A chicken mycoplasma synoviae fusion gene, a recombinant adenovirus and its application

Abstract

The invention discloses a fusion gene of Mycoplasma synoviae, a recombinant adenovirus and applications thereof. The fusion gene comprises a pdhβ gene sequence and a pdhD gene sequence. The vaccine of the invention can produce antibodies and cytokines at a relatively high level and can have a certain protective effect on Mycoplasma synoviae infection after immunization.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a chicken synoviae mycoplasma fusion gene, a recombinant adenovirus and applications thereof. Background Art

[0002] Mycoplasma synoviae (MS) is one of the important avian mycoplasmas. It can cause subclinical infection of the upper respiratory tract, and also lead to exudative synovitis, tenosynovitis and bursitis in the synovial bursa of the joints and tendon sheaths. Severe MS infection can cause poor growth and development of chickens, reduced carcass quality of broilers, reduced egg production, fertilization and hatching rates of breeder hens, resulting in significant economic losses. The pathogen can be transmitted both horizontally and vertically, and it is difficult to completely purify MS-positive chicken farms. MS is often mixed with other pathogens, resulting in complex and severe disease. MS infection is spread all over the world, and chicken flocks in many parts of China are also seriously infected.

[0003] The in vitro culture of MS requires the addition of nicotinamide adenine dinucleotide (NAD) and animal serum. Factors such as the long time and high cost of in vitro culture have limited the development and application of traditional vaccines. Therefore, it is very necessary to develop new MS vaccines. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, one of the objectives of the present invention is to provide a fusion gene of Mycoplasma synoviae.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0006] A fusion gene based on Mycoplasma synoviae, the fusion gene comprises a pdhβ gene sequence and a pdhD gene sequence.

[0007] Furthermore, the fusion gene also includes a Linker sequence for connecting the pdhβ gene sequence and the pdhD gene sequence.

[0008] Furthermore, the nucleotide sequence of the fusion gene is shown in SEQ ID NO.3.

[0009] The second object of the present invention is to provide a recombinant adenovirus shuttle plasmid, including the above-mentioned fusion gene.

[0010] The third object of the present invention is to provide a recombinant adenovirus vector, comprising the above-mentioned shuttle plasmid and backbone plasmid.

[0011] Furthermore, the backbone plasmid is a replication-defective adenoviral vector.

[0012] A fourth object of the present invention is to provide a method for constructing the above-mentioned recombinant adenovirus vector, comprising the following steps:

[0013] Step 1, using the Mycoplasma synoviae DNA as a template to perform PCR amplification to obtain the target gene;

[0014] Step 2: Connect the target gene and plasmid to obtain a recombinant shuttle plasmid;

[0015] Step 3: Recombining the recombinant shuttle plasmid and the backbone plasmid to obtain a recombinant adenovirus vector; preferably, transfecting the recombinant shuttle plasmid and the backbone plasmid into HEK 293A cells for homologous recombination.

[0016] A fifth object of the present invention is to provide a method for preparing a recombinant adenovirus, wherein the above-mentioned recombinant adenovirus vector is infected into cells for amplification to obtain the recombinant adenovirus; preferably, the recombinant adenovirus vector is infected into HEK 293A cells for amplification to obtain a large amount of recombinant adenovirus.

[0017] The sixth object of the present invention is to provide a recombinant adenovirus, which is obtained by the above-mentioned method for preparing the recombinant adenovirus.

[0018] The seventh object of the present invention is to provide an application of the above-mentioned recombinant adenovirus in the preparation of chicken synoviae Mycoplasma vaccine.

[0019] Beneficial effects of the present invention:

[0020] The vaccine of the invention can produce relatively high levels of antibodies and cytokines, and can have a certain protective effect on chicken synoviae Mycoplasma infection after immunization.

