CN116064668A - Recombinant adenovirus plasmid, preparation method and recombinant adenovirus - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a recombinant adenovirus plasmid, the nucleotide sequence of which is shown as SEQ NO. 1. The plasmid and the virus based on the plasmid have self-shearing function by utilizing P2A protein of the type 1 porcine teschovirus (Porcine Teschovirus-1), the P2A protein is used as a connecting peptide to connect VP2 of IBDV and Fiber2 protein coding genes of FAdV-4 in series, and the gene is placed in an expression box of a replication-defective human type 5 adenovirus vector, and can realize expression of 2 proteins at the same time through one-time transcription and translation, so that a new idea is provided for research and development of IBD and FAdV-4 bivalent candidate live vector vaccines. The virus can stably express VP2 and Fiber2 proteins, and has high virus titer. Meanwhile, the invention also discloses a preparation method of the plasmid.

Description

Recombinant adenovirus plasmid, preparation method and recombinant adenovirus

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a recombinant adenovirus plasmid, a preparation method and a recombinant adenovirus.

Background

Infectious bursal disease (Infectious bursal disease, IBD) and avian adenovirus type 4 (Fowl adenovirus serotype-4, FAdV-4) are two important infectious diseases that jeopardize the poultry industry, causing serious economic losses to the poultry industry and limiting the healthy sustainable development of the poultry industry.

IBD was first found in 1957 in the dry broussonetia area of the united states and is a serious infectious disease caused by infectious bursal virus (Infectious bursal disease virus, IBDV), mainly affecting chickens 3-6 weeks old, with mortality rates as high as 50% -60% that can cause strong immunosuppression. At present, no effective treatment method exists for the disease, and the prevention and control difficulty of the disease are increased due to the mutation of viruses and the emergence of novel virulent strains, so that the development of novel vaccines becomes a problem to be solved urgently. IBDV belongs to the genus avian Birna virus of the family Birna virus, has no bursa membrane and no hemagglutination, its genome comprises A, B two double-stranded RNA fragments, the two open reading frames (Open Reading Frames, ORFs) of the A fragment partially overlap, the larger ORF encodes VP2, VP3 and VP4 proteins, the smaller ORF encodes VP5 protein, and the B fragment encodes VP1 protein; wherein the trimeric subunits of VP2 and VP3 constitute the outer surface and the underwear shell of the virus particle, and the VP2 gene is the main virulence gene for controlling IBDV, the VP2 protein is the main structural protein of IBDV, accounting for about 51% of the total amount of viral proteins, and comprises a plurality of neutralizing epitopes of IBDV, which can induce the generation of neutralizing antibodies of animal bodies, and the variable regions of the neutralizing epitopes are closely related to the variation of IBDV antigens, so that the VP2 gene is the main object of the development of subunit vaccines and recombinant virus vaccines of IBDV.

The pericardial effusion-hepatitis syndrome (HHS) caused by FAdV-4 is largely outbreaked in China in 2015, the disease has no obvious seasonality, the death rate is not equal from 30% to 100%, the HHS is clinically frequently applied to broilers of 3-5 weeks old, virus particles can be vertically spread or horizontally spread, and a new challenge is brought to the development of poultry industry in China. FAdV-4 is an icosahedron without a capsule, the genome of the FAdV-4 is double-stranded DNA of 40-45 kb, and the FAdV-4 belongs to the species C of avian adenovirus in the family of adenoviridae, the genus avian adenovirus and the group I avian adenovirus; the virion consists of 252 capsomeres, including four major structural proteins, hexon (Hexon), penton (Penton) and two Fiber proteins (Fiber 1 and Fiber 2); the Fiber is firmly combined with the substrate of Penton in a non-covalent bond, and is used as the outermost protein of the virus, and the Fiber protein can be firstly contacted with and combined with host cells in the process of the invasion of the virus into animal organisms, so that the adsorption process of the virus is mediated. Wherein the Fiber2 protein mainly influences the replication and pathogenicity of FAdV-4, and researches show that constructing recombinant FAdV-4 of which the Fiber2 gene is deleted obviously reduces pathogenicity of chicken flocks, and obviously reduces the replication capacity of viruses after the interaction site of the Fiber2 protein and the host protein KPN3/4 is deleted; the Fiber2 protein has good antigenicity, can induce animal organisms to generate corresponding antibodies, can provide higher protection rate for FAdV-4 invasion, and is an ideal target protein for developing genetic engineering vaccines.

