CN117603920A - Recombinant serotype 4 avian adenovirus expressing serotype 11 avian adenovirus Fiber protein as well as construction method and application thereof - Google Patents

Recombinant serotype 4 avian adenovirus expressing serotype 11 avian adenovirus Fiber protein as well as construction method and application thereof Download PDF

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CN117603920A
CN117603920A CN202311597478.2A CN202311597478A CN117603920A CN 117603920 A CN117603920 A CN 117603920A CN 202311597478 A CN202311597478 A CN 202311597478A CN 117603920 A CN117603920 A CN 117603920A
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avian adenovirus
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叶建强
刘时
汤也
谢泉
秦爱建
邵红霞
万志敏
李拓凡
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Yangzhou University
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Abstract

The invention provides a recombinant serum type 4 avian adenovirus expressing serum type 11 avian adenovirus Fiber protein, and a construction method and application thereof, belonging to the technical field of genetic engineering. According to the invention, FAdV-4 is taken as a viral vector, and a recombinant avian adenovirus of which the Fiber-2 protein is targeted to knock out FAdV-4 and simultaneously the Fiber protein of the 11-type avian adenovirus of serum is expressed is constructed through a CRISPR/Cas9 technology and a Cre-loxP recombination system. The recombinant avian adenovirus not only can stably express the serotype 11 avian adenovirus Fiber protein, but also can replicate faster than the serotype 4 avian adenovirus by the measurement of a growth curve, has high titer and is expected to reduce the cost for the production of vaccines. Animal experiments also show that the inactivated recombinant avian adenovirus FAdV4-F11 can effectively induce the neutralizing antibodies against the serum 11 type avian adenovirus and FAdV4 in chicken bodies, and provides technical support and vaccine candidates for simultaneous immune prevention and control of the serum 4 type avian adenovirus and the serum 11 type avian adenovirus.

Description

Recombinant serotype 4 avian adenovirus expressing serotype 11 avian adenovirus Fiber protein as well as construction method and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant serum type 4 avian adenovirus for expressing a serum type 11 avian adenovirus Fiber protein, and a construction method and application thereof.
Background
Avian adenoviruses (Fowl adenoviruses, FAdV) belong to the genus aviadenovirus of the family adenoviridae. Currently, FAdV can be divided into 5 genotypes (A-E) based on restriction map analysis and hexon sequences; based on the serum cross-neutralization assay, 5 genotypes were subdivided into 12 serotypes (1-7, 8a, 8b, 9-11). Hepatitis-pericardial effusion syndrome (HHS) and Inclusion Body Hepatitis (IBH) caused by infection with avian adenovirus type 4 (FAdV-4) and avian adenovirus type 11 (FAdV-11) present a significant economic loss to the poultry industry.
At present, eukaryotic expression systems are used for expressing specific antigen proteins aiming at prevention and control of avian adenovirus, and the aim of resisting avian adenovirus infection is fulfilled by preparing subunit vaccine. For example, FAdV-4 can be controlled, and a baculovirus expression system is utilized to efficiently express the serum type 4 avian adenovirus Fiber-2 protein to prepare subunit vaccine, so that the viral infection transmission path (CN 112940084A) can be effectively blocked. Aiming at FAdV-11 prevention and control, the avian adenovirus fiber protein and the targeting peptide SP sequence of chicken bone marrow-derived dendritic cells are fused and expressed to prepare subunit vaccine so as to realize FAdV-11 precise prevention and control (CN 116789850A). Since avian adenovirus infection often occurs with multiple serotypes, it is desirable to prepare multiple vaccines against avian adenoviruses of multiple serotypes. For example, patent publication No. 202310094554.1 discloses a tetravalent chimeric virus-like particle of avian adenovirus disease, which is obtained by taking matrix protein M of newcastle disease virus NA-1 strain as a skeleton, respectively embedding Fiber-2 protein of group I serum 4 type avian adenovirus, fiber protein of group I serum 8a type avian adenovirus, fiber protein of group I serum 8b type avian adenovirus and Fiber protein of group I serum 11 type avian adenovirus on the surface of newcastle disease virus-like particle carrier NDVVLPs, and is named FAdV4-8a-8b-11cVLPs. The patent with publication number CN114214291A constructs an avian adenovirus serum 4 type recombinant virus for expressing avian adenovirus serum 8b type Fiber protein, which is obtained by replacing FAdV-4Fiber1 gene with FAdV-8b Fiber gene, and the recombinant virus can be used for preparing a bivalent vaccine for preventing and controlling chicken hepatitis-pericardial effusion syndrome and/or chicken inclusion body hepatitis, and the bivalent vaccine prepared by the recombinant virus can achieve the effect of injecting one-injection vaccine and simultaneously preventing two epidemic diseases. However, there is no report on related vaccines for simultaneous immunization against both serotype 4 and serotype 11 avian adenoviruses.
Disclosure of Invention
In view of the above, the invention aims to provide a recombinant serum type 4 avian adenovirus expressing serum type 11 avian adenovirus Fiber protein, which replaces Fiber-2 protein in the recombinant serum type 4 avian adenovirus based on CRISPR-Cas9 technology, and provides technical support and vaccine candidates for simultaneous immune prevention and control of the serum type 4 avian adenovirus and the serum type 11 avian adenovirus.
The invention provides a recombinant type 4 avian adenovirus expressing type 11 avian adenovirus Fiber protein, which replaces Fiber-2 protein in the type 4 avian adenovirus by the type 11 avian adenovirus Fiber protein on the basis of the type 4 avian adenovirus.
Preferably, the amino acid sequence of the serum 11 type avian adenovirus Fiber protein is shown as SEQ ID NO: 1.
