Recombinant vector for expressing OrfV truncated F1L protein, strain and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a recombinant vector for expressing OrfV truncated F1L protein, a strain and application of the recombinant vector. The preparation method comprises the following steps: truncating an F1L gene of an orf virus, introducing a His tag and a termination codon sequence, and constructing into a pYD1 plasmid to obtain a pYD1-F1L recombinant plasmid; and introducing the recombinant plasmid into competent cells of saccharomyces cerevisiae to obtain the recombinant yeast strain. According to the invention, the F1L gene of OrfV is truncated, so that the immunogenicity of the OrfV is effectively retained; the anchoring protein is displayed on the surface of the saccharomyces cerevisiae, so that the immune function of the saccharomyces cerevisiae can be developed; recombinant saccharomyces cerevisiae is adopted as a delivery carrier, the antigen protein can better play an immune role by means of the constant value of the recombinant saccharomyces cerevisiae in intestinal tracts, and meanwhile, the risk that the F1L protein is degraded by protease when being orally taken is further reduced. The invention provides a new direction for the development of OrfV oral vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and in particular relates to a recombinant vector and strain for expressing an OrfV truncated F1L protein and an application thereof. Background Art

[0002] Orf, also known as Contagious Ecthyma (CE), is a zoonosis caused by orf virus (OrfV). Orf is classified as a Class B animal infectious disease in my country. After sheep are infected, typical symptoms such as pustules, ulcers and nodules can be seen on the lips, tail base and udder. Orf is more common in autumn and winter, is highly contagious, spreads quickly, and can infect different breeds. The disease is most harmful to lambs and can cause the death of lambs. Due to the strong resistance of the virus, once the disease is prevalent, it is difficult to completely eliminate the pathogen in the epidemic area, which not only endangers the development of my country's animal husbandry and causes huge economic losses, but also directly threatens human health and public health safety. Therefore, it is of great significance to develop a safe and effective Orf vaccine.

[0003] At present, most reports on Orf focus on epidemic diagnosis, drug prevention and control, and etiology research, but there is still a lack of safe and effective vaccine products that can be used in clinical practice. It is known that there is only one live vaccine for sheep infectious pustular dermatitis that has obtained the approval number for veterinary products, and there are no other types of vaccine products. Traditional vaccines mainly include inactivated vaccines and live attenuated vaccines. Live attenuated vaccines have the advantages of low cost and good immune effect, but some microorganisms are not easy to attenuate, or they are easy to cause virulence to return after attenuation, which poses a safety risk. Although inactivated vaccines do not have the risk of virulence returning, they have a high preparation cost and relatively poor immune effect. Some microorganisms prepared into inactivated vaccines do not even have an immune protection effect. Nucleic acid vaccines refer to the use of recombinant plasmids containing antigen-encoding gene sequences as vaccines, which are directly introduced into animal cells, synthesize antigens with the help of the host cell's transcription system, and induce the host to produce an immune response to the antigen. However, long-term expression of nucleic acid vaccines in the body may induce immune tolerance in the body, ultimately leading to immunosuppression; continuous expression of exogenous antigens may stimulate the body to produce a strong CTL response, thereby killing other normal cells in the body; after entering the body, exogenous genes can be randomly integrated into the host's genome, causing gene breakage or rearrangement, thereby causing chromosomal instability.

[0004] In recent years, recombinant live vector vaccines have become a research hotspot. They overcome the shortcomings of conventional vaccines and have the advantages of both inactivated vaccines and attenuated vaccines. Genetically engineered recombinant live vector vaccines are a new type of vaccine that uses genetic engineering technology to use non-pathogenic microorganisms as vectors to express and deliver heterologous antigens. The way in which this type of vaccine immunizes animals to submit immunogenic proteins to the host immune system is close to the actual situation during natural infection, thereby inducing a wider range of immunity, including cellular immunity and humoral immunity, and even mucosal immunity, thus overcoming many of the shortcomings of recombinant subunit vaccines. Live vector vaccines have the advantages of high immune efficacy, low production cost and good safety performance, and have the advantages of both conventional live vaccines and inactivated vaccines.