Adenovirus vectors are widely used in the development of novel vaccines and gene therapy, and are widely used as human adenovirus type 5 (Human adenovirus serotype-5, HAdV-5) vectors and human adenovirus type 2 (Human adenovirus serotype-2, HAdV-2) vectors. At present, adenovirus vector vaccines have been tested in hundreds of clinical trials worldwide, and the adenovirus vector vaccine has the advantages of wide host range, high proliferation efficiency, no integration into chromosomes of host cells, large amount of antigen proteins with high immunogenicity, simultaneous insertion and expression of a plurality of exogenous genes, capability of stimulating humoral immunity and cellular immunity of animal organisms and the like, becomes one of high-efficiency vector systems in vaccine antigen delivery, and has good development and application prospects.

Therefore, the technical problem solved by the scheme is as follows: how to develop a plasmid and virus for expressing the two proteins.

Disclosure of Invention

The invention aims to provide a recombinant adenovirus plasmid, the plasmid and a virus based on the plasmid have a self-shearing function by utilizing P2A protein of a type 1 porcine teschovirus (Porcine Teschovirus-1), the recombinant adenovirus plasmid is used as a connecting peptide to connect the VP2 of IBDV and Fiber2 protein coding genes of FAdV-4 in series, and the recombinant adenovirus plasmid is placed in an expression box of a replication-defective human adenovirus type 5 vector, and can realize expression of 2 proteins simultaneously through one-time transcription and translation, so that a new idea is provided for research and development of a double candidate live vector vaccine of IBD and FAdV-4. The virus can stably express VP2 and Fiber2 proteins, and has high virus titer.

Meanwhile, the invention also discloses a preparation method of the plasmid.

In order to achieve the above purpose, the present invention provides the following technical solutions: a recombinant adenovirus plasmid has a nucleotide sequence shown in SEQ NO. 1.

Meanwhile, the invention also discloses a preparation method of the recombinant adenovirus plasmid, which comprises the following steps:

step 1: amplifying VP2 gene and Fiber2 gene; the VP2 gene contains gene of connecting peptide P2A protein; the nucleotide sequence of the VP2 gene is shown as SEQ NO. 2; the nucleotide sequence of the Fiber2 gene is shown as SEQ NO. 3;

step 2: carrying out homologous recombination on the VP2 gene, the Fiber2 gene and the shuttle plasmid pAdTrack-GOI, and transforming the homologous recombination into competent cells of escherichia coli Trans10 to obtain a recombinant shuttle plasmid pAdTrack-VP2-P2A-Fiber2; the nucleotide sequence of the recombinant shuttle plasmid pAdTrack-VP2-P2A-Fiber2 is shown as SEQ NO. 4;

step 3: linearizing the recombinant shuttle plasmid pAdTrack-VP2-P2A-Fiber2 with restriction enzyme Pme I to expose homology arm with adenovirus backbone plasmid pAdEasy-1 in BJ5183-AD-1 competent cells to obtain enzyme digestion product, converting enzyme digestion product into E.coli BJ5183-AD-1 competent cells, and extracting to obtain recombinant adenovirus plasmid pAd-VP2-P2A-Fiber2 according to claim 1.

In the preparation method of the recombinant adenovirus plasmid, the amplification method of the VP2 gene is as follows:

step 11: extracting RNA of IBDV and reversely transcribing the RNA into cDNA molecules, and amplifying the cDNA molecules by using specific primers VP2-F and VP2-R to obtain a recovered product;

step 12: cloning the recovered product in a PMD19-T vector, transforming the recovered product into a Trans10 competent cell, and picking positive clones and propagating to form a plasmid PMD19T-VP2;

step 13: plasmid PMD19T-VP2 is used as a template, H-VP2-F and H-VP2-R are used as homologous recombination upstream and downstream primers, and high-fidelity enzyme is used

GXL DNA Polymerase PCR amplification of VP2 gene carrying homology arms;

the nucleotide sequence of VP2-F, VP2-R, H-VP2-F, H-VP2-R is shown in SEQ NO. 5-8.

Finally, the invention also discloses a recombinant adenovirus, which is prepared by the following method:

the recombinant adenovirus plasmid pAd-VP2-P2A-Fiber2 is linearized by Pac I enzyme and then transfected into cells.

In the recombinant adenovirus, the cells are HEK293A cells

Compared with the prior art, the invention has the beneficial effects that:

the plasmid and virus based on the plasmid have self-shearing function by utilizing P2A protein (the nucleotide sequence of the corresponding P2A gene is shown as SEQ NO. 14) of the type 1 porcine teschovirus (Porcine Teschovirus-1), and the plasmid is used as a connecting peptide to connect VP2 of IBDV and Fiber2 protein coding genes of FAdV-4 in series, and are placed in an expression cassette of a replication-defective human adenovirus type 5 vector, and expression of 2 proteins can be simultaneously realized through primary transcription and translation, so that a new idea is provided for research and development of a dual candidate live vector vaccine of IBD and FAdV-4. The virus can stably express VP2 and Fiber2 proteins, and has high virus titer.

