CN116492456B - African swine fever virus D129L gene and application thereof in preparation of replication-defective African swine fever vaccine - Google Patents
African swine fever virus D129L gene and application thereof in preparation of replication-defective African swine fever vaccine Download PDFInfo
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Abstract
The invention discloses an African swine fever virus D129L gene and application thereof in preparation of replication-defective African swine fever vaccines, and belongs to the technical field of biological medicines. The invention aims to solve the technical problem of how to prevent African swine fever. In order to solve the technical problem, the invention provides application of a protein with an amino acid sequence of SEQ ID No.2 in preparation of replication-defective African swine fever vaccine. The invention discovers a key essential gene D129L for controlling ASFV replication, and after the D129L gene deletion replication defective virus is used for immunizing pigs, the ASFV virulent attack is carried out, so that 100% virus attack protection can be obtained for the immunized pigs. Therefore, the gene deletion virus can be used as replication-defective African swine fever vaccine for preventing African swine fever.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to an African swine fever virus D129L gene and application thereof in preparation of replication-defective African swine fever vaccines.
Background
African swine fever (AFRICAN SWINE FEVER, ASF) is a febrile hemorrhagic infectious disease of domestic pigs and European and Asia wild pigs caused by African swine fever Virus (AFRICAN SWINE FEVER Virus, ASFV) infection, the morbidity and mortality rate can be up to 100%, and the African swine fever is the first epidemic disease or the primary threat of the global pig industry, and can cause great economic loss. At present, no safe and effective African swine fever vaccine exists worldwide, and although some enterprises or research institutions strive to push gene-deficiency inactivated vaccines, clinical trials and large-area safety evaluation prove that the African swine fever virus gene-deficiency live vaccine has a great biosafety problem. Therefore, safe and effective African swine fever vaccine is still an important subject faced by African swine fever prevention and control.
African swine fever virus is a large complex arbovirus DNA and is the only member of the African swine fever virus genus of the African swine fever virus family. The major capsid protein p72 gene can be divided into 24 genotypes so far according to its difference in nucleotide sequence at its 3' end. The African swine fever virus has a structure which is divided into 5 layers of an outer envelope, a capsid, an inner envelope, a core envelope and a core from outside to inside, and has a virus genome length of 170-193 kb, and more than 150 encoded proteins, wherein more than 68 structural proteins. It is not clear which protective antigen or antigens of african swine fever virus are, and the function of most genes in the viral genome is not clear, and the complexity and unknown structure and function bring great difficulty to the development of african swine fever vaccines. In addition to the biological safety problems of the above-mentioned gene-deficient inactivated vaccines, african swine fever vaccines currently under study include inactivated vaccines, subunit vaccines, recombinant vector vaccines, etc., the safety of these vaccine candidates should not be so great, but the efficacy is not satisfactory, and no safe and effective vaccine has been marketed so far.
Disclosure of Invention
The invention aims to solve the technical problem of how to prevent African swine fever. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
In order to solve the above technical problems, the present invention provides first an application of a protein, wherein the application can be any of the following:
a1 Use in the preparation of a replication-defective african swine fever vaccine;
A2 Use in the preparation of replication-defective african swine fever virus;
A3 Use of the composition for inhibiting replication of african swine fever virus;
a4 Use of a polypeptide for the preparation of a product for propagation or cultivation of replication-defective african swine fever virus;
the protein may be any of the following:
B1 A protein having an amino acid sequence of SEQ ID No. 2;
B2 A protein which is obtained by substituting and/or deleting and/or adding the amino acid sequence of SEQ ID No.2, has more than 80 percent of identity (homology) with the protein shown in B1) and has the same function;
b3 A fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of B1) or B2).
The protein may be a viral protein, named pD129L.
The protein pD129L may be derived from African swine fever Virus (AFRICAN SWINE FEVER Virus, ASFV).
Further, the protein pD129L can be derived from African swine fever virus SY18 strain, and can also be derived from other African swine fever virus natural isolates and artificially attenuated strains.
The protein pD129L is a protein necessary for replication of African swine fever virus, and the virus cannot replicate and assemble into complete virus particles after the protein is deleted. The protein pD129L has a replicative switching function in cells.
In order to facilitate purification or detection of the protein of B1), a tag protein may be attached to the amino-or carboxy-terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing.
Such tag proteins include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his6 tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.
The nucleotide sequence encoding the protein pD129L of the present invention can be easily mutated by a person skilled in the art using a known method, for example, directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the isolated protein pD129L of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein pD129L and function as the protein pD 129L.
The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.
Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.
Herein, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
The invention also provides the use of a biological material related to said protein pD129L, which can be any of the following:
c1 Use in the preparation of a replication-defective african swine fever vaccine;
C2 Use in the preparation of replication-defective african swine fever virus;
C3 Use of the composition for inhibiting replication of african swine fever virus;
C4 Use of a polypeptide for the preparation of a product for propagation or cultivation of replication-defective african swine fever virus;
the biomaterial may be any one of the following D1) to D6):
D1 A nucleic acid molecule encoding said protein pD 129L;
D2 A nucleic acid molecule which inhibits or reduces the function of the gene encoding the protein pD 129L;
D3 An expression cassette containing the nucleic acid molecule of D1) and/or D2);
d4 A recombinant vector comprising D1) and/or D2) said nucleic acid molecule, or a recombinant vector comprising D3) said expression cassette;
D5 A recombinant microorganism comprising D1) and/or D2) said nucleic acid molecule, or a recombinant microorganism comprising D3) said expression cassette, or a recombinant microorganism comprising D4) said recombinant vector;
D6 A recombinant cell containing the nucleic acid molecule of D1) and/or D2), or a recombinant cell containing the expression cassette of D3), or a recombinant cell containing the recombinant vector of D4).
Further, the D2) may be a nucleic acid molecule that inhibits or reduces expression of a gene encoding the protein pD 129L.
In the above application, the nucleic acid molecule of D1) may be any of the following:
e1 A DNA molecule having a nucleotide sequence of SEQ ID No. 1;
e2 A DNA molecule whose coding sequence is SEQ ID No. 1;
E3 A DNA molecule whose coding sequence is the degenerate sequence of any genetic code in SEQ ID No. 1.
The DNA molecule shown in SEQ ID No.1 (D129L gene) encodes a protein pD129L whose amino acid sequence is SEQ ID No. 2.
The nucleotide sequence shown in SEQ ID NO.1 is the nucleotide sequence of the gene encoding the protein pD129L (CDS).
African swine fever virus with deletion of all or part of the sequence of the D129L gene or disruption of the Open Reading Frame (ORF) of the D129L gene, i.e., deletion of the function of the D129L gene, cannot replicate, and the virus can replicate in cells providing the expression protein of the D129L gene, and the D129L gene plays a key role in replication of the African swine fever virus.
An ASFV-sensitive cell line (helper cell line) stably expressing the D129L gene or an ASFV-sensitive cell line capable of providing the pD129L protein intracellularly is capable of supporting replication of any of the D129L gene-deleted replication-defective ASFV on the cell.
An ASFV-sensitive cell line carrying the D129L gene and expressing the pD129L protein or an ASFV-sensitive cell capable of providing the pD129L protein intracellularly is capable of supporting replication of african swine fever virus lacking the D129L gene function.
The African swine fever virus with the D129L gene function deletion replication defective which is proliferated in the auxiliary cell line can be used as a vaccine for preventing African swine fever virus infection, and can provide effective virus attack protection for immunized pigs.
D1 The nucleic acid molecules may also comprise nucleic acid molecules which have been modified by codon preference on the basis of the nucleotide sequence indicated in SEQ ID No. 1.
The coding gene of the protein pD129L (D129L gene) of the present invention may be any nucleotide sequence capable of coding the protein pD 129L. In view of the degeneracy of codons and the preferences of codons of different species, one skilled in the art can use codons appropriate for expression of a particular species as desired.
D2 The nucleic acid molecule may be a molecule that knocks out the gene encoding the protein pD 129L. The knockout can be achieved by homologous recombination.
Further, D2) the nucleic acid molecule may be a DNA molecule with a nucleotide sequence of SEQ ID No.7 and/or SEQ ID No.8 (for knocking out the D129L gene by means of homologous recombination).
The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be an RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or an antisense RNA.
D6 The recombinant cell may be any of the following:
F1 A recombinant cell stably expressing said protein pD 129L;
f2 Recombinant wild boar lung cells (WSL) stably expressing said protein pD 129L;
f3 Recombinant Porcine Alveolar Macrophages (PAMs) stably expressing the protein pD 129L.
