SNV strain full-length infectious clone primer group, construction method, mutant and application

Abstract

The invention relates to an SNV strain full-length infectious clone primer group, a construction method, a mutant and application, belongs to the technical field of virology and biology, and provides an SNV strain full-length infectious clone primer and a novel construction method thereof. And the 697th site of the Pol protein is mutated into glutamine or arginine through a site-specific mutagenesis technology to obtain two mutant infectious clones. According to the infectious clone pKS-REV-SNV constructed by the invention, filial generation viruses can be successfully rescued, and the filial generation viruses and female parent wild type viruses have the same growth characteristics; according to the present invention, with the mutated infectious clones pKS-REV-SNV-K697Q and pKS-REV-SNV-K697R, the acetylation modification site of the Pol protein has been mutated, such that the acetylation and deacetylation modification conditions can be artificially simulated, and the two mutants can be proliferated so as to provide the new platform for the REV pathogenicity, the acetylation modification function research and other aspects;

Description

Technical Field

[0001] The invention belongs to the field of virology and biotechnology, and discloses a full-length infectious clone primer set of an SNV strain, a construction method, a mutant and an application. Background Art

[0002] Reticuloendotheliosis (RE) is an immunosuppressive disease of poultry caused by the Reticuloendotheliosis Virus (REV). It belongs to the C-type retrovirus genus of the Retroviridae family and is a typical gamma-retrovirus. REV mainly causes a group of syndromes characterized by proliferation of reticuloendothelial cells, including acute reticulocytosis, dwarf syndrome, and chronic tumor proliferation diseases of lymphoid tissue and other tissues.

[0003] REV is a single-stranded positive-strand RNA virus, mainly including genes such as gag, pol, env and conserved region LTRs. The gag protein is a nucleocapsid protein, and its main function is to encode the core protein. It is relatively conservative and has strong antigenicity. It can be cleaved by the virus's own enzymes to form many mature structural proteins, including matrix protein p18 (MA), capsid protein p30 (CA), nucleocapsid protein p10 (NC) and many small proteins, among which p30 is the main group-specific antigen and has the function of assembling virus particles. The envelope protein (env) is a glycoprotein containing two peptide chains, gp20 and gp90. The smaller gp20 is a transmembrane protein (TM) that can pass through the viral envelope, while the larger gp90 is exposed outside the envelope and belongs to the envelope protein (SU). It is immunogenic and can cause the body to produce antibodies. The polymerase protein (pol) mainly encodes the viral protease (PR), reverse transcriptase (RT) and integrase (IN), which is the most conserved region in the entire REV genome. The function of the protease is to hydrolyze some precursor proteins. The reverse transcriptase has polymerase activity and can synthesize new DNA chains using DNA or RNA as templates. It also has Rnade H activity and can remove the reverse transcribed viral RNA template. The integrase can insert the circular proviral DNA into the host genome. The end of the genome is called the long terminal repeat sequence (LTRs), which is about 596 bases long and is a highly efficient promoter.

[0004] Acetylation is a common PTM (post-translational modification) that can regulate multiple processes such as transcription, chemotaxis, cell signal transduction, proteolysis, and apoptosis. A large number of studies have shown that protein acetylation modification has been used by a variety of viruses throughout every step of the virus life cycle. After invading cells, viruses use the host's acetylation network to disrupt cell metabolism and signaling pathways, thereby regulating their own replication and infection.

[0005] Mutation analysis is often used to determine the importance of acetylation sites: by mutating the desired lysine (Lysine, K) to arginine (Arginine, R) to simulate deacetylated lysine, which has a similar positive charge and structure to lysine and does not undergo acetylation; by mutating lysine to glutamine (Glutamine, Q) to simulate acetylated lysine, which can neutralize the positive charge and has a similar structure to lysine.

[0006] Reverse genetics technology is a technology that uses DNA recombination, nucleotide site-directed mutagenesis and other technologies to purposefully and accurately modify the viral gene structure, and then uses in vivo and in vitro transcription, transfection and other means to package and rescue the virus in the host cell. It can be applied to the fields of viral biology, immunology, pathogenicity research and new vaccine development. This technology is widely used in the research of DNA viruses, RNA viruses, etc., and reverse genetics is commonly used for viruses.

[0007] There are two transmission technologies: one is to insert the viral cDNA into the eukaryotic expression vector and then directly transfect mammalian cells to rescue the virus; the other is to transfect the viral RNA obtained by in vitro transcription of the full-length viral cDNA into mammalian cells to rescue the virus. Since the genome of the rescued virus comes from the full-length cDNA, the viral genome can be cut, modified or transformed at the DNA level using site-directed mutagenesis or homologous recombination technology, and the phenotypic changes of the rescued virus can be used to determine the viral gene function, viral characteristics, etc. Summary of the invention

[0008] In view of the gaps in the prior art, the inventors of the present invention, based on the characteristics of the REV-SNV genome and its own restriction sites, for the first time used the homologous recombination method to connect it to the cloning vector pBluescriptII KS(+) through five ligation transformations, and acetylated and deacetylated the K697 site through site-directed mutagenesis technology, constructed the full-length infectious clone PB-REV-SNV of the REV-SNV strain, mutants pKS-REV-SNV-K697Q and pKS-REV-SNV-K697R, and achieved in vitro virus rescue and pathogenicity research, providing a platform for the functional research on REV pathogenicity and acetylation of the 697 site.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The invention provides a full-length infectious clone primer set of avian reticuloendotheliosis virus SNV strain, and the specific sequences are shown in SEQ ID No.1-12.