Drawings

FIG. 1 shows the result of amplifying VP2 gene in example 1 of the present invention, M is Trans2K DNA Marker, lanes 1 and 2 represent VP2 gene PCR products.

FIG. 2 shows the amplification of VP2 gene carrying homology arms in example 1 of the present invention, M is Trans2K DNA Marker, lanes 1 and 2 represent PCR products of VP2 gene carrying homology arms, and lanes 3 and 4 represent PCR products of Fiber2 gene carrying homology arms.

FIG. 3 shows the result of amplification of the Fiber2 gene in example 1 of the present invention, M is a Trans2K DNA Marker, lanes 1 and 2 represent the PCR products of the Fiber2 gene.

FIG. 4 shows the results of double digestion of shuttle plasmids pAdTrack-GOI SalI and Xho I in example 1 of the present invention, M is the Trans15KDNA Marker, lanes 1 and 2 represent the corresponding double digested products.

FIG. 5 shows the results of double digestion identification of the recombinant shuttle plasmids pAdTrack-VP2-P2A-Fiber2 Kpn I and Nhe I in example 1 of the present invention, M is a Trans8K DNA Marker, lanes 1 and 2 represent the corresponding double digestion products.

FIG. 6 is a plasmid map of the recombinant shuttle plasmid pAdTrack-VP2-P2A-Fiber2 in example 1 of the present invention.

FIG. 7 shows the result of single cleavage assay of recombinant adenovirus plasmid pAd-VP2-P2A-Fiber2 Pac I in example 1 of the invention, M is Trans15K DNA Marker, lanes 1 and 2 represent the corresponding cleavage products.

FIG. 8B shows the lesion results of HEK293A cells infected with the recombinant adenovirus rAd5-VP2-P2A-Fiber2 packaged in example 2 of the invention, and FIG. A shows normal HEK293A cells.

FIG. 9A is a fluorescence of rAd5-VP2-P2A-Fiber2 of generation P0 in example 2 of the invention after 130h of packaging in HEK293A cells, and B is a HEK293A cell without virus infection.

FIG. 10 shows the identification of VP2 gene of recombinant adenovirus rAD5-VP2-P2A-Fiber2 in example 3 of the invention, M is Trans2K DNA Marker, lanes 1, 2, 3, 4 correspond to VP2 gene PCR amplification products of P1, P2, P3, P4 generation rAD5-VP2-P2A-Fiber2, lanes 5 and 6 represent rAD5-EGFP and HEK293A cell freeze-thawing solutions, respectively.

FIG. 11 shows the identification of the Fiber2 gene of recombinant adenovirus rAD5-VP2-P2A-Fiber2 in example 3 of the invention, M is a Trans2K DNA Marker, lanes 1, 2, 3, 4 correspond to the PCR amplification products of the Fiber2 gene of P1, P2, P3, P4 generation rAD5-VP2-P2A-Fiber2, respectively, lanes 5 and 6 represent rAD5-EGFP and HEK293A cell freeze-thawing solutions, respectively.

FIG. 12 shows the expression verification of recombinant adenovirus rAD5-VP2-P2A-Fiber2 VP2 protein in example 3 of the invention, wherein M is 180kDa Prestained Protein Marker, lanes 1, 2, 3 and 4 correspond to the expression of VP2 protein in HEK293A cells infected with P1, P2, P3 and P4-substituted rAD5-VP2-P2A-Fiber2, and lanes 5 and 6 represent rAD5-EGFP and HEK293A cell freeze-thawing solutions, respectively.

FIG. 13 shows the expression verification of recombinant adenovirus rAD5-VP2-P2A-Fiber2 protein in example 3 of the invention, M is 180kDa Prestained Protein Marker, lanes 1, 2, 3 and 4 correspond to the expression of Fiber2 protein in HEK293A cells infected with P1, P2, P3 and P4 rAD5-VP2-P2A-Fiber2, respectively, and lanes 5 and 6 represent rAD5-EGFP and HEK293A cell freeze-thawing solutions, respectively.

FIG. 14A shows rAD5-VP2-P2A-Fiber 2-infected HEK293A cells of example 3 according to the invention, with red arrows indicating brown positive signals after DAB development, and B as non-virus infected HEK293A cells.

