Construction method and application of bovine enterovirus VP1 gene recombinant adenovirus

Abstract

The invention provides a construction method and application of a recombinant adenovirus of a bovine enterovirus VP1 gene, and aims to construct a recombinant adenovirus rAdv-VP1 of a structural protein VP1 of the bovine enterovirus. According to the bovine enterovirus VP1 gene recombinant adenovirus constructed by the technical scheme provided by the invention, the VP1 protein is successfully expressed in BEV infected cells, the protein amount is obviously increased compared with that of uninfected cells, and mouse positive serum and the VP1 protein can be successfully expressed and reacted; the rAdv-VP1 can be used for immunizing mice, the serum antibody level of the organism can be remarkably improved, good body fluid and cellular immune response is caused, BEV in-vivo replication can be remarkably inhibited by using the rAdv-VP1 to immunize the mice, and the mice can be protected from being attacked by BVDV. The bovine enterovirus VP1 gene recombinant adenovirus rAdv-VP1 constructed by the technical scheme provided by the invention has an inhibition effect on BEV, can provide data for later application of the vaccine, and provides a basis for BEV live vector vaccine research.

Description

Technical Field

[0001] The invention belongs to the field of gene bioengineering and relates to a method for constructing a recombinant adenovirus, specifically, a method for constructing a recombinant adenovirus using the bovine enterovirus VP1 gene, the recombinant adenovirus and its application. Background Art

[0002] Bovine enterovirus (BEV) is an epidemic infectious disease that is widely present in animals and mainly affects the digestive tract and respiratory tract. Infection can cause diarrhea, runny nose, fever, cough, gastroenteritis, dyspnea and even sudden death in cattle. Some cattle infected with BEV are latently infected and do not show obvious clinical symptoms, which often leads to secondary infection and serious losses. In my country, due to the lack of prevention and control measures for bovine enterovirus disease and effective vaccination, the environmental conditions for raising animals are also limited. A large number of fresh breeds of cattle have to be introduced from abroad for hybridization, which has also led to the rapid spread of bovine enterovirus. BEV is being widely spread in China's cattle industry, greatly hindering the development of China's cattle industry, because it not only causes symptoms such as diarrhea in cattle, but also combines with other viruses to infect cattle, resulting in more serious clinical symptoms.

[0003] In recent years, the number of clinical diseases characterized by diarrhea has increased day by day in China. If it is not controlled, the situation will be irreversible. At present, most of the treatments for BEV on the market are drug treatments, but in fact, vaccination is currently the most economical and effective means to prevent infectious diseases. Disease pathogens or their related proteins, polysaccharides or nucleic acids constitute most vaccines, which can directly or through certain carriers cause specific humoral and cellular immunity in the body. However, there are many problems with the commonly used vaccines, such as low safety and poor immunogenicity, so it is imperative to develop a safe and efficient new vaccine. VP1 is a structural protein of BEV, which can induce infected animals to produce neutralizing antibodies, can participate in cell recognition and adsorption, and has the most important antigen-antibody binding site on the VP1 protein, which has an incomparable advantage in the field of immunity. Recombinant adenovirus vector vaccine is a new type of vector vaccine. It is a vaccine that expresses specific antigens in vivo by recombining vectors with gene proteins, and uses viruses as vectors. Compared with traditional vaccines, recombinant adenovirus vector vaccines have the advantages of better safety, higher recombinant virus titer, larger yield, and stronger immunogenicity. They also have multiple routes of administration and can infect different types of cells and tissues, making them very suitable for genetic engineering vaccines and gene therapy research.

[0004] How to provide a method for constructing a recombinant adenovirus of the bovine enterovirus VP1 gene that is convenient for large-scale production, has mature technology and good formula stability and has good immune effect is crucial for obtaining an effective vaccine to provide more effective protection against BEV, prevent and control BEV, and promote the healthy and sustainable growth of animal husbandry production and its products. Summary of the invention

[0005] In view of the technical problems that vaccines related to bovine enterovirus infection in the prior art have low safety and poor immunogenicity, and new safe and efficient vaccines are rarely reported, the present invention aims to provide a method for constructing a recombinant adenovirus of the VP1 gene of bovine enterovirus, a recombinant adenovirus and its application, combining the VP1 gene of BEV with the characteristics of the adenovirus vector system for efficient and stable expression of foreign genes, constructing a recombinant adenovirus capable of expressing the above gene, carrying out a BEV challenge test on immunized mice, and evaluating the effect of the vaccine on inhibiting BEV infection in vivo by detecting the BEV virus load of each tissue by fluorescent quantitative PCR to verify its immunogenicity. The immunogenicity and safety of the vaccine were preliminarily verified by immunizing mice, aiming to provide materials for the later application of the vaccine and provide a basis for the research of BEV virus live vector vaccine.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for constructing a recombinant adenovirus of bovine enterovirus VP1 gene, comprising the following steps:

[0007] (1) Amplify the VP1 gene sequence from bovine enterovirus strains;

