A nucleic acid construct for an insect cell-baculovirus expression system and uses thereof

Abstract

The present application relates to a nucleic acid construct for an insect cell-baculovirus expression system and its application, and belongs to the field of genetic engineering. The present application provides a novel nucleic acid construct, a corresponding insect cell-baculovirus expression system and its application. Specifically, the present application constructs a chimeric promoter capable of efficiently driving the transcription of an exogenous gene by creatively combining a specific enhancer and a promoter and designing the order and direction thereof, and further combines an independent polh promoter to construct a double-promoter system for forming two independent transcription units. The double-promoter system can be placed in the same vector to efficiently drive the expression of the same target gene at different stages of baculovirus infection of insect cells, greatly enhancing the expression intensity and persistence of the target gene at the transcription level, and ultimately improving the yield of the target protein.

Description

Technical Field

[0001] This invention relates to a nucleic acid construct for an insect cell-baculovirus expression system and its application, belonging to the field of genetic engineering. Background Technology

[0002] The insect cell-baculovirus expression system (BEVS) is a widely used eukaryotic expression platform for recombinant protein expression. It utilizes baculoviruses (such as AcMNPV, the nucleopolyhedrovirus of the alfalfa silver-striped moth) as vectors to efficiently express exogenous genes in insect cells (such as Sf9 and High Five cells). This system offers advantages such as high expression levels, post-translational modifications (e.g., glycosylation and phosphorylation), capacity for large exogenous gene fragments, and high biocompatibility, and has been widely applied in vaccine development, gene therapy vector production, membrane protein expression, and structural biology research. However, despite these advantages, the protein expression levels of the baculovirus expression system sometimes fall short of the requirements for industrial production or research on complex proteins (such as complex membrane proteins and multi-subunit complexes). Low expression levels often lead to increased downstream purification difficulties and costs, limiting the application of this technology in large-scale production.

[0003] To improve expression efficiency, researchers mainly employ methods such as viral genome modification, host cell modification, and promoter engineering. Among these, promoter selection and modification are key strategies. Traditionally, very late promoters, such as polyhedrin, are most commonly used. polh promoters and p10 Promoters drive extremely high levels of transcription in the later stages of infection, enabling the target protein to account for more than 30% of the total cellular protein. However, the expression phases of these promoters are highly synchronized, and their expression window is narrow due to limited cellular resources and cell lysis in the later stages of viral infection. Other promoters have also been developed and utilized, including late promoters such as... p6.9 VP39, and early or very early promoters (such as IE1, GP64, ... HSP70 These promoters are complementary to very late promoters in terms of expression phase, enabling phased expression or reducing resource competition. In addition, homologous regions (hr) such as hr1 sequences act as transcriptional enhancers, significantly increasing the activity of downstream promoters, and are often inserted into expression cassettes to enhance transcription (María Martínez-Solís et al., Appl MicrobiolBiotechnol. 2019 Jan;103(1):113-123).

[0004] In the prior art, there have been reports of forming multi-promoter expression cassettes by combining different promoters. For example, (1) CN121065099A constructed a dual-promoter tandem vector containing two Polyhedrin promoters, which are used to drive the transcription of human IgG1κ light chain gene and human IgG1 heavy chain gene, respectively; (2) CN101372684A constructed a recombinant baculovirus containing dual promoters, which contains p10 Promoters and polyhedral promoters are used to drive the transcription of reporter genes and foreign genes, respectively. In the above-mentioned prior art, the two promoters drive different genes and cannot achieve the effect of increasing the expression level of a single target protein. (3) CN110964749A uses a dual promoter ( p10 The recombinant baculovirus (rBac-Hr3-PH) produces the same recombinant protein (with two promoters each driving the transcription of one copy of the target gene), and the expression level can be increased by 100% or more (in Example 5, the yield increase of 10 μg / mL for rBac-Hr3-Fiber-2×2 is less than 200% compared to 6 μg / mL for rBac-Fiber-2×2), demonstrating the potential of the dual promoter system to increase protein expression. However, whether this dual promoter system can be used to express conformationally unstable complex proteins (especially complex proteins that need to be correctly assembled into trimers and maintain a specific conformation, such as RSV F) and the extent of yield increase after expression still need to be studied. Therefore, for insect cell-baculovirus expression systems, existing multi-promoter strategies still have shortcomings in terms of synergy, stability, and yield increase of complex proteins during expression, making it difficult to consistently achieve high yield increases and meet the needs of industrial production. Summary of the Invention

[0005] To address existing technical problems, this invention provides a novel expression system and its applications for significantly increasing the yield of recombinant proteins in insect cell-baculovirus expression systems. Specifically, this invention constructs a chimeric promoter that efficiently drives the transcription of exogenous genes by creatively combining specific enhancers and promoters and designing their sequence and orientation. Its core elements include an enhancer element (selected from hr1 or its reverse complementary sequence) and promoter X (selected from...). polh , p10 and p6.9 (or its inverse complementary sequence) and promoter Y (selected from HSP70 Based on this, a dual-boot subsystem was further constructed, which includes independent polh The promoter and a chimeric promoter described above are used to form two independent transcription units. This dual promoter system can be placed in the same expression vector to efficiently drive the expression of the same target gene at different stages of baculovirus infection of insect cells, thereby greatly enhancing the expression intensity and persistence of the target gene at the transcriptional level.