[0005] Saccharomyces cerevisiae (S. cerevisiae) is widely used in the food industry. It has a post-translational modification process, and the surface components of its cell wall, β-1,3-D-glucan and mannan, have strong adjuvant functions. Saccharomyces cerevisiae has a clear genetic background, strong operability of genetic level modification, and has advantages such as fast growth rate and strong tolerance to inhibitors. It is recognized as the safest yeast preparation. As a eukaryotic yeast, its display system is different from the previously commonly used Escherichia coli and bacteriophage. It has the organelles and enzymes required for post-translational modification of polypeptides, so that the antigen protein displayed by yeast will be close to its natural conformation and have better immunogenicity. Therefore, the food-grade Saccharomyces cerevisiae surface display system is very suitable for the development of oral vaccines.

[0006] In the prior art, some patents or literature have reported on the sheep sore vaccine. For example, the patent CN118561965A discloses an mRNA vaccine based on the sheep sore virus F1L gene. The invention optimizes the coding sequence of the sheep sore virus F1L protein based on codon degeneracy and sequence optimization to construct an mRNA vaccine for the sheep sore virus. For another example, the patent CN114807225A discloses a recombinant DNA vaccine against sheep sore and sheep pox and its recombinant plasmid. The invention selects the sheep sore virus B2L (011) gene and the sheep pox virus P32 gene, and uses the self-cleaving peptide P2A to connect the two genes in series. The self-cleaving peptide P2A undergoes self-cleavage in eukaryotic cells, so that the two antigen proteins are independently expressed on the eukaryotic expression vector pcDNA3.1 (+); the recombinant DNA vaccine has a good immune effect after immunizing mice.

[0007] So far, there are no reports on Orf recombinant live vector vaccines based on Saccharomyces cerevisiae. Summary of the invention

[0008] In view of this, the present invention provides a recombinant vector expressing a truncated F1L protein of OrfV, a recombinant yeast strain, a vaccine delivery system and its application. The present invention constructs a recombinant vector expressing a truncated F1L gene of OrfV and displays it on the surface of Saccharomyces cerevisiae, and the obtained recombinant yeast strain has good immunogenicity. The present invention provides a new idea for the development of OrfV vaccine.

[0009] One of the purposes of the present invention is to provide a recombinant vector for expressing the truncated F1L gene of oropharyngeal sore virus.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] A recombinant vector for expressing a truncated F1L gene of aphthous ulcer virus, wherein the recombinant vector comprises the truncated F1L gene of aphthous ulcer virus, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0012] The F1L gene is the 59th complete open reading frame of OrfV, encoding a 39kD protein, which is one of the components of the microtubules on the surface of the virus. It has good immunogenicity, can induce the body to produce neutralizing antibodies, and can be used as one of the candidate antigens for the development of Orf vaccines. The aa15-aa191 fragment encoded by the F1L gene has strong antigenicity, good hydrophilicity, and no transmembrane domain. The present invention truncates the F1L gene of OrfV and displays it on the surface of brewer's yeast, which has the advantages of good immunogenicity and low degradation risk, and is helpful for the subsequent development of OrfV oral vaccines.

[0013] Furthermore, a His tag and a stop codon sequence were introduced into the truncated F1L gene sequence, which is shown in SEQ ID NO.2.

[0014] Furthermore, the amino acid sequence of the truncated F1L gene of the oropharyngeal canker sore virus is shown in SEQ ID NO.3.

[0015] Furthermore, the recombinant vector also includes a plasmid vector, and the plasmid vector is a pYD1 plasmid.

[0016] Furthermore, the identification primers of the recombinant vector include a forward primer orf-F and a reverse primer orf-R, the nucleotide sequence of the orf-F is shown in SEQ ID NO.4, and the nucleotide sequence of the orf-R is shown in SEQ ID NO.5.