[0021] In addition, recombinant adenovirus vector vaccines can make up for the high cost of traditional vaccine MS cultivation and are safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram for the construction of recombinant adenovirus;

[0023] Figure 2 The electrophoresis results of PCR amplification of the target gene are shown in Figure 1, where M: DL 2000 Marker; A: 1: pdhβ gene amplification product; -: negative control; B: 1: pdhD gene amplification product, -: negative control; C: 1: pdhβ gene amplification product containing linker; -: negative control; D: 1: pdhD gene amplification product containing linker, -: negative control;

[0024] Figure 3 The figure shows the electrophoresis result of SOE-PCR amplification of pdhβ-pdhD fusion gene, where M: DL 2000Marker; 1: pdhβ-pdhD fusion gene amplification product; -: negative control;

[0025] Figure 4 PCR electrophoresis results for gene identification after cloning pdhβ gene, pdhD gene and pdhβ-pdhD fusion gene into pMD19-T vector; M: DL 2000Marker; A: 1: pMD-pdhβ; -: negative control; B: 1: pMD-pdhD; -: negative control; C: 1: pMD-pdhβ-pdhD; -: negative control;

[0026] Figure 5 The results of restriction enzyme electrophoresis for gene identification after cloning the pdhβ gene, pdhD gene and pdhβ-pdhD fusion gene into the pMD19-T vector; M: DL 5 000Marker; A: 1-2: pMD-pdhβ; B: 1-2: pMD-pdhD; C: 1-2: pMD-pdhβ-pdhD;

[0027] Figure 6 The results of PCR electrophoresis after the pdhβ gene, pdhD gene and pdhβ-pdhD fusion gene were connected to the pDC316-mCMV-EGFP adenovirus shuttle vector; M: DL 2000Marker; A: 1: pDC-pdhβ; -: negative control; B: 1: pDC-pdhD; -: negative control; C: 1: pDC-pdhβ-pdhD; -: negative control;

[0028] Figure 7 The results of restriction enzyme electrophoresis after the pdhβ gene, pdhD gene and pdhβ-pdhD fusion gene were connected to the pDC316-mCMV-EGFP adenovirus shuttle vector; M: DL 5 000Marker; A: 1-2: pDC-pdhβ; B: 1-2: pDC-pdhD; C: 1-2: pDC-pdhβ-pdhD;

[0029] Figure 8Figures are the packaging results of recombinant adenovirus, where A: normal cells for 2 days; B: fluorescence image of normal cells for 2 days; C: normal cells for 7 days; D: fluorescence image of normal cells for 7 days; E: recombinant adenovirus pBH-pdhβ packaging for 2 days; F: fluorescence image of recombinant adenovirus pBH-pdhβ packaging for 2 days; G: recombinant adenovirus pBH-pdhβ packaging for 7 days; H: fluorescence image of recombinant adenovirus pBH-pdhβ packaging for 7 days; I: recombinant adenovirus pBH-pdhD packaging for 2 days; J: fluorescence image of recombinant adenovirus pBH-pdhD packaging for 2 days; K: recombinant adenovirus pBH-pdhD packaging for 7 days; L: recombinant adenovirus p BH-pdhD packaging fluorescence image 7 days; M: recombinant adenovirus pBH-pdhβ-pdhD packaging image 2 days; N: recombinant adenovirus pBH-pdhβ-pdhD packaging fluorescence image 2 days; O: recombinant adenovirus pBH-pdhβ-pdhD packaging image 7 days; P: recombinant adenovirus pBH-pdhβ-pdhD packaging fluorescence image 7 days; U: recombinant adenovirus pBH-EGFP packaging image 2 days; V: recombinant adenovirus pBH-EGFP packaging fluorescence image 2 days; W: recombinant adenovirus pBH-EGFP packaging image 7 days; X: recombinant adenovirus pBH-EGFP packaging fluorescence image 7 days; Scale bar: 200μm;

[0030] Fig. 9 Figures are the results of recombinant adenovirus amplification, where A: 2-day image of normal cells; B: 2-day fluorescence image of normal cells; C: 4-day image of normal cells; D: 4-day fluorescence image of normal cells; E: 2-day image of recombinant adenovirus pBH-pdhβ amplification; F: 2-day fluorescence image of recombinant adenovirus pBH-pdhβ amplification; G: 4-day image of recombinant adenovirus pBH-pdhβ amplification; H: 4-day fluorescence image of recombinant adenovirus pBH-pdhβ amplification; I: 2-day image of recombinant adenovirus pBH-pdhD amplification; J: 2-day fluorescence image of recombinant adenovirus pBH-pdhD amplification; K: 4-day image of recombinant adenovirus pBH-pdhD amplification; L: recombinant adenovirus Fluorescence image of pBH-pdhD amplified for 4 days; M: Amplification image of recombinant adenovirus pBH-pdhβ-pdhD for 2 days; N: Amplification image of recombinant adenovirus pBH-pdhβ-pdhD for 2 days; O: Amplification image of recombinant adenovirus pBH-pdhβ-pdhD for 4 days; P: Amplification image of recombinant adenovirus pBH-pdhβ-pdhD for 4 days; U: Amplification image of recombinant adenovirus pBH-EGFP for 2 days; V: Amplification image of recombinant adenovirus pBH-EGFP for 2 days; W: Amplification image of recombinant adenovirus pBH-EGFP for 4 days; X: Amplification image of recombinant adenovirus pBH-EGFP for 4 days; Scale bar: 200μm;