The invention provides a construction method of recombinant serum 4 type avian adenovirus expressing serum 11 type avian adenovirus Fiber protein, which comprises the following steps:
constructing a donor plasmid containing a serotype 11 avian adenovirus Fiber protein gene, a left homologous arm and a right homologous arm of a serotype 4 avian adenovirus genome and an RFP expression cassette with LoxP sequences;
constructing a recombinant vector containing sgRNA of a targeted serum 4 type avian adenovirus fiber-2 gene;
transfecting cells with the donor plasmid and a recombinant vector containing sgRNA of a targeted serum 4 type avian adenovirus Fiber-2 gene, inoculating the transfected cells with serum 4 type avian adenovirus, culturing, screening cells which display red fluorescence, and culturing to obtain recombinant serum 4 type avian adenovirus with RFP expression cassette for expressing serum 11 type avian adenovirus Fiber protein;
and infecting cells transfected with Cre recombinase by the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein with the RFP expression cassette, and culturing the cells which are cytopathic and have no red fluorescence to obtain the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein.
Preferably, the construction method for constructing the donor plasmid containing the serum 11 type avian adenovirus Fiber protein gene, the left homologous arm and the right homologous arm of the serum 4 type avian adenovirus genome and the RFP expression cassette with LoxP sequence comprises the following steps:
amplifying a serotype 11 avian adenovirus Fiber protein gene, and inserting the gene into a donor plasmid with a left homologous arm and a right homologous arm of a serotype 4 avian adenovirus genome and an RFP expression cassette with LoxP sequences through a homologous recombination mode and a linearized serotype 11 avian adenovirus Fiber protein gene;
the primer for amplifying the serotype 11 avian adenovirus Fiber protein gene comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 3 and a forward primer with a nucleotide sequence shown as SEQ ID NO. 4;
the primer for linearization treatment of the donor plasmid with the homologous arm at the left end and the homologous arm at the right end of the serum 4-type avian adenovirus genome and the RFP expression cassette with LoxP sequences comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 5 and a forward primer with a nucleotide sequence shown as SEQ ID NO. 6.
Preferably, the sgRNA of the targeting serum 4 type avian adenovirus fiber-2 gene is obtained by annealing a forward primer with a nucleotide sequence shown as SEQ ID NO. 7 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 8.
Preferably, the serum type 4 avian adenovirus is a recombinant serum type 4 avian adenovirus expressing a green fluorescent protein;
the chimeric position of the gene encoding the green fluorescent protein in the recombinant serum 4-type avian adenovirus genome is upstream of the Fiber-2 protein gene.
The invention provides application of the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein or the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein obtained by the construction method in preparing vaccines or medicines for preventing and controlling hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis.
The invention provides a vaccine for preventing and controlling hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis, which comprises recombinant serum type 4 avian adenovirus expressing serum type 11 avian adenovirus Fiber protein or recombinant serum type 4 avian adenovirus expressing serum type 11 avian adenovirus Fiber protein and an adjuvant obtained by the construction method.
Preferably, the adjuvant comprises Freund's complete adjuvant and/or Freund's incomplete adjuvant.
The invention provides a medicine for preventing and/or treating hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis, which comprises recombinant serum 4 type avian adenovirus expressing serum 11 type avian adenovirus Fiber protein or recombinant serum 4 type avian adenovirus expressing serum 11 type avian adenovirus Fiber protein obtained by the construction method and pharmaceutically acceptable auxiliary materials.
The invention provides a recombinant type 4 avian adenovirus expressing type 11 avian adenovirus Fiber protein, which replaces Fiber-2 protein in the type 4 avian adenovirus by the type 11 avian adenovirus Fiber protein on the basis of the type 4 avian adenovirus. The experiment of the invention shows that the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein can efficiently replicate on LMH cells, and the virus titer can reach 10 at most on the 5 th day 9.6 TCID 50 The growth rate and titer of the strain were much higher than that of wild-type FAdV-4. The stability test result shows that the recombinant virus after serial passage can stably express the serotype 11 avian adenovirus F iber protein. Immunogenicity studies have shown that after inoculation of the inactivated oil emulsion prepared from recombinant avian adenovirus serotype 4, the average titers of neutralizing antibodies against FAdV-4 and FAdV-11 in animal serum were about 256 and 128, respectively, while the average titers of neutralizing antibodies in control chicken serum were less than 4. It can be seen that the inactivated FAdV4-F11 can induce high-level neutralizing antibodies to FAdV-4 and FAdV-11, and provides a basis for prevention, control and prevention of hepatitis-pericardial effusion syndrome (HHS) and Inclusion Body Hepatitis (IBH) caused by infection of serum type 4 avian adenovirus (FAdV-4) and serum type 11 avian adenovirus.
The invention provides a construction method of recombinant serum 4 type avian adenovirus expressing serum 11 type avian adenovirus Fiber protein, which utilizes currently popular serum 4 type avian adenovirus as a vector, inserts the serum 11 type avian adenovirus Fiber gene, successfully obtains the recombinant serum 4 type avian adenovirus expressing serum 11 type avian adenovirus Fiber protein, and provides technical support and vaccine candidates for simultaneous immune prevention and control of the serum 4 type avian adenovirus and the serum 11 type avian adenovirus. Different from the traditional reverse genetic construction method of recombinant avian adenovirus, FAdV-4 is used as a template virus, the sgRNA is utilized to target and knock out the virulence gene Fiber-2, and simultaneously the exogenous gene and loxP-RFP sequence of the serotype 11 avian adenovirus are introduced, so that the Fiber-2 of FAdV-4 is directly replaced by the exogenous gene and the RFP expression frame with loxP, and the purpose of virus purification is realized by the RFP protein. And then the Cre enzyme is utilized to cut off the RFP expression cassette, and finally the recombinant virus which stably expresses FAdV-11Fiber protein is obtained, so that technical support and vaccine candidates are provided for simultaneous immune prevention and control of the serum type 4 avian adenovirus and the serum type 11 avian adenovirus. Therefore, the invention uses a fluorescent system, integrates a CRISPR/Cas9 technology and a Cre-loxP recombination system, does not need to clone the whole genome of the avian adenovirus, and ensures that the construction, screening and purification of the recombinant avian adenovirus are very quick and efficient.