[0017] A second object of the present invention is to provide a recombinant yeast strain containing the aforementioned recombinant vector.

[0018] To achieve the above object, the present invention adopts the following technical solutions:

[0019] A recombinant yeast strain containing the aforementioned recombinant vector.

[0020] Furthermore, the recombinant yeast strain is obtained by introducing the recombinant vector into Saccharomyces cerevisiae competent cells.

[0021] Preferably, the Saccharomyces cerevisiae competent cells are EBY100 competent cells.

[0022] As a preferred solution, the preparation method of the recombinant yeast strain is as follows:

[0023] The F1L gene of the oropharyngeal sore virus was truncated, a His tag and a stop codon sequence were introduced, and then constructed into a pYD1 plasmid, and purified to obtain a pYD1-F1L recombinant plasmid; the pYD1-F1L recombinant plasmid was introduced into a Saccharomyces cerevisiae competent cell to obtain a recombinant yeast strain.

[0024] The third object of the present invention is to provide a vaccine delivery system of the truncated F1L protein of the oropharyngeal sore virus.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] A vaccine delivery system for a truncated F1L protein of aphthous ulcer virus, comprising a delivery vector and a target gene; the delivery vector is Saccharomyces cerevisiae, and the target gene is a truncated F1L gene of aphthous ulcer virus into which a His tag and a stop codon sequence are introduced; the nucleotide sequence of the truncated F1L gene of aphthous ulcer virus is shown in SEQ ID NO.1, and the nucleotide sequences of the His tag and the stop codon are shown in SEQ ID NO.2; and the target gene is displayed on the surface of Saccharomyces cerevisiae.

[0027] A fourth object of the present invention is to provide an OrfV oral vaccine.

[0028] To achieve the above object, the present invention adopts the following technical solutions:

[0029] An OrfV oral vaccine, comprising the aforementioned recombinant vector, the aforementioned recombinant yeast strain and / or the aforementioned vaccine delivery system.

[0030] A fifth object of the present invention is to provide a use of the aforementioned recombinant vector, the aforementioned recombinant yeast strain and / or the aforementioned vaccine delivery system in the preparation of a vaccine for preventing goat ulcer virus infection and / or a drug for treating goat ulcer.

[0031] The beneficial effects of the present invention are as follows: the present invention truncates the F1L gene of OrfV, which is more conducive to improving the expression amount and display efficiency of the target protein and retaining its immunogenicity; by displaying the anchor protein on the surface of brewer's yeast, the eukaryotic expression of the F1L gene is achieved, which is more conducive to its immune function. At the same time, as a live bacterial carrier, recombinant brewer's yeast can reduce the risk of F1L protein being degraded by proteases when taken orally, and the antigen protein can better play an immune role with the help of the colonization of the recombinant brewer's yeast in the intestine. As a probiotic, the recombinant brewer's yeast is more helpful in regulating the body's immunity, providing an effective technical means for the development of OrfV oral vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The figure is the identification result of the recombinant plasmid, where M is DNA MarkerDL2000, 1 is the recombinant plasmid pYD1-F1L, and 2 is the pYD1 plasmid negative control;

[0033] Figure 2 This is a diagram of the screening results of transformants;

[0034] Figure 3 The PCR identification result of the recombinant strain is shown in Figure 1, where M is DNA MarkerDL2000, 1-6 are positive recombinant strains, and 7 is a negative strain control;

[0035] Figure 4 This is the Dotblot test result of live bacteria;

[0036] Figure 5 This is the Western Blot identification result of the target protein, where M: Protein Marker (17-200kDa); 1-8 are recombinant proteins, and 9 is a negative control. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0038] Example 1. Construction of pYD1-F1L recombinant plasmid

[0039] (1) Design of target gene

[0040] The target gene was designed with reference to the full-length gene sequence of F1L published in NCBI (accession number: KX951408.1). The dominant antigen fragment was intercepted through bioinformatics analysis, and the corresponding gene sequence was optimized for yeast codon preference. The his tag sequence and the corresponding sequence of the stop codon were introduced at the 3' end.

