGGGGAATGGACACCGGAGAGTCACCAACTGATACAGTTGGACCAGCACTTGACTTTTGGAATTTATATAGGACAGTCAACACATTACAGACTGGTTCAACATCACAGGCCTATATTGCTCCGCTATTTTCCATAGAAGTGTTTACTGTCTATGTTTTTTCAGGCTACGAGCCTAAGCCTGCCCTTGCAACTATGACAAATTCAACCTTTGAAAGCCAGCAGGGTGTAACTATAACAAACGGCTCAAATGGAGAACTTTTGCTTAATGTTCCACAACAGTCAGGACTTGCTGAGCGGCTGCGTGAAAAAGAAGTCCCACAACGTGGCCAGAATCAGGCAGGTGGAGTTGGAGAGGTCCTCTGGGCAGTTGCATCAGGCGCCGTGGAAGGAGCGGCAGAAGCATTGGGCCCGTGGGGTTGGCTATTAAGAGGTGGCTGGTGGGTTATAAAGAAAATCTTTGGACAGAGCTCTGAAAATGCTAATGACGTTTATGTCATGTATTCGTCTATTGAAGACGCCAATAAGGACAGTAGAATTTATGGCGGCGGCGGCAGCAGCCACACTATGAGGTTAACTGGGTTTCCTGCCTCAAAAATGGTGACATCTACATCATCACAATGGTTGGGTACTGCTGATAAGAGCATTCAAGCAACAAAGTGGCTCATGTCTGACTATACTGATACAGGAGTGATATTTGGCTTCCCATACTCTAATGAAACTCCAGGAGAGACATTAGGCAATATTGGGGTGATACATACGGCAAAATCGCTCTTAAAAACAGTGATATCACGACACCAGCGTAGGCTGAGTATGACACCACTAAATTCGACCCCAATACCAGCAGCATCGCGGGGTCCAAATCAGATGCGTGGATGTTTTGACACGCCCTATTATTGGATAAGGGTTTGTGACAACATGTGTTCAAACAAGCCTACAAATGGACCAGTTACACACCGGTATGATGCATGGGGTATTATGGTGGTGAGTGTAATTCATAATAAAGTCTATGTACTAGCTGGGTACCCTGATAACCAAAATACGCCATCAAAACAACAGATGGTTTGGGACACTTTTAATTGGGATGACACTTTTCCCACTGGCAGGATTTATTATACAACCTGGCCAGGTGTTGAGCAGGAAAATGAAGATGAGACTGATGCTGATTCTGACATCTCTAGTCTTTTTGACCCAGTGAATGAGGTGGAAAATGACTTCCACTTCCAATGTAGTCTAAAAACATCCGACTACTTAAAAGAGGAGGCTGACTTTTGGAAAGCAAAAGCGCAACAGCTTCTCATGGAGAAGGCAATGGAAAAACCAGGAACCAATCCTCCTCTTGTCTGTTTGGGTGAGGGTGGAATAGAGCAGCAAAAACAACCTGCTAGCAGCCGCGGCCACGCCGAGTAG
The 3' -end of the target gene fragment encoding the CAstV-ORF2 recombinant protein is tagged with a3 XFlag tag. The fluorescent protein mNeonGreen gene was cloned into the vector pcADV-EF1-CMV-MCS (vector available from Heyuan biosome). The recombinant CAstV-ORF2 shuttle plasmid was obtained by ligating a gene fragment of interest encoding the CAstV-ORF2 recombinant protein to a vector using an In-Fusion HD Cloning Kit kit. The recombinant CAstV-ORF2 shuttle plasmid pcADV-EF1-mNeonGreen-CMV-CAstV-ORF2-SDAU-3×FLAG whole gene map is shown in FIG. 3.
The specific steps of the recombinant CAstV-ORF2 shuttle plasmid are as follows:
(1) The following reaction solutions were prepared in a 0.2mL PCR tube.
TABLE 1 seamless cloning System
(2) The reagents were mixed well and two 0.2mL centrifuge tubes were prepared, one for positive reaction preparation (number 1) and one for negative reaction preparation (number 2). Repeatedly blowing and mixing the mixture in the tube by using a pipette, centrifuging the blown mixture, and placing the mixture into a PCR instrument for reaction at 37 ℃ for 30min. The sample is taken out and put on ice, and the sample after the reaction is finished enters the next conversion operation.