Drawings
FIG. 1 is a flow chart of a rescue strategy for FAdV4-F11 recombinant viruses; wherein A: schematic diagram of construction strategy of FAdV4-F11 recombinant virus; b: constructing donor plasmid; m: DNAmarker;1: PCR amplification of linearized plasmid HR1-RFP-HR2inpMD 19;2: FAdV-11fiber gene;
FIG. 2, rescue and purification results of FAdV4-F11 recombinant virus; wherein A is a fluorescence diagram of the primary recombinant virus FAdV4-F11 RFP; a: recombinant FAdV4-F11 RFP; b: maternal virus FA4-EGFP; b: PCR identification of purity results of recombinant disease; m: DNAmarker;1: unpurified recombinant virus FAdV4-F11-RFP;2: purified recombinant virus FAdV4-F11-RFP;3: recombinant virus FAdV4-F11 excised from RFP; 4: maternal virus FA4-EGFP;5: wild type FAdV-4;6: a negative control;
FIG. 3 shows the results of the expression and identification of the serotype 11 avian adenosis Fiber protein in recombinant viruses; a: WB identifies the expression of the serotype 11 avian adenosis Fiber protein in recombinant viruses; 1: purified recombinant virus FAdV4-F11-RFP;2: purified recombinant virus FAdV4-F11;3: wild type FAdV-4; the IFA identifies the expression of the 11-type avian adenosis Fiber protein in the recombinant virus;
FIG. 4 shows the growth curve and stability results of recombinant virus FAdV4-F11; a: growth curve of recombinant virus FAdV4-F11; b and C: identification of FAdV4-F11 stabilization by PCR (B) and WB (C); m is marker;1 to 6: 2 nd generation, 4 th generation, 6 th generation, 8 th generation, 10 th generation and 12 th generation recombinant virus FAdV4-F11;
FIG. 5 shows the results of an inactivated FAdV4-F11 immune SPF chicken serum neutralizing antibody assay.
Detailed Description
The invention provides a recombinant type 4 avian adenovirus expressing type 11 avian adenovirus Fiber protein, which replaces Fiber-2 protein in the type 4 avian adenovirus by the type 11 avian adenovirus Fiber protein on the basis of the type 4 avian adenovirus.
In the present invention, the amino acid sequence of the serotype 11 avian adenovirus Fiber protein is preferably as set forth in SEQ ID NO:1 (MAKSTPFTFSMGQHSSRKRPADSENTQNASKVAKT QTSATRAGVDGNDDLNLVYPFWLQNSTSGGGGGGSGGNPSLNPPFIDPNGPLYVQNSLLYVKTTAPIEVENKSLALAYDSSLDVDAQNQLQVKVDAEGPIRISPDGLDIAVDPSTLEVDDEWELTVKLDPTGPISSSSAGINIRVDDTLLIEDDDTAQVKELGVHLNPNGPITADQDGLDLEVDPQTLTVTTSGATGGVLGVLLKPSGGLQTSIQGIGVAVADTLTISSNRVEVKTDPNGSIGSSSNGIAVVTDPAGPLTTSSNGLSLKLTPNGSIQSSSTGLSVQTDPAGPITSGANGLSLSYDTSDFTVSQGMLSIIRNPSAYPDAYLESGTNLLNNYTAYAENSSNYKFNCAYFLQSWYSNGLVTSSLYLKINRDNLTSLPSGQLSENAKYFTFWVPTYESMNLSNVATPTITPSSVPWGAFLPAQNCTSNPAFKYYLTQPPSIYFEPESGSVQTFQPVLTGDWDTNTYNPGTVQVCILPQTVVGGQSTFVNMTCYNFRCQNPGIFKVAASSGTFTIGPIFYSCPTNKLTQP). The nucleotide sequence of the gene of the serotype 11 avian adenovirus Fiber protein is shown as SEQ ID NO. 9 (ATGGCGAAATCGACTCCTTTCA CGTTCTCCATGGGTCAGCACTCCAGCCGAAAACGTCCCGCGGACAGCGAAAACACGCAAAATGCATCAAAAGTCGCCAAAACGCAGACTTCTGCCACGCGCGCCGGTGTCGACGGAAATGACGACCTAAACCTGGTGTACCCCTTTTGGCTCCAAAACAGCACTTCGGGGGGCGGAGGAGGAGGAAGCGGCGGAAACCCCTCCCTAAATCCCCCTTTTATCGACCCTAACGGACCCCTCTATGTTCAAAACAGTCTCCTTTATGTCAAAACTACTGCACCTATCGAGGTTGAAAACAAGTCACTCGCCCTAGCTTATGATTCCTCACTGGATGTGGACGCTCAAAATCAGCTACAAGTGAAGGTAGATGCCGAGGGACCCATCAGGATTTCCCCAGATGGGCTCGATATTGCTGTCGACCCATCGACGTTGGAGGTTGATGATGAATGGGAGCTGACCGTCAAACTCGACCCGACCGGACCCATATCCTCATCCTCTGCCGGAATCAACATACGAGTAGATGATACGCTCTTAATCGAAGACGATGACACCGCCCAAGTTAAAGAATTAGGCGTGCATCTCAACCCCAACGGCCCCATTACCGCTGACCAAGATGGGTTGGACTTAGAAGTGGACCCACAAACTTTAACCGTCACTACCAGCGGAGCCACGGGAGGAGTTCTAGGAGTACTTCTGAAACCCAGCGGGGGGTTACAGACAAGCATTCAGGGTATCGGAGTCGCCGTAGCAGATACTTTAACTATATCGAGTAACAGGGTCGAAGTAAAAACCGATCCCAACGGTTCTATTGGATCCTCTAGCAATGGGATAGCAGTAGTTACCGATCCCGCGGGTCCGCTCACTACTTCATCTAACGGTCTGTCTCTGAAATTAACACCGAACGGTTCCATCCAATCATCAAGTACGGGCCTATCCGTCCAGACCGATCCCGCGGGACCCATTACCTCCGGTGCCAACGGACTAAGCTTATCCTACGACACTTCTGACTTTACAGTAAGTCAAGGAATGCTCAGTATCATACGGAATCCAAGCGCCTATCCAGATGCTTATCTAGAATCGGGAACCAACTTACTGAATAATTACACGGCTTATGCCGAAAACTCCAGTAATTACAAGTTTAACTGCGCTTATTTTCTTCAGTCCTGGTATTCCAACGGACTAGTGACTTCCTCCCTTTATCTCAAAATCAACAGGGATAACCTCACTAGTCTACCTTCTGGTCAATTAAGTGAAAATGCCAAATATTTTACATTTTGGGTGCCCACCTATGAATCGATGAACCTTTCCAATGTTGCAACACCTACTATTACCCCTAGCAGCGTCCCGTGGGGAGCATTCTTACCCGCACAAAATTGCACGAGTAATCCCGCCTTTAAGTACTATCTCACGCAACCGCCAAGCATCTATTTCGAGCCAGAATCGGGTTCCGTGCAAACTTTCCAACCCGTATTGACAGGAGATTGGGATACCAACACCTACAACCCAGGAACCGTTCAAGTCTGCATACTGCCTCAAACCGTTGTGGGAGGCCAGTCGACCTTTGTTAACATGACATGTTATAACTTCCGGTGTCAAAATCCTGGAATATTCAAGGTTGCTGCTAGTAGCGGCACATTCACTATCGGACCCATTTTCTACTCCTGTCCAACCAACAAATTAACACAACCCTAA). The source of the serotype 11 avian adenovirus Fiber protein is a serotype 11 avian adenovirus 380 strain. The amino acid sequence of Fiber-2 protein in the avian adenovirus type 4 of serum is preferably shown as SEQ ID NO:2 (MAK STPFTFSMGQHSSRKRPADSENTQNASKVAKTQTSATRAGVDGNDDLNLVYPFWLQNSTSGGGGGGSGGNPSLNPPFIDPNGPLYVQNSLLYVKTTAPIEVENKSLALAYDSSLDVDAQNQLQVKVDAEGPIRISPDGLDIAVDPSTLEVDDEWELTVKLDPTGPISSSSAGINIRVDDTLLIEDDDTAQVKELGVHLNPNGPITADQDGLDLEVDPQTLTVTTSGATGGVLGVLLKPSGGLQTSIQGIGVAVADTLTISSNRVEVKTDPNGSIGSSSNGIAVVTDPAGPLTTSSNGLSLKLTPNGSIQSSSTGLSVQTDPAGPITSGANGLSLSYDTSDFTVSQGMLSIIRNPSAYPDAYLESGTNLLNNYTAYAENSSNYKFNCAYFLQSWYSNGLVTSSLYLKINRDNLTSLPSGQLSENAKYFTFWVPTYESMNLSNVATPTITPSSVPWGAFLPAQNCTSNPAFKYYLTQPPSIYFEPESGSVQTFQPVLTGDWDTNTYNPGTVQVCILPQTVVGGQSTFVNMTCYNFRCQNPGIFKVAASSGTFTIGPIFYSCPTNKLTQP) the nucleotide sequence of the Fiber-2 protein gene in the avian adenovirus type 4 serum is shown as SEQ ID NO. 10 (ATGCTCCGGGCCCCTAAAAGAAGACATTCCGAA AACGGGAAGCCCGAGACCGAAGCGGGACCTTCCCCGGCTCCAATCAAGCGCGCCAAACGCATGGTGAGAGCATCCCAGCTTGACCTGGTTTATCC
TTTCGATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCCTTTTTTG
GGAGGCTCAGGACCCCTAGTGGACCAGGGCGGACAGCTTACGCTCAA
CGTCACCGATCCCATCATCATCAAGAACAGATCGGTGGACTTGGCCCA
CGACCCCAGTCTCGATGTCAACGCCCAAGGTCAACTGGCGGTGGCCGT
TGACCCCGAAGGGGCCCTGGACATCACCCCCGATGGACTGGACGTCA
AGGTCGACGGAGTGACCGTAATGGTCAACGATGACTGGGAACTGGCC
GTAAAAGTCGACCCGTCCGGCGGATTGGATtCCACCGCGGGTGGACTG
GGGGTCAGCGTGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGG
CGTACACCTCAACCAACAAGGACCCATCACTGCCGATAGCAGTGGTAT
CGACCTCGAGATCAATCCTAACATGTTCACGGTCAACACCTCGACCGG
AAGCGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCATCCAAG
CCGACAGTTCGGGAGTGGGCGTTTCCGTGGATGAAAGCCTACAGATTG
TCAACAACACTCTGGAAGTGAAACCGGATCCCAGCGGACCGCTTACG
GTCTCCGCCAATGGCCTAGGGCTGAAGTACGACACTAATACCCTAGCG
GTGACCGCGGGCGCTTTAACCGTGGTCGGAGGGGGGAGCGTCTCCAC
ACCCATCGCTACTTTTGTCTCGGGAAGTCCCAGCCTCAACACCTACAAT
GCCACGACCGTCAATTCCAGCGCGAACGCCTTCTCTTGCGCCTACTAC
CTTCAACAGTGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTTGA
AATTGGACAGCGCCACCATGGGGAATCGCCCTGGGGACCTCAACTCCG
CCAATGCCAAATGGTTCACCTTTTGGGTGTCCGCCTATCTCCAGCAATG
CAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCCTCCACCGCCAC
CCTCACGGACTTTGAACCCATGGCCAATAGGAGCGTGACCAGCCCATG
GACGTACTCGGCCAATGGATACTATGAACCATCCATCGGGGAATTCCAA
GTGTTCAGCCCGGTGGTAACAGGTGCCTGGAACCCGGGAAACATAGG
GATCCGCGTCCTCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACAC
CCTTCTATGCTATAGTCTGCAGTGCACGAACGCGAGCATTTTTAATCCA
AACAACAGCGGAACCATGATCGTGGGACCCGTGCTCTACAGCTGTCCA
GCGGCCTCCCTCCCGTAA).
