A transport carrier

Abstract

The present invention provides a kind of transfer vector, for Bac-to-Bac baculovirus expression system, relate to biotechnology and virology technology field, described transport vector includes 3 expression cassettes that are independently regulated and expressed by 3 promoters, can express multiple foreign proteins independently simultaneously, wherein promoter OP166 is early and late promoter, VP39 and P6.9 are late promoters, relative to commonly used promoters P10 and PH, expression time is advanced by 8 hours. Through experimental verification, transport vector p-BT has diversified selection for foreign protein expression mode, and the foreign protein expressed has biological activity, and virus-like particles (VLP) can be formed. Therefore, the expression of transport vector p-BT is more diversified, sequential, multi-quantity, large capacity, is conducive to being applied to multiple fields such as genetic engineering, drug development, vaccine production, expression of immunocompetent molecules and some oncogenic virus proteins and gene expression regulation research.

Description

Technical Field

[0001] The present invention relates to the technical fields of biotechnology and virology, and in particular to a circular transport vector for a Bac-to-Bac baculovirus expression system, in particular to a transport vector comprising three promoters for regulating expression. Background Art

[0002] Baculovirus is an insect pathogen that regulates insect populations in nature and has been successfully used as a biopesticide for pest control. Insect cells are excellent factories for expressing foreign proteins, and baculovirus offers advantages such as high expression levels, large protein expression capacity, and the ability to perform post-translational protein modifications. Therefore, the ability of budding virus particles (BV) to infect insect cells can be transformed into eukaryotic expression vectors. The Insect Baculovirus Expression Vector System (IBEVS) has been widely used in the production of various biological products and in gene therapy.

[0003] Currently, the most commonly used baculovirus for exogenous gene expression is the model species of the alpha baculovirus, AcMNPV. Commonly used promoters for baculovirus vectors to express exogenous proteins in insect cells are the AcMNPV P10 and polyhedrin (PH) promoters. These two promoters, as strong promoters during the late stages of viral infection, can efficiently express exogenous genes. However, exogenous protein expression under the control of the P10 and PH promoters begins in the final stages of viral infection, a stage that also involves host cell death, resulting in unpredictable defects in the yield and modification of exogenous protein expression. However, the application of other baculovirus promoters is relatively limited.

[0004] In addition, general transfer vectors only contain one promoter, and pFastBacDual also has only two promoters (P10 and PH). Accordingly, only one or two exogenous genes can be expressed simultaneously. Although these commonly used transfer vectors have high expression efficiency of exogenous proteins, their disadvantages are: ① A maximum of two proteins can be expressed simultaneously in insect cells (pFastBac Dual); ② The expression of multiple tandem exogenous proteins under the regulation of the same promoter cannot achieve regulation of the expression intensity and timing of each protein; ③ The promoters are all late promoters, which may lead to defects in the expression modification of exogenous proteins. Summary of the Invention

[0005] The purpose of the present invention is to at least partially overcome the shortcomings of the existing technology, especially to overcome the limitations of expression vectors commonly used in baculovirus expression systems in terms of expression timing, and to provide a new transfer vector that creatively uses three promoters to independently regulate the expression of three expression cassettes. The effect of the transfer vector on multi-protein expression is further verified, and the transfer vector achieves the purpose of simultaneously expressing multiple exogenous proteins.

[0006] In order to achieve the above purpose or one of the purposes, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a transfer vector p-BT (p-Bac-triple), a circular vector for a Bac-to-Bac baculovirus expression system, comprising three expression cassettes whose expression is regulated by three promoters, namely: expression cassette one under the control of the OP166 promoter encoded by the nucleic acid sequence shown in SEQ ID NO: 29, expression cassette two under the control of the VP39 promoter encoded by the nucleic acid sequence shown in SEQ ID NO: 30, and expression cassette three under the control of the p6.9 promoter encoded by the nucleic acid sequence shown in SEQ ID NO: 31.

[0008] Preferably, the expression cassette one includes the OP166 promoter, the multiple cloning site one MCS1, and the terminator SV40; the expression cassette two includes the VP39 promoter, the multiple cloning site two MCS2, and the terminator bGH; the expression cassette three includes the p6.9 promoter, the multiple cloning site three MCS3, and the terminator HSV TK.

[0009] Further preferably, MCS1: includes but is not limited to SalⅠ, KasⅠ, NarⅠ, SfoⅠ, PluTⅠ, NdeⅠ, XbaⅠ, BstBⅠ, NsrⅡ, XmaⅠ, SmaⅠ, PstⅠ; MCS2: includes but is not limited to SpeⅠ, BamHⅠ, AgeⅠ, HindⅢ; MCS3: includes but is not limited to NotⅠ, Acc65Ⅰ, KpnⅠ, AlfⅡ, NotⅠ, PuvⅠ, Bsu36Ⅰ, EcoRⅠ.

[0010] Preferably, the expression frame one and expression frame three are arranged head to head in opposite directions, and the expression frame two is arranged sequentially after the expression frame three.

[0011] Preferably, the transport vector p-BT further comprises a chloramphenicol expression unit (CmR), a gentamicin expression unit (GmR) and a transposon element (transposon, Tn);

[0012] Further preferably, the transposon element is a Tn7 transposon, including Tn7-L and Tn7-R.

[0013] Preferably, the transfer vector p-BT further comprises a prokaryotic replication origin element (origin, ori).

[0014] Further preferably, the prokaryotic replication origin element includes an F1 phage replication origin element (f1 ori) and a high copy replication origin element (ori).

[0015] Preferably, the transport vector p-BT is encoded by the nucleic acid sequence shown in SEQ ID NO: 1.

[0016] In a second aspect, the present invention provides a polynucleotide constructed by recombinant cloning of a transport vector p-BT and a gene fragment of an exogenous protein.

[0017] Preferably, the gene fragment of the exogenous protein is the nucleic acid sequence shown in SEQ ID NO: 24 encoding firefly luciferase.

[0018] Preferably, the gene fragment of the exogenous protein is composed of at least one of the following three: the nucleic acid sequence shown in SEQ ID NO: 25 encoding the SARS-CoV-2 M protein, the nucleic acid sequence shown in SEQ ID NO: 26 encoding the SARS-CoV-2 E protein, and the nucleic acid sequence shown in SEQ ID NO: 27 encoding the SARS-CoV-2 S protein.