The vectors described herein refer to vectors capable of carrying exogenous DNA or genes of interest into host cells for amplification and expression, and may be cloning vectors or expression vectors, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), viral vectors (e.g., baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpesviruses (e.g., herpes simplex viruses), etc. In one or more embodiments of the invention, the vector is a pMD18-T vector, pMD18-T-eGFP, pacAd5 CMVK-NpA (human adenovirus type 5 expression vector), pacAd 5.9.2-100, and/or pcDNA3.1 vector.
The microorganism described herein may be a yeast, bacterium, algae, fungus or virus. Wherein the bacteria may be derived from Escherichia, erwinia, agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas, bacillus (Bacillus), etc. In one or more embodiments of the invention, the microorganism is escherichia coli dh5α.
The cells (host cells) described herein refer to cells that can be used to introduce vectors, including but not limited to: eukaryotic cells (e.g., yeast cells, aspergillus), animal cells (e.g., mammalian cells, insect cells), plant cells, or prokaryotic cells. In one or more embodiments of the invention, the cells are PAM cells, HEK293AD cells, wild boar lung cells (WSL) and/or Immortalized Pig Alveolar Macrophages (iPAM).
The recombinant vector as described herein refers to a recombinant DNA molecule constructed by connecting an exogenous gene of interest to a vector in vitro, and may be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous gene of interest into a recipient cell and provide the exogenous gene of interest with replication, integration, amplification and/or expression capabilities in the recipient cell. In one or more embodiments of the invention, the recombinant vector is pΔD129L-EGFP, pAdCMV-D129L and/or pcDNA3.1-D129L.
The recombinant vector pΔD129L-EGFP contains a D129L gene left homology arm D129L-Larm (SEQ ID No. 7) and a right homology arm D129L-Rarm (SEQ ID No. 8), and the construction method comprises the following steps: the recombinant expression vector is obtained by cloning a D129L homology arm D129L-Larm (SEQ ID No. 7) and a right homology arm D129L-Rarm (SEQ ID No. 8) of a D129L gene into a T-eGFP vector by adopting a seamless connection technology by taking pMD18-T-eGFP (abbreviated as T-eGFP) as a basic vector.
The base vector pMD18-T-eGFP may be a recombinant vector obtained by cloning the p72 promoter (SEQ ID No. 24) and eGFP-SV40polyA (SEQ ID No. 23) into the pMD18-T vector.
The recombinant vector pAdCMV-D129L is a recombinant vector obtained by connecting a D129L gene with a homology arm to a pacAd CMVK-NpA vector by using a homologous recombination method, specifically a vector obtained by inserting an ASFV D129L target gene into an EcoRI cleavage site of a pacAd CMVK-NpA plasmid through a homologous recombination gene fragment of the gene, and the recombinant plasmid pAdCMV-D129L contains a DNA molecule (D129L gene) shown in SEQ ID No.1 and expresses the D129L target gene.
The recombinant vector pcDNA3.1-D129L is obtained by connecting a D129L gene with a homology arm to a pcDNA3.1 vector through a seamless connection technology, the recombinant vector pcDNA3.1-D129L contains a DNA molecule (D129L gene) shown as SEQ ID No.1, and the expressed amino acid sequence is protein pD129L of SEQ ID No. 2.
The recombinant microorganism as used herein refers to a microorganism in which a gene of a microorganism of interest is manipulated and modified to thereby obtain a function change, or a recombinant microorganism in which an endogenous gene of a microorganism of interest is directly subjected to gene editing. Such as a recombinant microorganism obtained by introducing an exogenous gene of interest or a recombinant vector into a microorganism of interest. In one or more embodiments of the invention, the recombinant microorganism is ΔrAdv5-D129L.
The recombinant microorganism DeltarAdv 5-D129L is a recombinant adenovirus expressing the D129L gene. The recombinant adenovirus delta rAdv5-D129L is obtained by linearizing the recombinant vector pAdCMV-D129L and the skeleton plasmid pacAd 5.2-100 by PacI enzyme respectively, then co-transfecting HEK293AD cells, and carrying out rescue of the recombinant adenovirus. The recombinant adenovirus delta rAdv5-D129L can be used for preparing and amplifying replication-defective African swine fever virus.
Recombinant cells as used herein refer to cells that have been manipulated and modified in genes of host cells to produce altered functions. Such as a recombinant cell obtained by introducing a foreign gene of interest or a recombinant vector into a host cell. In one or more embodiments of the invention, the recombinant cell is WSL-D129L and/or iPAM-D129L.
The recombinant cell WSL-D129L is obtained by introducing the recombinant vector pcDNA3.1-D129L into wild boar lung cells (WSL) and screening the recombinant cell to stably express the protein pD 129L.
The recombinant cells iPAM-D129L are obtained by introducing the recombinant vector pcDNA3.1-D129L into Immortalized Pig Alveolar Macrophages (iPAM) and screening the recombinant cells to stably express the protein pD 129L.
The recombinant cells WSL-D129L and iPAM-D129L can be used as auxiliary cells for preparing (proliferating or culturing) replication-defective African swine fever virus with the D129L gene deletion.
The invention also provides replication-defective African swine fever virus, the replication-defective African swine fever virus genome lacks the gene encoding the protein pD129L, or the gene encoding the protein pD129L in the replication-defective African swine fever virus genome is functionally inactivated.
The replication-defective african swine fever virus is unable to replicate, losing replication functions in primary mononuclear-macrophages and in pigs.
The replication-defective african swine fever virus may be a replication-defective african swine fever virus Δsy18Δd129L strain.
The invention also provides a method for preparing replication-defective African swine fever virus (such as strain ΔSY18ΔD129L), which may comprise deleting or functionally inactivating the gene encoding the protein pD129L in the African swine fever virus genome to obtain replication-defective African swine fever virus.
The deletion or functional inactivation of the gene encoding the protein pD129L in the African swine fever virus genome can be accomplished by gene mutation, gene knockout, gene editing or gene knockdown techniques.
Further, the deletion or functional inactivation of the gene encoding the protein pD129L in the African swine fever virus genome can be performed by using a homologous recombination technique to knock out the D129L gene.
In the above method, the method may include the steps of:
g1 Constructing a recombinant vector containing left and right homology arms of the protein pD129L coding gene;
G2 Co-transfecting/infecting (transfecting and/or infecting) the recombinant vector described in G1) with african swine fever virus into a host cell;
g3 Culturing the host cell and screening to obtain replication-defective African swine fever virus.
Further, the left homology arm of the gene encoding the protein pD129L is named as D129L-Larm, and the nucleotide sequence of the D129L-Larm can be SEQ ID No.7. The right homology arm name of the protein pD129L coding gene is D129L-Rarm, and the nucleotide sequence of the D129L-Rarm can be SEQ ID No.8.
G1 The recombinant vector may be the recombinant vector pΔd129L-EGFP.
G2 The African swine fever virus may be African swine fever virus SY18 strain (ASFV SY18 strain).
G2 The host cell may be a PAM cell.
G3 The culture conditions may be 37℃under 5% CO 2 for 72 to 96 hours.
Although the examples provided herein utilize homologous recombination techniques to knock out the D129L gene, the invention is not limited to this particular method. The D129L gene in the African swine fever virus genome can be deleted or inactivated by one of skill in the art using other gene knockout, gene editing, gene mutation or gene knockdown methods known in the art. These methods can also be used in the present invention. Such alternatives are intended to be included within the scope of the invention.
The invention also provides the replication-defective African swine fever virus prepared by the method for preparing the replication-defective African swine fever virus.
The invention also provides a method of proliferation of any of the replication defective african swine fever viruses described herein, which method may comprise culturing any of the replication defective african swine fever viruses described herein in helper cells, which helper cells may be recombinant cells stably expressing the protein pD129L and/or cells containing the protein pD 129L.
The use of said helper cells (recombinant cells stably expressing said protein pD129L and/or cells containing said protein pD 129L) or said helper cells for the preparation of said replication-defective African swine fever virus is also within the scope of the present invention.
The replication-defective African swine fever virus genome lacks the gene encoding the protein pD129L, or the gene encoding the protein pD129L in the replication-defective African swine fever virus genome is functionally inactivated.
The helper cell may be obtained by stably expressing the protein pD129L in any one of the following recipient cells, or the helper cell may be any one of the following cells containing the protein pD 129L:
h1 Primary mononuclear-macrophages suitable for the proliferation of african swine fever virus;
H2 A passaged mononuclear-macrophage cell line suitable for the proliferation of African swine fever virus;
H3 Other passage cell lines suitable for the proliferation of African swine fever virus such as wild boar lung cells, vero cells, heLa cells or HEK293 cell lines, etc.