[0011] The present invention also provides a method for constructing a full-length infectious clone of the avian reticuloendotheliosis virus SNV strain, comprising the following steps:

[0012] (1) Extraction of total viral genome DNA of REV-SNV strain

[0013] (2) Design and synthesize primers covering the whole genome: primers were designed according to the full gene sequence of REV-SNV strain and the homologous recombination instructions. The amplification primers were G1-F, G1-R, G1+-R, G2-F, G2-R, G2+-R, G3-F, G3-R, G3+-R, G4-F, G4-R, G4+-R, wherein G1-F, G1+-R, G2+-R, G3-F, G3+-R, G4+-R contained homology arms of plasmid pBluescript IIKS(+), G2-F and G4-F contained homology arms with REV sequence, MluI restriction sites were introduced into primers G1-R, G2-R, G3-R, G4-R; genetic markers were introduced into the G3+ fragment, and the mutation primers were YCBJ-F / YCBJ-R; the specific primer sequences described above are shown in SEQ ID No. 1 to 14;

[0014] (3) using primers G1-F / G1-R, G2-F / G2-R, G3-F / G3-R, G4-F / G4-R, respectively, and using a high-fidelity enzyme to perform PCR amplification on the four fragments, the PCR amplification products are detected and purified to obtain four fragments G1, G2, G3, and G4; then using the obtained fragments as templates, using primers G1-F / G1+-R, G2-F / G2+-R, G3-F / G3+-R, G4-F / G4+-R, respectively, and using a high-fidelity enzyme to perform PCR amplification, the obtained amplification products are again detected and purified to obtain four fragments G1+, G2+, G3+, and G4+;

[0015] (4) First, the plasmid pBluescript II KS(+) was treated with Hind III and Kpn I for double restriction digestion and then purified to obtain the target fragment PB-1. Then, primers pKS1-F / pKS1-R were designed, and the fragment PB-1 was amplified with a high-fidelity enzyme. The PCR amplification product was detected and purified to obtain the pKS-1 fragment. The obtained pKS-1 fragment was homologously recombined with the G1+ fragment and transformed into DH5α competent cells. After the colonies were picked, the plasmid was extracted. After double restriction digestion and sequencing to identify the successful connection, the plasmid was expanded and the plasmid was extracted. The obtained plasmid was named pKS-G1;

[0016] (5) The recombinant plasmid pKS-G1 was double-digested with Hind III and Mlu I, and the target fragment PB-2 was recovered by gel electrophoresis. Then, primers pKS2-F / pKS2-R were designed, and the fragment PB-2 was amplified with a high-fidelity enzyme. The PCR amplification product was detected and purified and recovered to obtain the pKS-2 fragment. The obtained pKS-2 fragment was homologously recombined with the G2+ fragment and transformed into DH5α competent cells. After the colonies were picked, the plasmid was extracted. After double-digestion and sequencing to identify the successful connection, the plasmid was expanded and the plasmid was extracted. The obtained plasmid was named pKS-G1+G2;

[0017] (6) The pKS-1 fragment and the G3+ fragment were homologously recombined and transformed into DH5α competent cells. After the colonies were picked, the plasmids were extracted. After double restriction digestion and sequencing to identify the successful connection, the plasmids were expanded and the obtained plasmids were extracted. The obtained plasmids were named pKS-3. In order to distinguish the infectious clones constructed later from the wild strains, a pair of mutation primers YCBJ-F / YCBJ-R were designed to introduce a nonsense mutation (G5749→A) as a genetic marker. After the mutation was successfully identified by restriction digestion and sequencing, the plasmids were expanded and the obtained plasmids were extracted. The obtained plasmids were named pKS-G3;

[0018] (7) The recombinant plasmid pKS-G3 was double-digested with Hind III and Mlu I, and the fragment PB-3 was recovered after gel electrophoresis; then primers pKS3-F / pKS3-R were designed, and the fragment PB-3 was amplified with a high-fidelity enzyme. The PCR amplification product was detected and purified and recovered to obtain the pKS-3 fragment. The obtained pKS-3 fragment was homologously recombined with the G4+ fragment and then transformed into DH5α competent cells. After the colonies were picked, the plasmid was extracted, and after double-digestion and sequencing to identify the successful connection, it was expanded and the plasmid was extracted. The obtained plasmid was named pKS-G3+G4;

[0019] (8) The recombinant plasmid pKS-G1+G2 was double-digested with Hind III and Mlu I, and the fragment PB-4 was recovered from the gel after gel electrophoresis; then, primers pKS4-F / pKS4-R were designed, and the fragment PB-4 obtained in the previous step was amplified with a high-fidelity enzyme. The PCR amplification product was detected and purified and recovered to obtain the pKS-4 fragment. The recombinant plasmid pKS-G3+G4 was amplified with the designed primers G3-F and G4-R using a high-fidelity enzyme to obtain the G3+G4+ fragment. After agarose gel electrophoresis and gel recovery, the primers G3-F and G4+-R were used to amplify the G3+G4+ fragment required for the subsequent recombination reaction using a high-fidelity enzyme. The obtained pKS-4 fragment was homologously recombined with the G3+G4+ fragment and then transformed into DH5α competent cells. After picking the colonies, the plasmid was extracted. After double-digestion and sequencing to identify the successful connection, it was amplified and the plasmid was extracted. The obtained plasmid was named pKS-REV-SNV.

[0020] The present invention also provides two mutants of avian reticuloendotheliosis virus SNV strain, the mutants are pKS-REV-SNV-K697Q and pKS-REV-SNV-K697R, that is, the lysine at position 697 on pKS-REV-SNV is mutated to glutamine or arginine.

[0021] The present invention also provides a method for rescuing avian reticuloendotheliosis virus SNV strain and mutant strains. First, recombinant plasmids pKS-REV-SNV, pKS-REV-SNV-K697Q and pKS-REV-SNV-K697R are mixed with a transfection reagent, and DF-1 cells are transfected respectively. After 5 days, the cells and supernatant are collected. After repeated freezing and thawing three times, new DF-1 cells are inoculated, and the supernatant is harvested after 5 days to obtain the rescued virus.

