A method for constructing a recombinant adenovirus of a tp0326 antigen

Abstract

The application discloses a kind of Tp0326 antigen's recombinant adenovirus construction method, comprising the following steps S1, from Treponema pallidum genomic DNA, the gene fragment of Tp0326 antigen coding is amplified by PCR;S2, the Tp0326 antigen coding gene fragment amplified by step S1 is cloned into pshuttle-IRES-rGFP-1 shuttle expression vector by restriction enzyme cutting site, and recombinant shuttle plasmid is constructed;The application is formed by gene amplification, standardization operation process of vector construction to virus packaging, purification identification, and is constructed in direction relying on Treponema pallidum Nichols strain Tp0326 antigen coding gene, ensures the repeatability and stability of recombinant adenovirus construction, can be efficiently copied in laboratory, provides stable carrier preparation technical support for syphilis vaccine research and development, the used carrier (pshuttle-IRES-rGFP-1, pAdeasy-1) and cell (HEK293T) are all conventional commercial materials, easy to obtain, reduce construction cost;Tp0326 antigen coding gene and GFP gene are co-expressed by pshuttle-IRES-rGFP-1 shuttle vector, and preparation period is shortened.

Description

Technical Field

[0001] This invention relates to the field of recombinant adenovirus technology, specifically a method for constructing a recombinant adenovirus with Tp0326 antigen. Background Technology

[0002] Syphilis is a sexually transmitted and vertically transmitted infectious disease caused by Treponema pallidum subsp. pallidum. Its global incidence remains high, placing a heavy burden on public health. Although penicillin can be used to treat syphilis, it remains endemic in low- and middle-income countries, and the high prevalence of congenital syphilis poses a serious threat to newborn health. Therefore, the development of a preventative syphilis vaccine is of significant public health importance.

[0003] Current technologies for constructing recombinant adenoviruses targeting the Tp0326 antigen often employ non-standardized procedures. There are no standardized parameters for each stage, including antigen-encoding gene amplification, vector construction, virus packaging, purification, and identification. Furthermore, common cloning vectors and conventional competent cells are frequently used for gene recombination, and standard transfection reagents are employed without the introduction of reporter genes for real-time monitoring of viral infection efficiency. This lack of standardized procedures and parameters leads to significant variations in operation among different personnel and laboratories, resulting in poor reproducibility and stability of recombinant adenovirus construction. It is difficult to efficiently replicate in the laboratory. Additionally, the vectors and cells used often require special customization or modification, leading to high acquisition costs and construction costs. Moreover, the absence of dedicated shuttle vectors with reporter genes makes it impossible to directly monitor viral infection efficiency in real-time, requiring verification through cumbersome subsequent testing methods. Combined with the low homologous recombination efficiency of conventional competent cells and the poor transfection effect of conventional transfection reagents, the overall construction efficiency of recombinant adenoviruses is low, significantly extending the preparation cycle and failing to meet the high efficiency and stability requirements for vector preparation in syphilis vaccine development.

[0004] To this end, we have developed a method for constructing a recombinant adenovirus based on the Tp0326 antigen. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a recombinant adenovirus based on the Tp0326 antigen, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for constructing a recombinant adenovirus of Tp0326 antigen, comprising the following steps: S1, amplifying the gene fragment encoding Tp0326 antigen from the genomic DNA of Treponema pallidum by PCR; S2. The Tp0326 antigen encoding gene fragment obtained in step S1 is cloned into the pshuttle-IRES-rGFP-1 shuttle expression vector through restriction enzyme sites to construct a recombinant shuttle plasmid. S3. Perform DNA sequencing verification on the recombinant shuttle plasmid constructed in step S2, and screen for recombinant shuttle plasmids with correct sequencing results. S4. Linearize the correctly sequenced recombinant shuttle plasmid with restriction endonuclease PmeI, and then co-transform it with the pAdeasy-1 adenovirus backbone plasmid into E. coli BJ5183 competent cells for homologous recombination. S5. After homologous recombination, culture for 18-20 hours, extract plasmids from monoclonal cells, and verify by enzyme digestion and DNA sequencing to obtain recombinant adenovirus vectors. S6. Linearize the recombinant adenovirus vector obtained in step S5 with restriction endonuclease PacI, and recover the linearized plasmid by agarose gel electrophoresis. S7. Using Lipofectamine®LTX transfection reagent, the linearized plasmid recovered in step S6 was transfected into HEK293T cells; S8. After transfection, incubate for 6-7 days, observe GFP fluorescence production and cytopathic effects, and collect the virus solution. S9. The viral fluid collected in step S8 was amplified in HEK293T cells and purified by CsCl density gradient centrifugation to obtain purified recombinant adenovirus. S10. Identify the purified recombinant adenovirus, including observing the virus morphology under a transmission electron microscope, verifying the expression of Tp0326 antigen by Western blot, and determining the virus titer by TCID50. Once the identification is qualified, the recombinant adenovirus with Tp0326 antigen is obtained.