[0031] Fig.10The results of RT-PCR identification of recombinant adenovirus, where M: DL 2 000 Marker; A: 1: pBH-pdhβ; -: negative control; B: 1: pBH-pdhD; -: negative control; C: 1: pBH-pdhβ-pdhD; -: negative control;

[0032] Fig.11 Figure 1 is a graph showing the results of western blot identification of recombinant adenovirus; Figure A is a graph showing the results of recombinant adenovirus pBH-pdhβ, Figure B is a graph showing the results of recombinant adenovirus pBH-pdhD, and Figure C is a graph showing the results of recombinant adenovirus pBH-pdhβ-pdhD;

[0033] Fig.12 For recombinant adenovirus P1, P4, P8, P 12 Generation, P 15 The results of the genetic stability analysis of the next generation of genes; M: DL 2000 Marker; A: 1-5: pBH-pdhβ; -: negative control; B: 1-5: pBH-pdhD; -: negative control; C: 1-5: pBH-pdhβ-pdhD; -: negative control;

[0034] Fig.13 The results of antibody changes in different immune groups are shown in Figure 2. Figure 13-1 The graphs show the antibody changes in the PBS group, pBH-EGFP group, pBH-pdhβ-pdhD group, and pBH-pdhD group; Figure 13-2 The graphs show the antibody changes in the PBS group, pBH-EGFP group, pBH-pdhβ-pdhD group, and pBH-pdhβ group;

[0035] Fig.14 This is the result diagram of the changes in IL-2 secretion levels in immune chicken serum;

[0036] Fig.15 This is the result diagram of the changes in IFN-γ secretion levels in the serum of immune chickens;

[0037] Fig.16 This is the result diagram of the changes in IL-4 secretion levels in immune chicken serum;

[0038] Fig.17 This is a graph showing changes in serum IL-6 secretion levels in immune chickens. DETAILED DESCRIPTION

[0039] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0040] The experimental cells, strains and plasmids in the following experimental examples are from:

[0041] The chicken synoviae Mycoplasma MS-ZJ strain was isolated and preserved by the Preventive Veterinary Laboratory of Guizhou University; the pMD19-T vector was purchased from TaKaRa; the adenovirus AdMax packaging system (adenovirus shuttle vector pDC316-mCMV-EGFP and backbone plasmid pBHGlox (delta) E1,3Cre) was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; HEK 293A cells were purchased from Wuhan Pronocell Life Science Co., Ltd. The healthy chicks used in the experiment, 18 days old, were purchased from Guizhou Foster Biotechnology Co., Ltd.

[0042] Experimental Example 1: Primer design and gene amplification

[0043] The pyruvate dehydrogenase Elβ subunit (pdhβ) and dihydrolipoic acid dehydrogenase (pdhD) of Mycoplasma synoviae were used as antigen genes. The pdhβ and pdhD gene sequence information in the MS full gene sequence on GenBank (accession number: LS991953.1) was referenced, and specific primers for the pdhβ gene, pdhD gene, and pdhβ-pdhD fusion gene were designed using Primer Pemier 5.0. Adding a Kozak sequence before the start codon of the upstream primer can enhance the expression of the target protein; adding Not I and EcoR V restriction sites to the upstream and downstream of the primer gene respectively; designing a Linker sequence (GGGGS)2 to connect the pdhβ gene to the pdhD gene. All primer sequences are shown in Table 1, and the primers were synthesized by Shanghai Shenggong Biological Co., Ltd.

[0044] Table 1 Primer information

[0045]

[0046]

[0047] Note:" GCGGCCGC " indicates Not I restriction site, "GATATC" indicates EcoR V restriction site, It represents the linker sequence, and the italic bold letters are the Kozak sequence.