FIG. 15 shows the one-step growth curves of recombinant adenovirus rAd5-VP2-P2A-Fiber2 and empty rAd5-EGFP of example 3 of the invention, the abscissa indicates different time points of detoxification in h, and the ordinate indicates viral titer in Lg (IFU/mL).

Detailed Description

All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

IBDV strains and FAdV-4 strains were isolated and stored in the laboratory in the following examples; HEK293A cells (procall CL-0003) were purchased from wunpro life technologies limited; trans10 competent cells, DNA markers were purchased from Beijing all-gold biotechnology Co., ltdCompany limited; BJ5183-AD-1 competent cells were purchased from Shanghai Biotechnology Inc.; the shuttle plasmid pAdTrack-GOI (SEQ NO. 9) and the empty toxin rAD5-EGFP are given away by the institute of biological treatment of the university of medical science in the south; homologous recombinases were purchased from Beijing gold sand Biotechnology Co., ltd; high-fidelity enzyme

GXL DNA Polymerase、Premix Taq ^TM Enzymes, reverse transcriptase, PMD19T-Vector, adenovirus titer quick assay kit, restriction enzymes Xho I, sal I, kpn I, nhe I were all purchased from Takara doctor materials technology (Beijing); restriction enzymes Pme I and Pac I were purchased from New England Biolabs company; DNA gel recovery Kit, endo-Free BAC/PAC DNA Kit purchased from OMEGA company; plasmid miniprep kit was purchased from Tiangen Biochemical technologies Co., ltd; axyPrep humoral virus DNA/RNA minikit was purchased from AxyGen company; transporter ^TM 5Transfection Reagent from Polysciences; poly-D-lysine, SDS gel preparation kit, skimmed milk powder, 5 XSDS-Loading Buffer, NC membrane, 20 XTBS were purchased from Biyun Tian Biotechnology Co., ltd; the anti-IBDV VP2 monoclonal antibody and the anti-FAdV-4Fiber2 monoclonal antibody are prepared and stored by the experiment; IRDye 680RD coat anti-Mouse, available from LICOR; viraTrap ^TM Adenovirus bulk purification kits were purchased from BIOMIGA company; primer synthesis and sequencing were all done by Soujin Zhi Biotechnology Co.

EXAMPLE 1 construction of recombinant adenovirus plasmid pAd-VP2-P2A-Fiber2

1. PCR amplification of IBDV VP2 and FAdV-4Fiber2

Primer sequences synthesized by Jin Wei Intelligence company are shown in Table one (the underlined capital letters in the primers represent homology arms):

TABLE 1 all primer sequences in this experiment and amplified fragment sizes

1.1 Amplification of VP2 Gene

For IBDV, the Trizol method is used for extracting RNA in the disease material, the system is 8 mu L of product, 2 mu L of reverse transcription premix enzyme, the reaction conditions are 37-15 min, 85-5 s and 4-infinity, cDNA is used as a template, and specific primers VP2-F and VP2-R (table 1) are used for amplifying VP2 genes, and the PCR system is as follows:

Premix Taq ^TM PCR reaction conditions: pre-denaturation at 95℃for 3min; denaturation at 98℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 1min for 22s for 30 cycles; and then the extension is carried out for 5min at 72 ℃. The PCR products were identified by 1.0% agarose gel electrophoresis (FIG. 1) and gel recovered, premix Taq ^TM The 3' end of the PCR recovery product obtained by enzyme amplification is attached with an A base, and can be directly cloned in a PMD19-T carrier, and the connection system is as follows:

the system was allowed to react overnight at 16℃after which the ligation products were transformed into Trans10 competent cells, positive clones were picked and propagated to construct a new plasmid, which was sequenced correctly and designated PMD19T-VP2.

Then, PMD19T-VP2 is used as a template, H-VP2-F and H-VP2-R (table 1) are used as homologous recombination upstream and downstream primers, and high-fidelity enzyme is used

GXL DNA Polymerase PCR amplification of VP2 gene (SEQ NO. 2) carrying homology arms was performed as follows:

the reaction conditions are as follows: pre-denaturation at 95℃for 3min; denaturation at 98℃for 10s, annealing at 65℃for 15s, elongation at 68℃for 1min for 26s for 30 cycles; the extension was carried out at 68℃for a further 5min. Amplified products were identified by 1.0% agarose gel electrophoresis and recovered (FIG. 2) as fragments of homologous recombination.