[0008] The amplification of the VP1 gene comprises: using VP1-F SEQ ID NO.1 and VP1-R SEQ ID NO.2 as primers, using cDNA of bovine enterovirus strain BL311 as a template, and performing PCR amplification to obtain VP1 gene SEQ ID NO.3; the PCR reaction amplification system comprises: 2×Taq PCR Mix 10.0 μL, VP1-F 0.5 μL, VP1-R 0.5 μL, cDNA template 2.0 μL, ddH2O 7.0 μL; the PCR reaction procedure comprises: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 68°C annealing for 30 s, 72°C extension for 1 min, 38 cycles; 72°C extension for 10 min;

[0009] (2) The adenovirus empty vector pDC316-CMV-copGFP was digested with restriction endonucleases EcoR I and Sal I-HF. The reaction system was: EcoR I 1.0 μL, Sal I-HF 1.0 μL, CutSmart Buffer 1.0 μL, pDC316-CMV-copGFP 0.6 μL, ddH2O 6.4 μL; reaction procedure: incubate at 37°C for 4 h, inactivate at 85°C for 15 min;

[0010] (3) The VP1 gene amplified in step (1) and the adenovirus empty vector pDC316-CMV-copGFP in step (2) were respectively connected. The reaction system was 10×Ligase Buffer 1.0 μL, T4 DNA Ligase 1.0 μL, PDC316-CMV-copGFP 1.0 μL, and VP1 7.0 μL. The mixture was mixed and placed at 4°C overnight to obtain the recombinant shuttle plasmid pDC316-VP1-copGFP.

[0011] (3) The recombinant shuttle plasmid pDC316-VP1-copGFP and the adenovirus backbone plasmid pBHGlox-△E1,3cre were co-transfected into 293LP cells, and the recombinant adenovirus rAdv-VP1 was obtained by recombination and packaging.

[0012] Preferably, the large backbone plasmid and the shuttle plasmid are co-transfected at a ratio of (1-4):1.

[0013] More preferably, the large backbone plasmid and the shuttle plasmid are co-transfected at a ratio of 4:1.

[0014] Preferably, during the transfection process, the concentration of fetal bovine serum is 2-5%.

[0015] More preferably, during the transfection process, the concentration of fetal bovine serum is 2%.

[0016] The recombinant adenovirus is prepared by the construction method of the recombinant adenovirus of the bovine enterovirus VP1 gene.

[0017] The invention relates to the application of the recombinant adenovirus prepared by the construction method of the recombinant adenovirus of the bovine enterovirus VP1 gene in preventing and treating bovine enterovirus infection.

[0018] The recombinant adenovirus prepared by the construction method of the bovine enterovirus VP1 gene recombinant adenovirus is used in the preparation of vaccines and drugs related to the prevention and treatment of bovine enterovirus infection.

[0019] The dosage form of the vaccine for preventing and treating bovine enterovirus infection includes injection, nasal drops or spray.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) By adopting the technical scheme provided in this application to immunize mice with adenovirus, 10d and 15d after BVDV challenge, the relative copy number of BEV between the recombinant adenovirus immunized group and the control group gradually formed compared with the control group, and the BEV copy number of each tissue in the immunized group was significantly lower than that of the control group (P<0.0001). The results showed that starting from 10d and 15d after BEV challenge, the rAdv-VP1 immunized group could significantly reduce the BEV copy number compared with the control group, indicating that the use of rAdv-VP1 to immunize mice can significantly inhibit the replication of BEV in vivo.

[0022] (2) After immunization with recombinant adenovirus rAdv-VP1 and rAdv-copGFP, there was no significant difference in body weight between the experimental group mice and the control group mice vaccinated with PBS (P﹥0.05). There was no significant change in the body weight of the mice in the adenovirus immunization group after the virus attack, while the weight gain trend of the mice in the non-immunized group and the empty vector group was reduced. This indicates that vaccination with recombinant adenovirus vaccine has no side effects and can better protect mice from the attack of BEV virus.

[0023] (3) Compared with the empty vector group and the PBS control group, the corresponding specific antibodies in the recombinant adenovirus rAdv-VP1 group were detected 1-2 weeks after the first immunization. After three immunizations, the antibody titer of the recombinant adenovirus immunization group gradually increased, and higher antibody levels were produced after the second immunization on the 14th day and the third immunization on the 21st day, which were significantly higher than those in the control group (P<0.01).

[0024] (4) After three immunizations, the antibody level of the recombinant adenovirus group increased significantly (P<0.05). Among them, the antibody induction trends of the adenovirus empty group and the PBS control group were similar, and there was no significant difference in the ability of the two groups to induce antibody production (P>0.05). After the third immunization, the recombinant adenovirus vaccine group induced the highest VP1 antibody level, which was significantly higher than that of the adenovirus empty group and the PBS control group (P<0.01).