[0007] Preferably, the pale spirochetes in step S1 are Nichols strains.

[0008] Preferably, the restriction enzyme sites in step S2 are conventional restriction enzyme sites adapted to the Tp0326 antigen-encoding gene fragment and the pshuttle-IRES-rGFP-1 shuttle vector, and the Tp0326 antigen-encoding gene and GFP gene are co-expressed in the cloning recombinant shuttle plasmid.

[0009] Preferably, the culture conditions for the Escherichia coli BJ5183 competent cells in step S4 are 37°C with shaking culture, and the incubation conditions for homologous recombination are 37°C for 18-20 hours.

[0010] Preferably, the culture conditions for HEK293T cells in step S7 are 37°C and 5% CO2 humidification, and the culture medium is DuPont modified Eagle medium supplemented with 10% fetal bovine serum.

[0011] Preferably, the incubation conditions in step S8 are 37°C and a 5% CO2 humidified environment, and the GFP fluorescence is monitored by a confocal laser scanning microscope under 488nm excitation.

[0012] Preferably, the conditions for CsCl density gradient centrifugation in step S9 are: ultracentrifugation, collection of viral bands, and obtaining purified recombinant adenovirus particles.

[0013] Preferably, the specific method for Western blot verification in step S10 is as follows: HEK293T cells are infected with recombinant adenovirus at MOI=10, the cells are lysed after 48 hours, the protein is extracted, and the expression of Tp0326 antigen is detected using anti-Flag mouse monoclonal antibody as primary antibody, HRP-labeled goat anti-mouse IgG secondary antibody as secondary antibody, and GAPDH as internal control.

[0014] Preferably, in step S10, the TCID50 assay is performed using the HEK293T cell endpoint dilution method, and the viral titer is calculated using the Reed-Muench method, with the viral titer not less than 1×10⁻⁶. 10 VP / mL.

[0015] Preferably, the specific method for transmission electron microscopy observation in step S10 is as follows: the purified virus suspension is dropped onto a carbon-coated copper grid and adsorbed at room temperature for 5-10 minutes. Excess liquid is gently absorbed with filter paper, and the grid is negatively stained with 2% (w / v) phosphotungstic acid (pH 7.0) for 30-60 seconds. After air drying, the grid is examined under a transmission electron microscope to observe the morphology and structural integrity of the virus particles.

[0016] Compared with the prior art, the beneficial effects of the present invention are: (1) A standardized operating procedure has been formed from gene amplification, vector construction to virus packaging, purification and identification. The recombinant adenovirus is constructed by relying on the antigen encoding gene of the Nichols strain Tp0326. This ensures the reproducibility and stability of the recombinant adenovirus construction and can be efficiently replicated in the laboratory. This provides stable vector preparation technology support for the development of syphilis vaccines. The vectors (pshuttle-IRES-rGFP-1, pAdeasy-1) and cells (HEK293T) used are all conventional commercial materials that are easy to obtain and reduce the construction cost. (2) By using the pshuttle-IRES-rGFP-1 shuttle vector to achieve co-expression of the Tp0326 antigen encoding gene and the GFP gene, the viral infection efficiency can be directly monitored by confocal laser scanning microscopy. At the same time, efficient homologous recombination is achieved with the help of Escherichia coli BJ5183 competent cells. Combined with Lipofectamine®LTX transfection reagent, the linearized plasmid is efficiently transfected into HEK293T cells, which greatly improves the construction efficiency of recombinant adenovirus and shortens the preparation cycle. Attached Figure Description

[0017] Figure 1 This is a schematic block diagram of the method for constructing recombinant adenovirus of the Tp0326 antigen of the present invention; Figure 2 This is a schematic diagram illustrating the expression of the full-length recombinant TP0326 protein and the rPfTrx-TP0326ECL4 fusion protein of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1