Example 2: transformation, screening, purification and restriction enzyme identification of recombinant CAstV-ORF2 shuttle plasmid.
1. The recombinant CAstV-ORF2 shuttle plasmid was transformed into DH 5. Alpha. Competent cells as follows:
1) Taking out DH5 alpha competent cells at-80 ℃, placing the EP tube filled with the competent cells on ice, and naturally thawing the competent cells;
2) 1. Mu.L (concentration greater than 10 ng/. Mu.L) of the recombinant CAstV-ORF2 shuttle plasmid prepared in example 1 was taken using a pipette and slowly spun into DH 5. Alpha. Competent cells, ice-incubated for 15-30min;
3) Inserting the EP pipe with competence into the buoy, placing the buoy into a water bath kettle at 42 ℃ for heat shock for 90 seconds, and immediately transferring the buoy to ice for 2-3 minutes;
4) Adding 500 mu L of antibiotic-free culture medium into an EP pipe filled with competence in an ultra-clean workbench, and placing the mixture into a constant temperature oscillator at 37 ℃ and 220rpm for culturing for 40-60min;
5) Placing the flat plate into a 37 ℃ incubator for standby;
6) Igniting an alcohol lamp in an ultra-clean workbench, sterilizing a coating rod (glass beads after sterilization can be used), and naturally cooling to room temperature;
7) Taking out the flat plate from the incubator at 37 ℃ and putting the flat plate into an ultra-clean workbench for standby;
8) Taking out the transformation liquid EP tube, taking 80 mu L of transformation liquid by using a liquid-transferring gun, and uniformly coating the transformation liquid on a solid culture medium by using a coating rod;
9) The plates were inverted, incubated at 37℃in an incubator overnight (14 h).
2. The positive clone detection and plasmid DNA extraction steps are as follows:
1) White individual colonies on the plates were picked with sterilized 10. Mu.L tips in an ultra clean bench and placed into test tubes containing 10mL of LB medium at 100ng/L AMP, respectively.
2) Placing in a shaking table at 37deg.C and 200rpm for 12 hr, placing in an ultra-clean workbench, taking small amount of bacterial liquid, delivering to large gene limited company for sequencing, extracting plasmid DNA from positive clone with correct sequencing by using plasmid extraction kit, and storing the extracted DNA product at-20deg.C.
3. The single cleavage identification procedure for the recombinant CAstV-ORF2 shuttle plasmid was as follows:
1) Carrying out single enzyme digestion on recombinant adenovirus plasmid by using KPNI, wherein a single enzyme digestion reaction system is as follows;
TABLE 2 20. Mu.L Single cleavage reaction System
2) The above reagents were mixed in a PCR tube. Reaction conditions: after reaction for 5min at 37 ℃, 1% agarose gel electrophoresis is carried out, the constant voltage is 120V, the current is 110mA,1h is carried out, after electrophoresis is finished, the gel is put into a gel imager for photographing, and the result is shown in FIG. 4, and the enzyme digestion product of the recombinant plasmid accords with the size of the fragment of the expected purpose. The recombinant CAstV-ORF2 shuttle plasmid is completely correct in size of the inserted target fragment, and the plasmid construction is successful.
Example 3: packaging of CastV-ORF2 recombinant adenovirus vectors
The shuttle plasmid constructed in example 2 was co-transfected into 293T cells and recombined to obtain viruses, and the experimental procedure was as follows:
1) The day before transfection, cells are inoculated into a 6-well plate, and the density of the cells when transfected is controlled to be 70-80%;
2) Taking out the cell culture plate one hour before transfection, removing the original cell culture medium, adding 1.5mL of Opti-MEM culture medium, and placing the cells back into the incubator;
3) Preparing a complex of transfection reagent and plasmid:
4) The viral vector plasmid to be transfected was 4 μg (backbone plasmid: shuttle plasmid = 1:1) was dissolved in Opti-MEM medium in a total volume of 250 μl and gently mixed;
5) Dissolving the transfection reagent in Opti-MEM culture medium, and mixing gently, wherein the total volume is 250. Mu.L;
6) And (3) dripping the transfection reagent dilution obtained in the step (5) into the plasmid dilution obtained in the step (4), and standing for 20min at room temperature after gently mixing the dilution with the dilution, so that DNA and the transfection reagent are fully combined to form a stable transfection complex.