1.2 Amplification of Fiber2 Gene

For FAdV-4, the nucleic acid of virus in the disease is extracted by using AxyPrep humoral virus DNA/RNA small-scale kit, specific operation reference instruction, the extracted nucleic acid product is used as template, fiber2-F and Fiber2-R are used as specific primers (table 1) to carry out PCR amplification (figure 3), the PCR amplification system and reaction condition are referred to 1.1, as well as VP2 gene, it is cloned in PMD19-T vector so as to better preserve Fiber2 gene, the newly constructed plasmid is named PMD19T-Fiber2, then PMD19T-Fiber2 is used as template, H-Fiber2-F and H-Fiber2-R (table 1) are used as upstream and downstream primers for homologous recombination, high-fidelity enzyme is used

GXL DNA Polymerase the Fiber2 gene (SEQ NO. 4) carrying the homology arm was PCR amplified and the amplified product is shown in FIG. 2.

2. Construction of the recombinant shuttle plasmid pAdTrack-VP2-P2A-Fiber2

The shuttle plasmid pAdTrack-GOI was digested with Sal I and Xho I in the following manner:

carrying out metal bath constant temperature reaction at 37 ℃ for 2 hours, carrying out gel recovery after the product is completely linearized by 0.8% agarose gel electrophoresis identification (figure 4), taking VP2 genes and Fiber2 genes carrying homology arms obtained in 1.1 and 1.2 as homologous recombination fragments, carrying out reaction at 50 ℃ for 15 minutes under the action of homologous recombination enzymes, and then converting the products into competent cells of escherichia coli Trans10, wherein the specific steps of conversion are as follows:

taking 100 mu L of competent cells, melting on ice, adding 10 mu L of recombinant product, gently mixing, standing on ice for 30min, performing heat shock in a water bath at 42 ℃ for 45s, rapidly transferring to ice, standing for 2min, adding 800 mu L of SOC culture medium into an EP tube, resuscitating for 1h at 37 ℃ by using a shaking table at 200rpm, taking 100 mu L of bacterial liquid, coating on LB solid culture medium with Canada resistance, and culturing for 12-16h in an inverted incubator at 37 ℃. The monoclonal bacteria on the plate are selected, amplified and cultured after the bacteria liquid PCR is primarily identified to be correct, plasmids are extracted, the plasmids are subjected to double digestion by using Kpn I and Nhe I, the digestion products are identified by 1.0% agarose gel electrophoresis, three bands of 7318bp, 3771bp and 1009bp can be displayed (figure 5), and the plasmids with correct sequencing are sent to be sequenced and named pAdTrack-VP2-P2A-Fiber2 (figure 6).

3. Construction of recombinant adenovirus plasmid pAd-VP2-P2A-Fiber2

The recombinant shuttle plasmid pAdTrack-VP2-P2A-Fiber2 was linearized with the restriction enzyme Pme I to expose the homology arm with the adenovirus backbone plasmid pAdEasy-1 in BJ5183-AD-1 competent cells, the cleavage system was as follows:

carrying out metal bath constant temperature reaction at 37 ℃ for 2 hours, carrying out gel recovery after the complete linearization of the enzyme digestion product is determined by nucleic acid electrophoresis, taking 10ng of the enzyme digestion product, converting the enzyme digestion product into competent cells of escherichia coli BJ5183-AD-1, carrying out inversion culture by a 37 ℃ incubator, picking up relatively smaller colonies on a flat plate for expansion culture, carrying out enzyme digestion identification by Pac I after extracting plasmids, carrying out sequencing verification on the initially identified correct plasmids, and naming the sequenced correct plasmids as pAd-VP2-P2A-Fiber2, wherein a single enzyme digestion system is as follows:

a large band of about 30kb and a small band of about 4.5kb appeared after the cleavage products were subjected to nucleic acid electrophoresis (FIG. 7). Because BJ5183-AD-1 competent cells have low plasmid yield, the plasmids generated by the BJ5183-AD-1 competent cells need to be transformed into Trans10 competent cells with high copy capacity for propagation, and the extracted plasmids are stored at-20 ℃.

EXAMPLE 2 packaging of recombinant adenovirus rAd5-VP2-P2A-Fiber2

15. Mu.g of the constructed recombinant adenovirus plasmid pAd-VP2-P2A-Fiber2 was taken and linearized with Pac I enzyme, the cleavage system was increased in proportion to step 3 of example 1, under the same conditions as in this step, and the linearized DNA molecule was purified by ethanol precipitation:

1) Purified water was added to a final volume of 90 μl of the whole cleavage system, and 10 μl of 3M sodium acetate (ph=5.2) and 200-250 μl of absolute ethanol were added to the EP tube.