[0041] (2) Truncated F1L gene sequence information

[0042] The dominant antigen fragment of the aa15-aa191 fragment was intercepted, and the corresponding gene sequence was optimized for yeast codon preference. The optimized gene sequence is shown in SEQ ID NO.1, and the sequence length is 531 bp.

[0043] (3) His tag sequence

[0044] The full length of the his tag sequence (1bp-18bp in SEQ ID NO.2) and the sequence corresponding to the stop codon (19bp-21bp in SEQ ID NO.2).

[0045] (4) Amino acid sequence information corresponding to the target gene

[0046] The amino acid sequence is mainly composed of a truncated F1L fragment (aa1-aa177) and a His (aa178-183aa) tag, and the specific sequence is shown in SEQ ID NO.3.

[0047] (5) Construction and synthesis of recombinant plasmids

[0048] The designed target gene sequence was handed over to Sangon Biotechnology (Shanghai) Technology Co., Ltd. for synthesis and constructed into the pYD1 plasmid provided by the Veterinary and Veterinary Drug Research Institute of Chongqing Academy of Animal Husbandry Sciences, and the pYD1-F1L recombinant plasmid was purified.

[0049] Example 2: Identification of recombinant plasmids and construction of recombinant yeast strains

[0050] (1) Extraction and identification of recombinant plasmids

[0051] Use a plasmid extraction kit to extract the pYD1-F1L plasmid from the recombinant Escherichia coli, and use the F1L identification primers orf-F / orf-R shown in Table 1 to amplify the target gene. PCR reaction system: 3μL DNA template, 0.2μL each of primers orf-F / orf-R (10pmol / μL), 10μL 2×Taq Master Mix, and ddH2O to make up to 20μL. Reaction conditions: 94℃5min; 94℃40sec, 59℃40sec, 72℃40sec, 30 cycles in total; 72℃ extension for 10min. After the reaction, the expected band appeared at about 531bp by agarose gel electrophoresis analysis, see for details. Figure 1 The purified PCR product was sent to Shanghai Biotechnology Co., Ltd. for sequencing analysis, which was consistent with the expected results.

[0052] Table 1. PCR amplification primers

[0053] F1L identification primers Sequence (5'-3') orf-F SEQ ID NO.4 orf-R SEQ ID NO.5

[0054] (2) Preparation of competent cells

[0055] A single colony of EBY100 purchased from Shanghai Qincheng Biotechnology Co., Ltd. was picked from the YPDA plate and inoculated into 4 ml of YPDA liquid culture medium, and cultured with shaking at 30°C, 225 rpm for 18-20 h (overnight) until OD600>1.5; transferred to YPDA liquid culture medium, the culture volume was 50 ml, the initial OD600=0.2, 30°C, 225 rpm, and cultured with shaking for 4-5 h until OD600=0.6; centrifuged at 4000 rpm for 5 min, and the supernatant was discarded; the bacteria were resuspended with 20 ml of sterile water, mixed, centrifuged at 4000 rpm for 5 min, and the supernatant was discarded; the bacteria were resuspended with 5 ml of 0.1 M LiAc, mixed, centrifuged at 4000 rpm for 5 min, and the supernatant was discarded; the bacteria were resuspended with 500 ul of 0.1 M LiAc, mixed, 50 ul / tube, and divided for use.

[0056] (3) Conversion

[0057] Take the competent cells in the aliquots, add each reagent in order according to Table 2, mix well by pipetting; incubate in a 30℃ water bath for 30min; heat shock in a 42℃ water bath for 25min; recover in a 30℃ water bath for 30min; centrifuge at 4000rpm for 5min, discard the supernatant; resuspend the cells in 200ul sterile water for each transformation, mix gently, and spread on the SD-Trp screening plate; culture at 30℃ for 4d. The screening results of the transformants are as follows: Figure 2 shown.