7) Taking out the cell culture plate, adding the DNA-transfection reagent complex prepared above into the cell culture plate, marking, and placing back into the incubator;
8) After 6h, the medium was aspirated, washed once with PBS, and 2mL of fresh complete medium was added for cultivation;
9) Taking photo record by fluorescent microscope, preserving white light, and naming green fluorescent photo as shown in FIG. 5, observing cells every day, taking photo every other day, observing cell state, changing liquid in time when cell state is bad, packaging for about seven to fifteen days, observing whether CPE appears, and taking poison when cell leaves wall about 70%.
Example 4: obtaining high quality recombinant adenovirus of sufficient purity and titer
1. Massive amplification and purification of viruses
293T cells were plated in 30-40 10cm dish, and when cells grew to 70-80%, 10. Mu.L of virus (about 10 7-108 PFU/mL) was added to each plate to infect the cells, and after all lesions of the cells (2-3 days), about 500. Mu.L of 10% Nonidet P40 (NP 40) was added to each plate to lyse the cells. Cell lysates were collected, centrifuged at 12000rpm for 10min, and cell debris was discarded to collect the supernatant. 50mL of virus pellet (20% PEG8000,2.5 MNacl) was added to each 100mL of supernatant and placed on ice for 1h to pellet the virus. The mixture was centrifuged at 12000rpm for 20min, the supernatant was discarded, and the pellet was suspended in 10mL of CsCl solution (solvent 20mM Tris-HCl, pH 8.0) having a density of 1.10g/mL, centrifuged at 7000rpm for 5min at 4℃and the virus suspension was collected.
2.0ML of 1.40g/mL CsCl solution (solvent supra) was added to the Beckman ultracentrifuge tube. 3.0mL of 1.30g/mL CsCl solution was added. Finally, 5mL of virus suspension was added. 22800rpm,4℃for 2.5h. Viral bands were collected at a density between 1.30-1.40g/mL into dialysis bags (10 mM EDTA Na 2 boiled for 10min before use). The dialysis was performed overnight at 4℃in dialysis buffer (50 g sucrose, 10mL 1M Tris-HCl pH 8.0, 2mL1M MgCl 2 to 1000 mL) with a single exchange of dialysate. The virus was collected and stored at-80 ℃.
2. Virus titration assay
The adenovirus titer assay kit (TaKaRa) was used for titer detection.
1) HEK293 cells in good condition were selected and whole medium (10% dmem: DMEM+fetal bovine serum+green-streptomycin double antibody) was used to resuspend cells to prepare a 5.0X10 5/mL cell suspension, 1mL cells were added to each well of a 24-well plate, and cultured at 37℃in 5% CO 2.
2) 10-Fold gradient dilutions of virus samples were prepared and 10 -5 to 10 -8 dilutions of virus solution were then added sequentially to 24 well plates, 100 μl per well.
3) Infection is carried out at 37 ℃ for 48h by 5% CO 2. The culture broth was gently removed and 500. Mu.L of pre-chilled methanol was slowly added along the 24-well plate side wall and fixed at-20℃for 20min.
4) Cells were gently rinsed 3 times with PBS for 5min each.
5) 200. Mu.L of 1% BSA was added thereto and the mixture was blocked at 37℃for 1 hour.
6) 200. Mu.L of primary antibody solution was added to each well and incubated at 37℃for 1h.
7) Cells were gently rinsed 3 times with PBS for 5min each.
8) 200. Mu.L of secondary antibody was added to each well and incubated at 37℃for 1h.
9) Cells were gently rinsed 3 times with PBS for 5min each.
10 200. Mu.L of freshly prepared working fluid was added to each well and incubated for 5-10min at room temperature.
11 The working fluid was discarded, washed 2 times with PBS, and 1000. Mu.L of PBS was added to each well.
12 Randomly selecting 5 fields per well, and calculating the number of positive cells under a 10 x objective lens using an optical microscope. The titers of the different viral dilutions are shown in fig. 6, with an average number of positives of 8 when the dilution is 10 7.
13 Using the formula pfu/ml= [ (average number of positive cells per field) × (field number per well) × (dilution) ]/viral volume, the viral titer was calculated to be 6.32x10 10 (pfu/mL).
Example 5: and (3) detecting the expression of rAd5-CastV-ORF2 recombinant adenovirus target genes.