[0019] In a third aspect, the present invention provides a host cell comprising a polynucleotide constructed by recombinant cloning of a transport vector p-BT and a gene fragment of an exogenous protein, a recombinant plasmid obtained by transformation, or a recombinant virus obtained by transfection or infection.

[0020] In a fourth aspect, the present invention provides a transport vector p-BT, and its use in the preparation of antigens, antibodies, drugs or subunit vaccines.

[0021] Beneficial effects of the present invention:

[0022] The present invention has developed a novel transport vector, p-BT (p-Bac-triple), for use in baculovirus expression systems. It contains three expression cassettes, each independently regulated by three promoters. These three promoters have different strengths and independently regulate the expression of exogenous proteins. Specific examples of multi-protein expression demonstrate that this transport vector has the following advantages:

[0023] 1. The transport vector p-BT of the present invention has diverse expression sequences. This invention utilizes, for the first time, three promoters from the baculovirus AcMNPV in combination: an early and late promoter, the OP166 promoter (OP166), and two late promoters, the VP39 promoter (VP39) and the P6.9 promoter (P6.9), respectively, in combination with three terminators: the SV40 poly(A) signal (SV40), the bGH poly(A) signal (bGH), and the HSV TK poly(A) signal (HSV), forming three independent expression cassettes. By inserting three exogenous protein genes into each of the three expression cassettes using a simple method, the simultaneous expression of all three exogenous proteins can be achieved. By inserting multiple exogenous protein genes in series into one of the expression cassettes, the number of expressed genes can be further increased. The present invention realizes the expression of at least three exogenous proteins in a baculovirus expression system and is no longer limited to the late stage, and can provide diverse options for the quantity and timing of exogenous protein expression.

[0024] 2. The three expression cassettes formed by the three promoters in the p-BT transport vector not only increase the number of exogenous proteins that can be expressed simultaneously (at least three), but also contain three independent multiple cloning sites (MCS)—MCS1, MCS2, and MCS3—allowing simple cloning techniques to insert gene fragments of exogenous proteins and generate recombinant plasmids. Compared to existing transport vectors, this transport vector has a greater exogenous gene carrying capacity and is suitable for the expression of multi-protein complexes, further facilitating the development, production, and application of related vaccines, such as multivalent vaccines, protein complexes, and virus-like particles (VLPs).

[0025] 3. The novel, circular transport vector p-BT, obtained through artificial synthesis, also includes a prokaryotic replication origin element (ori) and a transposon (Tn). The prokaryotic replication origin element includes an F1 bacteriophage replication origin element (f1 ori) and a high-copy replication origin element (ori), which enable the transport vector p-BT to retain basic replication capability in bacteria, facilitating the construction and amplification of recombinant vectors. The transposon utilizes the site-specific transposon Tn7 transposase (including Tn7-L and Tn7-R) from Escherichia coli. The function of the Tn7 transposase is to, when the constructed recombinant transport vector is transformed into DH10Bac, a helper plasmid (pMON7124) in DH10Bac provides a transposase, which, with the assistance of the Tn7 transposase, allows the transposition process to be completed, introducing the target gene fragment containing the exogenous protein into the bacmid, thereby obtaining a recombinant bacmid containing the exogenous protein.

[0026] 4. The present invention verifies the feasibility of the combined effect of three promoters. In the baculovirus expression system, the present invention not only expresses good biological activity of firefly luciferase (FLUC), but also expresses multiple exogenous proteins through the combination of three promoters, and successfully expresses and packages SARS-CoV-2 virus-like particles (virus-like particles, VLPs). Further electron microscopy results also observed a complete virus-like particle structure. Therefore, the present invention also provides a method for constructing and applying a transport vector that can simultaneously express three proteins, which can not only be used to construct a recombinant baculovirus for multi-gene expression, but also can further expand the scope of application of the baculovirus expression system.

[0027] 5. The present invention adopts two resistance selection markers, which is conducive to reducing pollution and improving construction efficiency. Chloramphenicol (C) is newly selected to form a chloramphenicol resistance element (CmR), combined with the original commonly used gentamicin (G) resistance element (GmR) as a screening marker. Not only can the dual resistance reduce the contamination of exogenous proteins during the construction process, but also the success rate of screening can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the map of the transport vector p-BT of the present invention;

[0029] Figure 2The figure is a schematic diagram of the structure of six recombinant Bacmid transfer sites containing the firefly luciferase gene constructed using the transfer vector p-BT of the present invention, wherein Bac-Syn, Bac-FLUC(VP39, Bac-FLUC(P6.9), Bac-FLUC(OP166), Bac-FLUC(P10) and Bac-FLUC(PH) represent the structures of the transfer sites of six recombinant Bacmids (Bac-Syn, Bac-VP39-FLUC, Bac-P6.9-FLUC, Bac-OP166-FLUC, Bac-p10-FLUC, Bac-PH-FLUC), respectively;

[0030] Figure 3 Schematic diagram of the structure of the recombinant Bacmid transfer site formed by expressing SARS-CoV-2 virus-like particle protein and enhanced green fluorescent protein using the transport vector p-BT of the present invention, wherein AcBac, AcBac-MSE and AcBac-EGFP represent the structures of the recombinant Bacmid: AcBac, AcBac-MSE and AcBac-EGFP transfer site, respectively;

[0031] Figure 4 for Figure 2 Comparison of the relative activities of firefly luciferase expressed under the regulation of different promoters for the five constructed expression cassettes. The ordinate represents the fluorescence intensity values ​​obtained by luciferase detection reagent for the five recombinant baculoviruses corresponding to the five expression cassettes; the abscissa represents the time at which the five recombinant baculoviruses corresponding to the five expression cassettes were synchronously infected with Sf9 cells. Panel A shows the detection results after 6 hours of infection, and Panel B shows the detection results after 24 hours, 48 ​​hours, 72 hours, and 96 hours of infection, respectively.