[0025] (5) After the second immunization, the cytokine levels of the adenovirus immunization group began to differ significantly from those of the empty vector group and the control group. The IFN-γ level of rAdv-VP1 in the adenovirus immunization group was significantly higher than that in the other groups (P<0.05). After the third immunization, the concentration could reach 13.85 pg / mL. The IFN-γ level of rAdv-VP1 in the immunization group was extremely significantly higher than that in the adenovirus empty vector group and the PBS control group (P<0.01). Similarly, the IL-4 level of rAdv-VP1 in the adenovirus immunization group was also extremely significantly higher than that in the other groups (P<0.01). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Shown is the result of PCR amplification of BEVVP1 gene.

[0027] Figure 2 Shown is the result of restriction digestion of adenovirus empty vector pDC316-CMV-copGFP.

[0028] Figure 3 Shown is the result of identification of adenovirus shuttle plasmid pDC316-VP1-copGFP bacterial solution.

[0029] Figure 4 Shown is the identification result of adenovirus shuttle plasmid pDC316-VP1-copGFP plasmid.

[0030] Figure 5 Shown is the plasmid map result of adenovirus shuttle plasmid pDC316-VP1-copGFP.

[0031] Figure 6 Shown is the double enzyme digestion identification result of the recombinant plasmid pDC316-VP1-copGFP.

[0032] Figure 7 Shown is the green fluorescence expression of 293LP cells transfected with different plasmid ratios and different serum concentrations for 48 hours.

[0033] Figure 8 Shown is a diagram of recombinant adenovirus rAdv-VP1 infecting 293LP cells.

[0034] Fig. 9 Shown is a picture of 293LP cells infected with recombinant adenovirus rAdv-copGFP.

[0035] Fig.10 Shown are the RT-PCR results of recombinant adenovirus rAdv-VP1.

[0036] Fig.11 Shown is the bacterial solution and plasmid identification diagram of the recombinant plasmid pET-32a-BEV-VP1.

[0037] Fig.12 Shown is a graph showing the changing trend of the effect of different induction times on the expression level of the fusion protein.

[0038] Fig.13 The figure shows the changing trend of the effect of different induction temperatures on the expression level of the recombinant protein.

[0039] Fig.14 Shown is the identification diagram of the induced expression of the recombinant protein pET-32a-BEV-VP1.

[0040] Fig.15Shown is a solubility analysis graph of the recombinant protein pET-32a-BEV-VP1.

[0041] Fig.16 Shown is the purification diagram of pET-32a-BEV-VP1 recombinant protein.

[0042] Fig.17 Shown is the Western-blot identification of pET-32a-BEV-VP1 recombinant protein.

[0043] Fig.18 Shown are the pathological changes of BEV-uninfected and BEV-infected MDBK cells.

[0044] Among them, Figure A shows MDBK cells before BEV infection; Figure B shows MDBK cells after BEV infection.

[0045] Fig.19 Shown are VP1 protein levels in BEV-uninfected and BEV-infected MDBK cells.

[0046] Fig. 20 Shown is the fluorescence quantitative RT-PCR detection of viral load in different tissues of BALB / c mice at different times after adenovirus immunization.

[0047] Among them, Figure A shows the viral load graph on day 0; Figure B shows the viral load graph on day 5; Figure C shows the viral load graph on day 10; and Figure D shows the viral load graph on day 15.

[0048] Fig.21 Shown is the trend graph of mouse body weight changes.

[0049] Fig. 22 Shown are graphs of serum-specific antibody levels.

[0050] Fig.23 Shown is a graph showing serum IgG antibody level detection.

[0051] Fig.24 Shown is the detection graph of serum IFN-γ cytokine level.

[0052] Fig.25 Shown is the detection graph of serum IL-4 cytokine level. Specific embodiments

[0053] The present invention will be described below with reference to examples; however, the present invention is not limited to the following examples.

[0054] 293LP cells were purchased from the Cell Bank of Shanghai Institutes for Biological Studies, Chinese Academy of Sciences; Escherichia coli Stbl3 competent cells (BC108-01) were purchased from Beijing Bomade Company; pBHGlox-△E1,3cre and pDC316-CMV-copGFP were purchased from Shanghai Haijihaoge Company; BEV strain BL311 was isolated, identified and retained by our research group and deposited in the China Center for Type Culture Collection on January 9, 2021, with the accession number CCTCC NO: V202098. The prokaryotic expression vector pET-32a(+) and prokaryotic expression plasmid pET-32a-BEV-VP1 and puncture bacteria were purchased from Beijing Qingke Biotechnology Co., Ltd.; Escherichia coli DH5α and BL21(DE3) competent cells were purchased from Beijing Quanshijin Company; MDBK cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, the BEV strain BL311 was isolated, identified and retained by our research group, the recombinant adenovirus vector vaccine rAdv-VP1 and adenovirus empty vector rAdv-copGFP were constructed and preserved in the early stage of the experiment; the BEV virus was preserved in the laboratory; 6-8 week old male SPF-grade BALB / c mice were purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.