[0020] Please see Figure 1-2 This invention provides a technical solution: a method for constructing a recombinant adenovirus with Tp0326 antigen, comprising the following steps: Material preparation Genomic DNA of *Treponema pallidum* strain Nichols, pshuttle-IRES-rGFP-1 shuttle vector, pAdeasy-1 adenovirus backbone plasmid, *Escherichia coli* BJ5183 competent cells, HEK293T cells, restriction endonucleases PmeI and PacI, Lipofectamine® LTX transfection reagent, anti-Flag mouse monoclonal antibody, HRP-labeled goat anti-mouse IgG secondary antibody, CsCl, TMB substrate solution, etc.

[0021] Experimental steps (1) PCR amplification of the Tp0326 antigen-encoding gene: Using the genomic DNA of the Nichols strain of Treponema pallidum as a template, PCR amplification was performed using the primers recorded in the supplementary table. The Tp0326 antigen-encoding gene fragment was amplified, and the PCR product was verified by agarose gel electrophoresis. The target fragment was recovered.

[0022] (2) Construction of recombinant shuttle plasmid: The recovered Tp0326 antigen encoding gene fragment and the pshuttle-IRES-rGFP-1 shuttle vector were digested with the same restriction endonuclease. After purification of the digestion products, they were ligated with T4 DNA ligase to construct the recombinant shuttle plasmid pshuttle-IRES-rGFP-1-Tp0326.

[0023] (3) Sequencing verification of recombinant shuttle plasmid: The recombinant shuttle plasmid was extracted and sent to a sequencing company for DNA sequencing. The sequencing results were compared with the Tp0326 antigen encoding gene sequence to confirm that there were no mutations, deletions or insertions. The recombinant shuttle plasmid with the correct sequencing was then screened.

[0024] (4) Homologous recombination of recombinant adenovirus vector: The correctly sequenced recombinant shuttle plasmid was linearized with PmeI and co-transformed with pAdeasy-1 adenovirus backbone plasmid into Escherichia coli BJ5183 competent cells. After incubation at 37°C for 18 hours, single colonies were picked and the plasmid was extracted after culturing.

[0025] (5) Validation of recombinant adenovirus vector: The extracted plasmid was analyzed by double digestion with PmeI and PacI. The digestion products were verified by agarose gel electrophoresis and DNA sequencing was performed to confirm that the recombinant adenovirus vector pAdeasy-1-Tp0326 was constructed correctly.

[0026] (6) Linearization and recovery of recombinant adenovirus vector: pAdeasy-1-Tp0326 was linearized with PacI, separated by agarose gel electrophoresis, and the linearized plasmid fragment was recovered.

[0027] (7) Transfection of HEK293T cells: HEK293T cells were seeded into 6-well plates and cultured to 80% confluence. The linearized plasmid was transfected into HEK293T cells using Lipofectamine® LTX transfection reagent. After transfection, the cells were incubated at 37°C and 5% CO2.

[0028] (8) Virus packaging and collection: After incubation for 6 days after transfection, obvious cytopathic effect and GFP fluorescence were observed. Cells and culture supernatant were collected, and the cells were repeatedly frozen and thawed 3 times. The supernatant was collected by centrifugation, which is the recombinant adenovirus stock solution.

[0029] (9) Virus amplification and purification: HEK293T cells were infected with recombinant adenovirus stock solution. After amplification, the virus solution was collected and purified by CsCl density gradient centrifugation. Viral bands were collected and CsCl was removed by dialysis to obtain purified recombinant adenovirus.

[0030] (10) Identification of recombinant adenovirus: ① Transmission electron microscopy: The purified virus suspension was dropped onto a carbon-coated copper grid and observed after negative staining. Typical icosahedral particles of adenovirus were visible, with intact structure. ② Western blot verification: After infecting HEK293T cells, a specific band of Tp0326 antigen was detected, and the GAPDH internal control band was clear. ③ TCID50 determination: The viral titer was calculated to be 1.2 × 10⁻⁶. 10 VP / mL, which meets the requirements.