[0048] The lyophilized powder of Mycoplasma synoviae (MS-ZJ strain) was revived, cultured in avian Frey modified liquid medium and passed for 3 generations. 2 mL of bacterial liquid was taken to extract DNA according to the instructions of the DNA extraction kit. The extracted MS DNA was used as a template, and the pdhβ and pdhD genes were PCR amplified using the primers in Table 1. After the amplification, the products were subjected to 1% agarose gel electrophoresis and the results were observed. The pdhβ gene of about 993 bp was obtained (see Figure 2 A in Figure ), pdhD gene of about 975 bp in size (see Figure 2 Figure B in Figure 2), a pdhβ gene fragment of approximately 1 023 bp containing a linker (see Figure 2 C in Figure 3) and a pdhD gene fragment of approximately 1005 bp containing a linker (see Figure 2 The pdhβ and pdhD gene fragments containing the linker were recovered using a gel recovery kit, and the pdhβ-pdhD fusion gene was amplified by SOE-PCR using the pdhβ-F / pdhD-R primers with the recovered corresponding gene fragments as templates. After the amplification, the product was subjected to 1% agarose gel electrophoresis and the results were observed, and a pdhβ-pdhD fusion gene with a fragment size of about 1998 bp was obtained (see Figure 3 ).

[0049] Experimental Example 2: Identification results of recombinant cloned plasmids

[0050] After the pdhβ, pdhD and pdhβ-pdhD genes were recovered by gelatinization, they were connected to pMD19-T and then transformed into competent E. coli cells. After constant temperature shaking at 37°C for 1 hour in LB liquid medium, an appropriate amount was evenly spread on LB solid medium and cultured for 12 hours. A single colony was picked and cultured in LB liquid medium for 12 hours. After the plasmid was extracted, PCR identification was performed, and then double enzyme digestion identification was performed using restriction endonucleases BamH I and Kpn I. The results were respectively Figure 4 and Figure 5 ,from Figure 4 It can be seen that the recombinant plasmids pMD-pdhβ, pMD-pdhD and pMD-pdhβ-pdhD amplified target bands of 993bp, 975bp and 1998bp respectively, and the size of the target bands was consistent with the expectations, indicating that the target gene has been successfully cloned into the pMD19-T vector; Figure 5 As can be seen, the recombinant plasmids pMD-pdhβ, pMD-pdhD and pMD-pdhβ-pdhD obtained target bands of 993 bp, 975 bp and 1998 bp respectively, and the sequences consistent with the expectations were obtained.

[0051] Then the correctly identified recombinant cloning plasmids pMD-pdhβ, pMD-pdhD, and pMD-pdhβ-pdhD were sent to Shanghai Biotech Co., Ltd. for sequencing. The pdhβ gene sequence obtained by sequencing was SEQ ID NO.1, the pdhD gene sequence obtained was SEQ ID NO.2, and the pdhβ-pdhD gene sequence obtained was SEQ ID NO.3. Among them, the sequence 1-6bp in SEQ ID NO.3 was the Kozak sequence, 7-996bp was the pdhβ gene sequence, 997-1026bp was the linker sequence, and 1027-1998bp was the pdhD gene sequence. The pdhβ and pdhD gene sequences were compared with the gene sequences in GenBank, and the consistency was 100%, indicating that the pMD-pdhβ, pMD-pdhD, and pMD-pdhβ-pdhD recombinant cloning plasmids were successfully constructed.

[0052] pdhβ gene sequence:

[0053] (SEQ IDNO.1);

[0054] pdhD gene sequence:

[0055] (SEQID NO.2);

[0056] pdhβ-pdhD gene sequence:

[0057]

[0058] Experimental Example 3: Construction of recombinant adenovirus shuttle plasmid

[0059] pMD-pdhβ, pMD-pdhD, pMD-pdhβ-pdhD and adenovirus shuttle vector pDC316-mCMV-EGFP were digested with Not I and EcoRV endonucleases, and the digested products were subjected to agarose gel electrophoresis and then recovered. The pdhβ, pdhD and pdhβ-pdhD gene fragments and the large pDC316-mCMV-EGFP fragment were ligated and transformed into competent Escherichia coli cells. After constant temperature shaking at 37°C for 1 hour in LB liquid culture medium, an appropriate amount was evenly spread on LB solid culture medium and cultured for 12 hours. A single colony was picked and cultured in LB liquid culture medium for 12 hours. After extracting the plasmid, PCR identification was performed using primers, and double digestion identification was performed using Not I and EcoRV endonucleases. The results were as follows: Figure 6 and Figure 7 ,from Figure 6 As can be seen, the recombinant shuttle plasmids pDC-pdhβ, pDC-pdhD and pDC-pdhβ-pdhD amplified target bands of 993bp, 975bp and 1998bp, respectively, indicating that the target gene has been successfully connected to the adenovirus shuttle vector pDC316-mCMV-EGFP; Figure 7 As can be seen, the recombinant shuttle plasmids pDC-pdhβ, pDC-pdhD, and pDC-pdhβ-pdhD obtained target bands of 993 bp, 975 bp, and 1998 bp, respectively.