[0055] The reagents used in the present invention include: minimum basal medium (MEM); fetal bovine serum (FBS); penicillin-streptomycin (Penicillin-Streptomycin); trypsin (Trypsin-EDTA); polyethyleneimine (PEI) Endo-Free Plasmid Mini Kit (50); endotoxin-free small amount plasmid extraction kit; ordinary agarose gel DNA recovery kit; 2×Taq PCR Mix; restriction endonuclease EcoR I; restriction endonuclease BamH I; penicillin-streptomycin (Penicillin-Streptomycin) isopropyl-β-D-thiogalactoside (IPTG); Coomassie Brilliant Blue R250; 10-180kDa protein marker; ECL chemiluminescence kit; rapid plasmid mini-extraction kit; HRP-labeled goat anti-mouse IgG (H+L) antibody; BCA protein concentration determination kit; urea; imidazole; NaCl; Tris; Ni-NTA agarose resin; bovine serum albumin BSA; TRIzol; RT Easy TMⅡ reverse transcription kit; high glucose medium DMEM; TMB colorimetric solution; HRP-labeled goat anti-mouse IgG (H+L) antibody; GAPDH Antibody 10494-1-AP antibody; SYBR Green fluorescence quantitative PCR kit; RIPA lysis solution (P0013B); PMSF; mouse IFN-γ and IL-4 ELISA detection kit; IgG antibody ELISA detection kit; disposable medical sterile syringe. The reagents and materials can be purchased through public channels, and the equipment and instruments used in the process are all common equipment in this field.

[0056] The instruments used in the present invention are: a fluorescent inverted microscope (TE2000) gradient PCR instrument; an electrophoresis gel imager (Gel Doc XP); a Nano Drop 2000C ultra-micro spectrophotometer; a low-temperature high-speed centrifuge; an ultra-clean workbench SW-CJ-2F; an ELISA reader; a protein electrophoresis instrument; a cell ultrasonic disruptor; a constant temperature metal bath; a 4°C high-speed centrifuge; an ultraviolet spectrophotometer; a Real-Time PCR System (7500Fast); a low-temperature high-speed centrifuge; and a biological safety cabinet.

[0057] All materials, reagents and instruments used in the present invention are well known in the art, but do not limit the implementation of the present invention. Other reagents and equipment well known in the art can be applied to the implementation of the following embodiments of the present invention.

[0058] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0059] Example 1: Construction method and application of a recombinant adenovirus containing the VP1 gene of bovine enterovirus

[0060] The present application provides a method for constructing a recombinant adenovirus of the bovine enterovirus VP1 gene, which specifically comprises the following steps:

[0061] (1) Amplify the VP1 gene sequence from bovine enterovirus strains;

[0062] The amplification of the VP1 gene comprises: using VP1-F SEQ ID NO.1 and VP1-R SEQ ID NO.2 as primers, using cDNA of bovine enterovirus strain BL311 as a template, and performing PCR amplification to obtain VP1 gene SEQ ID NO.3; the PCR reaction amplification system comprises: 2×Taq PCR Mix 10.0 μL, VP1-F 0.5 μL, VP1-R 0.5 μL, cDNA template 2.0 μL, ddH2O 7.0 μL; the PCR reaction procedure comprises: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 68°C annealing for 30 s, 72°C extension for 1 min, 38 cycles; 72°C extension for 10 min;

[0063] (2) The adenovirus empty vector pDC316-CMV-copGFP was digested with restriction endonucleases EcoR I and Sal I-HF. The reaction system was: EcoR I 1.0 μL, Sal I-HF 1.0 μL, CutSmart Buffer 1.0 μL, pDC316-CMV-copGFP 0.6 μL, ddH2O 6.4 μL; reaction procedure: incubate at 37°C for 4 h, inactivate at 85°C for 15 min;

[0064] (3) The VP1 gene amplified in step (1) and the adenovirus empty vector pDC316-CMV-copGFP in step (2) were respectively connected. The reaction system was 10×Ligase Buffer 1.0 μL, T4 DNA Ligase 1.0 μL, PDC316-CMV-copGFP 1.0 μL, and VP1 7.0 μL. The mixture was mixed and placed at 4°C overnight to obtain the recombinant shuttle plasmid pDC316-VP1-copGFP.

[0065] (3) The recombinant shuttle plasmid pDC316-VP1-copGFP and the adenovirus backbone plasmid pBHGlox-△E1,3cre were co-transfected into 293LP cells, and the recombinant adenovirus rAdv-VP1 was obtained by recombination and packaging.

[0066] Preferably, the large backbone plasmid and the shuttle plasmid are co-transfected at a ratio of (1-4):1.

[0067] Preferably, during the transfection process, the concentration of fetal bovine serum is 2-5%.

[0068] The recombinant adenovirus is prepared by the construction method of the recombinant adenovirus of the bovine enterovirus VP1 gene.

[0069] The invention relates to the application of the recombinant adenovirus prepared by the construction method of the recombinant adenovirus of the bovine enterovirus VP1 gene in preventing and treating bovine enterovirus infection.