In the embodiment of the invention, the research result shows that the substitution of the Fiber-2 protein in the serum 11 type avian adenovirus Fiber protein for the serum 4 type avian adenovirus does not influence the replication performance of the serum 11 type avian adenovirus, and the replication capacity of the serum 11 type avian adenovirus is slightly improved compared with that of the serum 4 type avian adenovirus. The recombinant avian adenovirus FAdV4-F11 not only can stably express the avian adenovirus Fiber protein of the 11-type serum, but also has high virus titer. Animal experiments also show that the inactivated recombinant avian adenovirus FAdV4-F11 can effectively induce the neutralizing antibodies aiming at the serotype 11 avian adenovirus 380 strain and FAdV4 in chicken bodies, and provides technical support and vaccine candidates for simultaneous immune prevention and control of the serotype 4 avian adenovirus and the serotype 11 avian adenovirus.
The invention provides a construction method of recombinant serum 4 type avian adenovirus expressing serum 11 type avian adenovirus Fiber protein, which comprises the following steps:
constructing a donor plasmid containing a serotype 11 avian adenovirus Fiber protein gene, a left homologous arm and a right homologous arm of a serotype 4 avian adenovirus genome and an RFP expression cassette with LoxP sequences;
constructing a recombinant vector containing sgRNA of a targeted serum 4 type avian adenovirus fiber-2 gene;
transfecting cells with the donor plasmid and a recombinant vector containing sgRNA of a targeted serum 4 type avian adenovirus Fiber-2 gene, inoculating the transfected cells with serum 4 type avian adenovirus, culturing, screening cells which display red fluorescence, and culturing to obtain recombinant serum 4 type avian adenovirus with RFP expression cassette for expressing serum 11 type avian adenovirus Fiber protein;
and infecting cells transfected with Cre recombinase by the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein with the RFP expression cassette, and culturing the cells which are cytopathic and have no red fluorescence to obtain the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein.
In the present invention, the construction method of the donor plasmid containing the serotype 11 avian adenovirus Fiber protein gene, the left homologous arm and the right homologous arm of the serotype 4 avian adenovirus genome and the RFP expression cassette with LoxP sequence preferably comprises the following steps:
amplifying the serotype 11 avian adenovirus Fiber protein gene, and inserting the linear serotype 11 avian adenovirus Fiber protein gene into a donor plasmid with a left homologous arm and a right homologous arm of a serotype 4 avian adenovirus genome and an RFP expression cassette with LoxP sequences by a homologous recombination mode.
In the invention, the donor plasmid of the serum 11 type avian adenovirus Fiber protein gene inserted with the left homologous arm, the right homologous arm and the RFP expression cassette with LoxP sequence of the serum 4 type avian adenovirus genome is HR1-RFP-HR2in pMD 19, which is constructed and stored in the laboratory, and the specific construction method is shown in patent CN116286685A.
In the invention, the primer for amplifying the serotype 11 avian adenovirus Fiber protein gene comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 3 (CCCATCATCATCAAGAACGAGACAGTATCA CTAATAAC) and a forward primer with a nucleotide sequence shown as SEQ ID NO. 4 (CTTTCTAGACTC GAGTTATATACAAATGTTGCATC). The primer for linearization treatment of the donor plasmid with the homologous arm at the left end and the homologous arm at the right end of the serum 4-type avian adenovirus genome and the RFP expression cassette with LoxP sequence comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 5 (GTGTATCGCTCTCCG GAGGGGTGACCTACTGACCCTC) and a forward primer with a nucleotide sequence shown as SEQ ID NO. 6 (GGTGTATCGCTCTCCGGAGGTTCGTGGAACGCTCCGTCAG).
In the invention, the sgRNA of the targeting serum 4 type avian adenovirus fiber-2 gene is preferably obtained by annealing a forward primer with a nucleotide sequence shown as SEQ ID NO. 7 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 8. The skeleton carrier of the recombinant carrier containing the sgRNA of the targeting serum 4 type avian adenovirus fiber-2 gene is preferably a lentiCRISPRv2 plasmid. The construction method of the recombinant vector is not particularly limited, and construction methods well known in the art may be employed.