2) Placing in a refrigerator at-80deg.C for 3 hr, thawing the above product, centrifuging at room temperature for 15min at maximum speed, and observing white DNA precipitate on the tube wall.

3) Removing the supernatant, adding 70% ethanol for washing once, centrifuging, sucking all liquid as much as possible, opening the cover of the EP tube, and air-drying in a biosafety cabinet for 5min.

4) 20. Mu.L of pre-heated ultrapure water was added to dissolve the linearized product, and the measured concentration was kept at-20 ℃.

The procedure for transfection of linearized pAd-VP2-P2A-Fiber2 into HEK293A cells and packaging rAD5-VP2-P2A-Fiber2 was as follows:

1) HEK293A cells were plated in 6-well plates 18-24h prior to transfection and transfection was started at a cell density of around 70%.

2) 1-2h prior to transfection the medium in the wells requiring transfection was replaced with 3mL fresh medium containing 2% low or no serum.

3) Using 150mM NaCl solution as diluent, 300. Mu.L of diluent was added to the EP tube and 2. Mu.g of Pac I-linearized pAd-VP2-P2A-Fiber2 was added, after gentle mixing, 8. Mu.L of PEI transfection reagent was added, vortexed for 5s, and the EP tube was allowed to stand in a biosafety cabinet for 15min to promote PEI-DNA complex formation.

4) The mixture was gently swirled up and down 3 times with a pipette, and PEI-DNA mixture was added to the transfection wells with medium change while shaking the cell culture plate to ensure uniform distribution of PEI-DNA complexes and prevent local concentration from becoming too high.

5) The 6-well plate was placed at 37℃with 5% CO ₂ Culturing in incubator for 7-10d, observing cytopathic effect (cytopathic effect, CPE) and green fluorescence every day, and timely supplementing liquid.

6) When the cells are observed to be round under an optical microscope, show a beaded shape, have reduced refractive index and have typical CPE (FIG. 8B) with signs of shedding and the like, have fluorescence under a fluorescence microscope, and a negative control group has no fluorescence (FIG. 9), the cells are repeatedly frozen and thawed for 3 times at-80 ℃ and normal temperature, centrifuged at 12000r/min at 4 ℃ for 8min, and the supernatant is taken, namely P0 generation rAD5-VP2-P2A-Fiber2, and stored at-80 ℃.

7) Adjusting cell density to 80%, inoculating HEK293A cells with P0 generation rAD5-VP2-P2A-Fiber2 at a ratio of 1% (v/v), and inoculating at 37deg.C and 5% CO ₂ Culturing in an incubator for 2-3d until obvious CPE and clustered fluorescence appear on the cells, repeatedly freezing and thawing the cells for 3 times at-80 ℃ and normal temperature, centrifuging to obtain a supernatant which is P1 generation rAD5-VP2-P2A-Fiber2, and continuously transferring the supernatant to P4 generation according to the same method.

Example 3 identification of recombinant adenovirus rAd5-VP2-P2A-Fiber2 and growth kinetics detection

1. PCR detection of VP2 Gene and Fiber2 Gene

200 mu L of each of P1, P2, P3 and P4 generation cell freeze-thawing liquid is taken, nucleic acid is extracted by using AxyPrep humoral virus DNA/RNA small-scale kit, VP2-F and VP2-R are used as identification primers of VP2 genes, fiber2-F and Fiber2-R are used as identification primers of Fiber2 genes, and Premix Taq is used ^TM The enzyme was amplified by PCR in a 20. Mu.L volume under the conditions and procedures set as 1.1 in example 1, and the VP2 gene of 1356bp (FIG. 10) and the Fiber2 gene of 1440bp (FIG. 11) were detected simultaneously by 1.0% agarose gel electrophoresis, and the negative control rAD 5-EGFP-infected cell freeze-thaw and the non-toxic HEK293A cell freeze-thaw were not provided with corresponding bands.