[0070] The recombinant adenovirus prepared by the construction method of the bovine enterovirus VP1 gene recombinant adenovirus is used in the preparation of vaccines and drugs related to the prevention and treatment of bovine enterovirus infection.

[0071] The dosage form of the vaccine for preventing and treating bovine enterovirus infection includes injection, nasal drops or spray.

[0072] Example 2: Construction of adenovirus shuttle plasmid pDC316-VP1-copGFP

[0073] (1) Primer design and synthesis

[0074] According to the BEVVP1 gene sequence published by NCBI, two specific primers were designed, namely the bovine enterovirus VP1 gene-specific upstream primer BEVVP1 (ad, EcoR I)-F SEQ ID NO.1 and the bovine enterovirus VP1 gene-specific downstream primer BEV VP1 (ad, Sal I)-R SEQ ID NO.2, which were synthesized by Suzhou Jinweizhi Company. The design principle follows that the product length is between 100bp-300bp, the primer length is between 18bp-25bp, and there are no three consecutive identical bases. The pre-amplified fragment BEVVP1 is 849bp in length, and its primer sequence is shown in Table 1.

[0075] Table 1: Primer sequences

[0076]

[0077] (2) PCR amplification results of target gene

[0078] The VP1 gene of BEVVP1 template was amplified by PCR using upstream and downstream primers designed according to the BEV VP1 gene sequence in GenBank. The results are shown in the attached figure. Figure 1 As shown, the amplification result obtained a target band of about 849 bp in size, which was consistent with the expected size, indicating that the BEVVP1 gene was successfully amplified, and its gene sequence is SEQ ID NO.3.

[0079] (3) pDC316-CMV-copGFP digestion results

[0080] The adenovirus-loaded pDC316-CMV-copGFP was digested with EcoRI and SalI-HF restriction endonucleases and then detected by 1% agarose gel electrophoresis. The results are shown in the attached figure. Figure 2 As shown, two bands were observed at approximately 1493 bp and 4999 bp, which were consistent with the expected size. The 4999 bp band was recovered from the gel.

[0081] (4) Identification of the bacterial culture and plasmid of the recombinant adenovirus shuttle plasmid pDC316-VP1-copGFP

[0082] The monoclonal colonies picked after ligation and transformation and the plasmids without endotoxin were identified by PCR. The results are shown in the attached figure. Figure 3 , Attachment Figure 4 As shown, a band was found at 849 bp, which was consistent with the expected result. The positive colonies were sent to Suzhou Genewise for sequencing. The plasmid map is shown in the attached Figure 5 As shown, the sequencing results were correct, indicating that the recombinant adenovirus shuttle plasmid was successfully constructed.

[0083] (5) Double restriction enzyme digestion identification of the recombinant adenovirus shuttle plasmid pDC316-VP1-copGFP

[0084] The recombinant adenovirus shuttle plasmid was digested with restriction endonucleases EcoR I and BamH I. After the digestion reaction was completed, 10 μL of the digestion product was mixed with 2 μL of 10× DNA Buffer and then subjected to 1% agarose gel electrophoresis and UV analysis. The results are shown in the attached figure. Figure 6 As shown, the two bands were approximately 5365 bp and 849 bp, respectively, which were consistent with the size of VP1 gene and pDC316-CMV-copGFP, indicating that the recombinant adenovirus shuttle plasmid pDC316-VP1-copGFP was successfully constructed.

[0085] Example 3: Packaging, amplification and identification of recombinant adenovirus

[0086] (1) Optimization of recombinant adenovirus packaging conditions

[0087] The recombinant adenovirus empty vector control plasmid pDC316-CMV-copGFP with a green fluorescent label and the adenovirus backbone plasmid pBHGlox-△E1,3cre were co-transfected into 293LP cells according to different conditions. The serum of different concentrations was replaced every 24 hours. After 48 hours of culture, the green fluorescence of the cells was observed under a fluorescence microscope. The results are shown in the attached figure. Figure 7 As shown, when the concentration of the large backbone plasmid: shuttle plasmid is 4:1 and the concentration of fetal bovine serum is 2%, the green fluorescence is the highest. As the concentration of the large backbone plasmid decreases, the green fluorescence gradually decreases, indicating that the adenovirus transfection efficiency is highest when the ratio of the backbone plasmid: shuttle plasmid is 4:1 and 2% FBS medium is used for culture.

[0088] (2) Packaging and amplification of recombinant adenovirus rAdv-E2

[0089] According to the optimized conditions of plasmid concentration 4:1 and 2% FBS, the recombinant adenovirus rAdv-VP1 and rAdv-copGFP were used to infect 293LP cells with a fusion degree of 70% to 80%, and the pathological changes were observed. After the cell lesions appeared, the diseased cells and supernatant were repeatedly frozen and thawed at -80℃ / 37℃ for 3 times, and the supernatant was centrifuged and added to MEM without double antibodies. The virus solution was added to the 293LP culture dish, and the serum was replaced with 2% FBS after 24 hours. The cell status was observed every day until the cells were completely diseased. The results are shown in the attached figure. Figure 8 , Attachment Fig. 9 As shown, a large number of lesions and green fluorescence appeared in the cells infected with BEV, indicating that the recombinant adenovirus can continuously infect 293LP cells and has infection activity.