[0032] Figure 5 The results of western blot identification of the recombinant baculovirus AcBac-MSE, AcBac-EGFP and purified SARS-CoV-2 virus-like particles constructed using the transport vector p-BT of the present invention, wherein WT is a Calu-3 cell sample infected with SARS-CoV-2, Anti-S represents the RBD antibody as the primary antibody, Anti-M represents the S-tag antibody as the primary antibody, Anti-GP64 represents the GP64 antibody as the primary antibody, and MSE-purified represents the purified SARS-CoV-2 virus-like particles;

[0033] Figure 6 This is a western blot identification image of virus-like particles after purification of the recombinant baculovirus AcBac-MSE constructed using the transport vector p-BT of the present invention;

[0034] Figure 7 These are morphological images of virus-like particles observed under an electron microscope at different magnifications after negative staining of the purified virus-like particles expressed by the recombinant baculovirus AcBac-MSE constructed using the transport vector p-BT of the present invention. DETAILED DESCRIPTION

[0035] The following is a detailed description of exemplary embodiments of the present invention in conjunction with the accompanying drawings, wherein the same or similar represents the same concept, such as firefly luciferase (and its gene fragment) = FLUC, new coronavirus = SARS-CoV-2, spike protein (Spike, S) and its gene fragment = S, membrane protein (membrane, M) and its gene fragment = M, enhanced green fluorescent protein = EGFP, SARS-CoV-2 virus-like particles = new coronavirus VLP, etc.

[0036] In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. The following examples are intended to illustrate the present invention only and are not to be construed as limiting the scope of the present invention.

[0037] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified. Among them, the plasmid p-Bac-triple (abbreviated as pBT) was commissioned to Beijing Qingke Biotechnology Co., Ltd. (Wuhan Branch) for synthesis and stored and cultured by the applicant's laboratory; Sf9 cells (Spodoptera frugiperda, fall armyworm cells) were stored and cultured by this laboratory; fetal bovine serum (FBS), Grace's insect culture medium, transfection reagent Cellfectin, etc. were purchased from GIBCO in the United States; the recombinant baculovirus Bac-Syn was prepared according to the reference (Shang et al. Construction and Characterization of a Novel BacmidAcBac-Syn Based on a SynthesizedBaculovirus Genome.2021) synthesis; PCR enzyme, recombinase (infusion) and luciferase detection reagents were purchased from Nanjing Novigene Biotechnology Co., Ltd. (Vazyme); T4 DNA ligase was purchased from NEB, USA; restriction endonucleases were purchased from Takara; plasmid extraction kits were purchased from QIAGEN, Germany; baculovirus plasmid AcBac-bMON14272 (Luckow et al., 1993) and auxiliary plasmid pMON7124 were purchased from Thermo Fisher, USA; the primers involved in the present invention were commissioned to Shanghai Sangon Biotechnology Co., Ltd. (Shanghai Sangon for short) for synthesis; the sequence determination of the present invention was commissioned to Beijing Qingke Biotechnology Co., Ltd. (Wuhan Branch). However, it is obvious that one or more embodiments can also be implemented without these specific details. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0038] The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book "Molecular Cloning Laboratory Manual" (3rd edition) by J. Sambrook, or in accordance with the kit and product instructions. The objects of detection in the implementation of the present invention are derived from microorganisms (including viruses) preserved and constructed in the applicant's laboratory, and do not involve living human or animal bodies; the direct purpose of the detection is to verify the beneficial effects of the transport vector p-BT described in the present invention, and there is no process of obtaining the diagnosis results of the disease or the health status. Therefore, the detection involved in the present invention does not belong to the diagnostic method of the disease and meets the basic requirements of the Patent Law for the subject of patent protection.

[0039] The present invention provides a new transfer vector p-BT (p-Bac-triple) for a baculovirus expression system. Through in-depth research on the commercial transfer vector pFastBackDual, the use of two late promoters (P10 and PH) is creatively abandoned. The newly designed transfer vector p-BT includes three independent expression cassette sequences regulated by three promoters: VP39, P6.9 and OP166; two replication elements: an F1 bacteriophage replication origin element (f1 ori) and a high-copy replication origin element (ori); a transposon; and two resistance selection markers: a chloramphenicol resistance element (CmR) and a gentamicin resistance element (GmR). The transport vector p-BT of the present invention was constructed with gene fragments of three types of exogenous proteins: firefly luciferase, the new coronavirus SARS-CoV-2 related structural protein MSE, and enhanced green fluorescent protein, to obtain a series of recombinant baculoviruses. Further experiments verified the effective expression of exogenous proteins (with biological activity) and the diverse selection of exogenous proteins expressed using the transport vector p-BT. The following describes a specific embodiment of the present invention, and the specific content is as follows:

[0040] Example 1. Design of p-BT plasmid

[0041] The basic design ideas of the transport carrier p-BT of the present invention are as follows:

[0042] 1) Select three promoters, preferably OP166, VP39, and P6.9. The OP166 promoter (OP166 promoter) is an early-late promoter; VP39 is a late promoter, beginning expression 24 hours after viral infection; and P6.9 is a late promoter, with peak expression between 12 and 20 hours after viral infection, more than eight hours earlier than the expression of promoters of very late genes (such as PH). Therefore, the two late promoters, VP39 and P6.9, express earlier than traditional very late promoters (P10 and PH), which helps ensure the activity and correct folding of exogenous proteins.

[0043] 2) Select terminators corresponding to three promoters: SV40, bGH, and HSV;

[0044] 3) Based on the sequences of the three promoters and the corresponding three terminators, design three appropriate multiple cloning sites (MCS: MCS1, MCS2 and MCS3) as the insertion sites for the gene fragments of foreign proteins. The restriction endonucleases that can be recognized by the sequences of these three MCSs are:

[0045] MCS1: including but not limited to SalⅠ, KasⅠ, NarⅠ, SfoⅠ, PluTⅠ, NdeⅠ, XbaⅠ, BstBI, NsrⅡ, XmaⅠ, SmaⅠ, PstⅠ;

[0046] MCS2: including but not limited to SpeⅠ, BamHI, AgeⅠ, HindⅢ;

[0047] MCS3: includes but is not limited to NotⅠ, Acc65Ⅰ, KpnⅠ, AlfⅡ, NotⅠ, PuvⅠ, Bsu36Ⅰ, EcoRⅠ.

[0048] 4) Connect to form three independent expression cassettes (expression cassette 1, expression cassette 2, and expression cassette 3). Each expression cassette consists of a promoter, a multiple cloning site, and a terminator. The sequence characteristics are as follows:

[0049] Expression cassette 1: OP166+MCS1+SV40, the complementary chain is the nucleic acid sequence shown in SEQ ID NO: 29.

[0050] Expression cassette two: VP39+MCS2+bGH, encoded by the nucleic acid sequence shown in SEQ ID NO:30.

[0051] Expression cassette three: P6.9+MCS3+HSV, encoded by the nucleic acid sequence shown in SEQ ID NO:31.