[0031] The region encoding the recombinant protein was amplified from the genomic DNA of *Treponema pallidum* by PCR and cloned into the expression vector pshuttle-IRES-rGFP-1 via restriction enzyme digestion. Primers used are listed in the supplementary table. The sequences of all constructs were confirmed by DNA sequencing. The correctly sequenced recombinant shuttle plasmid was linearized with the restriction endonuclease pmeI and co-transformed with the backbone plasmid pAdeasy-1 into competent *E. coli* BJ5183 cells. After incubation for 18-20 hours, plasmids were extracted from single-clone cells and confirmed by enzyme digestion and DNA sequencing. The recombinant adenovirus vector was linearized with the restriction endonuclease pacI and recovered by agarose gel electrophoresis. Transfection with the linearized plasmid was performed using Lipofectamine® LTX (Invitrogen). After incubation for 6-7 days, fluorescence production and cytopathic effects were continuously observed. Wild-type adenovirus type 5 [Ad5(wt)] was previously prepared and preserved by the Dermatology Hospital of Southern Medical University.

[0032] Validation of viral protein expression HEK293T and Sf1Ep cells were infected with wild-type adenovirus (Ad5(wt)) or rAd5-Tp0326 at a multiplicity of infection (MOI) of 10 and incubated for 48 hours. GFP fluorescence was monitored at 488 nm excitation using a confocal laser scanning microscope to assess infection efficiency. Cells and culture supernatant were collected by centrifugation after infection. Cell pellets were lysed on ice with RIPA buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China), and protein extracts were denatured in 5×SDS sample buffer at 100°C for 10 min. Primary antibody was anti-Flag mouse monoclonal antibody (1:5000; Sangon Biotech Co., Ltd., Shanghai, China), followed by incubation with horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (1:5000; Sangon Biotech Co., Ltd., Shanghai, China). Western blot analysis confirmed Tp0326 expression in rAd5-Tp0326-infected cells. GAPDH was used as an internal control.

[0033] Transmission electron microscope To examine the morphology and structural integrity of Ad5(wt) and rAd5-Tp0326 viral particles, purified viral samples were prepared for transmission electron microscopy (TEM) analysis. Aliquots of the CsCl-purified viral suspension were dropped onto a carbon-coated copper grid and allowed to adsorb for 5-10 minutes at room temperature. Excess liquid was gently blotted away with filter paper, and the grid was negatively stained with 2% (w / v) phosphotungstic acid (pH 7.0) for 30-60 seconds. After air drying, the grid was examined under a TEM microscope running at an appropriate accelerating voltage. Representative images were acquired to assess viral particle size, morphology, and capsid integrity.

[0034] TCID50 assay was performed using the HEK293T cell endpoint dilution assay to determine the 50% tissue culture infectious dose (TCID50) of the virus. Briefly, HEK293T cells were seeded into 96-well plates and cultured overnight to approximately 80-90% confluence. Virus samples were serially diluted 10-fold in serum-free DuPont modified Eagle medium (DMEM), with each dilution added to multiple replicate wells (8 wells per dilution). Control wells were added only with medium. After adsorption at 37°C for 2 hours, the inoculum was removed and replaced with fresh DMEM supplemented with 5% fetal bovine serum. Cells were then incubated at 37°C under 5% CO2 humidification for up to 5 days, with virus-induced cytopathic effect (CPE) observed daily. Wells were rated as positive or negative based on the presence or absence of CPE, and TCID50 was calculated using the Reed-Muench method. Viral titers are expressed as TCID50 per milliliter (TCID50 / mL).

[0035] Figure 2 The full-length recombinant proteins TP0326 and rPfTrx-Tp0326ECL4 fusion proteins were expressed using an E. coli expression system. (a) rpfTrx-TP0326ECL4 fusion protein purified by Coomassie Brilliant Blue staining was identified; (b) rpfTrx-TP0326ECL4 fusion protein purified by Ni-NAT resin column was identified by Western blot analysis; (c) rTP0326 recombinant protein purified by Ni-NAT resin column was identified by Coomassie Brilliant Blue staining; (d) rTPO326 recombinant protein purified by Ni-NAT resin column was identified by Western blot analysis.