[0090] (3) Titer determination of recombinant adenovirus rAdv-E2 and rAdv-EGFP

[0091] The purified recombinant adenovirus rAdv-VP1 and rAdv-copGFP were diluted in a multiple ratio of 10-2 to 10-11 and added to the cultured 96-well plate. The cell half infection dose was determined by the TCID50 method, and the recombinant adenovirus titer was calculated according to the KaBer method. The calculation results are shown in Table 2. The titers of rAdv-VP1 and rAdv-copGFP were 1×10 10.6 TCID 50 / mL, 1×10 8.5 TCID 50 / mL, which meets the virus titer requirements for subsequent animal tests.

[0092] Table 2: TCID 50 calculate

[0093]

[0094]

[0095] (4) RT-PCR identification of recombinant adenovirus rAdv-E2

[0096] The recombinant adenovirus rAdv-VP1 was used to infect 293LP cells. When the cells showed obvious CPE, the virus was harvested and the total RNA of the cells was extracted. RT-PCR was performed to identify the expression of VP1 gene in 293LP cells. The results are shown in the attached figure. Fig.10 As shown, a band with the same size as the VP1 gene appeared at 849 bp, indicating that the target gene was successfully expressed in the recombinant adenovirus.

[0097] Example 4: Prokaryotic expression and purification of bovine enterovirus VP1 protein

[0098] (1) Identification of the bacterial culture and plasmid of the prokaryotic expression vector recombinant plasmid pET-32a-BEV-VP1

[0099] Using the prokaryotic expression plasmid constructed by the company and the transformed bacterial solution as templates, PCR amplified the BEV-VP1 gene fragment. The fragment size shown on 1% agarose gel electrophoresis was about 861 bp, which was consistent with the expected result. Fig.11 As shown, it was shown that the pET-32a-BEV-VP1 prokaryotic expression vector was successfully constructed.

[0100] (2) Effects of different induction conditions on recombinant protein expression

[0101] 1. Influence of induction time

[0102] In order to explore the relationship between the expression level of recombinant protein and induction time, 9 time gradients were set in this experiment. The results are shown in the attached figure. Fig.12 As shown, a band at a molecular mass of 48 kDa that is more obvious and wider than other bands is consistent with the expected size of the recombinant protein pET-32a-BEV-VP1. It can be observed that the expression level of the protein first increases and then stabilizes with the induction time, indicating that the expression level of the pET-32a-BEV-VP1 recombinant protein is higher when the induction time is 8 h.

[0103] 2. Influence of induced temperature

[0104] In order to explore the relationship between the expression of recombinant protein and induction temperature, three temperature gradients were set in this experiment. Fig.13 As shown, a target band appeared at 48 kDa after induction, which was consistent with expectations, indicating that the expression level of pET-32a-BEV-VP1 recombinant protein was high at 37°C.

[0105] (3) Expression and identification of recombinant protein pET-32a-BEV-VP1

[0106] The recombinant plasmid pET-32a-BEV-VP1 that was successfully verified by enzyme digestion was transformed with pET-32a and then expanded for culture. The bacteria were harvested after induction for 8 h at 1.0 mM IPTG, 37°C, and 200 rpm. The results of SDS-PAGE identification are shown in the attached figure. Fig.14 As shown in the figure, the recombinant plasmid pET-32a-VP1 showed a specific protein band between the standard molecular weight of 45 and 60 kDa, which was consistent with the expected protein size of 48 kDa, and the protein amount after induction increased significantly compared with that before induction, while the pET-32a empty vector had no obvious change before and after induction. This shows that the recombinant plasmid pET-32a-BEV-VP1 was successfully constructed and can be expressed in large quantities in E. coli, while the protein without IPTG induction and the empty vector protein

[0107] (4) Solubility analysis of recombinant protein pET-32a-VP1

[0108] The pET-32a-BEV-VP1 recombinant protein was induced by adding 1.0 mM IPTG at 37°C for 8 h, then resuspended in PBS and ultrasonically disrupted. The supernatant and precipitate were centrifuged and subjected to SDS-PAGE gel electrophoresis. The results are shown in the attached figure. Fig.15 As shown, the recombinant protein was almost absent in the supernatant, and a large amount of protein was present in the inclusion bodies. After the inclusion bodies were dissolved with 8M urea and then ultrasonically broken again, most of the protein was transferred to the supernatant and expressed in large quantities. The molecular size was about 48kDa, which was consistent with the expected result. This indicates that the recombinant protein pET-32a-VP1 belongs to the inclusion body protein, and only a faint protein band exists in the supernatant.