[0022] The composition and final concentration of the transfection reagent are: LipofectamineTM 3000: plasmid: P3000 is 6:5:5.

[0023] The present invention also provides an identification primer for reticuloendotheliosis virus SNV strain rescue virus, and the sequence of the primer is shown as SEQ ID No.7-8.

[0024] Compared with the prior art, the present invention has the following technical effects:

[0025] (1) The present invention provides a full-length infectious clone of the avian reticuloendotheliosis virus SNV strain and mutant strain, through which the virus can be successfully rescued, and a genetic marker is introduced into the full-length infectious clone, and the marker can still exist stably after passage, and can be used as a genetic marker to distinguish wild strains from rescued viruses.

[0026] (2) The present invention constructs the full-length infectious clone of the avian reticuloendotheliosis virus SNV strain in steps through the method of homologous recombination, thereby ensuring the integrity of the gene sequence of the avian reticuloendotheliosis virus SNV strain in the obtained plasmid, and uses the method of homologous recombination to construct an intermediate plasmid, so that there is no need to consider the influence of restriction sites on the construction during the construction process. At the same time, the constructed intermediate plasmid is conducive to the modification of a series of gene sites in the later stage.

[0027] (3) The present invention obtains two mutant recombinant viruses pKS-REV-SNV-K697Q and pKS-REV-SNV-K697R through site-directed mutagenesis technology, in which the acetylation modification site of the Pol protein has been mutated, which can artificially simulate the acetylation and deacetylation modifications, and both mutants can proliferate, which can provide a new platform for the study of REV pathogenicity and acetylation modification function.

[0028] (4) The reverse genetic manipulation method of the avian reticuloendotheliosis virus SNV strain established by the present invention can restore the recombinant viral plasmid into a pathogenic virus, and can be used for virulence analysis, cross-species transmission, and related vaccine development of avian reticuloendotheliosis virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the construction strategy of the full-length infectious clone of the avian reticuloendotheliosis virus SNV strain in Example 1;

[0030] Figure 2 This is the gel electrophoresis diagram of the segmented amplification products of the REV-SNV strain in Example 1;

[0031] Figure 3 The gel electrophoresis diagram of the enzyme digestion identification of the recombinant plasmid pKS-G1 in Example 1;

[0032] Figure 4 This is the gel electrophoresis diagram of the enzyme digestion identification of the recombinant plasmid pKS-G1+G2 in Example 1;

[0033] Figure 5 The gel electrophoresis diagram of the enzyme digestion identification of the recombinant plasmid pKS-G3 in Example 1;

[0034] Figure 6 This is the gel electrophoresis diagram of the enzyme digestion identification of the recombinant plasmid pKS-G3+G4 in Example 1;

[0035] Figure 7 This is the gel electrophoresis diagram of the restriction enzyme digestion identification of the recombinant plasmid pKS-REV-SNV in Example 1;

[0036] Figure 8This is a gel electrophoresis diagram for identifying the genetic markers of the rescued virus in Example 2;

[0037] Fig. 9 A comparison diagram of the genetic marker sequences of the rescued virus and the wild virus in Example 2;

[0038] Fig.10 This is a comparison of the growth curves of the rescued virus and the wild virus in Example 2;

[0039] Fig.11 This is a WB verification image of the rescued virus, wild virus and empty vector in Example 3;

[0040] Fig.12 This is a validation diagram of site-directed mutation sequencing of acetylation and deacetylation of the rescued mutant virus and the non-mutation rescued virus in Example 3;

[0041] Fig.13 This is a comparison of REV mRNA expression levels between rescued mutant virus and rescued virus without mutation in Example 3;

[0042] Fig.14 This is a diagram of the acetylation modification of the rescued viruses of different groups in Example 3 verified by WB;

[0043] Fig.15 This is a comparison chart of the effects of different groups of rescued viruses on cell viability in Example 3. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are used to explain the present invention, but not to limit the present invention. Except for special instructions, all other embodiments in this embodiment are completed using existing technologies.

[0045] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0046] Example 1: Construction of full-length infectious clones of avian reticuloendotheliosis virus SNV strain and mutant strains

[0047] Schematic diagram of the construction strategy of the full-length infectious clone of avian reticuloendotheliosis virus SNV strain Figure 1 The construction of mutant strains uses the recombinant plasmids in the construction process for site-directed mutagenesis, and after normal connection, the infectious clones of the mutant strains are obtained. The specific process is as follows:

[0048] 1.1 Materials:

[0049] Experimental cell line: Chicken embryo fibroblast cell line (DF-1) was purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd.

[0050] Plasmid: cloning vector pBluescript II KS(+)

[0051] Viruses: The avian reticuloendotheliosis virus (SNV) strain was kindly donated by Professor Cui Zhizhong of Shandong Agricultural University (GenBank accession number: DQ003591).

[0052] 1.2 Methods:

[0053] 1.2.1 Extraction of proviral genomic DNA

[0054] Take 1 mL of REV-SNV virus solution, inoculate a bottle of 25 cm2 DF-1 cell monolayer, adsorb at 37°C for 1 hour, add 1% serum DMEM maintenance solution, continue to culture at 37°C for 3 days, digest with EDTA-trypsin, and collect cells by centrifugation. Use the cell / tissue genomic DNA extraction kit (centrifugal column type) produced by Tiangen Company to extract the REV proviral genome according to the cell genome extraction method in the manual, and store at -20°C for use as an amplification template.

[0055] 1.2.2 Design and synthesis of primers for segmented amplification of the REV whole genome

[0056] Based on the obtained REV-SNV proviral genomic DNA, PrimerVersion 5.0 and Snap Gene software were used for analysis. Combined with the requirements for inserting fragments in the homologous recombination instructions, primers containing homologous arms covering the full-length genome and primers introducing restriction sites were designed.