[0060] Then, the correctly identified recombinant shuttle plasmid pDC316-pdhβ-pdhD was sent to Biotech (Shanghai) Co., Ltd. for sequencing, and the results were consistent with expectations, indicating that the pDC-pdhβ, pDC-pdhD, and pDC-pdhβ-pdhD recombinant shuttle plasmids were successfully constructed.

[0061] Experimental Example 4: Construction and packaging of recombinant adenovirus

[0062] Resuscitate the recombinant shuttle plasmids pDC-pdhβ, pDC-pdhD, pDC-pdhβ-pdhD and adenovirus backbone plasmid pBHGlox(delta)E1,3Cre and extract them with an endotoxin-free large-scale extraction kit. Resuscitate HEK 293A cells and pass them to the third generation; pretreat the 6-well plate with cell wall adhesive for 30 minutes; plate HEK 293A cells with a confluency of 90% on a 6-well plate (treated) and culture them in a cell culture incubator; when the cell confluency reaches 60% to 70%, transfection can be performed. Before transfection, replace the cell culture medium with double-antibody-free serum-free DMEM medium; take out the endotoxin-free recombinant shuttle plasmids and adenovirus backbone plasmids from the -80℃ refrigerator; and transfer the Neofect transfection reagent and the recombinant shuttle plasmids pDC-pdhβ, pDC-pdhD, pDC-pdhβ-pdhD and adenovirus backbone plasmids pBHGlox(delta)E1, 3Cre returned to room temperature; serum DMEM was used to dilute the large backbone plasmid and the recombinant shuttle plasmid, and incubated at room temperature in the dark for 5 minutes, and Neofect transfection reagent was added, and mixed by pipetting, and incubated at room temperature in the dark for 25 minutes; after the incubation, the mixture of plasmid and transfection reagent in the centrifuge tube was evenly added dropwise to 1 well of 293A cells, and after shaking, the cells were returned to the incubator for continued culture. After 10 hours of transfection, fresh DMEM complete medium was replaced for continued culture. After 48 hours, fluorescence observation could be performed to determine the transfection efficiency, which was about 80% ( Figure 8 ). 10-14 days after transfection, half of the culture medium was replaced with fresh medium every 2 days, and the culture medium was no longer replaced after 7 days; when more than half of the cells floated up and the remaining cells were fluorescent, the cells could be seen to be rounded, swollen and other lesions (CPE), and all cells and culture medium were collected into a centrifuge tube; frozen and thawed repeatedly in a -80℃ / 37℃ water bath for 3 times to destroy the cell structure and release adenovirus particles; centrifuged at 4℃, 6 000×g for 5 minutes, and the supernatant after 0.22μm filtration was the primary (P0 generation) virus stock solution.

[0063] The packaging results of recombinant adenovirus are detailed in Figure 8 ,from Figure 8 It can be seen that the cells showed green fluorescence under the fluorescence microscope on the 2nd and 7th day after transfection, and the cell density was very high on the 7th day, and a small number of cells showed cytopathic changes such as swelling, rounding, and floating like grapes; while the untransfected normal cells did not show green fluorescence under the fluorescence microscope, and the normal cells only showed increased density on the 7th day without cytopathic changes. The above results show that the recombinant adenovirus pBH-pdhβ, pBH-pdhD, pBH-pdhβ-pdhD and pBH-EGFP were successfully packaged as primary (P0 generation) recombinant adenovirus.