[0109] (5) Recombinant protein purification results and Western-blot identification

[0110] The purified recombinant protein pET-32a-BEV-VP1 was analyzed by SDS-PAGE. Fig.16 As shown in the figure, only a small amount or no target protein band appeared in the flow-through and washing solution, while a single and clear target band was obtained in the 5 tubes of eluate, and the band size was about 48kDa, which was consistent with the expected size. This shows that all the proteins were eluted and the purified target protein was successfully expressed. The purified recombinant protein was identified by Western blot as shown in the attached figure. Fig.17 As shown, a specific color band appeared at 48 kDa, confirming that the induced expressed protein could specifically bind to goat anti-mouse antibody and had immunological activity.

[0111] Example 5: Evaluation of the immune effect of recombinant adenovirus carrying the bovine enterovirus VP1 gene

[0112] (1) Pathogenicity of MDBK cells infected with BEV

[0113] BEV was infected into MDBK cells grown to 70%-80%, and the cell cytopathic effect was observed after 24 hours. The results are shown in the attached figure. Fig.18 As shown, the cells showed a large number of lesions, and the cells appeared wrinkled and vacuolized, indicating that BEV successfully infected MDBK cells.

[0114] (2)TCID 50 Determination of BEV virus titer

[0115] TCID 50 The BEV virus purified by PEG-6000 was measured by the method, and the titer measurement results are shown in Table 3 below:

[0116] Table 3: TCID50 calculation

[0117]

[0118]

[0119] The titers of the concentrated viruses were calculated. The results showed that the titers of BEV were 1×10 11.75 TCID 50 / 0.1mL, which meets the virus titer requirements for subsequent animal tests.

[0120] (2) Western blot detection of vp1 protein levels in MDBK cells

[0121] Western blot was used to detect the expression of VP1 protein in BEV-infected MDBK cells to observe whether BVDV would affect the expression of VP1 protein. The results are shown in the attached figure. Fig.19 As shown, using GAPDH as an internal reference, compared with uninfected MDBK cells, the expression of VP1 in BVDV-infected MDBK cells was significantly upregulated.

[0122] (3) qPCR detection of BEV viral load in mouse tissues

[0123] On the 0th, 5th, 10th and 15th days after the three immunizations, the mice were dissected after sterile treatment, and the liver, spleen, lung, kidney and small intestine were taken. The RNA of the tissues was routinely extracted and reverse transcribed into cDNA for fluorescence quantitative RT-PCR to detect the BEV copy number in each tissue of BALB / c mice at different days after BEV infection. The results are shown in the attached figure. Fig. 20As shown in the figure, compared with the negative control group and the empty group after BVDV challenge, the relative copy number of BEV in each tissue of the adenovirus immunized group was lower than that of the control group and the empty group at 0d and 5d, and the difference was not significant (P>0.05); at 5d, there was a slight and significant difference in the copy number compared with the control group (P<0.01); 10d and 15d after BVDV challenge, compared with the control group, the relative copy number of BEV between the recombinant adenovirus immunized group and the control group gradually formed, and the copy number of BEV in each tissue of the immunized group was significantly lower than that of the control group (P<0.0001). The results showed that starting from 10d and 15d after BEV challenge, the rAdv-VP1 immunized group could significantly reduce the BEV copy number compared with the control group, indicating that the use of rAdv-VP1 to immunize mice can significantly inhibit the replication of BEV in vivo.

[0124] (4) Observation results of clinical symptoms in mice

[0125] During the experimental phase, the mice's mental state, body temperature, weight, diet, drinking water, and behavioral activities were all normal. No abnormal clinical symptoms or adverse reactions were observed in the mice immunized with the recombinant adenovirus. The results of weight changes are shown in the attached Fig.21 As shown. After immunization with recombinant adenovirus rAdv-VP1 and rAdv-copGFP, there was no significant difference in body weight between the experimental group mice and the PBS-vaccinated control group mice (P>0.05). After the virus attack, there was no significant change in the body weight of the adenovirus-immunized mice, and the weight growth trend of the non-immunized group and the empty group mice was reduced, which shows that the recombinant adenovirus vaccine has no side effects and can better protect mice from the attack of BEV virus.

[0126] (5) Serum specific antibody detection

[0127] According to the optimal antigen coating concentration and the most suitable serum dilution ratio screened in the preliminary experiment, the purified pET-32a-BEV-VP1 recombinant protein was used as the antigen coating layer, and the antibody level of mouse serum at different times was detected by indirect ELISA method. The results are shown in the attached figure. Fig. 22 As shown, compared with the empty group and PBS control group, the recombinant adenovirus rAdv-VP1 group could detect corresponding specific antibodies 1-2 weeks after the first immunization. After three immunizations of mice, the antibody titer of the recombinant adenovirus immunization group gradually increased, and higher antibody levels were produced after the second immunization on the 14th day and the third immunization on the 21st day, which was significantly higher than the control group (P<0.01). There was no significant difference between the two control groups (P>0.05).