[0057] Primer design should ensure that at least 15bp-20bp sequences at both ends of the target fragment are homologous to the two ends of the linearized vector. The length of each PCR primer should be at least 35-40bp, including 15bp-20bp at the 5' and 3' ends that are homologous to the linear vector (excluding the restriction site) and 20-25bp of the target fragment-specific sequence.

[0058] The extracted DNA was used as a template, and primers G1-F / G1-R, G2-F / G2-R, G3-F / G3-R, and G4-F / G4-R were used to perform PCR amplification on the four fragments using a high-fidelity enzyme. The PCR amplification products were detected and purified to obtain four fragments, G1, G2, G3, and G4. The electrophoresis diagrams are shown as follows: Figure 2 As shown, M is DL5000 Marker; G1 is the G1 fragment (2298 bp) amplified by G1-F / G1-R; G2 is the G2 fragment (1929 bp) amplified by G2-F / G2-R; G3 is the G3 fragment (2510 bp) amplified by G3-F / G3-R; G4 is the G4 fragment (1853 bp) amplified by G4-F / G4-R.

[0059] Table 1 Primer sequence details of amplification primers, molecular markers and site-directed mutagenesis primers

[0060]

[0061]

[0062] (Note: The horizontal lines are homology arms, the italics are the introduced MluI restriction sites, and the lowercase letters are the REV gene sequences)

[0063] 1.2.3 Construction of infectious clone of avian reticuloendotheliosis virus SNV strain by homologous recombination

[0064] Because the viral pre-genome is long, it needs to be recombined and connected multiple times:

[0065] (1) First connection

[0066] ① Linearized vector preparation

[0067] The plasmid pBluescript II KS(+) was double-digested with Hind III and Kpn I and then purified to obtain the target fragment PB-1. Subsequently, primers pKS1-F / pKS1-R were designed, and the specific sequences were shown in SEQ ID No.19-20. The fragment PB-1 was amplified with a high-fidelity enzyme, and the PCR amplification product was detected and purified to obtain the pKS-1 fragment.

[0068] ② Insert fragment acquisition

[0069] The designed primers G1-F and G1-R were used to amplify the G1 fragment using a high-fidelity enzyme. After agarose gel electrophoresis, the gel was recovered and then the primers G1-F and G1+-R were used to amplify the G1+ fragment required for the subsequent recombination reaction using a high-fidelity enzyme.

[0070] ③Recombination reaction:

[0071] a. Calculation of vector fragment usage:

[0072] Optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)

[0073] Optimal amount of insert fragment = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

[0074] b. Prepare the following reaction system on ice as shown in Table 1;

[0075] Table 1 Recombination reaction system

[0076]

[0077]

[0078] Note: X / Y is calculated according to the formula to obtain the amount of vector and insert. Negative control 1 can be used to confirm whether there is any circular plasmid residue in the linearized cloning vector, and it is recommended. Negative control 2 is recommended when the insert amplification template is a circular plasmid with the same resistance as the cloning vector. Positive control reaction can be used to exclude the influence of other experimental materials and operating factors.

[0079] Use a pipette to gently mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0080] c. React at 37℃ for 30 min; cool to 4℃ or immediately place on ice to cool.

[0081] ④ Transformation of recombinant products

[0082] a. Thaw DH5α competent cells on ice;

[0083] b. Take 10 μL of the recombinant product and add it to 100 μL of competent cells, flick the tube wall to mix, and place on ice for 30 minutes;

[0084] c. After heat shock at 42℃ water bath for 90 seconds, immediately place on ice to cool for 2-3 minutes;

[0085] d. Add 900 μL LB medium (without antibiotics) and shake at 37°C for 1 h (speed 150-200 rpm);

[0086] e. Preheat the Amp-resistant LB solid medium plate prepared in advance in a 37°C incubator. Centrifuge at 5,000rpm (2,400×g) for 5 minutes and discard 900μL of supernatant. Resuspend the bacteria with the remaining medium and spread it evenly on the plate containing the correct resistance using a sterile spreader.

[0087] f. Incubate inverted in a 37°C incubator for 12-16 hours.

[0088] ⑤Positive colony screening

[0089] a. Use an inoculation loop to pick up a single colony and place it in the pre-prepared LB liquid medium containing Amp;

[0090] b. Shake at 37℃ for 12-16h (speed 200-220rpm);

[0091] c. Use a plasmid extraction kit to extract the plasmid according to the instructions and send it for sequencing identification.

[0092] ⑥ Identification of recombinant plasmid (pKS-G1) by double restriction digestion with Hind III and Kpn I

[0093] a. Prepare the reaction system shown in Table 2 on ice;

[0094] Table 2 shows the dual enzyme system

[0095]

[0096] b. Gently pipette or tap the tube wall to mix (do not vortex), then centrifuge briefly to collect the droplets on the wall;

[0097] c. After incubation at 37°C for 15 min, perform agarose gel electrophoresis.

[0098] The recombinant plasmid pKS-G1 and the empty vector were double-digested with Hind III and Kpn I. The results are as follows Figure 3 As shown, the Marker is DL5000 Marker, the empty vector is the DNA fragment (2929 bp) produced by the empty vector double enzyme digestion, and 1, 2, and 3 are the DNA fragments (3659 bp and 1570 bp) produced by the recombinant plasmid double enzyme digestion.

[0099] The results showed that all the bands after enzyme digestion were correct bands.

[0100] (2) Second connection

[0101] ①Preparation of linearized vector:

[0102] In order to reduce the false positive situation in subsequent experiments, the recombinant plasmid pKS-G1 was double-digested with Hind III and Mlu I, and the target fragment PB-2 was recovered after gel electrophoresis; a pair of primers pKS2-F / pKS2-R were designed, and the specific sequences were shown in SEQ ID No.21-22. The target fragment PB-2 was amplified with a high-fidelity enzyme, and the PCR amplification product was detected and purified and recovered to obtain the pKS-2 fragment.