[0052] Among them, the three expression frames are preferably arranged in the following order: expression frame one and expression frame three are arranged head to head, and expression frame two is arranged after expression frame three in the same direction.

[0053] 5) Dual resistance marker sequences: The chloramphenicol resistance element (CmR) and the gentamicin resistance element (GmR, 534 bp) were selected. The CmR is encoded by the chloramphenicol (C) nucleic acid sequence and the upstream CAT promoter (a synthetic promoter designed by CN108795946A); the GmR is encoded by the gentamicin (G) nucleic acid sequence and the upstream Pc promoter sequence.

[0054] 6) Tn7 attachmet site (attTn7) on the transfer vector: Select the minimum unit sequence required for T7 transposon transposition: Tn7L (166 bp) and Tn7R (225 bp);

[0055] The sequence composition of attTn7 is: Tn7R, complementary strand sequence of expression cassette 1, expression cassette 3, expression cassette 2 and Tn7L;

[0056] 7) Replication origin element: Select the F1 phage replication origin element (f1 ori, 456 bp) and the high copy replication origin element (ori, 589 bp);

[0057] 8) The sequence composition outside the attTn7 region is: Tn7L, f1 ori, GmR, ori and Tn7R.

[0058] 9) Finally, according to the above design, the sequence of attTn7 and the sequence outside the attTn7 region are formed into a ring, and the transport vector p-BT (p-Bac-triple) sequence is displayed with the terminator of expression frame 2 as the starting point, such as Figure 1 Shown: that is, the nucleic acid sequence shown by SEQ ID NO: 1.

[0059] The final components of the p-BT sequence are: terminator of expression cassette two, Tn7L, f1 ori, GmR, ori, Tn7R, complementary chain sequence of expression cassette one, expression cassette three, VP39 and MCS2 of expression cassette two.

[0060] The transport vector p-BT sequence (SEQ ID NO: 1) of the present invention was commissioned to Shanghai Bioengineering to synthesize a circular plasmid DNA, and was used for subsequent functional verification and application of the transport vector.

[0061] in addition, Figure 1 There are other replacement options for the transport vector p-BT shown, for example: the positions of the three expression frames can also be interchanged, ensuring that two of the expression frames are arranged head to head; other resistance screening markers can also be selected for the double-antibody marker sequence; among the replication initiation elements, at least one must be retained to enable the transport vector p-BT to still have basic replication ability in bacteria.

[0062] During the application of the transport vector p-BT described in the present invention, it was found that the p-BT sequence (SEQ ID NO: 1) had a low proportion of radical mutations, with particular attention paid to the radical mutations present in the attTn7 transposable unit. As long as the basic functions of p-BT for diversified and sequential activation of the three promoters during the construction process are ensured, the basic design purpose of the transport vector p-BT can be achieved.

[0063] In addition, according to the basic idea of ​​the present invention, the composition of each element in the transport carrier can also be achieved by modifying the transfer carrier pFastBackDual.

[0064] Example 2: Multiplex protein expression of p-BT Firefly luciferase (FLUC)

[0065] Using p-BT as the transport vector, firefly luciferase (FLUC) as the exogenous protein, and competent cells Bac-Syn as the recipient bacteria, five protein expression methods were designed. The specific designs and experimental steps are as follows:

[0066] 1) Construction of five recombinant p-BT plasmids

[0067] ① Primer design and gene amplification:

[0068] According to the restriction endonuclease sites of three MCS on the transport vector p-BT and the gene sequence of firefly luciferase (FLUC), three recombinant transport plasmids expressing FLUC downstream of three promoters were constructed respectively.

[0069] The FLUC gene sequence is shown in SEQ ID NO:24. Amplification primers were designed as shown in Table 1 below. Primers were synthesized by Qingke Biotechnology. Three fragments containing the FLUC gene were amplified by PCR using a plasmid (containing FLUC) stored in our laboratory as a template: Spe I-FLUC-Sac I, Sal I-FLUC-Nde I, and Not I-FLUC-Ecor I.

[0070] PCR amplification program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, 30 cycles; final extension at 72°C for 5 min.

[0071] Table 1: Primers for amplification of the FLUC gene

[0072]

[0073] ②Molecular cloning:

[0074] After separating the three PCR products on an agarose gel, the gel containing the target band was excised under UV light for gel recovery. The recovered products and the transfer vector p-BT were digested with the following enzymes: NdeⅠ / SalⅠ, SpeⅠ / SacⅠ, and EcoRI / NotⅠ, respectively. Refer to the enzyme instructions for the enzyme digestion system. The digested products were ligated downstream of the VP39, P6.9, and OP166 promoters of the p-BT vector, respectively, using the ligation system specified in the T4 DNA ligase instructions. After the ligation reaction, the ligation product was electrotransformed into DH10B competent cells, and the cells were resuspended in 500 μL SOC. After recovery at 37°C for 40 min, the transformation product was spread onto a solid LB plate containing 50 μg / mL ampicillin (Amp) and 25 μg / mL chloramphenicol (C). After the positive strains were selected and expanded, three recombinant plasmids pBT-VP39-FLUC, pBT-P6.9-FLUC and pBT-OP166-FLUC were extracted and obtained.

[0075] ③ Amplification of control plasmid:

[0076] Control plasmid: Design a recombinant plasmid expressing FLUC regulated by the PH and P10 promoters. The specific design and amplification are as follows:

[0077] Amplification primers (T-PRO, Table 1) were designed and synthesized by Qingke Biotechnology. Using the recombinant plasmid pBT-OP166-FLUC obtained above as the template, amplification of the vector fragment pBT-FLUC, excluding the OP166 promoter, was performed using PCR.

[0078] Simultaneously, primers for the P10 and PH genes were designed and synthesized by Qingke Biotechnology (Table 1). Using the pFBD plasmid as a template, two amplified fragments of the P10 and PH genes were amplified: PH-PRO and P10-PRO.