293T cells with good growth vigor were inoculated into 24-well plates, and after digestion, the cell concentration was adjusted to 1X 10 5 cells/mL and added at 500. Mu.L/well. The number of inoculated cells varies slightly with the growth rate of the cells, generally ensuring that the cell confluency is between 70-80% when the virus infection is performed the next day.
293T cells were infected with the recombinant adenovirus, and after 48 hours the cells were diseased and all 1ml cells were collected.
Extracting cellular proteins, adding loading buffer, and preparing samples. Western blot detection is performed. The CAstV-ORF2-SDAU protein was about 78kDa, and the experimental results showed that a protein band was detected between Marker 70-100kDa (FIG. 7), indicating successful expression of the gene of interest in the recombinant adenovirus.
Example 6: clinical application of recombinant adenovirus vector vaccine rAd5-CastV-ORF 2.
1. Animal immunogenicity experiments
(1) Body weight changes after immunization of SPF chickens with rAd5-CastV-ORF2 vaccine.
3D SPF chickens were randomly divided into four groups of 15, and the inner muscles of hind limbs were injected with rAD5-CAstV-ORF2 vaccine 10 9PFU/mL、108 PFU/mL and 10 7 PFU/mL 100. Mu.L and DMEM 100. Mu.L (Mock group), respectively, and the chickens were weighed and observed for behavioral status of the chickens at 7d, 14d, 21d, 28d, 35d before and after immunization. The experimental results show that: the differences in weight change were not significant in the vaccine groups (10 9 PFU/mL, 10 8 PFU/mL, and 10 7 PFU/mL) compared to chickens in the Mock group, and vaccination had substantially no effect on chicken growth (FIG. 8).
(2) And (3) detecting the immune efficacy of the rAd5-CastV-ORF2 vaccine and the content of cytokines (IFN-gamma and IL-4) in serum.
The day-old SPF chicks were immunized with DMEM and rAD5-CastV-ORF2 vaccine at doses of 10 9PFU/mL、108 PFU/mL and 10 7 PFU/mL for 100. Mu.L, jugular vein blood collection was performed on all the tested chickens at 4d, 7d, 14d, 21d, 28d and 35d after immunization, serum was separated in time, and the levels of CastV-ORF 2-specific antibodies in the immunized chicken serum and the cytokine IL-4 and IFN-gamma content in the chicken serum were detected by using an indirect ELISA method.
The experimental results of the CAstV-ORF2 specific antibody level in chicken serum show that: starting at 4d post immunization, the antibodies produced by the chickens in the vaccine groups (10 9 PFU/mL, 10 8 PFU/mL, and 10 7 PFU/mL) began to rise and were significantly higher than in the Mock group. And chickens remained at higher antibody levels after immunization, with antibody levels reaching a maximum at 14d, and then antibody levels began to drop (fig. 9).
The detection results of the content of cytokines (IFN-gamma and IL-4) in chicken serum show that: after 4d of initial immunization, the vaccine groups (10 9 PFU/mL, 10 8 PFU/mL, and 10 7 PFU/mL) had higher levels of IL-4 and IFN-gamma cytokines than the Mock group (FIG. 10).
2 Animal protection experiment
3D SPF chickens 80 were randomly divided into 4 groups: mock group, rAD5-CastV-ORF2 group (abbreviated as vaccine group), rAD5-CastV-ORF2+CastV group (abbreviated as vaccine+virus attack group), castV group (abbreviated as virus attack group), 20 of which are subjected to virus attack when SPF chicken 7d in the CastV group are subjected to virus attack, wherein the virus attack dose is 200 mu L of CastV virus liquid stored in 10 4TCID50 laboratories; the rAD5-CAstV-ORF2 vaccine is inoculated at a dose of 10 9 PFU/mL and 100 mu L when the SPF chickens in the rAD5-CAstV-ORF2+CAstV group are 3d, and is subjected to CAstV challenge at the age of 7d, wherein the challenge dose is 200 mu L of laboratory-stored CAstV virus liquid with the dose of 10 4TCID50; the rAD5-CastV-ORF2 vaccine was vaccinated at a dose of 10 9 PFU/mL and 100. Mu.L at 3d in the SPF chickens of the rAD5-CastV-ORF2 group, while Mock was used as a control. In detail, as shown in Table 3, the growth of the chicken flocks was observed at 7d, 14d, 21d, 28d, 35d, and 42d after the completion of each group of the breeding.