Further, the helper cell (genetically stable transgenic cell) may be the recombinant cell WSL-D129L and/or iPAM-D129L.
In one embodiment of the present invention, the replication defective african swine fever virus propagation method comprises the steps of:
Inoculating the replication-defective African swine fever virus into recombinant cells iPAM-D129L according to a proportion of 1%, culturing for 72-96 hours under the condition of 5% CO 2 at 37 ℃, harvesting the cultured cells after more than 90% of the cells show fluorescence, and preserving below-15 ℃ after freeze thawing and mixing.
The invention also provides replication-defective african swine fever vaccines, which may include any of the replication-defective african swine fever viruses described herein.
The active ingredient in the replication-defective African swine fever vaccine is any one of the replication-defective African swine fever viruses.
Further, the replication defective african swine fever vaccine may further comprise an adjuvant (adjuvant) and/or a vaccine delivery system (VACCINE DELIVERY SYSTEM).
Further, the replication defective african swine fever vaccine may further include one or more pharmaceutically acceptable carriers.
The adjuvant is a substance that stimulates the body to mount a more intense humoral and/or cellular immune response against the antigen with which it is vaccinated. Adjuvants described herein are well known to those skilled in the art and include, but are not limited to: plant adjuvants (e.g., alkylamines, phenolic components, quinines, sapocins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, and the like), bacterial adjuvants (e.g., cholera toxin, escherichia coli heat labile toxin, bacterial lipopolysaccharide, and the like), aluminum adjuvants and other inorganic component adjuvants (e.g., calcium adjuvants), cytokines and nucleic acid adjuvants (e.g., monocyte clone stimulating factors, leukocyte factors IL-1, IL-2, IL-4, IL-5, IL-6, IFN-gamma, cpG motifs, nucleic acid vectors, and the like), emulsion adjuvants (e.g., freund's adjuvant).
The vaccine delivery system is a class of substances that are capable of carrying antigenic substances to the immune system of the body and storing and exerting their antigenic effects therein for a prolonged period of time. The vaccine delivery system described herein may be an aluminum salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system, or a nanoadjuvant vaccine delivery system.
The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant, or lubricant.
The replication-defective african swine fever virus described herein may be a D129L gene deletion replication-defective african swine fever virus, and the replication-defective african swine fever vaccine may be a D129L gene deletion replication-defective african swine fever vaccine.
The replication-defective African swine fever vaccine adopts a genetic engineering method, and some essential genes in the replication process of African swine fever virus are deleted or normal coding sequences are destroyed, so that the African swine fever virus can be effectively replicated only in an auxiliary cell line for providing the replication essential genes, and cannot be effectively replicated after an infected animal, so that the vaccine strain furthest maintains the original state of the virus, maintains the immunogenicity of complete virus particles, and cannot generate immune side effects such as detoxification and the like in the infected animal.
The invention aims at providing a functional identification of African swine fever virus protein-pD 129L protein and application thereof in preparation of replication-defective African swine fever vaccine.
The experiment proves that the protein encoded by the African swine fever virus D129L gene is a protein necessary for virus replication, and the virus cannot replicate and assemble into complete virus particles after the protein is deleted, but can assemble into complete virus particles when the virus without the protein is coexistent; african swine fever virus lacking the gene can replicate and assemble into viral particles in ASFV-sensitive cell (helper cell) lines stably expressing the protein or ASFV-sensitive cells transiently expressing the pD129L protein, and can proliferate continuously in cell lines stably expressing the protein, but not in cells not expressing the D129L gene; after immunization of pigs with a D129L gene-deleted replication-defective ASFV that proliferated in ASFV-sensitive cells stably or transiently expressing the pD129L protein, the latter may provide a robust immunoprotection against ASFV virulence challenge. The above shows that the D129L gene is one of the key essential genes for replication and assembly of African swine fever virus, and reveals the switching function of the D129L gene expression protein on replication of African swine fever virus in cells. Accordingly, the invention further proves that the D129L gene deletion replication-defective ASFV immune pig can enable immune animals to generate strong immune functions aiming at African swine fever virus infection, and is an effective African swine fever vaccine.
The experiment proves that after the D129L gene of the African swine fever virus is deleted by adopting a homologous recombination technology, the recombinant genome cannot be replicated into infectious virus particles, and a recombinant virus which carries fluorescent protein and can only proliferate in auxiliary cells is obtained by screening and culturing on iPAM expressing the pD129L protein and/or a WSL auxiliary cell line established by using the D129L gene, wherein the D129L gene-deleted virus cannot be replicated in primary PAM cells or non-transformed (not expressing the D129L gene) iPAM and WSL cells, and belongs to replication-defective viruses; after the replication-defective D129L gene deletion virus is immunized twice, ASFV SY18 strain virulent attack is used for the pig, and 100% of virus attack protection can be obtained for the immunized pig. In conclusion, the invention discovers a key essential gene D129L for controlling ASFV replication, and the gene deletion virus can be used as a replication-defective African swine fever vaccine for preventing African swine fever.
Drawings
FIG. 1 shows amplification primers for homologous recombination fragments on both sides of the D129L gene in example 1.
FIG. 2 shows the result of electrophoresis of the homologous recombination arm amplification product in example 1. Wherein, 1: a Marker;2: amplification results of the homologous arm on the left; 3: right homology arm amplification results.
FIG. 3 shows the amplification results of linearized T-eGFP in example 1. Wherein, 1: a Marker; 2. 3: linearized T-eGFP.
FIG. 4 is the identification of pΔD129L-Larm recombinant plasmid in example 1. Wherein, 1: a Marker;2 to 9: positive recombinants.
FIG. 5 shows the result of linearization amplification of the pΔD129L-Larm recombinant plasmid of example 1. Wherein, 1: linearizing the p.DELTA.D129L-Larm recombinant plasmid; 2: and (5) Marker.
FIG. 6 shows the results of identifying pΔD129L-EGFP recombinant plasmid in example 1. Wherein, 1: a Marker;2: negative clone bacteria; 3-10: positive clone.
FIG. 7 is a schematic representation of the homologous recombinant plasmid pΔD129L-EGFP of example 1.
FIG. 8 shows the result of homologous recombination (appearance of green fluorescent cells) in PAM cells transfected with the homologous recombinant plasmid pΔD129L-EGFP/SY 18 of example 1.
FIG. 9 shows fluorescent clusters formed by PAM cells infected with ASFV SY18 wild type virus and SY18ΔD129L in example 1.
FIG. 10 shows the results of PCR identification of the culture supernatant for 16 cells in example 1. Wherein, lane M: a Marker; lanes 1-16: PCR identification results corresponding to 16 single fluorescence focus cell holes respectively; lane 17: is wild poison control; lane 18: is a negative template control.
FIG. 11 shows the results of PCR identification of the culture supernatant of single fluorescent foci cell well for further cloning in example 1. Wherein, lane 1: a Marker; lanes 2-17: PCR identification results corresponding to 16 single fluorescence focus cell holes respectively; lane 18: is wild poison control; lane 19: is a negative template control.
FIG. 12 is a diagram of construction of recombinant human adenovirus type 5 expressing the D129L gene in example 2. ASFV gene and X refer to the D129L gene in the figure.
FIG. 13 shows the size of the D129L gene amplification primers and the expected product in example 2.
FIG. 14 shows the fluorescence observation result of the mixed toxin in example 2. Wherein +: is fluorescent; -: is non-fluorescent.
FIG. 15 shows the construction primers and expected product sizes of the recombinant D129L eukaryotic expression vector of example 3.
FIG. 16 shows the results of partial detection of gene integration in different transformed cell lines in example 3. Wherein, lane M: a Marker; lane 1: WSL-D129L-1; lane 2: iPAM-D129L-1; lane 3: positive control with pcDNA3.1-D129L plasmid as template; lane 4: negative control without template added.
FIG. 17 shows the analysis of the expression level of the desired protein pD129L in the cell clone of example 3.
FIG. 18 shows single foci in iPAM-D129L cells of example 3.
FIG. 19 shows the results of the culture of infected cells collected in the single fluorescence focal cell well of example 3 after freeze thawing and inoculation with iPAM-D129L cell line. The left panel shows the results of the fluorescent microscope of iPAM-D129L stable expression cell line inoculated with the D129L gene deletion replication defective virus; the right panel shows the results of primary porcine PAM cell fluorescence.
FIG. 20 shows the results of determining the defective toxicity of the replication defective D129L gene in example 3. Wherein +: is fluorescent; -: is non-fluorescent.
FIG. 21 shows the results of the D129L gene deletion replication defective African swine fever virus ΔSY18ΔD129L immunized swine challenge in example 4. And (3) injection: the real-time qPCR detection results of fever or morbidity pigs are positive.