[0128] (6) Serum total IgG antibody level detection

[0129] The indirect ELISA method was used to measure the levels of total serum antibodies. The results are shown in the attached Fig.23After three immunizations, the antibody level of the recombinant adenovirus group increased significantly (P<0.05), among which the adenovirus empty group and the PBS control group had a similar trend in inducing antibodies, and there was no significant difference in the ability of the two groups to induce antibody production (P>0.05); after the third immunization, the recombinant adenovirus vaccine group induced the highest VP1 antibody level, which was significantly higher than the adenovirus empty group and the PBS control group (P<0.01).

[0130] (7) Cytokine level detection

[0131] The levels of IFN-γ and IL-4 cytokines in the serum of mice at 0, 7, 14, and 21 days after immunization were detected, as shown in the attached figure. Fig.24 As shown in the figure, at 7 days, there was no significant difference in the cytokine level between the adenovirus immunization group and the empty group and the control group. After the second immunization, the difference began to be significant. The IFN-γ level of rAdv-VP1 in the adenovirus immunization group was significantly higher than that in other groups (P<0.05). After the third immunization, the concentration could reach 13.85 pg / mL. The IFN-γ level of rAdv-VP1 in the immunization group was extremely significantly higher than that in the adenovirus empty group and the PBS control group (P<0.01). Fig.25 As shown, the IL-4 level of rAdv-VP1 in the adenovirus immunization group was also significantly higher than that in other groups (P<0.01), but there was no significant difference between the empty group and the control group (P>0.05).

[0132] As described above, the present invention can be well implemented. The above embodiments are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A method for constructing a recombinant adenovirus of bovine enterovirus VP1 gene, characterized in that: The specific steps include: (1) Amplify the VP1 gene sequence from bovine enterovirus strains; The amplification of the VP1 gene comprises: using VP1-F SEQ ID NO.1 and VP1-R SEQ ID NO.2 as primers, using cDNA of bovine enterovirus strain BL311 as a template, and performing PCR amplification to obtain VP1 gene SEQ ID NO.3; the PCR reaction amplification system comprises: 2×Taq PCR Mix 10.0 μL, VP1-F 0.5 μL, VP1-R 0.5 μL, cDNA template 2.0 μL, ddH2O 7.0 μL; the PCR reaction procedure comprises: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 68°C annealing for 30 s, 72°C extension for 1 min, 38 cycles; 72°C extension for 10 min; (2) The adenovirus empty vector pDC316-CMV-copGFP was digested with restriction endonucleases EcoRI and SalI-HF. The reaction system was: EcoRI 1.0 μL, SalI-HF 1.0 μL, CutSmart Buffer 1.0 μL, pDC316-CMV-copGFP 0.6 μL, ddH2O 6.4 μL; reaction procedure: incubate at 37°C for 4 h, inactivate at 85°C for 15 min; (3) The VP1 gene amplified in step (1), i.e., the adenovirus empty vector pDC316-CMV-copGFP in step (2), was ligated respectively. The reaction system was 10×Ligase Buffer 1.0 μL, T4 DNA Ligase 1.0 μL, PDC316-CMV-copGFP 1.0 μL, and VP1 7.0 μL. The mixture was mixed and placed at 4°C overnight for ligation to obtain the recombinant shuttle plasmid pDC316-VP1-copGFP. (3) The recombinant shuttle plasmid pDC316-VP1-copGFP and the adenovirus backbone plasmid pBHGlox-△E1,3cre were co-transfected into 293LP cells, and the recombinant adenovirus rAdv-VP1 was obtained by recombination and packaging.

2. The method for constructing a recombinant adenovirus of bovine enterovirus VP1 gene according to claim 1, characterized in that: The large backbone plasmid and the shuttle plasmid were co-transfected at a ratio of (1-4):

1.

3. The method for constructing a recombinant adenovirus of bovine enterovirus VP1 gene according to claim 1, characterized in that: The large backbone plasmid and the shuttle plasmid were co-transfected at a ratio of 4:

1.

4. The method for constructing a recombinant adenovirus of bovine enterovirus VP1 gene according to claim 1, characterized in that: During the transfection process, the concentration of fetal bovine serum is 2-5%.

5. The method for constructing a recombinant adenovirus of bovine enterovirus VP1 gene according to claim 1, characterized in that: During the transfection process, the concentration of fetal bovine serum was 2%.

6. The recombinant adenovirus prepared by the method for constructing a recombinant adenovirus containing the bovine enterovirus VP1 gene as described in any one of claims 1 to 5.

7. Use of the recombinant adenovirus prepared by the method for constructing a recombinant adenovirus carrying the bovine enterovirus VP1 gene according to any one of claim 1 or claim 5 in preventing and treating bovine enterovirus infection.

8. Use of the recombinant adenovirus prepared by the method for constructing a recombinant adenovirus of bovine enterovirus VP1 gene as claimed in claim 1 in the preparation of vaccines and drugs related to the prevention and treatment of bovine enterovirus infection.

9. Use of the recombinant adenovirus prepared by the construction method of the recombinant adenovirus of bovine enterovirus VP1 gene as claimed in claim 1 in preventing and treating bovine enterovirus infection, wherein the dosage form of the vaccine for preventing and treating bovine enterovirus infection includes injection, nasal drops or spray.

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