In the invention, when the donor plasmid and the recombinant vector containing the sgRNA of the targeting serum 4 type avian adenovirus fiber-2 gene transfect cells, the donor plasmid and the recombinant vector containing the sgRNA of the targeting serum 4 type avian adenovirus fiber-2 gene are preferably mixed in an equal mass ratio. The cells are preferably LMH cells. After transfection, the cells were cultured for 6h, and the system solution was replaced with cell growth solution. The avian adenovirus serotype 4 was inoculated again 12h after transfection and replaced with cell maintenance fluid 2h after infection.
In the present invention, the serotype 4 avian adenovirus is preferably a recombinant serotype 4 avian adenovirus (FA 4-EGFP) expressing a green fluorescent protein. The chimeric position of the gene encoding the green fluorescent protein in the recombinant serum 4-type avian adenovirus genome is upstream of the Fiber-2 protein gene. The construction method of the recombinant serum 4-type avian adenovirus expressing the green fluorescent protein is described in the prior art (Xie Q, cao S, zhang W, wang W, li L, kan Q, fu H, geng T, li T, wan Z, gao W, shao H, qiaN, ye J.A non-tissue-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4fowl adenovirus.Vet Res.2021Feb 27;52 (1): 35.).
In the invention, a method for screening cells which show red fluorescence is carried out, preferably after FA4-EGFP is infected for 3 days, the cells are blindly transferred to new LMH cells, a plurality of rounds of limiting dilution are utilized on a 96-well cell plate, and cells which have no green fluorescence and have red fluorescence are selected each time until the green fluorescence completely disappears, thus obtaining the recombinant serum 4 type avian adenovirus which has RFP expression cassette and expresses the serum 11 type avian adenovirus Fiber protein.
In the present invention, the construction method of the recombinant expression vector expressing Cre recombinase is not particularly limited, and construction methods well known in the art can be used, and specific examples thereof include the prior art (Lu Y, yuan Y, jiang H, xu Z, guo Y, cao X, li T, wan Z, shao H, qiaN, xie Q, ye J.effective cross-protection against serotype/8 a fowl adenoviruses (FAdVs): recombiant FAdV-4with FAdV-8a Fiber.Microbiol Spectr.2023Nov15:e0246223). The method of the present invention for transfecting the recombinant expression vector expressing Cre recombinase into LMH cells is not particularly limited, and transfection methods well known in the art may be employed.
In the invention, after 2-3d infection of the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein with the RFP expression cassette is preferably observed by a fluorescence microscope, the cell with cytopathy and no red fluorescence is selected, and LMH cells transfected with Cre recombinase are continuously inoculated until the red fluorescence is completely disappeared. Recombinant serotype 4 avian adenoviruses expressing the serotype 11 avian adenovirus Fiber protein from which the RFP expression cassette was deleted are preferably identified using PCR, IFA and WesternBlot methods. The results of the examples show that.
In the embodiment of the invention, the FAdV4-F11-RFP and the FAdV4-F11 can detect the avian adenovirus type 11Fiber protein and the Hexon protein. Whereas the negative control FAdV4-EGFP only detected the expression of the Hexon protein.
In view of the fact that the recombinant serum 4 type avian adenovirus immune animal can simultaneously generate neutralizing antibodies aiming at FAdV-4 and FAdV-11 and can block the infection of FAdV-4 and FAdV-11, the invention provides the application of the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein or the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein obtained by the construction method in preparing vaccines or medicines for preventing and controlling hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis.
The invention provides a vaccine for preventing and controlling hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis, which comprises recombinant serum type 4 avian adenovirus expressing serum type 11 avian adenovirus Fiber protein or recombinant serum type 4 avian adenovirus expressing serum type 11 avian adenovirus Fiber protein and an adjuvant obtained by the construction method.
In the present invention, the adjuvant preferably includes Freund's complete adjuvant and/or Freund's incomplete adjuvant. The vaccine is an inactivated vaccine. In an embodiment of the invention, the vaccine is an inactivated oil emulsion. The method for preparing the vaccine is not particularly limited in the present invention, and the method for preparing the vaccine known in the art may be used. The vaccine is preferably administered to the chicken at an immunizing dose of 0.3 mL/min.
The invention provides a medicine for preventing and/or treating hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis, which comprises recombinant serum 4 type avian adenovirus expressing serum 11 type avian adenovirus Fiber protein or recombinant serum 4 type avian adenovirus expressing serum 11 type avian adenovirus Fiber protein obtained by the construction method and pharmaceutically acceptable auxiliary materials.
The invention is not particularly limited in the types of the auxiliary materials, and the auxiliary materials of the virus drugs known in the art can be used. The medicament is used for preventing and/or treating hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis.
The following examples are provided to illustrate in detail a recombinant avian adenovirus type 4 expressing a Fiber protein of avian adenovirus type 11, and a method of constructing and using the same, but are not to be construed as limiting the scope of the invention.
Example 1
Construction method of recombinant serum 4 type avian adenovirus for expressing serum 11 type avian adenovirus Fiber protein
1. Preparation of viral genome: the DNA extraction kit of Tiangen is used for extraction, 200 mu L of serum 11 type avian adenovirus supernatant is taken in a 1.5mL finger tube, and the extraction of serum 11 type avian adenovirus genome is carried out according to the specification of the DNA extraction kit.