[0103] ② Insert fragment acquisition

[0104] The recombinant plasmid pKS-G2 was digested with Kpn I. Figure 4 As shown, the Marker is DL10000 Marker, and samples 1, 2, 3, and 4 are DNA fragments (4209 bp and 2915 bp) generated by single enzyme digestion of the recombinant plasmid.

[0105] The results showed that all the bands after enzyme digestion were correct. The designed primers G2-F and G2-R were used to amplify the G1 fragment using a high-fidelity enzyme, and the gel was recovered after agarose gel electrophoresis, and then the primers G2-F and G2+-R were used to amplify the G2+ fragment required for the subsequent recombination reaction using a high-fidelity enzyme.

[0106] ③ Recombination reaction

[0107] Same as first connection

[0108] ④ Transformation of recombinant products

[0109] Same as first connection

[0110] ⑤Positive colony screening

[0111] Same as first connection

[0112] ⑥ Identification of recombinant plasmid (pKS-G2) by Kpn I single restriction enzyme digestion

[0113] The recombinant plasmid pKS-G2 was digested with Kpn I. The results are shown in the attached figure. Figure 4 As shown, the Marker is DL10000 Marker, and samples 1, 2, 3, and 4 are DNA fragments (4209 bp and 2915 bp) generated by single enzyme digestion of the recombinant plasmid.

[0114] The results showed that all the bands after enzyme digestion were correct bands.

[0115] (3) The third connection

[0116] ① Linearized vector preparation

[0117] The plasmid pBluescript II KS(+) was double-digested with Hind III and Kpn I and then purified to obtain the target fragment PB-1. Subsequently, primers pKS1-F / pKS1-R were designed, and the specific sequences were shown in SEQ ID No.19-20. The fragment PB-1 was amplified with a high-fidelity enzyme, and the PCR amplification product was detected and purified to obtain the pKS-1 fragment.

[0118] ② Insert fragment acquisition

[0119] The G3 fragment was amplified using the designed primers G3-F and G3-R using a high-fidelity enzyme. After agarose gel electrophoresis, the gel was recovered and then the G3+ fragment required for the subsequent recombination reaction was amplified using primers G3-F and G3+-R using a high-fidelity enzyme.

[0120] ③ Recombination reaction

[0121] Same as first connection

[0122] ④ Transformation of recombinant products

[0123] Same as first connection

[0124] ⑤Positive colony screening

[0125] Same as first connection

[0126] ⑥ Identification of recombinant plasmid (pKS-3) by Pst I single enzyme digestion

[0127] The recombinant plasmid pKS-3 was digested with PstI. Figure 5 As shown, the Marker is DL5000Marker, and 1, 2, and 3 are DNA fragments (4417 bp and 970 bp) produced by single enzyme digestion of the recombinant plasmid.

[0128] The results showed that all the bands after enzyme digestion were correct bands.

[0129] ⑦ Introduction of genetic markers and acetylation / deacetylation mutation sites

[0130] a. Primer design

[0131] According to the instructions of the site-directed mutagenesis kit, three pairs of primers were designed, and the specific sequences are shown in SEQ ID No. 13 to 18.

[0132] b. Establish the PCR system as shown in Table 3, and the PCR reaction procedure as shown in Table 4;

[0133] Table 3 PCR system

[0134]

[0135] Table 4. PCR reaction program

[0136]

[0138] c. Digestion of template plasmid

[0139] Configure the digests as described in Table 5;

[0140] Table 5. Enzyme digestion system

[0141]

[0143] After thorough mixing, the above system was digested at 37°C for 1 h.

[0144] d. Thaw three FDM competent cells from the -90-65℃ refrigerator on ice;

[0145] After the competent cells were thawed, 5 μL of DpnⅠ digestion product was added (add the product when the competent cells were just thawed) and the ice bath was continued for 30 min;

[0146] Heat shock at 42℃ for 90 seconds, and immediately place on ice for 2 minutes;

[0147] Add 350 μL of sterile SOC or LB medium (without antibiotics), mix well, and place in a 37°C shaker for 45-60 min (150 rpm) to allow the bacteria to recover.

[0148] After the incubation, mix the bacterial solution in the centrifuge tube and spread all the bacterial solution on the LB solid agar medium containing the corresponding antibiotics. Place the plate at room temperature until the liquid is absorbed, invert the plate, and culture at 37°C for 12 to 16 hours.

[0149] After the colonies grow, mutant clones are screened.

[0150] e. Detection of mutations in recombinant plasmids (pKS-G3, pKS-G3-697Q, pKS-G3-697R)

[0151] The sequencing results showed that the genetic marker (G5749→A) was successfully introduced. Fig. 9 The acetylation and deacetylation site mutations were successful, and the results are shown in Fig.12

[0152] (4) Fourth connection

[0153] ① Linearized vector preparation

[0154] In order to reduce the false positive situation in subsequent experiments, the recombinant plasmids pKS-G3, pKS-G3-697Q and pKS-G3-697R were double-digested with Hind III and Mlu I, and the gel was recovered after gel electrophoresis to obtain the target fragments PB-3, PB-3-6977Q and PB-3-697R; a pair of primers pKS3-F / pKS3-R were designed, and the specific sequences were shown in SEQ ID No.23-24. The target fragments PB-3, PB-3-6977Q and PB-3-697R were amplified respectively with a high-fidelity enzyme, and the PCR amplification products were detected and purified and recovered to obtain pKS-3, pKS-3-697Q and pKS-3-697R fragments.

[0155] ② Insert fragment acquisition

[0156] The designed primers G4-F and G4-R were used to amplify the G4 fragment using a high-fidelity enzyme. After agarose gel electrophoresis, the gel was recovered and then primers G4-F and G2+-R were used to amplify the G4+ fragment required for the subsequent recombination reaction using a high-fidelity enzyme.