[0064] The primary virus solution was used to infect new HEK 293A cells for passage. The results of recombinant adenovirus proliferation are detailed in Fig. 9 ;from Fig. 9 It can be seen that after infection, the cells showed CPE phenomena such as rounding and shedding. On the second day, the cells showed a comet-like green fluorescence diffusion trend under the fluorescence microscope. On the fourth day, the fluorescence had spread to all cells. The uninfected cells were in a normal state, and no green fluorescence was observed under the fluorescence microscope. The above results show that the packaged recombinant adenovirus pBH-pdhβ, pBH-pdhD, pBH-pdhβ-pdhD and pBH-EGFP can proliferate in HEK 293A cells.

[0065] Experimental Example 5: Identification of recombinant adenovirus

[0066] The packaged P3 recombinant adenovirus was used to infect HEK 293A cells cultured in 60 mm dishes. After about 48 hours, when most cells had fluorescence but did not float, the cell RNA was extracted using the Trizol method and reversely transcribed into cDNA. Using it as a template and primers, RT-PCR was performed to identify the pdhβ, pdhD and pdhβ-pdhD genes. The products were subjected to agarose gel electrophoresis and the results were observed. Fig.10 ,from Fig.10 It can be seen that the recombinant adenovirus pBH-pdhβ, pBH-pdhD and pBH-pdhβ-pdhD amplified target gene fragments of 993bp, 975bp and 1998bp, respectively, which are consistent with the expected size. The results show that the constructed recombinant adenovirus contains the target gene, and the target gene can undergo reverse transcription.

[0067] The packaged P3 generation recombinant adenovirus pBH-pdhβ, pBH-pdhD, pBH-pdhβ-pdhD and pBH-EGFP were used to infect HEK 293A cells cultured in 60 mm dishes. When most cells were fluorescent but not floating, the old culture medium was discarded, and 1 mL of pre-cooled Hanks was added to gently wash the cells, and the washing was repeated twice. 200 μL of pre-cooled lysis buffer (RIPA: PMSF = 99:1) was evenly added to the 60 mm dish and placed on ice for 2 min. The cells were blown to mix, and the cell lysate was collected in a 1.5 mL centrifuge tube and placed on ice for 20 min. The tube was centrifuged at 4°C and 12000 × g for 15 min. The supernatant was collected to obtain total protein, and the target proteins pdhβ, pdhD and pdhβ-pdhD were identified by Western blot. The primary antibody used was MS self-made positive serum, and the secondary antibody used was 1:1 000 dilution of HRP-labeled goat anti-chicken IgG (H+L) antibody. Fig.11 .

[0068] from Fig.11It can be seen that the recombinant adenovirus pBH-pdhβ has a specific band at 36KDa that is consistent with the size of the pdhβ protein ( Fig.11 A in Figure 1); The recombinant adenovirus pBH-pdhD showed a specific band at 34.5KDa that was consistent with the size of the pdhD protein ( Fig.11 B in Figure 1); the recombinant adenovirus pBH-pdhβ-pdhD showed a pdhβ-pdhD fusion protein band at 70.8KDa ( Fig.11 The above results show that the packaged recombinant adenoviruses pBH-pdhβ, pBH-pdhD and pBH-pdhβ-pdhD can express the corresponding target proteins.

[0069] Experimental Example 6: Genetic Stability Analysis

[0070] Extraction of P1, P4, P8, P 12 Generation, P 15 The DNA of the recombinant adenovirus pBH-pdhβ, pBH-pdhD, and pBH-pdhβ-pdhD of each generation was amplified by PCR using primers. The products were subjected to agarose gel electrophoresis and the results were observed. Fig.12 ;from Fig.12 It can be seen that the 1st, 4th, 8th, 12th and 15th generation recombinant adenovirus pBH-pdhβ, pBH-pdhD and pBH-pdhβ-pdhD can all obtain target fragments consistent with the expected size, indicating that the constructed recombinant adenovirus pBH-pdhβ, pBH-pdhD and pBH-pdhβ-pdhD have good genetic stability.

[0071] Experimental Example 7: Purification and titer determination of recombinant adenovirus

[0072] Low-generation HEK 293A cells were planted in 100 mm cell culture dishes and P3 adenovirus was amplified when the cell confluence was 90%. When most of the HEK 293A cells had obvious fluorescence, the cells and cell fluid were collected and centrifuged at 3 500 × g for 15 min. Only a small amount of supernatant was used to resuspend the cells, and the cells were repeatedly frozen and thawed three times at -80°C / 37°C, centrifuged at 3 500 × g for 15 min, and the virus supernatant was collected. The P4 recombinant adenovirus was purified using the Vivapure AdenoPACKTM adenovirus purification kit to obtain concentrated virus fluid.