2. Construction of donor plasmid: the donor plasmid HR1-RFP-HR2inpMD 19 with the left homology arm HAL and right homology arm HAR of the genome of the avian adenovirus type 4 serum and the RFP expression cassette with LoxP sequence is constructed and stored in the laboratory, and the specific construction method is shown in patent CN116286685A. Then taking HR1-RFP-HR2in pMD 19 plasmid vector as template, linearizing the vector by linearization primer (as shown in figure 1B), amplifying fiber gene of serum 11 type avian adenovirus by using DNA with plasmid partial sequence serum 11 type fiber gene primer and serum 11 type avian adenovirus 380 strain as template (as shown in figure 1B), recovering gel after agarose gel electrophoresis, connecting fiber gene to HR1-RFP-HR2in pMD 19 plasmid under homologous recombinase by the obtained linearization vector and fiber gene of serum 11 type avian adenovirus, and finally obtaining donor plasmid carrying fiber gene of serum 11 type avian adenovirus and RFP expression cassette. The final donor plasmid was sequenced and stored in the laboratory by Nanjing qingke biosciences. The primer sequences used to construct the donor plasmid are shown in Table 1.
TABLE 1 PCR primers used to construct donor plasmids
Construction of sgrna expression vector: the design of the sgRNA was carried out using the on-line design site for sgRNA (http:// crispor. Tefor. Net /) based on the fiber-2 gene sequence of FAdV-4. The designed sgrnas were cloned to the lentiCRISPR v2 plasmid and verification of the sgRNA expression vector was performed by sequencing. Specific sgRNA sequences are shown in table 2, synthesized by south jingqing biotechnology limited.
TABLE 2 sgRNA sequences for Fiber-2 genes
Rescue of FAdV4-F11 recombinant virus: the construction strategy is as in fig. 1 a. LMH cells were plated in 6-well plates, and the next day, 3. Mu.g of sgRNA, 3. Mu.g of donor plasmid, and 6. Mu.L of transfection reagent Mirus were added to 200. Mu.L of Opti-MEM and incubated at room temperature for 45min. Then adding into LMH cells, and changing into cell growth liquid after transfection for 6 h. After 12h of transfection, the cell growth fluid was discarded, LMH cells were infected with 0.1MOI of FA4-EGFP, and after 2h of infection, they were changed to cell maintenance fluid. After 3 days of FA4-EGFP infection, new LMH cells are blindly transferred, and after a few days of culture, the cells are observed by a fluorescence microscope, and if red fluorescent clusters appear (shown as A in figure 2), the primary construction of the recombinant virus is proved to be successful.
And then, taking out cells without green fluorescence and with red fluorescence on a 96-well cell plate by utilizing multiple rounds of limiting dilution until the green fluorescence completely disappears, obtaining the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein with the RFP expression cassette, and naming the recombinant serum 4 type avian adenovirus as FAdV4-F11-RFP. The purified red fluorescent recombinant virus is diluted by a multiple ratio and then inoculated on a 96-hole LMH cell plate transfected with Cre recombinase, cells with cytopathy but without red fluorescence are selected and inoculated on the 96-hole LMH cell plate transfected with Cre recombinase continuously for several times after 2-3d and then the recombinant virus with the RFP expression cassette deleted is obtained after the red fluorescence is completely disappeared, and the recombinant virus is named FAdV4-F11. The recombinant virus FAdV4-F11 obtained was identified by PCR (see FIG. 2B) using the primers shown in Table 3.
TABLE 3 primers for PCR identification of recombinant viruses
5. Expression identification of serotype 11Fiber protein in recombinant virus: LMH cells were infected with 0.1MOI recombinant virus and changed to cell maintenance fluid 2h after infection. 3 days after virus infection, cells are lysed by using cell lysate containing protease phosphatase inhibitor, protein loading buff is added to cook the sample, and WB verification is carried out by using the serum 11 type avian adenovirus Fiber murine polyclonal antibody and the Hexon monoclonal antibody.
As a result, both FAdV4-F11-RFP and FAdV4-F11 were able to detect the serotype 11 avian adenovirus Fiber protein and the Hexon protein as shown in FIG. 3A. Whereas the negative control FAdV4-EGFP only detected the expression of the Hexon protein.
LMH cells were further inoculated with 0.01MOI purified recombinant virus without RFP expression cassette, fixed after 3 days, and IFA identified using polyclonal antibody against serotype 11Fiber mice and monoclonal antibody 3B5 against FAdV-4Fiber-1 protein.
As a result, as shown in FIG. 3B, recombinant virus FAdV4-F11 can detect the type 11 avian adenovirus Fiber protein and FAdV-4Fiber-1 protein, indicating that the type 11 avian adenovirus Fiber protein was successfully inserted into FAdV-4 and good expression was obtained.
6. Growth curve assay of recombinant virus deleted RFP expression cassette: LMH cells were plated in 6-well plates, and LMH cells were inoculated with 0.1MOI of wild-type FAdV-4 and recombinant virus, respectively, the next day, 2h later and 1% maintenance solution was changed, and cell supernatants were harvested 24h, 48h, 72h, 96h and 120h after infection. Thereafter utilize TCID 50 The viral titer was measured at each time point of the virus and a growth curve was drawn for the virus.
As a result, as shown in FIG. 4A, recombinant virus FAdV4-F11 replicated efficiently on LMH cells, and virus titers of up to 10 at day 5 were achieved 9.6 TCID 50 The growth rate and titer of the strain were much higher than that of wild-type FAdV-4.
7. Stability test of recombinant virus deleted RFP expression cassette: recombinant virus FAdV4-F11 was serially passaged for 12 passages, genome extraction and WB identification were performed every 2 passages.
As shown in FIGS. 4B and C, recombinant virus FAdV4-F11 was stably replicated in LMH cells, and serum 11-type avian adenovirus Fiber protein was detected by WB experiments with murine polyclonal antibodies against serum 11-type avian adenovirus Fiber protein at passages 2,4,6,8, 10, and 12, indicating that recombinant virus after serial passages stably expressed serum 11-type avian adenovirus Fiber protein.