FIG. 22 shows ASFV nucleic acid detection results of ASFV, ΔSY18ΔD129L immunized pigs, mouth, nose, anal swab, at the end of the observation in example 4. Determination criteria: judging that the sample is less than or equal to 35 and positive; the CT values are more than 35 and less than 40, and are judged to be suspicious (CT values are given); and judging that no numerical value is negative.
FIG. 23 shows the results of ASFV nucleic acid detection in example 4 in 28-day section after challenge in ΔSY18ΔD129L immunized pigs. "-" is no numerical value. Determination criteria: judging that the sample is less than or equal to 35 and positive; the CT values are more than 35 and less than 40, and are judged to be suspicious (CT values are given); and judging that no numerical value is negative.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
African swine fever virus SY18 strain (ASFV SY18 strain, ASFV-SY 18) in the following examples is described in ：Zhou X,Li N,Luo Y,Liu Y,Miao F,Chen T,Zhang S,Cao P,Li X,Tian K,Qiu HJ,Hu R(2018)Emergence of African Swine Fever in China,2018.Transboundary and emerging diseases 65(6):1482-1484.doi:10.1111/tbed.12989), in the following literature isolated by the epidemiological laboratory of the military veterinary institute in 2018. GenBank number of genomic sequence of the virus: MH766894.2 (27-JAN-2021).
The expression plasmid vector pacAd CMVK-NpA and backbone plasmid pacAd 5.2-100 in the examples described below were purchased from Invitrogen, inc., U.S.A.
The pcDNA3.1 vector in the examples described below is a product of Invitrogen corporation.
HEK293AD cells in the examples below were purchased from Invitrogen, usa.
The wild boar lung cells (WSLs) in the examples below were purchased from Ulva test biotechnology Co.
IPAM cells in the following examples were a passaged porcine alveolar macrophage line, purchased from North Narax technologies Co.
PAM cells in the examples below were primary porcine alveolar macrophages derived from swine infected with no specific pathogen, obtained by flushing the lungs with sterile PBS (0.012 mol/L, pH 7.2), lysing the erythrocytes, and centrifuging.
The ASFV replication-related genes and the expressed proteins are more, such as p72, p30 and the like are indispensable components in the ASFV virus replication process, and histone-like pA104R, p (pK 78R), pG1340L, pD1133L, B962L, Q706L and the like are also related to the virus replication, transcription initiation and the like, but key protein components influencing the ASFV replication are not clear. In the construction process of the African swine fever virus gene deletion virus, the invention discovers a gene (namely a D129L gene) capable of limiting virus replication, the African swine fever virus genome lacking the gene can not complete the virus replication process to assemble into virus particles, and the gene deletion virus coexisting with wild virus can assemble into virus particles and express the green fluorescent protein gene carried by the virus particles. An auxiliary cell line expressing the D129L gene is constructed, and after the pig is immunized by the replication-defective African swine fever virus saved by the cell line, the immune pig can be provided with a strong protection effect against ASFV virulent attack. The D129L gene, an African swine fever virus sensitive cell line expressing the gene and the replication-defective African swine fever virus lacking the gene are described as being used for developing and preparing replication-defective African swine fever vaccines.
The nucleotide sequence of the D129L gene is SEQ ID No.1, the encoded protein name is pD129L, and the amino acid sequence of the protein pD129L is SEQ ID No.2. The protein pD129L is a protein necessary for replication of African swine fever virus, and the virus cannot replicate and assemble into complete virus particles after the protein is deleted. The protein pD129L has a switching function on the replication of African swine fever virus in cells.
EXAMPLE 1 rescue of D129L Gene-deleted replication-defective ASFV
1. Cloning of D129L Gene deletion homologous recombination arm nucleic acid fragment
Two pairs of primers were designed on the left and right (upstream and downstream) sides of the D129L gene, respectively, and the primer sequences (SEQ ID No.3-SEQ ID No. 6) are shown in FIG. 1, and two homologous recombination fragments were amplified by ordinary PCR, respectively: the left homology arm of the D129L gene (named D129L-Larm, SEQ ID No. 7) and the right homology arm of the D129L gene (named D129L-Rarm, SEQ ID No. 8). The amplification products were recovered and then electrophoresed on 10% agarose gel to give the results shown in FIG. 2.
2. Construction of D129L Gene deletion homologous recombination arm plasmid
Construction of pMD18-T-eGFP plasmid
The pMD18-T-eGFP plasmid was obtained by cloning the p72 promoter (SEQ ID No. 24) and eGFP-SV40polyA (SEQ ID No. 23) into the pMD18-T vector (Takara Corp. Product, cat. No. 6011).
2.1.1, Acquisition of eGFP-SV40polyA sequence: the DNA fragment 1 (1036 bp) containing eGFP-SV40polyA (SEQ ID No. 23) was amplified from the peGFP-C3 plasmid (HonorGene company product, cat# HG-VPH 0002) with the design of primer (eGFP F：5'-GCCTGCAGGTCGACGATTATGGTGAGC AAGGGCGAGGA-3'(SEQ ID No.25);polyA R：5'-CGGGGATCCTCTAGAGATACCACA ACTAGAATGCAGTG-3'(SEQ ID No.26)), and the nucleotide sequence of the eGFP-SV40polyA was shown as SEQ ID No. 23.
2.1.2 Construction of pMD18T-eGFP-SV40polyA plasmid: the DNA fragment 1 is amplified, purified and recovered by PCR and then mixed with pMD18-T vector In-HD Cloning Plus (Takara Bio Inc. product, cat# 638911, according to the reagent operating instructions) under the action of connection to form pMD18T-eGFP-SV40polyA recombinant plasmid, the connection product is transformed into E.coli DH5 alpha competent cells, after 16-18 h culture, several colonies are selected and cultured in 200 mu L liquid LB medium containing ampicillin resistance, after 6h culture in a shaking table at 37 ℃,1 mu L bacterial solution is used as a template, bacterial solution identification is carried out, after identification correct colony expansion culture, the plasmid is extracted by using an Axygen plasmid miniextraction kit, and the plasmid which is correct by sequence determination is named pMD18T-eGFP-SV40polyA.
2.1.3, Acquisition of p72 promoter sequence: the p72 promoter sequence is obtained by amplifying ASFV SY18 strain genome, a primer (p72 F：5'-GCCTGCAGGTCGACGATTAAGGGTCGCCGGAGGAAA-3'(SEQ IDNo.27);p72 R：5'-TCCTCGCCCTTGCTCACCATATATAATGTTATAAAAATAAT-3'(SEQ IDNo.28)) is designed to amplify a DNA fragment 2 (237 bp) containing the p72 promoter (SEQ ID No. 24), and the nucleotide sequence of the p72 promoter is shown as SEQ ID No. 24.
2.1.4 Construction of pMD18-T-eGFP plasmid: designing primer (F: 5'-ATGGTGAGCAAGGGCGAGGA-3' (SEQ ID No. 29); R:5'-AATCGTCGACCTGCAGGC-3' (SEQ ID No. 30)) to amplify pMD18T-eGFP-SV40polyA recombinant plasmid, linearizing the plasmid to obtain 3778bp fragment, purifying and recovering, and then using In-HD Cloning Plus kit (used according to the operation instructions) is connected with p72 promoter fragment (DNA fragment 2), the connection product is transformed into E.coli DH5 alpha competent cells, after 16-18 h culture, a plurality of colonies are picked up and cultured in 200 mu L of liquid LB culture medium containing ampicillin resistance, after 6h culture in a shaking table at 37 ℃,1 mu L of bacterial liquid is used as a template, bacterial liquid identification is carried out, and pMD18T-p72 promoter-eGFP-SV 40polyA recombinant plasmid, abbreviated pMD18-T-eGFP or T-eGFP is obtained.
2.2 Construction of homologous recombination arm plasmids
The recombinant plasmid containing nucleotide fragments of both side sequences of the D129L gene (namely D129L-Larm and D129L-Rarm) is constructed by using a pMD18-T-eGFP plasmid (T-eGFP) containing the promoter sequence of the ASFV p72 protein gene and the SV40 polyA sequence as a vector and adopting a seamless connection technology, and the specific operation is as follows:
2.2.1, left homology arm ligation and identification: the primers p72 promoter-F and T-R are used, the T-eGFP is used as a template for amplification, the vector is linearized in front of the p72 promoter, the vector with the linearized left side locus is obtained, the length is 3890bp, and the amplification result is shown in figure 3. Then use In- HD Cloning Plus (Takara Bio Inc. product, cat# 638911), connecting the recovered D129L gene left homology arm D129L-Larm with a vector linearized at the left site according to the operation instructions, then transforming the connection product into E.coli DH5 alpha competent cells, culturing for 16-18 h, picking up a plurality of colonies, culturing in 200 μl of liquid LB medium containing ampicillin resistance, culturing in a shaking table at 37 ℃ for 6h, using 1 μl of bacterial liquid as a template, carrying out bacterial liquid identification (FIG. 4), extracting plasmids by using an Axygen plasmid miniextraction kit after the identification of correct colonies, and obtaining the correct plasmid named pDeltaD 129L-Larm after sequence determination, namely the recombinant plasmid inserted into the left homology arm.