Table 3: animal experiment design of recombinant adenovirus vector vaccine
After the groups are completed, the state of the chicken flock is observed for 7d, and as a result, the chicken feather of the vaccine+virus attack group, the vaccine group and the Mock group is found to be compliant, and the mental state is not changed abnormally.
The weight of the chicken groups was observed for 7d, 14d, 21d, 28d, 35d and 42d after completion of each group, and as a result, no difference in weight was found in the vaccine+challenge group and the vaccine group chicken compared with the Mock group chicken. Starting from 21d, the challenged group chickens grew slowly, with significantly lower body weight than the Mock group compared to the vaccine + challenged group (fig. 11).
Two weeks after each group was completed, DNA was extracted from the serum of each group of chickens, and PCR detection was performed using the CAstV identification primer (510 bp) stored in the present laboratory to verify whether chickens were infected with CAstV. Wherein, the nucleotide sequence of the CAstV identification primer is shown as SEQ ID NO. 5-SEQ ID NO. 6.
SEQ ID NO:5：KCATGGCTYCACCGYAADCA；
SEQ ID NO:6：CGGTCCATCCCTCTAC-CAGATTT。
Experimental results show that the positive number of vaccine plus challenge group is less than that of challenge group, the positive rate of serum virus is 15%, and the effective neutralization of CAstV by the induced antibody after vaccine immunization is shown, and the protection rate is up to 85% (Table 4).
TABLE 4 statistics of Positive Rate after challenge
3. Histopathological lesions
Paraffin sections were prepared by dissecting organs fixed by Mock group, rAd5-CAstV-ORF2 group (abbreviated as vaccine group), rAd5-CAstV-ORF2+castv group (abbreviated as vaccine+challenge group), CAstV group (abbreviated as challenge group), and pathological changes of bursa of fabricius, spleen, thymus, kidney and duodenum of each group were analyzed under a microscope after HE staining. The results show that: the tissue lesions of the challenge group chicken were severe, and the vaccine + challenge group chicken had no apparent lesions (fig. 12).
The rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine prepared by the application has excellent chicken group protection effect through clinical application detection experiments on the recombinant adenovirus vector vaccine rAD5-CastV-ORF2, and provides a new means for preventing and controlling chicken astrovirus infection, and the prepared vaccine has wide development and application prospects.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (8)
1. The CAstV-ORF2 recombinant protein is characterized in that the amino acid sequence of the CAstV-ORF2 recombinant protein is shown as SEQ ID NO. 3.
2. The target gene for encoding the CAstV-ORF2 recombinant protein according to claim 1, wherein the nucleotide sequence of the target gene is shown in SEQ ID NO. 4.
3. Use of the recombinant CAstV-ORF2 protein of claim 1 or the gene of interest encoding the recombinant CAstV-ORF2 protein of claim 1 of claim 2 in the preparation of a vaccine for preventing chicken astrovirus.
4. The use according to claim 3, wherein the vaccine against chicken astrovirus is a recombinant adenovirus vector live vaccine.
5. The rAd5-CastV-ORF2 recombinant adenovirus vector live vaccine is characterized by being prepared by the following steps:
(1) 3 xFlag tag is added at the 3' end of the target gene as claimed in claim 2, the target gene is cloned to a vector pcADV-EF1-CMV-MCS inserted with fluorescent protein mNeonGreen gene, and a recombinant pcADV-EF1-mNeonGreen-CMV-CAstV-ORF2-3 xFLAG shuttle plasmid is obtained;
(2) And (3) co-transfecting the shuttle plasmid obtained in the step (1) and the adenovirus skeleton vector into 293T cells for packaging, and recombining to obtain rAD5-CastV-ORF2 recombinant adenovirus vector live vaccine.
6. The vaccine of claim 5, wherein the adenovirus backbone vector is a human replication defective adenovirus type 5 vector.
7. The vaccine of claim 5, wherein the rAd5-CastV-ORF2 recombinant adenovirus vector live vaccine has a viral titer of greater than or equal to 6.32x10 10 (pfu/mL).
8. Use of a rAd5-CastV-ORF2 recombinant adenovirus vector live vaccine according to any one of claims 5-7 in the preparation of a biologic for preventing chicken astrovirus infection.
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