[0058] Table 2. Transformation reagents iBAO

[0059] Reagents 50% PEG3350 1MLiAc ssDNA (20mg / ml) Plasmid DNA Dosage 240ul 36ul 5ul 5ul

[0060] (4) Screening of positive clones

[0061] Single clones were randomly selected from the plate and identified by colony PCR. The PCR reaction system was: 3 μL DNA template, 0.2 μL primers orf-F / orf-R (10 pmol / μL), 10 μL 2×Taq Master Mix, and ddH2O was added to 20 μL. Reaction conditions: 94°C for 5 min; 94°C for 40 sec, 59°C for 40 sec, 72°C for 40 sec, for a total of 30 cycles; 72°C for 10 min extension; after the reaction, the expected band appeared at about 531 bp by agarose gel electrophoresis analysis; after the PCR, 1% agarose gel electrophoresis detection, such as Figure 3 As shown, the product size is about 531 bp, which is consistent with the expected result.

[0062] Example 3: Induced expression and identification of recombinant strains

[0063] (1) Inducible expression of target protein

[0064] Collect all positive clones, dilute and inoculate in YNB-CAA liquid culture medium containing 2% glucose, and culture at 30°C, 225rpm, with shaking until OD600 = 2-5; centrifuge at 4000rpm for 5min, and discard the supernatant; add YNB-CAA medium containing 2% galactose, mix by pipetting, adjust the culture solution to OD600 = 0.5-1, and culture at 30°C, 225rpm, with shaking for 72h to induce the expression of the target protein.

[0065] (2) Magnetic bead sorting and induced expression of yeast cells

[0066] Refer to the instructions for use of the Anti-His magnetic beads of Biyuntian Company, use a pipette to gently blow and resuspend the Anti-His magnetic beads, according to 10ul magnetic bead suspension for every 500ul sample, take an appropriate volume of magnetic beads to a clean centrifuge tube, add 500ul 1×TBS; gently blow and resuspend the magnetic beads with a pipette, separate on the magnetic stand for 30s, and discard the supernatant. Repeat the above steps twice; add 500ul1×TBS to resuspend the magnetic beads for use; centrifuge at 4000rpm for 5min, discard the supernatant, add the above-treated magnetic beads to the bacteria, and resuspend the bacteria; place the centrifuge tube on a rotating mixer and incubate at room temperature for 2h or at 4℃ overnight; after incubation, place on the magnetic stand for separation for 30s, and discard the supernatant; add 500ul 1×TBS to resuspend, place on the magnetic stand for separation for 30s, and discard the supernatant. Repeat washing six times; resuspend in 100ul 1×TBS and spread on SD-Trp screening plate; culture at 30°C for 4 days; collect clones on the plate and induce expression of the target protein according to the steps in (1).

[0067] (3) Dotblot detection of live bacteria

[0068] Take an appropriate amount of bacterial solution, select mouse anti-His-Tag as the primary antibody, dilute and incubate at room temperature for 2 hours; centrifuge at 4000rpm for 5 minutes, discard the supernatant; wash the bacteria 3 times with 1×TBST; select HRP-labeled goat anti-mouse IgG (H&L) as the secondary antibody, dilute and incubate at room temperature for 45 minutes; wash the bacteria 3 times with 1×TBST; take ECL luminescent liquid A and B, mix them in equal amounts and resuspend the bacteria, and take an appropriate amount of bacterial solution to expose the membrane after color development for 5 minutes. The results are as follows Figure 4 As shown, spots were formed in the sample spotting area, while no spots were formed in the control group spotting area, indicating that the protein was successfully displayed on the bacterial surface.