Primer p72 promoter-F:5'-AAGGGTCGCCGGAGGA-3' (SEQ ID No. 9),
Primer T-R:5'-AATCGTCGACCTGCAGGCATG-3' (SEQ ID No. 10).
2.2.2 Ligation of right homology arms: linearizing the recombinant plasmid pDeltaD 129L-Larm obtained in the step 2.2.1 from the right side locus by using linearizing primers T-F and poly-R to obtain a right side locus linearizing vector, and recovering by gel to obtain a purified linearizing recombinant plasmid to obtain a 5059bp product, wherein the identification result is shown in figure 5. According to the same method as that In the step 2.2.1, the right homology arm D129L-Rarm of the D129L gene is connected with a right site linearization vector by using an In-fusion Kit, after the connection product is transformed into E.coli DH5 alpha competent cells, colonies are picked up for identification, 3555bp products are obtained, the identification result is shown In FIG. 6, the positive colonies are amplified for culture, plasmids are extracted, and the plasmids are sent to Jilin provincial treasury Mei Biotechnology Co., ltd for sequencing. The plasmid with correct sequence is named p delta D129L-EGFP, namely the homologous recombinant plasmid containing a D129L homology arm D129L-Larm (SEQ ID No. 7) and a right homology arm D129L-Rarm (SEQ ID No. 8) of the D129L gene, and strains and plasmids are preserved. The schematic diagram of the recombinant plasmid is shown in FIG. 7.
Primer T-F:5'-ATCTCTAGAGGATCCCCG-3' (SEQ ID No. 11),
Primer poly-R:5'-ACCACAACTAGAATGCAG-3' (SEQ ID No. 12).
3. Rescue of D129L Gene deleted African swine fever recombinant Virus
The PAM cells were inoculated with ASFV SY18 strain 10 4.0TCID50 to obtain PAM cells inoculated with ASFV SY18 strain, and 5. Mu.g of the homologous recombinant plasmid pΔD129L-EGFP was transfected into PAM cells inoculated with ASFV SY18 strain with Jet-Macrophage transfection agent, and cultured at 37℃for 72 to 96 hours under 5% CO 2. When green fluorescent cells were present (as shown in FIG. 8), fluorescent cells were picked up by a sterile pipette, rinsed 3 times in PBS (0.012 mol/L, pH 7.2), transferred to a 0.5ml Eppendorf tube, freeze-thawed 2 times, serial diluted, and inoculated with PAM cells.
The fluorescent cells were selected and rinsed 3 to 6 times in this way, and when more green fluorescent cells were reached (as shown in FIG. 9). The titer of virus (green fluorescent virus) in the freeze-thaw was determined by serial dilution of the virus and inoculation of PAM cells. As a result, the green fluorescent virus titer was measured to be 10 3.2TCID50.
According to the measurement results, the green fluorescent virus was subjected to 10 3.2 -fold dilution and then to 2-fold dilution in succession, and each dilution was inoculated with 1 96-well PAM cell culture plate. Culturing at 37 deg.C under 5% CO 2 for 72-96 hr. As a result, 16 wells containing a single fluorescent focus were present in total in the 96-well plate.
For the culture supernatant of 16 cells wells (abbreviated as virus culture), primer D129L-I-F was used: 5'-GCACACGCACAAGAATAC-3' (SEQ ID No. 13) and primer D129L-I-R:5'-AACGCCACTGAATTACCTAT-3' (SEQ ID No. 14) in which 2 nucleic acid amplification bands were present in 15 wells (wells numbered 2-16 in FIG. 10), an amplification band of wild type virus (ASFV SY18 strain) (1090 bp in size) and an amplification band of recombinant type virus (1922 bp in size), and the other well (well numbered 17 in FIG. 10) was a wild type virus band. The results are shown in FIG. 10.
To confirm the rescue effect of the virus, a single well virus culture, which was identified as positive by PCR (the amplified bands of the wild virus and the recombinant virus were obtained as a result of PCR detection), was subjected to TCID 50 assay, diluted, inoculated into 96-well PAM cell culture plates, and single fluorescent foci cell wells were observed. PCR detection amplification is carried out on single fluorescent focal cell hole culture supernatant by using a primer D129L-I-F (SEQ ID No. 13) and a primer D129L-I-R (SEQ ID No. 14), and as a result, 2 amplification bands still appear, namely an amplification band of a gene deletion replication defect recombinant virus (the size is 1922 bp) and an amplification band of a wild virus (the size is 1090 bp). As shown in fig. 11.
The above results indicate that the D129L gene deletion recombinant toxin cannot be replicated alone and can be replicated with the aid of wild toxin, so that a mixture-mixed toxin containing both wild toxin and D129L gene deletion recombinant toxin is obtained through the experiment. The culture supernatant of the amplified band (1922 bp) and amplified band (1090 bp) of the recombinant virus having the gene deletion obtained by PCR detection with the primers D129L-I-F and D129L-I-R was designated as mixed virus Mix-V containing wild virus (ASFV SY18 strain) and D129L recombinant virus having the gene deletion. The D129L gene-deleted African swine fever recombinant virus is not rescueable, and belongs to replication-defective African swine fever viruses.
EXAMPLE 2 construction of recombinant human adenovirus type 5 expressing the D129L Gene and acquisition of the D129L Gene deletion replication defective African swine fever Virus
1. Construction of recombinant human adenovirus type 5 expressing D129L Gene
The construction scheme of the recombinant virus is shown in FIG. 12.
1.1 Construction of homologous recombinant plasmids
Extracting genome of SY18 strain of African swine fever virus by adopting a conventional method, designing primers of ASFV D129L target genes with expression plasmid vectors pacAd CMV K-NpA (human type 5 adenovirus expression vectors) homology arms, respectively D129L-F：5'-GCTTGATATCGAATTGCCACCATGGATATAAATCCCTTACTGTATTTGCAAGCA-3'(SEQ ID No.15);D129L-R：5'-TCCCCCGGGCTGCAGGTTAATCTTCATCATCTGT-3'(SEQ IDNo.16)., amplifying the genome of SY18 strain serving as a template by using primers D129L-F and D129L-R by adopting a PCR method to obtain homologous recombination gene fragments (fragment size is 427bp, italic marked part in the primers is homology arm sequence, fragment size is target gene plus homology arm) of the D129L gene. The following recombinant plasmid was obtained by inserting a desired gene fragment between EcoRI sites of plasmid pa cAd5 CMVK-NpA.
Connecting the homologous recombination gene fragment of the D129L gene with a linearized pacAd CMVK-NpA plasmid obtained by EcoRI enzyme digestion, then transforming competent cells of escherichia coli, and carrying out homologous recombination to obtain a recombinant plasmid: pAdCMV-D129L.
Recombinant plasmid pAdCMV-D129L (also called recombinant vector pAdCMV-D129L) is a recombinant vector obtained by connecting a D129L gene with a homology arm to a pacAd CMVK-NpA vector by using a homologous recombination method, specifically a vector obtained by inserting an ASFV D129L target gene into an EcoRI cleavage site of pacAd CMVK-NpA plasmid through a homologous recombination gene fragment of the gene, and recombinant plasmid pAdCMV-D129L contains a DNA molecule (D129L gene) shown in SEQ ID No.1 to express the D129L target gene.
1.2 Rescue and identification of recombinant viruses
Linearizing the recombinant plasmid pAdCMV-D129L and the backbone plasmid pacAd 5.2-100 by PacI enzyme to obtain the digested recombinant plasmid and the digested backbone plasmid pacAd 5.5.2-100.
Mixing the recombinant plasmid with the digested skeleton plasmid pacAd 5.2-100 (4 micrograms) at a certain ratio (2 micrograms), and then mixing with 12 microliters of transfection reagent2000 2Ml of cell culture medium was added to the monolayer, and the monolayer-grown HEK293AD cells of 25cm 2 were transfected, and after 3 days, the transfected cells were passaged to a monolayer, and after growing to a second transfection according to the above procedure.