[0036] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a recombinant adenovirus with Tp0326 antigen, characterized in that, Includes the following steps: S1. The gene fragment encoding the Tp0326 antigen was amplified from the genomic DNA of Treponema pallidum by PCR. S2. The Tp0326 antigen encoding gene fragment obtained in step S1 is cloned into the pshuttle-IRES-rGFP-1 shuttle expression vector through restriction enzyme sites to construct a recombinant shuttle plasmid. S3. Perform DNA sequencing verification on the recombinant shuttle plasmid constructed in step S2, and screen for recombinant shuttle plasmids with correct sequencing results. S4. Linearize the correctly sequenced recombinant shuttle plasmid with restriction endonuclease PmeI, and then co-transform it with the pAdeasy-1 adenovirus backbone plasmid into E. coli BJ5183 competent cells for homologous recombination. S5. After homologous recombination, culture for 18-20 hours, extract plasmids from monoclonal cells, and verify by enzyme digestion and DNA sequencing to obtain recombinant adenovirus vectors. S6. Linearize the recombinant adenovirus vector obtained in step S5 with restriction endonuclease PacI, and recover the linearized plasmid by agarose gel electrophoresis. S7. Using Lipofectamine® LTX transfection reagent, the linearized plasmid recovered in step S6 was transfected into HEK293T cells; S8. After transfection, incubate for 6-7 days, observe GFP fluorescence production and cytopathic effects, and collect the virus solution. S9. The viral fluid collected in step S8 was amplified in HEK293T cells and purified by CsCl density gradient centrifugation to obtain purified recombinant adenovirus. S10. Identify the purified recombinant adenovirus, including observing the virus morphology under a transmission electron microscope, verifying the expression of Tp0326 antigen by Western blot, and determining the virus titer by TCID50. Once the identification is qualified, the recombinant adenovirus with Tp0326 antigen is obtained.

2. The method for constructing a recombinant adenovirus of Tp0326 antigen according to claim 1, characterized in that: The pale spirochetes mentioned in step S1 are Nichols strains.

3. The method for constructing a recombinant adenovirus based on Tp0326 antigen according to claim 1, characterized in that: In step S2, the restriction enzyme sites are the conventional restriction enzyme sites adapted to the Tp0326 antigen-encoding gene fragment and the pshuttle-IRES-rGFP-1 shuttle vector. After cloning, the Tp0326 antigen-encoding gene and the GFP gene are co-expressed in the recombinant shuttle plasmid.

4. The method for constructing a recombinant adenovirus based on Tp0326 antigen according to claim 1, characterized in that: The culture conditions for the Escherichia coli BJ5183 competent cells in step S4 are 37°C with shaking culture, and the incubation conditions for homologous recombination are 37°C for 18-20 hours.

5. The method for constructing a recombinant adenovirus of Tp0326 antigen according to claim 1, characterized in that: The HEK293T cells in step S7 are cultured at 37°C in a humid environment with 5% CO2, and the culture medium is DuPont modified Eagle medium supplemented with 10% fetal bovine serum.

6. The method for constructing a recombinant adenovirus of Tp0326 antigen according to claim 1, characterized in that: The incubation conditions in step S8 are 37°C and 5% CO2 humidification. GFP fluorescence is monitored by confocal laser scanning microscopy under 488nm excitation.

7. The method for constructing a recombinant adenovirus based on Tp0326 antigen according to claim 1, characterized in that: The conditions for CsCl density gradient centrifugation in step S9 are: ultracentrifugation, collection of viral bands, and obtaining purified recombinant adenovirus particles.

8. The method for constructing a recombinant adenovirus of Tp0326 antigen according to claim 1, characterized in that: The specific method for Western blot verification in step S10 is as follows: HEK293T cells are infected with recombinant adenovirus at MOI=10. After 48 hours, the cells are lysed, and proteins are extracted. The expression of Tp0326 antigen is detected using anti-Flag mouse monoclonal antibody as the primary antibody, HRP-labeled goat anti-mouse IgG secondary antibody as the secondary antibody, and GAPDH as an internal control.

9. The method for constructing a recombinant adenovirus based on Tp0326 antigen according to claim 1, characterized in that: In step S10, the TCID50 assay was performed using the HEK293T cell endpoint dilution method, and the viral titer was calculated using the Reed-Muench method, with the viral titer not less than 1 × 10⁻⁶. 10 VP / mL.

10. The method for constructing a recombinant adenovirus of Tp0326 antigen according to claim 1, characterized in that: The specific method for transmission electron microscopy observation in step S10 is as follows: the purified virus suspension is dropped onto a carbon-coated copper grid and adsorbed at room temperature for 5-10 minutes. Excess liquid is gently absorbed with filter paper, and the grid is negatively stained with 2% (w / v) phosphotungstic acid (pH 7.0) for 30-60 seconds. After air drying, the grid is examined under a transmission electron microscope to observe the morphology and structural integrity of the virus particles.

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