[0069] (4) Target protein extraction and Western Blot identification

[0070] The induced bacterial solution was centrifuged at 4000rpm for 5min, and the supernatant was discarded; the bacterial solution was resuspended in 0.1M NaOH solution, mixed by blowing, and incubated at room temperature for 5min; the solution was centrifuged at 12000rpm for 10min, and the supernatant was discarded; the bacterial solution was resuspended in 100mM Tris-HCl (pH6.8), 4% SDS (sodium dodecyl sulfate), 0.2% bromophenol blue and 20% glycerol, and incubated at 95℃ for 5min; the solution was centrifuged at 3000rpm for 10min, and the supernatant was collected, which contained the target protein; 20ul of protein solution was taken, and an appropriate amount of protein Loading buffer, boil at 100℃ for 5-10min; centrifuge at 12000rpm for 2min, draw 20ul for protein electrophoresis; cut PVDF membrane of appropriate size, transfer protein to membrane by wet transfer; wash membrane with 1×TBST, 50rpm for 10min; take appropriate amount of blocking solution and block at room temperature at 50rpm for 10min; wash membrane with 1×TBST, 50rpm for 5min; select mouse anti-His-Tag as primary antibody, incubate at room temperature at 50rpm for more than 2h after dilution; wash membrane with 1×TBST, 50rpm for 10min, wash 3 times in total; select HRP-labeled goat anti-mouse IgG (H&L) as secondary antibody, incubate at room temperature at 50rpm for 45min after dilution; wash membrane with 1×TBST, 50rpm for 10min, wash 3 times in total; take ECL luminescent solution A and B, mix them in equal amounts and drop them on the front of the membrane, expose after 5min of color development, and the target band of about 26kDa appears, and there is no band in the negative control. Detection results are shown in Figure 5 .

Claims

1. A recombinant vector expressing a truncated F1L gene of aphthous ulcer virus, characterized in that: The recombinant vector contains the truncated F1L gene of the aphthous ulcer virus, and the nucleotide sequence thereof is shown in SEQ ID NO.

1.

2. The recombinant vector according to claim 1, characterized in that The truncated F1L gene sequence introduced a His tag and a stop codon sequence, which is shown in SEQ ID NO.

2.

3. The recombinant vector according to claim 1, characterized in that The amino acid sequence of the truncated F1L gene of the oropharyngeal sore virus is shown in SEQ ID NO.

3.

4. The recombinant vector according to claim 1, characterized in that The recombinant vector also includes a plasmid vector, and the plasmid vector is a pYD1 plasmid.

5. The recombinant vector according to claim 1, characterized in that The identification primers of the recombinant vector include a forward primer orf-F and a reverse primer orf-R. The nucleotide sequence of orf-F is shown in SEQ ID NO.4, and the nucleotide sequence of orf-R is shown in SEQ ID NO.

5.

6. A recombinant yeast strain containing the recombinant vector according to any one of claims 1 to 5.

7. The recombinant yeast strain according to claim 6, characterized in that: The recombinant yeast strain is obtained by introducing the recombinant vector into Saccharomyces cerevisiae competent cells.

8. A vaccine delivery system of truncated F1L protein of oropharyngeal sore virus, characterized in that: The vaccine delivery system comprises a delivery vector and a target gene; the delivery vector is Saccharomyces cerevisiae, and the target gene is a truncated F1L gene of the stomatitis virus into which a His tag and a stop codon sequence are introduced; the nucleotide sequence of the truncated F1L gene of the stomatitis virus is shown in SEQ ID NO.1, and the nucleotide sequence of the His tag and the stop codon is shown in SEQ ID NO.2; The target gene is displayed on the surface of Saccharomyces cerevisiae.

9. OrfV oral vaccine, characterized in that The OrfV oral vaccine contains the recombinant vector described in any one of claims 1 to 5, the recombinant yeast strain described in any one of claims 6 to 7 and / or the vaccine delivery system described in claim 8.

10. Use of the recombinant vector according to any one of claims 1 to 5, the recombinant yeast strain according to any one of claims 6 to 7 and / or the vaccine delivery system according to claim 8 in the preparation of a vaccine for preventing aphthous tussock virus infection and / or a drug for treating aphthous tussock.

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