Transfecting for 3 times according to the steps, when a large number of lesions such as a circle and the like are observed in the cells, freezing and thawing the cells, collecting and storing the freezing and thawing liquid, and respectively marking the freezing and thawing liquid as P0 generation recombinant adenovirus; the nucleic acid of the P0 generation recombinant adenovirus is extracted, an identification primer (an upstream primer: 5'-CGCAAATGGGCGGTAGGCGTG-3' (SEQ ID No. 17), a downstream primer: 5'-CACTGCATTCTAGTT GTGGTTT-3' (SEQ ID No. 18)) is used for identifying the recombinant adenovirus, the size of the expected identification product is shown in FIG. 13, and the target size fragment is amplified to obtain the target recombinant adenovirus.
After PCR was confirmed to be correct, the recombinant adenovirus expressing the D129L gene was designated ΔrAdv5-D129L. The P0 generation recombinant adenovirus delta rAdv5-D129L is passaged 3 times in HEK293AD cells according to 1% (V/V) and the freeze thawing liquid is collected for preservation, thus obtaining the P1, P2 and P3 generation recombinant adenovirus delta rAdv5-D129L.
1.3 Determination and identification of Virus titre
The titer of the P3 generation recombinant adenovirus delta rAdv5-D129L obtained in the step 1.2 is measured by the following method: 10 times serial dilutions are carried out on the virus culture solution, 12 times dilutions are carried out, 8 repeated holes are inoculated on HEK293AD cells of a 96-well plate at each dilution, the inoculum size is 0.1 ml/hole, the cells are cultivated for 5 to 6 days at 37 ℃ after inoculation, cytopathy is observed under a light microscope, and the TCID 50 of the recombinant adenovirus is calculated according to a Reed-Muench method, and the result is 10 8.4TCID50/ml.
2. Mixed toxin Mix-V TCID 50 assay
The harvested mixed toxin Mix-V was subjected to 10-fold serial dilutions, 6 dilutions of 10 1.0～106.0 were inoculated into a PAM cell 96-well plate, 8 cell wells were inoculated per dilution, 100. Mu.l of each well, and the cells were observed for fluorescence under an inverted fluorescence microscope after culturing for 72 hours under the condition of 5% CO 2 at 37℃with the complete medium RPMI 1640 (product of Gibco Co., ltd., product No. C11875500 BT) being supplemented, and the content of the mixed toxin (recombinant virus expressing fluorescent protein) was roughly calculated. The observation results of the mixed toxin of wild toxin and recombinant toxin are shown in FIG. 14. The rough content of mixed toxin Mix-V calculated in this way is 10 3.84TCID50/ml.
3. Co-infection of D129L gene-recombinant human adenovirus 5 and diluted mixed virus Mix-V and selection of purified virus
According to the measured virus titer of mixed virus Mix-V, wild boar lung cells (WSL) were inoculated with D129L gene-recombinant human adenovirus type 5 (i.e., recombinant adenovirus Δradv 5-D129L) at moi=1 to obtain recombinant adenovirus-infected WSL cells, and diluted mixed virus Mix-V100 μl containing 10TCID 50 per ml was added simultaneously to each well to infect recombinant adenovirus-infected WSL cells. Culturing at 37deg.C under 5% CO 2 for 3-5 days, and freezing and thawing to collect single-hole fluorescence foci when the number of fluorescence cells reaches above 80%. Wherein, part is used for the expansion culture and the preparation of viruses; another part was inoculated with primary porcine alveolar macrophages (PAM cells). The results showed that after the diluted Mix-V was inoculated with recombinant adenovirus-infected WSL cells, a total of 13 single fluorescent foci cells were present, designated Mix-V-1-13, respectively. After the cell freeze-thawing liquid is collected and transferred to the primary pig alveolar macrophage culture hole, only Mix-V-6 freeze-thawing liquid is inoculated, and fluorescence does not appear. The remaining Mix-V-6 virus was stored as a 0-generation D129L gene deletion replication defective African swine fever virus (strain ΔSY18ΔD129L).
4. Preparation of D129L Gene deletion replication-defective African swine fever Virus (ΔSY18ΔD129L Strain) and Virus titre measurement
4.1 Preparation
Purified clone cell cultures of the D129L gene deletion replication defective African swine fever virus (DeltaSY 18 DeltaD 129L strain) and D129L gene-recombinant human adenovirus type 5 (i.e. recombinant adenovirus DeltarAdv 5-D129L) were simultaneously CO-infected with a wild boar lung cell line (WSL) monolayer at a dose of MOI=1, cultured at 37 ℃ under 5% CO 2 condition, and when fluorescence reaches more than 60% (3-5 days), freeze-thawed cells were frozen, and virus-infected cell lysates were collected.
4.2 Titre determination
The collected virus culture solutions were subjected to 10-fold serial dilutions, 0.1ml of each dilution was simultaneously inoculated with a wild boar lung cell line (WSL) monolayer at a dose, with D129L gene-recombinant human adenovirus type 5 (i.e., recombinant adenovirus Δradv 5-D129L) having moi=1, each dilution was inoculated with 8 cell wells, and cultured at 37 ℃ under 5% CO 2 for 4 days, and fluorescence in each dilution cell well was observed. TCID 50 was calculated according to Spearman Karber method. The results showed that the prepared purified D129L gene deletion replication defective african swine fever virus (Δsy18Δd129L strain) had a titer of 10 5.64TCID50.
EXAMPLE 3 construction of African swine fever Virus D129L Gene stable expression cell line and rescue of replication defective D129L Gene deleted African swine fever Virus
1. African swine fever virus D129L gene fragment acquisition and construction of expression plasmid thereof
The invention uses the extracted African swine fever virus ASFV SY18 genome as a template, and uses D129L-Fin (SEQ ID No. 19) and D129L-Rin (SEQ ID No. 20) as primers, the sequences of which are shown in figure 15. The D129L gene fragment D129L-in with a left homology arm and a right homology arm is obtained through amplification and recovery, the D129L gene fragment D129L-in is constructed to an EcoRV site of a pcDNA3.1 vector through a seamless connection technology, bacterial colonies are picked for expansion culture after competent cells of escherichia coli DH5 alpha are transformed, and plasmids with correct PCR identification and sequence determination are named pcDNA3.1-D129L and are used for carrying out construction experiments of subsequent cell lines.
The plasmid pcDNA3.1-D129L (recombinant vector pcDNA3.1-D129L) is a recombinant vector obtained by linking a D129L gene with a homology arm to a pcDNA3.1 vector by a seamless connection technology, and the recombinant vector pcDNA3.1-D129L contains a DNA molecule (D129L gene) shown in SEQ ID No.1 and expresses a protein pD129L with an amino acid sequence of SEQ ID No. 2.
2. Transfection of cell lines
2.1 Screening of cells for sensitivity to G418
2.1.1 Wild boar Lung cells (WSL) were purchased from Shanghai Ulva test biotechnology Co., ltd. Immortalized pig alveolar macrophages (iPAM), a pig alveolar macrophage line, from North Narah Ind Co., ltd. Which may also employ other ASFV sensitive cell lines. The cells were passaged into 24 wells of a 24-well plate, 10 5 cells/well. The medium in the wells was 1640 medium (Gibco company, USA, cat# C11875500 BT).
2.1.2 After cell attachment, the medium in the wells was replaced with medium containing G418 at the specified concentration (made of G418 and 1640 medium). The concentration gradient is: 0. 100, 200, 400, 600, 800, 1000, 1200 μg/mL. Each concentration set 4 replicates.
2.1.3 Changing fresh culture medium with corresponding concentration G418 every other day, and observing and recording the growth condition of cells in each hole every day. Cells with high concentrations of G418 initially begin to grow slowly and die. On day 14, the G418 concentration of the cell wells where WSL or iPAM cells all died was determined and on this basis a gradient was increased as G418 usage concentration for later pressure screening of the cell lines.
The results showed that when cultured for 14 days, WSL or iPAM cells cultured in G418 medium at concentrations of 200 and 400. Mu.g/ml and above all died, and at 100 and 200. Mu.g/ml, partially surviving cells were seen. Thus, the concentrations of WSL and iPAM cells for subsequent pressure screening were 400 μg/ml and 600 μg/ml, respectively.