[0157] ③ Recombination reaction

[0158] Same as first connection

[0159] ④ Transformation of recombinant products

[0160] Same as first connection

[0161] ⑤Positive colony screening

[0162] Same as first connection

[0163] ⑥ Identification of recombinant plasmids (pKS-G3+G4, pKS-G3-697Q+G4, pKS-DG3-697R+G4) by Pst I single enzyme digestion

[0164] The recombinant plasmids pKS-G3+G4, pKS-G3-697Q+G4, and pKS-G3-697R+G4 were digested with PstI. The results were as follows: Figure 6 As shown, the Marker is DL10000Marker, and 1, 2, and 3 are DNA fragments (4417 bp and 2789 bp) produced by single enzyme digestion of the recombinant plasmid.

[0165] The results showed that all the bands after enzyme digestion were correct bands.

[0166] (5) Fifth connection

[0167] ① Linearized vector preparation

[0168] In order to reduce the false positive situation in subsequent experiments, the recombinant plasmid pKS-G1+G2 was double-digested with Hind III and MluI, and the fragment PB-4 was recovered from the gel after gel electrophoresis; a pair of primers pKS4-F / pKS4-R was designed, and the specific sequence was shown in SEQ ID No. 25-26. The target fragment obtained in the previous step was amplified using a high-fidelity enzyme, and the PCR amplification product was detected and purified and recovered to obtain the pKS-4 fragment.

[0169] ② Insert fragment acquisition

[0170] The designed primers G3-F and G4-R were used to amplify G3+G4, G3-697Q+G4, and G3-697R+G4 fragments using high-fidelity enzymes. After agarose gel electrophoresis, the gel was recovered and primers G3-F and G4+-R were used to amplify G3+G4+, G3-697Q+G4+, and G3-697R+G4+ fragments required for subsequent recombination reactions using high-fidelity enzymes.

[0171] ③ Recombination reaction

[0172] Same as first connection

[0173] ④ Transformation of recombinant products

[0174] Same as first connection

[0175] ⑤Positive colony screening

[0176] Same as first connection;

[0177] ⑥ Single enzyme digestion identification of recombinant plasmids (pKS-REV-SNV, pKS-REV-SNV-K697Q, pKS-REV-SNV-K697R):

[0178] The recombinant plasmids pKS-REV-SNV and pKS-REV-SNV-K697Q were digested with Kpn I. The results are as follows Figure 7 As shown, the marker is DL10000 marker, samples 1 and 2 are DNA fragments (8490 bp and 2915 bp) generated by single restriction digestion with Kpn I; the recombinant plasmids pKS-REV-SNV and pKS-REV-SNV-K697R were single-digested with Hind III, and the results are shown in Figure 7 As shown, the marker is DL10000 Marker, and samples 3 and 4 are DNA fragments (11405 bp) generated by single enzyme digestion with Hind III.

[0179] The results showed that all the bands after enzyme digestion were correct bands.

[0180] Example 2: Rescue of the full-length infectious clone of avian reticuloendotheliosis virus SNV strain pKS-REV-SNV

[0181] 2.1 Transfection

[0182] DF-1 cells were cultured in six-well plates, and transfection was performed when the cells grew to the logarithmic phase. The transfection system was configured as follows: LipofectamineTM 3000 was used as the transfection reagent, two sterilized centrifuge tubes were taken, 36 μL of LipofectamineTM 3000 and 750 μL of Opti-MEMTM were added to one tube, and 750 μL of Opti-MEMTM, 15 μg of plasmid and 30 μL of P3000 were added to the other tube, the liquids in the two tubes were mixed evenly, and the mixture was placed at room temperature for 10 to 15 minutes, 250 μL of liposome mixture was added to each well, 2 ml of DMEM culture medium containing 1% fetal bovine serum was added to each well, and the six-well plate was placed in a cell culture incubator at 37°C containing 5% CO2 for culture, and the cells and supernatant were collected after 5 days, and new DF-1 cells were inoculated after repeated freezing and thawing three times. The supernatant was harvested after 5 days to obtain the rescued virus.

[0183] 2.2 Enzyme digestion sequencing and sequencing verification of rescued virus genetic markers

[0184] The supernatant of the rescued virus of the F3 generation was collected and inoculated into DF-1 together with the wild strain. After 3 days, the cells were collected and DNA was extracted. The DNA was amplified using G3-F / G3-R primers and subjected to agarose gel electrophoresis. A portion of the gel was cut and sent for sequencing, and a portion of the target product was recovered and identified by single enzyme digestion using Hind III restriction endonuclease.

[0185] The results showed that the wild strain was amplified by G3-F / G3-R primers and digested by Hind III to produce two DNA fragments, while the rescued virus was amplified by G3-F / G3-R primers and digested by Hind III to produce one DNA fragment. Figure 8 As shown, the sequencing results also proved that the genetic marker was indeed introduced. Fig. 9 As shown, it shows that the genetic marker can still stably exist after the passage of the rescued virus, and can be used as a reliable genetic marker for identifying wild strains and rescued viruses. The rescued virus has the same sequence as the parental virus, except for the only base (G5749→A) introduced as a genetic marker in the rescued virus, indicating that the virus is the rescued virus.

[0186] 2.3 Determination of growth curve of rescued virus

[0187] The wild-type virus and the rescued virus were inoculated into DF-1 cells in a 12-well cell culture plate at the P0 generation with an MOI of 2. The cells were collected at different time points, RNA was extracted, and after reverse transcription into cDNA, the one-step growth curve of the virus was drawn using fluorescence quantification. Fig.10 As shown, the wild strain and the rescued virus showed similar growth curves, indicating that the rescued virus and the wild strain have similar biological properties and can be used for subsequent basic and applied research.