[0079] ④Molecular cloning of control plasmid:

[0080] The amplified fragments obtained by PCR amplification (pBT-FLUC, PH-PRO, and P10-PRO) were separated on an agarose gel, and the gel containing the target band was excised under UV light for gel recovery. The two promoter fragments (PH-PRO and P10-PRO) were respectively inserted upstream of the FLUC gene in the vector fragment pBT-FLUC using homologous recombinase (infusion). The ligation system was described in the instructions for the recombinase ligase. After the ligation reaction, the ligation products were electroporated into DH10B competent cells. The cells were resuspended in 500 μL of SOC and allowed to recover at 37°C for 40 minutes. The transformation products were then plated onto solid LB plates containing 50 μg / mL ampicillin (Amp) and 25 μg / mL chloramphenicol (C). Positive strains were selected and expanded, and the two recombinant plasmids, pBT-PH-FLUC and pBT-P10-FLUC, were isolated and obtained.

[0081] 2) Construction of five recombinant bacmids

[0082] Take 300 μg of each of the five recombinant plasmids pBT-VP39-FLUC, pBT-P6.9-FLUC, pBT-OP166-FLUC, pBT-PH-FLUC, and pBT-P10-FLUC obtained in step 1) and co-electroporate with the helper plasmid pMON7124 into Bac-Syn competent cells. Resuspend the cells in 500 μL SOC and resuspend them at 37°C for 40 minutes. Then, spread the transformation products on solid LB plates containing 50 μg / mL tetracycline (Tet), 50 μg / mL kanamycin (K), and 25 μg / mL chloramphenicol (C) for blue-white screening. Pick white colonies and expand them in LB liquid medium containing 50 μg / mL tetracycline (Tet), 50 μg / mL kanamycin (K), and 25 μg / mL chloramphenicol (C) to obtain positive bacterial liquid. Five recombinant Bacmids were extracted according to the instructions of the plasmid extraction kit: Bac-VP39-FLUC, Bac-P6.9-FLUC, Bac-OP166-FLUC, Bac-P10-FLUC, and Bac-PH-FLUC.

[0083] Among the five recombinant Bacmids obtained by the transfer vector pBT, the transposition site structures are as follows Figure 2As shown in the figure, FLUC can be seen downstream of the three promoters VP39, P6.9 and OP166 selected in the present invention, respectively. The two control groups are FLUC downstream of two commonly used late promoters P10 and PH, respectively. The structural abbreviations of the five recombinant Bacmid translocation sites in the figure are: Bac-FLUC (VP39), Bac-FLUC (P6.9), Bac-FLUC (OP166), Bac-FLUC (P10), and Bac-FLUC (PH).

[0084] 3) Acquisition and amplification of five recombinant baculoviruses

[0085] The five recombinant Bacmids obtained in step 2) were taken and transfected into insect cells Sf9 (2×10 6 ) and cultured at 27°C for 120 h. The cell culture supernatant was collected, which contained five primary (P0) recombinant baculoviruses: Bac-VP39-FLUC, Bac-P6.9-FLUC, Bac-OP166-FLUC, Bac-P10-FLUC, and Bac-PH-FLUC.

[0086] Take 200 μL of cell culture supernatant containing P0 recombinant virus and add it to healthy Sf9 cells (1×10 7 ) and cultured at 27°C. The infected state of the cells was observed under a fluorescence microscope. After 120 h, the supernatant of the infected cells was collected to obtain the P1 generation recombinant baculovirus.

[0087] 4) Obtaining and amplifying the parental recombinant baculovirus

[0088] The recombinant baculovirus Bac-syn of the parent strain Bac-syn was obtained by referring to the above similar method and used as a negative control for the above five recombinant baculoviruses.

[0089] 5) Titer determination of 6 recombinant baculoviruses

[0090] The virus titers of the six recombinant baculoviruses amplified above were measured: Bac-syn, Bac-VP39-FLUC, Bac-P6.9-FLUC, Bac-OP166-FLUC, Bac-P10-FLUC, and Bac-PH-FLUC. The determination method is as follows:

[0091] ① Packing EP tubes: 8 gradients of 10 for each virus -1 to 10 -8 , each concentration gradient was repeated 3 times, and there were 24 EP tubes in total for the three recombinant baculoviruses; 24 empty EP tubes were taken respectively, and 90 μL of culture medium containing 2% serum was added to each EP tube;

[0092] ② Dilute the virus: Take 10 μL of each recombinant baculovirus (BV) and add it to the labeled concentration of 10 -1 EP tube, vortex more than 10 times, each time for 0.5 to 1s; then take 10 μL of each and add it to the labeled concentration of 10 -2 EP tube, vortex more than 10 times, each time for 0.5 to 1s; refer to the above steps to dilute in sequence, and finally start from the marked concentration of 10 -8 Take 10 μL of the solution from each EP tube and discard it, ensuring that the final volume of each tube is 90 μL.

[0093] Take a T25 vial of cells and resuspend them in 6 ml of 10% serum medium. Add K, streptomycin (St), and G to the medium at a 1:50 ratio. Three recombinant viruses can be tested using one T25 flask of cells. Add the resuspended cell suspension to the virus dilution solution in each of the 24 EP tubes mentioned above, adding 90 μL of the cell suspension to each EP tube.

[0094] ③ Transfer the above cell and virus suspensions to 60-well plates respectively: vortex each EP tube more than 10 times, use a 20μL pipette to draw 10μL of the suspension into the culture wells of the 60-well plate, and repeat 6 wells of each virus concentration gradient on the 60-well plate, for a total of 6 rows and 8 columns. After wrapping with sealing film (make sure it is airtight), put it in a disposable lunch box, and put a wet paper at the bottom of the box. After culturing at 27°C for 5 days, measure the fluorescence number of each virus dilution concentration gradient and calculate the virus titer: AcBac-syn: TCID 50 / ml=8.65×10 7 , AcBac-VP39-FLUC:TCID 50 / ml=8.68×10 7 , AcBac-P6.9-FLUC:TCID 50 / ml=1.54×10 8 , AcBac-OP166-FLUC:TCID 50 / ml=2.23×10 8 , AcBac-P10-FLUC:TCID 50 / ml=4.82×10 8 , AcBac-PH-FLUC:TCID 50 / ml=8.88×10 8 .

[0095] 6) Identification of the biological activity of FLUC

[0096] According to the titer of the recombinant baculovirus determined above, 1×10 cells were infected with Bac-VP39-FLUC, Bac-OP166-FLUC, Bac-P6.9-FLUC, Bac-P10-FLUC, Bac-PH-FLUC and Bac-syn at an MOI of 1. 4 Sf9 cells were used to measure the luciferase activity at 6h, 24h, 48h, 72h and 96h. The detection method was based on the instructions of the luciferase detection reagent. Three replicates were set for each recombinant virus at each time phase. The test results were as follows: Figure 4 The figure shows that all three promoters (OP166, VP39, and P6.9) are capable of initiating the expression of exogenous proteins, and the promoter and terminator functions in the transfer vector p-BT are effective. Comparing the three promoters, the VP39 promoter exhibited the highest FLUC expression activity, indicating that when expressing a single protein, the VP39 promoter produces higher expression of the exogenous gene downstream of the promoter, while the expression activities of the OP166 and P6.9 promoters showed little difference.