2.2 Cell transfection and the obtaining of stable cell lines
2.2.1, About 12h in advance, WSL cells (recipient cells) or iPAM cells (recipient cells) were passaged into 6-well plates, 10 6 cells/well. After normal culture until cells grow to a monolayer, transfection is ready: mu.g of plasmid pcDNA3.1-D129L was added to 100. Mu.L of OPTI-MEM medium; 100. Mu.L of OPTI-MEM medium was added-LT1Reagent about 4 μl, each standing for 5min, mixing together, standing at room temperature for 15-20 min; after the transfected cells change a fresh OPTI-MEM culture medium, adding a transfection system, and uniformly mixing; incubating in a 5% CO 2 cell incubator at 37 ℃ for 4-6 h; discarding the transfection mixture in the cell holes, replacing a new culture solution, and placing the cell holes in a 5% CO 2 cell incubator for continuous culture at 37 ℃; after 24h of culture, passaging to 96-well cell culture plates at 1:500 while supplementing small amounts of normal cells; the G418 was added to 1640 medium at a final concentration of 400. Mu.g/mL (WSL) or 600. Mu.g/mL (iPAM) to select cell lines with G418 resistance under pressure.
2.2.2 During cell drug screening, cell survival in each well was observed daily. About 7 days, death of cells in some wells began to occur; around 14 days, most cells in the cell wells all died, and 1 or more clustered cell clones appeared in some of the cell wells.
2.2.3, Selecting single cell clone holes, digesting the cells in the single cell clone holes, and then, transferring the single cell clone holes into a new 24-hole plate for continuous pressurized culture; cell growth in wells was observed daily; after the cells grow to a monolayer, they are digested, partially passaged into 6-well plates, and then expanded, and partially passaged into 24-well plates, ready for identification.
2.3 Identification of D129L Gene expression in a Stable cell line
2.3.1, Detection of gene integration: a plurality of single cell clones, designated WSL-D129L-1 to WSL-D129L-6 (single cell clone obtained from WSL as a receptor cell) and iPAM-D129L-1 to iPAM-D129L-5 (single cell clone obtained from iPAM as a receptor cell), were picked up, and the chromosome genome was extracted for these cell clones, and PCR amplification was performed using the D129L gene primer (D129L-F1：5'-TTAATCTTCATCATCTGTTT-3'(SEQ ID No.21),D129L-R1：5'-ATGGATATAAATCCCTTACT-3'(SEQ ID No.22)). FIG. 16 shows the results of nucleic acid integration assays for a portion of cells of two cell clones.
2.3.2 Detection of expression level of the target protein pD129L in cell clone: collecting the single cell clone culture supernatant, coating an enzyme-linked plate, respectively reacting with anti-ASFV positive serum in a 37 ℃ wet box for 1 hour according to an indirect ELISA operation program, leaching by PBS-Tween for 3 times (3 min/time), and then respectively reacting with HRP-labeled rabbit anti-pig secondary antibody with working concentration in a 37 ℃ wet box for 1 hour, leaching by PBS-Tween for 3 times each for 3min. A blank PBS, a blank cell culture supernatant (Control 1), and a medium DMEM (Gibco product, cat. No. C11995500 BT) Control (Control 2) were also set. After the reaction, color development was performed. The results showed that all controls were true. Specific immune responses were generated between 11 cell clone supernatants and anti-ASFV positive serum, and the results are shown in fig. 17. The cell clones WSL-D129L-4 and iPAM-D129L-1, which had the highest expression level of pD129L protein, were kept as target cell lines, and were designated WSL-D129L (recombinant cells expressing pD129L protein obtained from WSL as receptor cells) and iPAM-D129L (recombinant cells expressing pD129L protein obtained from iPAM as receptor cells), respectively.
2.4 Preparation and preservation of the D129L Gene-Stable expression cell line WSL (or iPAM) -D129L
Monoclonal WSL-D129L and iPAM-D129L were stably expressed against the D129L proteins identified above, passaged for extended culture, and stored in liquid nitrogen.
3. Preparation of D129L Gene deletion replication defective African swine fever Virus Using D129L protein stable expression Single cell clones WSL-D129L and iPAM-D129L cell lines
3.1 Selection of D129L Gene deletion replication defective African swine fever Virus
3.1.1, Determination of Mixed toxin Mix-V TCID 50: as in example 2, "Mix-V TCID 50 determination", the crude content of the mixed toxin was 10 3.84TCID50/ml.
3.1.2 Purification, identification and seed toxicity preparation of D129L Gene deletion toxicity
The mixed toxin Mix-V was diluted 10 3.84 to give a diluted mixed toxin suspension containing 1 TCID 50 per 100. Mu.l. 100 μl of the diluted mixed suspension was inoculated into a D129L gene stable expression cell line (WSL-D129L or iPAM-D129L) cultured in a 96-well plate, and after culturing at 37℃under 5% CO 2 for 72-96 hours, the infected cell wells with only single fluorescent cells or single foci cells were observed and labeled under an inverted fluorescent microscope. Cells Kong Dongrong containing single fluorescent cell clones were inoculated with the freeze-thawing solution respectively into a D129L gene stable expression cell line and primary porcine PAM cells, and fluorescence was observed in the cells under an inverted fluorescence microscope. The results of fluorescence microscopy of infected cell wells of single fluorescent foci cells obtained by mixing toxic Mix-V infection iPAM-D129L are shown in FIG. 18. FIG. 19 shows the results of culture of single fluorescent focal cells obtained by mixed toxin Mix-V infection iPAM-D129L after freeze thawing and inoculation of iPAM-D129L cell line, with significant fluorescence in iPAM-D129L cell wells and no fluorescence in porcine PAM cells. It was demonstrated that the virus obtained in single fluorescent cells or single fluorescent cell foci was purified D129L gene-deleted virus, which was replication defective. Cells in the infected cell wells of single fluorescent foci cells obtained by infecting iPAM-D129L with the mixed toxin Mix-V were freeze-thawed, and a freeze-thawing solution was collected, which contained only the virus, which was replication-defective D129L gene, and was designated as DeltaSY18DeltaD 129L.
3.2 Preparation of replication defective D129L Gene
3.1 Of freeze-thawing solution containing DeltaSY 18 DeltaD 129L of the deletion replication defective African swine fever virus of the D129L gene is harvested, iPAM-D129L gene stable expression transgenic cell lines are inoculated according to a proportion of 1%, 1640 medium (product of Gibco company, product number C11875500 BT) is used for culturing for 72-96 hours under the condition of 5% CO 2 at 37 ℃, after more than 90% of cells show fluorescence, the cultured cells are harvested, and after freeze-thawing mixing, the freeze-thawing solution containing DeltaSY 18 DeltaD 129L is obtained, and the cells are preserved below-15 ℃ and marked as DeltaSY 18 DeltaD 129L-202201.
3.3 Titration of the content of replication defective D129L Gene deficiency
3.2 Freeze-thaw containing D129L gene deletion replication-defective African swine fever virus ΔSY18ΔD129L was harvested, serial 10-fold dilutions were made, 10 4.0～108.0 dilutions were inoculated into a D129L gene stable expression transgenic cell line (iPAM-D129L) 96-well plate, 8 wells were inoculated per dilution, 100 μl per well was supplemented with 1640 medium (Gibco Co., ltd., product No. C11875500 BT) containing 5% calf serum, and cultured at 37℃for 72 hours under 5% CO 2. Fluorescent cell wells were observed under an inverted fluorescent micromirror, and D129L gene deletion replication defective african swine fever virus titers were calculated. The results of the determination of the D129L gene deletion replication defective African swine fever virus content are shown in FIG. 20, and the result of the determination of the virus titer is 10 7.64TCID50/mL.
EXAMPLE 4D 129L Gene deletion replication defective African swine fever Virus immunoassay
1. Virus (virus)
1.1, Example 3 step 3.1.2, D129L Gene deletion replication defective African swine fever Virus (ΔSY18ΔD129L Strain)
The ASFV SY18ΔMGF/CD2v strain is a double gene fragment deleted African swine fever virus, and is described in the following documents: zhang Yanyan, chen Teng, zhang Jingyuan, ji Yu, mui invention, bao Zongyi, wang Lidong, guo Xiaoyu, zhou Xintao, yang Jinmei, wang Xiaohu, gao Yulong, wang Chunyu, bao Chen, mi Lijuan, sun Xuefei, feng Na, yang Jinjin, wang Chengyu, mo Zhonghai, li Jiping, meng Keyin, field much, li Nan, wang Shuchao, wang Ying, zhang Fei, zhang Jinxia, jiang Yiqian, liu, zhang Shoufeng, zhang Zhongyang, qian Yingjuan, zhu Hongfei, gao Yuwei, chen Hongjun, li Jinxiang, hu Rongliang. Construction of african swine fever virus gene deleted vaccine strain and immunoprotection properties. Chinese veterinary school, 2019;39 1421-1427 constructed, identified, stored and supplied by the military veterinary institute of the military medical institute of the academy of sciences.