2. Western blot detection of expression of VP2 protein and Fiber2 protein

Adding 2 XLD into cells of 1, 2, 3 and 4 th generation recombinant adenovirus, scraping the cells by using the back of a yellow gun head, adding 1/10 volume of DTT of 2 XLD, and blowing and mixing uniformly by a liquid-transferring gun; boiling at 98deg.C for 10min, sampling 15 μL, performing SDS-PAGE electrophoresis under 80V for 20min and 120V for 1h, transferring onto NC membrane under 80V for 2h, sealing with TBST containing 5% skimmed milk for 1.5h, washing with TBST for 3 times and 5min each time, respectively adding 1:2000 diluted anti-IBDV VP2 monoclonal antibody and 1:1000 diluted anti-FAdV-4Fiber2 monoclonal antibody, incubating at normal temperature for 3h, and washing with TBST for 4 times and 10min each time; IRDye 680RD coat anti-Mouse is added as a secondary antibody, the membrane is incubated for 1h at normal temperature, TBST is washed for 4 times, each time for 10min, and an Azure Bio-systems C600 multifunctional molecular imaging system is used for imaging. The results showed that the rAD5-VP2-P2A-Fiber2 of the P1-P4 generation showed a VP2 protein band of about 41kDa and a Fiber2 protein band of about 60kDa (FIGS. 12 and 13), and a weak, uncleaved, complete VP2-P2A-Fiber2 protein band of about 100kDa, but the band color was light compared with the VP2 protein and Fiber2 protein, indicating that the P2A cleavage efficiency was high in this experiment.

3. Growth kinetics detection of recombinant adenovirus rAd5-VP2-P2A-Fiber2

Determination of titres of P4-substituted rAd5-VP2-P2A-Fiber2 and empty adenovirus rAd5-EGFP at different time points: spreading the cells into a small dish of 35mm 24h before the inoculation, starting the inoculation according to the amount of 1% (v/v) when the cells grow to 80%, collecting viruses respectively at 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h and 108h after the inoculation, and preserving at-80 ℃ for later use after freeze thawing. After the completion of the virus collection for these 9 time periods, the adenovirus titer rapid assay kit was used to determine the titer of each time period, while rAd5-EGFP was used as a control, and a growth kinetics curve was plotted, as follows:

1) Spreading the cells into 24-well plate 24 hr before measuring virus titer, inoculating virus after cell density reaches about 80%, and 10-fold gradient diluting (10 ^-1 -10 ^-6 ) 2 replicates were made for each gradient and incubated for 48h.

2) The medium was aspirated, the cells were dried appropriately in a biosafety cabinet, 500. Mu.L of pre-chilled methanol was gently added to each well, and the cells were fixed at-20℃for 10min.

3) Methanol was pipetted off, the cells were gently rinsed 3 times with 500. Mu.L of 1% BSA in PBS for 3min each, then 250. Mu.L of Mouse anti-Hexon anti (1% BSA in 1:1000) was added to each well and incubated for 1h in a 5% CO2 incubator at 37 ℃.

4) The primary antibody was blotted off, cells were gently rinsed 3 times per well with PBS containing 1% BSA for 3min, then 250. Mu.L of Rabbit anti-Mouse anti-body (containing 1% BSA, 1:500) was added to each well and incubated for 1h in a 5% CO2 incubator at 37 ℃.

5) 10 XDAB Substrate was diluted to 1 XDAB Substrate with 1X Stable Peroxidase Buffer, the secondary antibody was blotted off, the cells were gently rinsed 3 times with PBS containing 1% BSA for 3min each well, and 250. Mu.L of 1 XDAB working solution was added to each well and incubated at room temperature for 10min.

6) DAB working fluid was pipetted off, 500 μl PBS was added per well, and observed using a 10-fold objective light microscope, and dilutions were selected containing at least 5-50 brown positive signals in one field of view, no brown signal was found for negative control cells (fig. 14), and titers were calculated according to the formula: viral titer (IFU/mL) = [ number of positive cells per field (cells/field) ×number of fields per well (fields/well) ]/[ volume of added virus solution (mL) ×dilution factor (/ mL) ].

7) Titers of rAd5-VP2-P2A-Fiber2 and rAd5-EGFP at 9 time points were calculated and counted and growth curves were plotted (FIG. 15).

As can be seen from the growth kinetics curve, the rAd5-VP2-P2A-Fiber2 and rAd5-EGFP have similar trends, and the virus titer reaches the highest point at 72h, wherein rAd5-VP2-P2A-Fiber2 is 3.86 multiplied by 10 ⁸ IFU/mL, rAd5-EGFP 3.53X10 ⁸ IFU/mL, the titer of the virus slightly decreased after 72 hours. Then, 6 HEK293A cells of T75 are infected with rAd5-VP2-P2A-Fiber2 of the generation P4, when obvious CPE appears on the cells, viruses are harvested at about 72h, freeze thawing is repeated for 3 times at-80 ℃ and normal temperature, the supernatant is centrifugally taken, filtered by a 0.45 mu m filter, and ViraTrap is used ^TM The adenovirus mass purification kit is used for purifying virus liquid, and the specific operation steps are as follows:

1) Balance column: before the experiment, 10×av Wash Buffer was diluted to 1×av Wash Buffer and 2× AV Elution Buffer was diluted to 1× AV Elution Buffer with ultrapure water, respectively; the AV Maxi Column is placed at 50mL Centrifugal Tube,4 ℃, centrifuged for 2min at 500x g, then the AV Maxi Column is held by a clip or other support, the bottom tip is broken off, the cap is released, liquid is discharged from the AV Maxi Column along with gravity, once the liquid stops dripping, 4mL of ultrapure water is slowly added, the solution is allowed to flow out of the AV Maxi Column, 10mL of 1XAV Wash Buffer is added, and the solution is allowed to flow continuously.

2) And (3) column loading: transfer 15mL of supernatant to AV Maxi Column, flow under gravity until all viral supernatants are transferred.

3) Rinsing and eluting: adding 10mL 1xAV Wash Buffer to AV Maxi Column, repeating for 5min by gravity flow or 4deg.C, 1000x g, and centrifuging; 4mL of 1X AV Elution Buffer was added, the virus eluted, and 4mL of the eluate was collected.

4) Desalination and buffer exchange: transferring 4mL of the sample collected in step 3) to Centrifugal Filter, centrifuging at 3000rpm for 10-15min until about 500. Mu.L of the solution remains in Centrifugal Filter, discarding the filtrate, adding 3.5mL of PBS to Centrifugal Filter, centrifuging at 3000rpm for 10-15min until about 400-500. Mu.L of the solution remains in Centrifugal Filter, blowing up and down the pipette a few times in Centrifugal Filter, transferring the virus-containing solution to a clean tube, and sub-packaging into small portions for storage in a refrigerator at-80℃for later use.

The viral titer of the purified rAd5-VP2-P2A-Fiber2 was 7.54X10 according to the 3 rd step viral titer calculation formula in example 3 ⁹ IFU/mL。

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (5)

1. A recombinant adenovirus plasmid is characterized in that the nucleotide sequence is shown in SEQ NO. 1.

2. A method for preparing recombinant adenovirus plasmid, which is characterized by comprising the following steps:

step 1: amplifying VP2 gene and Fiber2 gene; the VP2 gene contains gene of connecting peptide P2A protein; the nucleotide sequence of the VP2 gene is shown as SEQ NO. 2; the nucleotide sequence of the Fiber2 gene is shown as SEQ NO. 3;

step 2: carrying out homologous recombination on the VP2 gene, the Fiber2 gene and the shuttle plasmid pAdTrack-GOI, and transforming the homologous recombination into competent cells of escherichia coli Trans10 to obtain a recombinant shuttle plasmid pAdTrack-VP2-P2A-Fiber2; the nucleotide sequence of the recombinant shuttle plasmid pAdTrack-VP2-P2A-Fiber2 is shown as SEQ NO. 4; the nucleotide sequence of the shuttle plasmid pAdTrack-GOI is shown as SEQ NO. 9;

step 3: linearizing the recombinant shuttle plasmid pAdTrack-VP2-P2A-Fiber2 with restriction enzyme Pme I to expose homology arm with adenovirus backbone plasmid pAdEasy-1 in BJ5183-AD-1 competent cells to obtain enzyme digestion product, converting enzyme digestion product into E.coli BJ5183-AD-1 competent cells, and extracting to obtain recombinant adenovirus plasmid pAd-VP2-P2A-Fiber2 according to claim 1.

3. The method for preparing recombinant adenovirus plasmid according to claim 2, wherein the VP2 gene amplification method comprises:

step 11: extracting RNA of IBDV, and amplifying by using specific primers VP2-F and VP2-R to obtain a recovered product;

step 12: cloning the recovered product in a PMD19-T vector, transforming the recovered product into a Trans10 competent cell, and picking positive clones and propagating to form a plasmid PMD19T-VP2;

step 13: plasmid PMD19T-VP2 is used as a template, H-VP2-F and H-VP2-R are used as homologous recombination upstream and downstream primers, and high-fidelity enzyme is used

GXL DNA Polymerase PCR amplification of VP2 gene carrying homology arms;

the nucleotide sequence of VP2-F, VP2-R, H-VP2-F, H-VP2-R is shown in SEQ NO. 5-8.

4. The recombinant adenovirus is characterized by being prepared by the following method:

the recombinant adenovirus plasmid pAd-VP2-P2A-Fiber2 is linearized by Pac I enzyme and then transfected into cells.

5. The recombinant adenovirus according to claim 4, wherein the cell is a HEK293A cell.

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