[0097] In addition, the comparison in the figure also shows that FLUC expression can be detected in cells infected with Bac-OP166-FLUC and Bac-P6.9-FLUC at 6 hours, and the expression level is significantly higher than that of Bac-P10-FLUC and Bac-PH-FLUC, further proving that the expression time of the OP166 and P6.9 promoters is earlier than that of the PH and P10 promoters.

[0098] A comparison of the biological activities of six recombinant baculoviruses showed that the three promoters (OP166, VP39, and P6.9) selected for the transport vector p-BT of the present invention all had promoter activity, and could efficiently express genes of other exogenous proteins through the baculovirus expression system. In addition, the early expression of promoters OP166 and P6.9 was more conducive to the correct folding and early detection of exogenous proteins.

[0099] Example 3. Application of p-BT (SARS-CoV-2)

[0100] Using p-BT as the transport vector, the Spike (S) protein, membrane (M) protein and E unit of the new coronavirus (SARS-CoV-2) as foreign proteins, and the competent cell DH10Bac as the recipient bacteria, the S protein, M protein and E unit were set downstream of the promoters P6.9, VP39 and OP166 in p-BT, respectively, as shown in the following example: Figure 3 As shown. At the same time, Enhanced Green Fluorescent Protein (EGFP) was used as an exogenous protein and placed downstream of the promoter P6.9 in p-BT as a control. The specific steps of the experiment are as follows:

[0101] 1) Construction of recombinant p-BT plasmid co-expressing three proteins of the new coronavirus

[0102] ① Primer design and gene amplification:

[0103] Based on the sequence information of the S protein, M protein, and E unit, as well as the restriction endonuclease sites of the three MCSs on the p-BT transport vector, amplification primers (see Table 2 below) were designed and synthesized by Qingke Biotechnology. The goal was to construct the S protein, M protein, and E unit downstream of the promoters P6.9, VP39, and OP166, respectively, into the p-BT transport vector, thereby generating a recombinant transport plasmid capable of co-expressing the three foreign language proteins.

[0104] The M gene sequence is shown in SEQ ID NO: 25, and the E gene sequence is shown in SEQ ID NO: 26. A plasmid stored in our laboratory (containing the structural proteins of the novel coronavirus (SARS-CoV-2)) was used as an amplification template by PCR to obtain two amplified fragments: SpeⅠ-M-HindⅢ and SalⅠ-E-PstⅠ. S tag and Flag-tag were then added downstream of the M protein and E protein genes, respectively, by overlap PCR.

[0105] The S protein is a codon-optimized, outsourced sequence, see SEQ ID NO: 27. The amplified fragment S was amplified from the synthesized DNA fragment by PCR.

[0106] The EGFP gene sequence is shown in SEQ ID NO: 28. The target fragment, EGFP, was amplified by PCR using plasmid pIZ-V5-his-EGFP (stored in this laboratory, containing EGFP) as the amplification template.

[0107] Table 2 Amplification primers for SARS-CoV-2 protein and EGFP PCR amplification program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 4 min, 30 to 35 cycles; final extension at 72°C for 5 min.

[0108] ②Molecular cloning:

[0109] The amplified products obtained by the above PCR amplification were separated by agarose gel, and the gel containing the target band was cut out under ultraviolet light for gel recovery to obtain the amplified fragments: SpeⅠ-M-HindⅢ, SalⅠ-E-PstⅠ, S and EGFP, which were used in the following cloning and recombination processes respectively.

[0110] First, the SpeⅠ-M-HindⅢ and p-BT vector recovered from the gel were double-digested with SpeⅠ / HindⅢ respectively. The enzyme digestion system referred to the enzyme instructions, and the enzyme digestion product was ligated to the downstream of the promoter VP39 promoter of the p-BT vector using T4 DNA ligase; the ligation product was electroporated into DH10B competent cells, and the cells were resuspended in 500μL SOC. After resuscitation at 37℃ for 40min, the transformation product was spread on a solid LB plate containing 50μg / ml ampicillin (Amp) and 25μg / ml chloramphenicol (C), and the positive strain was selected for expansion and culture, and the recombinant plasmid pBT-M was extracted.

[0111] Second, the SalⅠ-E-PstⅠ recovered from the gel and the recombinant plasmid pBT-M obtained by extraction were double-digested with PstⅠ / PstⅠ, respectively. The enzyme digestion system was determined according to the enzyme instructions. The digestion products were ligated to the downstream of the OP166 promoter of the pBT-M vector using T4 DNA ligase. The ligation products were electroporated into DH10B competent cells, resuspended in 500 μL SOC, and revived at 37°C for 40 minutes. The transformation products were then plated onto solid LB plates containing 50 μg / ml ampicillin (Amp) and 25 μg / ml chloramphenicol (C). Positive strains were selected for expansion and culture, and the recombinant plasmid pBT-ME was obtained for the second time.

[0112] Third, the recombinant plasmid pBT-ME was amplified by PCR using the primers (TSF and TSR) shown in Table 2. The PCR products were separated by agarose gel, and the gel containing the target band was cut out under ultraviolet light for gel recovery.

[0113] Fourth, the amplified fragment S and pBT-ME recovered from the gel were taken and recombined using infusion recombinase. After electroporation into DH10B competent cells, the cells were resuspended in 500 μL of SOC and revived at 37°C for 40 minutes. The transformation product was then plated onto a solid LB plate containing 50 μg / ml ampicillin (Amp) and 25 μg / ml chloramphenicol (C). After selecting positive strains for expansion and culture, the third recombinant plasmid pBT-CoV-2-MSE was extracted.

[0114] The recombinant plasmid pBT-MSE is first amplified and cultured in Escherichia coli, and then the plasmid is extracted and a correct positive clone is identified by PCR amplification and used for later use.