1.3 African swine fever virus virulent SY18 strain (ASFV SY18 strain, ASFV-SY 18).
2. Animals
1 Month-old healthy piglets (Dunaliella long ternary hybrid piglets, weight 6.0-7.5 kg) are several. Serum was collected before purchase for detection of african swine fever virus nucleic acid and antibodies, and clinically healthy pigs negative for both ASFV nucleic acid and antibodies were used in this experiment.
3. Virus preparation
Preparation and determination of iPAM-D129L helper cell culture of ASFV SY18 ΔD129L strain: as in examples 3, 3.2 and 3.3.
Preparation of PAM cell culture of ASFV SY18 Δmgf/CD2v strain: the preparation of the vaccine strain for the deletion of genes of African swine fever virus and the immunoprotection property thereof in the same documents (Zhang Yanyan, chen Teng, zhang Jingyuan, ji Yu, muesli invention, bao Zongyi, wang Lidong, guo Xiaoyu, zhou Xintao, yang Jinmei, wang Xiaohu, gao Yulong, wang Chunyu, bao Chen, yi, mi Lijuan, sun Xuefei, feng Na, yang Jinjin, wang Chengyu, mo Zhonghai, li Jiping, meng Keyin, tianduo, li Nan, wang Shuchao, wang Ying, zhang Fei, zhang Jinxia, jiang Yiqian, liu, zhang Shoufeng, zhang Zhongyang, qian Yingjuan, zhu Hongfei, gao Yuwei, chen Hongjun, li Jinxiang, hu Rongliang, the construction of the vaccine strain for the deletion of genes of African swine fever virus and the immunoprotection property thereof in Chinese veterinary school newspaper, 2019;39 (8): 1421-1427).
4. Immunization and infection test
4.1 Immunization
15 Healthy piglets are randomly divided into 3 groups, 5 groups each, 1ml (10 7.0TCID50/ml) of iPAM-D129L auxiliary cell culture of 1 st immune ASFV SY18 delta D129L strain is immunized 2 times at intervals of 14 days; group 2 immunized ASFV, SY 18. Delta. MGF/CD2v strain, PAM cell culture 1ml (10 7.0TCID50/ml), immunization 2 times, 14 days apart; group 3 was not immunized (no safety evaluation was performed and no fluid was injected) as a blank challenge control. After immunization, the groups were kept isolated.
4.2 Attack on toxic infection
All 3 pigs were challenged with ASFV SY18 at 14 days post 2 nd immunization of the immunized group, with an intramuscular challenge dose of 25TCID 50 per pig. And after toxin expelling, normal feeding is performed. The observation was carried out for 28 days. ASFV real-time PCR detection was performed on febrile and sick pigs according to the world animal health Organization (OIE) recommendation method. Morbidity or protection is judged according to the following criteria:
(1) Criterion of morbidity
1) Death;
2) High fever, body temperature not less than 41 ℃ and more than 3 days;
3) Depression, anorexia or overwhelming for more than 3 days, and typical clinical symptoms of African swine fever such as diarrhea, dyspnea, skin bleeding point and the like appear in the later stage;
4) ASFV virus in mouth, nose and anal swabs of febrile or ill or dead pigs tested positive.
And 1) +4) or 2) +3) +4), and the disease is judged.
(2) Toxicity attack protection judgment standard
1) The body temperature is continuously raised to be more than or equal to 41 ℃ for no more than 3 days;
2) No obvious clinical symptoms (typical clinical symptoms of African swine fever such as depression of spirit, anorexia or abstinence, diarrhea, dyspnea, skin bleeding spot and the like) appear;
3) At the end of observation, collecting mouth, nose and anus swabs, and detecting viruses should be negative.
4. Results
4.1, Immune toxicity attack protection result: the observation results show that 8-13 days after the toxin is attacked, the group 3 and the group 5 blank toxin attacking controls all die, mental depression, anorexia and lying on the ground appear in the period of onset, fever lasts for 2-6 days, and finally all die (100% mortality rate). All individuals 5-8 had more obvious fever, 1 died and 4 were resistant to oversrotection after the group 2 pigs had been challenged. No abnormal clinical manifestation appears after the group 1 attack, all the patients are healthy and alive, and the protection rate is 100% (5/5). The results are shown in FIG. 21.
The results show that the D129L gene deletion replication-defective African swine fever virus delta SY18 delta D129L immunized pigs can generate complete virus attack protection after 2 times of immunization, and when the double gene fragment deletion virus SY18 delta MGF/CD2v strain attacks the virus after two times of immunization, the immunized pigs generate obvious fever and 1 pig dies.
4.2, At the end of the observation period of the immune virus attack group, the ASFV nucleic acid detection result in the immune swine swab and the dissecting tissue of the African swine fever virus defective in the D129L gene deletion replication: specific results are shown in fig. 22 (judgment standard: 35 or less is positive, CT values greater than 35 and less than 40 are suspicious (both give CT values), innumerable values are negative.) and fig. 23 ("-" is no value. Judgment standard: 35 or less is positive, CT values greater than 35 and less than 40 are suspicious (both give CT values), innumerable values are negative).
Aiming at a swab positive to a nucleic acid detection result, inoculating BMDM cells for virus separation after filtering and sterilizing, carrying out blind transmission for one generation, and carrying out dyeing by using a FITC-p30 fluorescent antibody and real-time PCR detection, wherein the results are negative.
And inoculating BMDM cells to the tissue suspension with positive nucleic acid detection results for virus separation, carrying out blind transmission for 2 generations, and carrying out staining by using a FITC-p30 fluorescent antibody and real-time PCR detection, wherein the results are negative.
According to the toxicity attacking results, the D129L gene deletion replication defective African swine fever virus can generate complete immune toxicity attacking protection after two times of pig immunization, and no toxicity expelling phenomenon occurs. The D129L gene deletion replication defective African swine fever virus has good immune protection effect, and is an ideal replication defective vaccine.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (7)
1. The use of a protein-related biomaterial, characterized in that,
The application is any one of the following:
c1 Use in the preparation of a replication-defective african swine fever vaccine;
C2 Use in the preparation of replication-defective african swine fever virus;
c3 The use of the composition for the preparation of a product for inhibiting replication of African swine fever virus in vitro;
the protein is a protein with an amino acid sequence of SEQ ID No. 2;
The biomaterial is any one of the following D1) to D5):
d1 Knocking out nucleic acid molecules of the protein coding genes; the nucleic acid molecule sequentially comprises a left homology arm, a reporter gene and a right homology arm; the left homology arm is a DNA molecule shown as SEQ ID No. 7, and the right homology arm is a DNA molecule shown as SEQ ID No. 8;
D2 An expression cassette comprising D1) said nucleic acid molecule;
d3 A recombinant vector comprising D1) said nucleic acid molecule, or a recombinant vector comprising D2) said expression cassette;
d4 A recombinant microorganism comprising D1) said nucleic acid molecule, or a recombinant microorganism comprising D2) said expression cassette, or a recombinant microorganism comprising D3) said recombinant vector;
D5 A recombinant cell comprising D1) said nucleic acid molecule, or a recombinant cell comprising D2) said expression cassette, or a recombinant cell comprising D3) said recombinant vector.
2. Replication-defective African swine fever virus, characterized in that,
The replication defective african swine fever virus genome lacks the gene encoding the protein of claim 1, or the gene encoding the protein of claim 1 in the replication defective african swine fever virus genome is functionally inactivated;
Functional inactivation of the gene encoding the protein of claim 1 deleted or the gene encoding the protein of claim 1 is achieved by using the nucleic acid molecule of claim 1.
3. A method for producing replication-defective African swine fever virus, characterized in that,
The method comprises deleting or functionally inactivating the encoding gene of the protein of claim 1 in the genome of african swine fever virus to obtain replication-defective african swine fever virus;
The deletion or functional inactivation of the gene encoding the protein of claim 1 in the genome of african swine fever virus is achieved by using the nucleic acid molecule of claim 1.
4. The method of claim 3, wherein the step of,
The method comprises the following steps:
g1 Constructing a recombinant vector comprising the nucleic acid molecule of claim 1;
G2 Co-transfecting/infecting the recombinant vector described in G1) with african swine fever virus into a host cell;
g3 Culturing the host cell and screening to obtain replication-defective African swine fever virus.
5. Replication-defective african swine fever virus produced by the method of claim 3 or 4.
6. A method of propagation of replication defective african swine fever virus according to claim 2 or 5, characterized in that the method comprises culturing the replication defective african swine fever virus according to claim 2 or 5 in helper cells, which are recombinant cells stably expressing the protein of claim 1 and/or cells containing the protein of claim 1.
7. A replication-defective african swine fever vaccine, characterized in that the replication-defective african swine fever vaccine comprises the replication-defective african swine fever virus of claim 2 or 5.
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