[0073] Digest HEK 293A cells into single 2 × 10 5100 μL of purified virus solution was evenly planted in a 96-well plate and cultured in a cell culture incubator. When the HEK 293A cells grew to a confluence of 80% to 90%, 100 μL of purified virus solution was diluted 10-fold with DMEM complete medium, respectively: 1×10 -1 , 1×10 -2 , 1×10 -3 ......1×10 -12 , and set up a negative control at the same time; add 100 μL of diluted virus solution to each well according to the dilution gradient, shake well and continue to culture until the 7th day, observe the cell growth and cell fluorescence under a fluorescence microscope every day; judgment standard: under a fluorescence microscope, HEK 293A cells show CPE and fluorescence within 7 days, which is positive, and no CPE and fluorescence is negative. Record the fluorescence and CPE of each well in the 96-well plate to judge the positive and negative, the results are shown in Table 2, and the virus titer (50% tissue culture infective dose, TCID 50 ), the titer of pBH-pdhβ-pdhD was calculated to be 10 9.11 TCID 50 / 100μL.

[0074] Table 2 Titer determination of recombinant adenovirus

[0075]

[0076]

[0077] Experimental Example 8: Animal Immunization Experiment

[0078] Fifty healthy chicks at 18 days of age were randomly divided into PBS group, pBH-EGFP group, pBH-pdhβ-pdhD group, pBH-pdhβ and pBH-pdhD group, with 10 chicks in each group. The total amount of virus injected into the leg muscle of the chicks in the recombinant adenovirus group was 10 8 TCID 50 / mouse, PBS group was injected with PBS 0.2mL / mouse, and immunized once every 2 weeks. Blood was collected on 7d, 14d, 21d, 35d and 42d after the first immunization. The indirect ELISA method was used to determine the serum antibody levels on 7d, 14d, 21d, 35d and 42d. The results are shown in the table. Fig.13 ;from Fig.13 It can be seen that the pBH-pdhβ-pdhD group has a higher antibody level than pBH-pdhβ and pBH-pdhD.

[0079] The levels of cytokines such as IL-2, IFN-γ, IL-4 and IL-6 in serum at 7d, 14d, 21d, 35d and 42d were detected by ELISA kits. Figure 14-17 .from Figure 14-17 It can be seen that after chickens were immunized with each recombinant adenovirus, the secretion of specific antibodies and cytokines (IL-2, IFN-γ, IL-4 and IL-6) in the body serum gradually increased from 7 days after the first immunization, reaching the highest level at 28 days after immunization, and then gradually decreased. The secretion of antibodies and cytokines in each recombinant adenovirus group was significantly higher than that in the PBS group and pBH-EGFP group (P < 0.01); the overall trend was pBH-pdhβ-pdhD group > pBH-pdhD group > pBH-pdhβ group > PBS group and pBH-EGFP group; the results showed that each recombinant adenovirus group could stimulate the chicken body to produce a good humoral and cellular immune response.

[0080] 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 fusion gene of Mycoplasma synoviae, characterized in that: The nucleotide sequence of the fusion gene is shown in SEQ ID NO.

3.

2. A recombinant adenovirus shuttle plasmid, characterized in that: Comprising the fusion gene according to claim 1.

3. A recombinant adenovirus vector, characterized in that: It comprises the shuttle plasmid and backbone plasmid described in claim 2.

4. The recombinant adenovirus vector according to claim 3, characterized in that The backbone plasmid is a replication-defective adenoviral vector.

5. The method for constructing the recombinant adenovirus vector according to claim 3 or 4, characterized in that: The following steps are involved: Step 1, using the Mycoplasma synoviae DNA as a template to perform PCR amplification to obtain the target gene; Step 2: Connect the target gene and plasmid to obtain a recombinant shuttle plasmid; Step 3: Recombining the recombinant shuttle plasmid and the backbone plasmid to obtain a recombinant adenovirus vector.

6. A method for preparing a recombinant adenovirus, characterized in that: The recombinant adenovirus vector according to claim 3 or 4 is infected into cells for amplification and expression to obtain the recombinant adenovirus.

7. The recombinant adenovirus obtained by the method for preparing the recombinant adenovirus according to claim 6.

8. Use of the recombinant adenovirus according to claim 7 in the preparation of a Mycoplasma synoviae vaccine.

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