[0188] The full-length infectious clone sequence of the above-mentioned avian reticuloendotheliosis virus SNV strain (SEQ ID NO.27) is as follows (lowercase letters represent the virus genome itself, and lowercase bold italic letters represent the pBluescript II KS(+) vector sequence)

[0189]

[0190] Example 3: Rescue and identification of biological characteristics of avian reticuloendotheliosis virus acetylation and deacetylation recombinant viruses pKS-REV-SNV-K697Q and pKS-REV-SNV-K697R

[0191] 3.1 Rescue of acetylated and deacetylated recombinant viruses;

[0192] DF-1 cells were fed in a six-well plate, and transfection was performed when the cells grew to the logarithmic phase. The transfection system was configured as follows: LipofectamineTM 3000 was used as the transfection reagent, and four sterilized centrifuge tubes were taken. 36μL of LipofectamineTM 3000 and 750μL of Opti-MEMTM were added to one tube, and 750μL of Opti-MEMTM, 15μg of plasmid and 30μL of P3000 were added to the other tube. The liquids in the two tubes were mixed evenly, and placed at room temperature for 10-15min, 250μL of liposome mixture was added to each well, and 2ml of DMEM medium containing 1% fetal bovine serum was added to each well. The six-well plate was placed in a cell culture incubator at 37℃ and 5% CO2 for culture. After 5d, the cells and supernatant were collected, and new DF-1 cells were inoculated after repeated freezing and thawing three times. The supernatant was harvested after 5d to obtain the rescued virus. Both plasmids were operated as above.

[0193] 3.2WB detection of viral protein expression;

[0194] Cells transfected with empty vector, pKS-REV-SNV, pKS-REV-SNV-K697Q, pKS-REV-SNV-K697R, and cells inoculated with wild-type REV were collected, lysed, and the protein expression of the virus was verified by WB using the monoclonal antibody against REV stored in the laboratory.

[0195] The results showed that pKS-REV-SNV, pKS-REV-SNV-K697Q, pKS-REV-SNV-K697R, and REV-WT all had bands at 55 kDa, while the empty vector group had no bands. All five groups of internal controls had bands, proving that the constructed infectious clones could be expressed and translated normally. The WB results were as follows: Fig.11 .

[0196] 3.3 Sequencing of acetylated and deacetylated recombinant virus mutation sites;

[0197] The supernatant of the rescued virus of the F3 generation was collected and inoculated into DF-1. After 3 days, the cells were collected and DNA was extracted. The DNA was amplified using G3-F / G3-R primers and subjected to agarose gel electrophoresis. The gel was cut and sent for sequencing.

[0198] The results showed that the sequencing results showed that the acetylation and deacetylation recombinant virus K697 site was confirmed to have acetylation or deacetylation mutations. Fig.12 .

[0199] 3.4 Determination of the mRNA expression level of the rescue mutant viruses pKS-REV-SNV-K697Q and pKS-REV-SNV-K697R relative to the pKS-REV-SNV rescue virus REV;

[0200] The mutant virus and common rescue virus obtained by rescue were inoculated into DF-1 in 12-well cell culture plates at P0 generation with 2 MOI. Cells were collected at different time points, RNA was extracted, and after reverse transcription into cDNA, the expression levels of REV mRNA in different groups at 24h, 48h, and 72h were determined by fluorescence quantitative analysis. The results were compared as shown in Figure 2. Fig.13 .

[0201] The results showed that the REV mRNA expression level of the recombinant virus in the deacetylation group increased compared with that of the non-mutated virus at different time points, and the REV mRNA expression level of the recombinant virus in the acetylation group decreased compared with that of the non-mutated virus at different time points, indicating that the acetylation modification of the K697 site has an effect on the replication of the virus.

[0202] 3.5 Verification of acetylation modification level of acetylated and deacetylated recombinant viruses;

[0203] The mutant recombinant viruses were inoculated in 6-well plates, and empty transfected cells were used as controls. The cells were collected after 72 hours, and the total cell protein was extracted. The pan-acetylated antibody was used for verification by WB. The results are as follows: Fig.14 .

[0204] The results showed that there was no band at around 130 kDa in the empty-transfected cells, while there were bands at around 130 kDa in both the acetylation group and the deacetylation group. The grayscale value was analyzed using software, and the degree of acetylation modification in the acetylation group was lower than that in the deacetylation group, proving that the acetylation level of the virus in the acetylation group was higher than that in the deacetylation group.

[0205] 3.6 Determination of the effects of acetylated and deacetylated recombinant viruses on cell viability;

[0206] DF-1 cells were cultured in 96-well plates. When the cell density reached about 70%, the common infectious clone recombinant virus, acetylated recombinant virus, and deacetylated recombinant virus were inoculated respectively. Six replicate wells were made for each group. The activity of the cells was detected at 24h and 48h using the CCK-8 kit. The analysis results were as follows: Fig.15 .

[0207] The results showed that the viability of cells infected with the deacetylated virus decreased after 48 hours, but the viability of cells infected with the acetylated virus did not decrease, proving that the virus modified by acetylation at the K697 site would affect the viability of cells after infecting cells.

[0208] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer set for full-length infectious clone of avian reticuloendotheliosis virus SNV strain, characterized in that: The specific sequences of the primer set are shown in SEQ ID No. 1-12.