8. Immunogenicity study of inactivated recombinant virus FAdV4-F11 in SPF chickens: to evaluate the immunogenicity of the inactivated recombinant virus, 12 14 day-old SPF chickens were randomly divided into two groups (6 per group). Each chicken of the experimental group had an intramuscular injection of 10 6 TCID 50 FAdV4-F110.3mL, and intramuscular injection of the cell culture medium containing the oil emulsion into the control group of chickens. Two groups of chickens were monitored daily for clinical symptoms and mortality, and blood samples were taken at 14dpi and used to detect neutralizing antibodies to FAdV-4 and FAdV-11.
As a result, as shown in FIG. 5, the average titers of neutralizing antibodies against FAdV-4 and FAdV-11 in the chicken serum inoculated with FAdV4-F11 were about 256 and 128, respectively, whereas the average titers of neutralizing antibodies in the chicken serum of the control group were less than 4. These data indicate that inactivated FAdV4-F11 can induce high levels of neutralizing antibodies to FAdV-4 and FAdV-11.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A recombinant serotype 4 avian adenovirus expressing a serotype 11 avian adenovirus Fiber protein, wherein the serotype 11 avian adenovirus Fiber protein replaces the Fiber-2 protein in the serotype 4 avian adenovirus on the basis of the serotype 4 avian adenovirus.
2. The recombinant serotype 4 avian adenovirus expressing a serotype 11 avian adenovirus Fiber protein of claim 1, wherein the amino acid sequence of the serotype 11 avian adenovirus Fiber protein is as set forth in SEQ ID NO: 1.
3. A method of constructing a recombinant avian adenovirus type 4 expressing the Fiber protein of avian adenovirus type 11 as claimed in claim 1 or 2, comprising the steps of:
constructing a donor plasmid containing a serotype 11 avian adenovirus Fiber protein gene, a left homologous arm and a right homologous arm of a serotype 4 avian adenovirus genome and an RFP expression cassette with LoxP sequences;
constructing a recombinant vector containing sgRNA of a targeted serum 4 type avian adenovirus fiber-2 gene;
transfecting cells with the donor plasmid and a recombinant vector containing sgRNA of a targeted serum 4 type avian adenovirus Fiber-2 gene, inoculating the transfected cells with serum 4 type avian adenovirus, culturing, screening cells which display red fluorescence, and culturing to obtain recombinant serum 4 type avian adenovirus with RFP expression cassette for expressing serum 11 type avian adenovirus Fiber protein;
and infecting cells transfected with Cre recombinase by the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein with the RFP expression cassette, and culturing the cells which are cytopathic and have no red fluorescence to obtain the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein.
4. The construction method according to claim 3, wherein the construction method for constructing the donor plasmid containing the serotype 11 avian adenovirus Fiber protein gene, the left homology arm with the serotype 4 avian adenovirus genome, the right homology arm and the RFP expression cassette with LoxP sequence comprises the steps of:
amplifying a serotype 11 avian adenovirus Fiber protein gene, and inserting the gene into a donor plasmid with a left homologous arm and a right homologous arm of a serotype 4 avian adenovirus genome and an RFP expression cassette with LoxP sequences through a homologous recombination mode and a linearized serotype 11 avian adenovirus Fiber protein gene;
the primer for amplifying the serotype 11 avian adenovirus Fiber protein gene comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 3 and a forward primer with a nucleotide sequence shown as SEQ ID NO. 4;
the primer for linearization treatment of the donor plasmid with the homologous arm at the left end and the homologous arm at the right end of the serum 4-type avian adenovirus genome and the RFP expression cassette with LoxP sequences comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 5 and a forward primer with a nucleotide sequence shown as SEQ ID NO. 6.
5. The construction method according to claim 3, wherein the sgRNA of the targeting serotype 4 avian adenovirus fiber-2 gene is annealed using a forward primer having a nucleotide sequence shown as SEQ ID NO. 7 and a reverse primer having a nucleotide sequence shown as SEQ ID NO. 8.
6. The method of claim 3, wherein the avian adenovirus serotype 4 is recombinant avian adenovirus serotype 4 expressing green fluorescent protein;
the chimeric position of the gene encoding the green fluorescent protein in the recombinant serum 4-type avian adenovirus genome is upstream of the Fiber-2 protein gene.
7. Use of a recombinant avian adenovirus type 4 expressing a avian adenovirus type 11Fiber protein according to claim 1 or 2 or a recombinant avian adenovirus type 4 expressing a avian adenovirus type 11Fiber protein according to the construction method of any one of claims 3 to 6 in the preparation of a vaccine or medicament for the prevention and control of hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis.
8. A vaccine for preventing and controlling hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis, characterized by comprising a recombinant serotype 4 avian adenovirus expressing a serotype 11 avian adenovirus Fiber protein according to claim 1 or 2 or a recombinant serotype 4 avian adenovirus expressing a serotype 11 avian adenovirus Fiber protein obtained by a construction method according to any one of claims 3 to 6 and an adjuvant.
9. The vaccine of claim 8, wherein the adjuvant comprises freunds complete adjuvant and/or freunds incomplete adjuvant.
10. A medicament for preventing and/or treating hepatitis-pericardial effusion syndrome and/or inclusion body hepatitis, which is characterized by comprising the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein according to claim 1 or 2 or the recombinant serum 4 type avian adenovirus expressing the serum 11 type avian adenovirus Fiber protein obtained by the construction method according to any one of claims 3 to 6 and pharmaceutically acceptable auxiliary materials.
CN202311597478.2A 2023-11-28 2023-11-28 Recombinant serotype 4 avian adenovirus expressing serotype 11 avian adenovirus Fiber protein as well as construction method and application thereof Pending CN117603920A (en)

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