[0115] Fifth, the amplified fragment EGFP recovered from the gel and the p-BT vector were double-digested with NotⅠ / EcoRI. The enzyme digestion system referred to the enzyme instructions, and the enzyme digestion product was used to connect EGFP to the downstream of the promoter P6.9 of p-BT using T4 DNA ligase; the ligation product was electroporated into DH10B competent cells, and the cells were resuspended in 500μL SOC. After recovery at 37℃ for 40min, the transformation product was spread on a solid LB plate containing 50μg / ml ampicillin (Amp) and 25μg / mL chloramphenicol (C). After selecting the positive strain for expansion and culture, the control recombinant plasmid: pBT-EGFP was extracted and obtained.

[0116] 2) Construction of two recombinant bacmids

[0117] The resulting recombinant plasmids pBT-CoV-2-MSE and pBT-EGFP were co-transfected with 300 ng of the helper plasmid pMON7124 into AcBac (bMON14272) competent cells. The cells were resuspended in 500 μL of SOC and allowed to recover at 37°C for 40 minutes. The transformed products were then plated onto solid LB plates containing 50 μg / ml tetracycline (Tet), 50 μg / ml kanamycin (K), and 25 μg / ml chloramphenicol (C) for blue-white colony selection. White colonies were selected and expanded in LB liquid medium containing 50 μg / ml tetracycline (Tet), 50 μg / ml kanamycin (K), and 25 μg / ml chloramphenicol (C) to obtain positive bacterial cultures. Two recombinant bacmids, AcBac-MSE and AcBac-EGFP, were extracted according to the instructions of the plasmid extraction kit.

[0118] In the two recombinant Bacmids obtained by the transfer vector pBT, the transposition site structure is as follows Figure 3 As shown in the figure, the M protein, S protein and E protein of the new coronavirus are respectively downstream of the three promoters VP39, P6.9 and OP166 selected by the present invention, and the exogenous protein EGFP of the control group is downstream of the promoter P6.9 of the present invention. The structural abbreviations of the two recombinant Bacmid transfer sites in the figure are: Bac-MSE and Bac-EGFP.

[0119] 3) Acquisition and amplification of two recombinant baculoviruses

[0120] The two recombinant Bacmids (AcBac-MSE and AcBac-EGFP) obtained in step 2) were taken and transfected into insect cells Sf9 (2×10 6) and cultured at 27° C. for about 120 h. The cell culture supernatant was collected, which contained primary generation (P0 generation) recombinant baculovirus: AcBac-MSE and AcBac-EGFP.

[0121] Take 200 μL of cell culture supernatant containing P0 recombinant virus and add it to healthy Sf9 cells (1×10 7 ) and cultured at 27°C. The infected state of the cells was observed under a fluorescence microscope. After about 120 h, the supernatant of the infected cells was collected to obtain the P1 generation recombinant baculoviruses AcBac-MSE and AcBac-EGFP, respectively.

[0122] 4) Preliminary identification of recombinant baculovirus

[0123] Referring to the titer determination method in Example 2, the titers of the obtained recombinant baculoviruses were: AcBac-MSE: TCID 50 / ml=3.17×10 6 , AcBac-EGFP: TCID 50 / ml=1.83×10 8 .

[0124] The P1 recombinant baculovirus AcBac-MSE and AcBac-EGFP were taken and infected into Sf9 cells (2×10 6 ), 96 hours after infection, the cell culture supernatant was discarded, and the infected Sf9 cells were resuspended in loading buffer to obtain a cell suspension. After the cell suspension was boiled at 100°C for 10 minutes, 5 μL was taken for Western blot analysis. The primary antibodies were RBD antibody, S-tag antibody and GP64 antibody, respectively. RBD antibody detected S protein, S-tag antibody detected M protein, and GP64 antibody detected EGFP protein. The Western blot verification results are shown in Figure 2. Figure 5 As shown in the figure. It can be seen that the recombinant virus AcBac-MSE obtained using the transport vector p-BT of the present invention can simultaneously express two important proteins M and S and EGFP protein required for the assembly of the new coronavirus (SARS-CoV-2), among which AcBac-MSE has the virus-like particles (VLP) structure of SARS-CoV-2. Therefore, the three promoters (VP39, P6.9 and OP166) in p-BT have the ability to co-promote the independent expression of their downstream exogenous proteins.

[0125] 5) Purification and identification of recombinant baculovirus

[0126] The P1 generation recombinant baculovirus AcBac-MSE was used to infect the suspended cultured Sf9 cells at an MOI of 0.05. After 96 hours of infection, the cells were collected by centrifugation at 3000 rpm for 10 minutes. The collected cells were resuspended with PBS and then ultrasonically disrupted. The disrupted sample was centrifuged at 8000 rpm for 30 minutes, and the supernatant was ultracentrifuged at 100,000 g (g is the centrifugal speed unit set by the Beckman ultracentrifuge and SW41 rotor used) for 1 hour. The obtained precipitate was fully resuspended with PBS and subjected to discontinuous sucrose density gradient centrifugation. 30%, 40%, and 50% sucrose were spread. After adding the sample, the sample was centrifuged at 100,000 g for 2 hours. The white band on the upper layer of the ultracentrifuge tube was added to PBS and mixed, and then centrifuged at 100,000 g for 2 hours for desugaring and concentration. Finally, the supernatant was discarded and the precipitate was resuspended with PBS to obtain preliminary purified SARS-CoV-2 virus-like particles, referred to as new crown VLP. The purified sample was run on SDS-PAGE gel and then identified by western blot. The results are as follows Figure 6 As shown, the S protein (~180kDa) of the new coronavirus VLP obtained by the RBD antibody as the primary antibody and the M protein (~20kDa) obtained by the S-tag antibody as the primary antibody are clearly visible.

[0127] The purified novel coronavirus VLPs were resuspended in loading buffer and boiled at 100°C for 10 min. The purified novel coronavirus VLPs were identified by Western blot according to step 4). The Western blot verification results are as follows: Figure 5 As shown in the figure, important proteins M and S that can be used to identify SARS-CoV-2 virus-like particles (COVID-19 VLPs) can be seen (the bands are not very obvious due to the small amount of S loaded).