2. A method for constructing a full-length infectious clone of avian reticuloendotheliosis virus SNV strain, characterized in that: The method comprises the following steps: (1) Extracting total viral genome DNA of REV-SNV strain; (2) Design and synthesize primers covering the whole genome: primers were designed according to the whole gene sequence of REV-SNV strain, the amplification primers were G1-F, G1-R, G1+-R, G2-F, G2-R, G2+-R, G3-F, G3-R, G3+-R, G4-F, G4-R, G4+-R, wherein G1-F, G1+-R, G2+-R, G3-F, G3+-R, G4+-R contained homology arms of plasmid pBluescript II KS(+), G2-F and G4-F contained homology arms with REV sequence, Mlu I restriction sites were introduced into primers G1-R, G2-R, G3-R, G4-R; in order to distinguish wild strains, genetic markers were introduced into the G3+ fragment, and the mutation primers were YCBJ-F and YCBJ-R; the specific primer sequences described above are shown in SEQ ID No. 1 to 14; (3) using primers G1-F / G1-R, G2-F / G2-R, G3-F / G3-R, G4-F / G4-R, respectively, and using a high-fidelity enzyme to perform PCR amplification on the four fragments, the PCR amplification products are detected and purified to obtain four fragments G1, G2, G3, and G4; then using the obtained fragments as templates, using primers G1-F / G1+-R, G2-F / G2+-R, G3-F / G3+-R, G4-F / G4+-R, respectively, and using a high-fidelity enzyme to perform PCR amplification, the obtained amplification products are again detected and purified to obtain four fragments G1+, G2+, G3+, and G4+; (4) First, the plasmid pBluescript II KS(+) was treated with Hind III and Kpn I for double restriction digestion and then purified to obtain the target fragment PB-1. Then, primers pKS1-F / pKS1-R were designed, and the fragment PB-1 was amplified with a high-fidelity enzyme. The PCR amplification product was detected and purified to obtain the pKS-1 fragment. The obtained pKS-1 fragment was homologously recombined with the G1+ fragment and transformed into DH5α competent cells. After the colonies were picked, the plasmid was extracted. After double restriction digestion and sequencing to identify the successful connection, the plasmid was expanded and the plasmid was extracted. The obtained plasmid was named pKS-G1; (5) The recombinant plasmid pKS-G1 was double-digested with Hind III and Mlu I, and the target fragment PB-2 was recovered by gel electrophoresis. Then, primers pKS2-F / pKS2-R were designed, and the fragment PB-2 was amplified with a high-fidelity enzyme. The PCR amplification product was detected and purified and recovered to obtain the pKS-2 fragment. The obtained pKS-2 fragment was homologously recombined with the G2+ fragment and transformed into DH5α competent cells. After the colonies were picked, the plasmid was extracted. After double-digestion and sequencing to identify the successful connection, the plasmid was expanded and the plasmid was extracted. The obtained plasmid was named pKS-G1+G2; (6) The pKS-1 fragment and the G3+ fragment were homologously recombined and transformed into DH5α competent cells. The plasmid was extracted after the colony was picked. After double restriction digestion and sequencing to confirm the successful connection, the plasmid was expanded and extracted. The obtained plasmid was named pKS-3. In order to distinguish the infectious clone constructed later from the wild strain, a pair of mutation primers YCBJ-F / YCBJ-R were designed to introduce a nonsense mutation (G5749→A) as a genetic marker. After the mutation was successfully confirmed by restriction digestion and sequencing, the plasmid was expanded and extracted. The obtained plasmid was named pKS-G3; (7) The recombinant plasmid pKS-G3 was double-digested with Hind III and Mlu I, and the fragment PB-3 was recovered after gel electrophoresis; then primers pKS3-F / pKS3-R were designed, and the fragment PB-3 was amplified with a high-fidelity enzyme. The PCR amplification product was detected and purified and recovered to obtain the pKS-3 fragment. The obtained pKS-3 fragment was homologously recombined with the G4+ fragment and then transformed into DH5α competent cells. After the colonies were picked, the plasmid was extracted, and after double-digestion and sequencing to identify the successful connection, it was expanded and the plasmid was extracted. The obtained plasmid was named pKS-G3+G4; (8) The recombinant plasmid pKS-G1+G2 was double-digested with Hind III and Mlu I, and the fragment PB-4 was recovered from the gel after gel electrophoresis; then, primers pKS4-F / pKS4-R were designed, and the fragment PB-4 obtained in the previous step was amplified with a high-fidelity enzyme. The PCR amplification product was detected and purified and recovered to obtain the pKS-4 fragment. The recombinant plasmid pKS-G3+G4 was amplified with the designed primers G3-F and G4-R using a high-fidelity enzyme to obtain the G3+G4+ fragment. After agarose gel electrophoresis and gel recovery, the primers G3-F and G4+-R were used to amplify the G3+G4+ fragment required for the subsequent recombination reaction using a high-fidelity enzyme. The obtained pKS-4 fragment was homologously recombined with the G3+G4+ fragment and then transformed into DH5α competent cells. After picking the colonies, the plasmid was extracted. After double-digestion and sequencing to identify the successful connection, it was amplified and the plasmid was extracted. The obtained plasmid was named pKS-REV-SNV.

3. Two mutants of the avian reticuloendotheliosis virus SNV strain, characterized in that: The mutants are pKS-REV-SNV-K697Q and pKS-REV-SNV-K697R, that is, the lysine at position 697 in the amino acid sequence of the plasmid pKS-REV-SNV prepared in claim 1 is mutated to glutamine or arginine.

4. A method for rescuing avian reticuloendotheliosis virus SNV strain and mutant strains, characterized in that: First, the recombinant plasmid pKS-REV-SNV prepared in claim 1 is mixed with the transfection reagent LipofectamineTM 3000, and DF-1 cells are transfected respectively. After 5 days, the cells and supernatant are collected, and new DF-1 cells are inoculated after repeated freezing and thawing three times. After 5 days, the supernatant is harvested to obtain the rescued virus.

5. A rescue method according to claim 4, characterized in that: The composition and final concentration of the transfection reagent are: LipofectamineTM 3000: plasmid: P3000 is 6:5:

5.

6. A primer for identifying a rescued virus of the avian reticuloendotheliosis virus SNV strain, characterized in that: The sequences of the primers are shown in SEQ ID No. 13-14, and can distinguish between artificial strain SNV and wild strain SNV.

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