[0128] Electron microscopy observation: Take 10 μL of purified new crown VLP, adsorb it on a copper mesh, stain it with phosphotungstic acid, and observe it under transmission electron microscopy after drying. The purified new crown VLP was observed and analyzed by transmission electron microscopy. Figure 7 As shown in the figure. It can be seen that the recombinant baculovirus AcBac-MSE obtained using the transport vector p-BT of the present invention successfully expressed granular substances, namely SARS-CoV-2 virus-like particles (novel coronavirus VLPs), with a diameter distribution of about 60-100nm. The observation results showed that the expression and preliminary purification of the novel coronavirus VLP were successful, verifying that the three-promoter expression system has the ability to express multiple exogenous proteins in a temporal manner, providing a research basis for the feasibility of producing multivalent vaccines and VLP vaccines.

[0129] Although there are documents that have successfully expressed SARS-CoV-2 virus-like particles using P10 and PH promoters (Youjun Mi.et al, Production of SARS-CoV-2 Virus-Like Particles in Insect Cells.2021), the use of three strong promoters to express SARS-CoV-2 S, M, and E proteins in this study can cause redundancy and waste of easily expressed proteins, and excessive E protein expression may cause premature cell apoptosis and reduce production (Yu yang.etal, Bcl-xL inhibits T-cell apoptosis induced by expression of SARS coronavirus E protein in the absence of growth factors.2005). In this embodiment, by regulating protein expression by different promoter strengths, it is possible not only to scientifically construct an efficient expression system based on the molecular composition of virus-like particles, but also to reduce the possibility of the above-mentioned problems, thereby achieving better vaccine production effects.

[0130] The present invention provides a transfer vector p-BT with three promoters. Due to the independent regulation of these three promoters, it is widely used in multiple fields such as genetic engineering, drug development, vaccine production, expression of immunologically active molecules and certain oncogenic viral proteins, and research on gene expression regulation. It has been verified that the transport vector p-BT of the present invention has the ability to simultaneously and independently express multiple exogenous proteins, diversify the selection of exogenous expression methods, form VLPs, and the exogenous proteins expressed using the transfer vector p-BT have biological activity. In addition, electron microscopy observation of SARS-CoV-2 virus-like particles constructed using the transport vector p-BT showed that the particle morphology was basically correct. Therefore, the p-BT of the present invention provides a basic means and tool for the diversified, temporal, large-scale, and large-capacity expression of exogenous proteins in eukaryotic expression, functional research, and vaccine drug research.

[0131] The specific embodiments of the present invention have been described in detail to facilitate understanding by those skilled in the art. However, based on the overall description disclosed herein, various modifications or substitutions may be made to certain details, and such modifications are intended to be within the scope of the present invention, which is defined by the appended claims and any equivalents thereof.

Claims

1. A transport carrier, characterized in that: A circular vector for a Bac-to-Bac baculovirus expression system comprises three expression cassettes whose expression is regulated by three promoters: expression cassette 1 under the control of the OP166 promoter encoded by the nucleic acid sequence shown in SEQ ID NO: 29, expression cassette 2 under the control of the VP39 promoter encoded by the nucleic acid sequence shown in SEQ ID NO: 30, and expression cassette 3 under the control of the p6.9 promoter encoded by the nucleic acid sequence shown in SEQ ID NO: 31; wherein, The three expression frames contain three independent multiple cloning sites for inserting the genes of three foreign proteins respectively; Three promoters are used in conjunction with three terminators to form three independent expression cassettes; The expression cassette one and the expression cassette three are arranged head to head and oppositely, and the expression cassette two is arranged sequentially after the expression cassette three; the expression cassette one includes the OP166 promoter, the multiple cloning site MCS1, and the terminator SV40; the expression cassette two includes the VP39 promoter, the multiple cloning site MCS2, and the terminator bGH; the expression cassette three includes the p6.9 promoter, the multiple cloning site MCS3, and the terminator HSV TK; The transfer vector further comprises two different prokaryotic replication origin elements: an F1 phage replication origin element f1 ori and another high-copy replication origin element ori that is different from the F1 phage replication origin element f1 ori.

2. The transport carrier according to claim 1, characterized in that: The multiple cloning site MCS1 contains SalⅠ, KasⅠ, NarⅠ, SfoⅠ, PluTⅠ, NdeⅠ, XbaⅠ, BstBⅠ, NsrⅡ, XmaⅠ, SmaⅠ, and PstⅠ; the multiple cloning site MCS2 contains SpeⅠ, BamHⅠ, AgeⅠ, and HindⅢ; the multiple cloning site MCS3 contains NotⅠ, Acc65Ⅰ, KpnⅠ, AlfⅡ, NotⅠ, PuvⅠ, Bsu36Ⅰ, and EcoRⅠ.

3. The transport carrier according to claim 1, characterized in that: The transfer vector also includes a chloramphenicol resistance element, a gentamicin resistance element and a Tn7 transposon element.

4. The transport carrier according to claim 3, characterized in that: The sequence composition outside the transposition site region of the Tn7 transposon element is: Tn7L, f1 ori, GmR, ori and Tn7R.

5. The transport carrier according to claim 3, characterized in that: The elements in the transfer vector sequence are composed of: terminator bGH of expression cassette two, Tn7L, F1 phage replication initiation element 0f1 ori, gentamicin resistance element GmR, high copy replication initiation element ori, Tn7R, chloramphenicol resistance element CmR, complementary chain sequence of expression cassette one, expression cassette three, VP39 promoter of expression cassette two and multiple cloning site two MCS2.

6. The transport carrier according to claim 1, characterized in that: The transport vector is encoded by the nucleic acid sequence shown in SEQ ID NO:

1.

7. A polynucleotide constructed by recombinant cloning of the transport vector according to any one of claims 1 to 6 and a gene fragment of an exogenous protein.

8. The polynucleotide according to claim 7, characterized in that: The gene fragment of the exogenous protein is the nucleic acid sequence shown in SEQ ID NO: 24 encoding firefly luciferase.

9. The polynucleotide according to claim 7, characterized in that: The gene fragment of the exogenous protein is composed of at least one of the following three: the nucleic acid sequence shown in SEQ ID NO:25 encoding the M protein of the new coronavirus, the nucleic acid sequence shown in SEQ ID NO:26 encoding the E protein of the new coronavirus, and the nucleic acid sequence shown in SEQ ID NO:27 encoding the S protein of the new coronavirus.

10. A host cell comprising the polynucleotide according to claim 7; or a recombinant plasmid obtained by transformation of the polynucleotide; or a recombinant virus obtained by transfection or infection of the recombinant plasmid.

11. Use of the transport vector according to any one of claims 1 to 6 in the preparation of antigens, antibodies or subunit vaccines.

Images