[0006] The specific technical solution provided by this invention is as follows:

[0007] In a first aspect, the present invention provides a nucleic acid construct, characterized in that the nucleic acid construct comprises a chimeric promoter, the chimeric promoter comprising an enhancer element, a promoter X, and a promoter Y connected in series, wherein:

[0008] a) Enhancer element: selected from hr1 or its inverse complementary sequence (i.e., Rhr1), the sequence of which is shown in SEQ ID NO: 3;

[0009] b) Promoter X: selected from polh , p10 , p6.9 The promoter, or the inverse complementary sequence of the three promoters mentioned above (i.e., R). polh R p10 R p6.9 ), the polh , p10 , p6.9 The sequences of the promoters are shown in SEQ ID NO: 4, 5, and 6, respectively;

[0010] c) Promoter Y: using HSP70 The promoter, the sequence of which is shown in SEQ ID NO: 7.

[0011] The term "sequentially connected in series" means that the reinforcing element is located upstream of the promoter X, and the promoter X is located upstream of the promoter Y. The elements can be directly connected end to end or connected through connectors.

[0012] Preferably, the chimeric promoter comprises any one of the following sets of elements connected in series:

[0013] Group 1: hr1+ polh + HSP70 (BV43);

[0014] Group 2: hr1+R polh + HSP70 (BV44);

[0015] Group 3: Rhr1+R polh + HSP70 (BV46);

[0016] Group 4: hr1+ p10 + HSP70 (BV47);

[0017] Group 5: hr1+ p6.9 + HSP70 (BV51).

[0018] Preferably, it is selected from any one of the groups 1, 2, 3, and 5; more preferably, it is selected from group 3 or group 5.

[0019] Preferably, the structure of the chimeric promoter is: linker I - enhancer element - linker II - promoter X - linker III - promoter Y (i.e., linker I, enhancer element, linker II, promoter X, linker III, and promoter Y are sequentially tandem). Linkers I, II, and III are only used to distinguish linkers at different positions; each is an independent nucleotide spacer sequence. The specific sequences of the linkers can be selected by those skilled in the art using conventional methods, provided that they do not negatively affect the function of the functional elements. The hyphen "-" indicates a covalent link (i.e., a phosphodiester bond). The elements before and after each "-" can be directly connected end-to-end, or additional restriction endonuclease recognition sequences can be introduced for ligation, allowing for flexible manipulation of each element through enzyme digestion in subsequent studies. When the chimeric promoter is used to drive the transcription of a target gene, the target gene is linked downstream of promoter Y in the chimeric promoter, with an appropriate spacer sequence maintained between them to ensure suitable spacing, thereby facilitating effective transcription driven by the promoter.

[0020] Regarding the connectors I, II, and III, the preferred options are:

[0021] (1) Connector I, II: Connector I uses sequence a, and connector II uses sequence b; or, connector I uses sequence b and connector II uses sequence a; wherein: sequence a is selected from SEQ ID NO: 8 or its reverse complementary sequence, and sequence b is selected from SEQ ID NO: 9 or its reverse complementary sequence;

[0022] (2) Connector III: selected from SEQ ID NO: 10 or its reverse complementary sequence.

[0023] Regarding the connectors I, II, and III, further preferred options are:

[0024] (1) Linkers I and II: ① When the enhancer element is hr1 (hr1 is inserted into the nucleic acid construct in the forward orientation, as in the aforementioned BV51): Linker I uses SEQ ID NO: 8, and Linker II uses SEQ ID NO: 9; ② ​​When the enhancer element is Rhr1 (hr1 is inserted into the nucleic acid construct in the reverse orientation, as in the aforementioned BV46): Linker I uses the reverse complementary sequence of SEQ ID NO: 9, and Linker II uses the reverse complementary sequence of SEQ ID NO: 8;

[0025] (2) Connector III: SEQ ID NO: 10.

[0026] Further preferably, the chimeric promoter comprises any of the sequences shown in SEQ ID NO: 17, 18, 20, 21, 25, or is composed of any of the above sequences; wherein, preferably any of the sequences shown in SEQ ID NO: 17, 18, 20, 25, and even more preferably the sequences shown in SEQ ID NO: 20 or SEQ ID NO: 25.

[0027] Preferably, the nucleic acid construct further includes a recombinant protein-coding gene, which is connected (i.e. located) downstream of the chimeric promoter.

[0028] Preferably, the recombinant protein is RSV F recombinant protein.

[0029] More preferably, the amino acid sequence of the RSV F recombinant protein is shown in SEQ ID NO: 1.

[0030] More preferably, the sequence of the RSV F recombinant protein encoding gene is shown in SEQ ID NO: 2.

[0031] In a second aspect, the present invention provides a nucleic acid construct, characterized in that the nucleic acid construct comprises a dual promoter subsystem, wherein the dual promoter subsystem comprises a first promoter and a second promoter connected in series, wherein:

[0032] i) The first promoter: is a single polh promoter;

[0033] ii) The second promoter: is the chimeric promoter described in the first aspect;

[0034] The first promoter and the second promoter each have an insertion site downstream (for enabling the recombinant protein-coding gene to be operatively linked downstream of the promoter to form an independent transcription unit).

[0035] Preferably, the nucleic acid construct is a baculovirus transfer vector.

[0036] More preferably, the baculovirus transfer vector is constructed using pOET1.1 as the backbone (initial vector). For example, (1) one specific embodiment is to utilize the features inherent in pOET1.1. polh The promoter is used as the first promoter, and the chimeric promoter is connected to a region in pOET1.1 other than the transcription unit where the first promoter is located as the second promoter; (2) Another optional implementation is to insert another polh The promoter is connected to pOET1.1 as the first promoter, and the chimeric promoter is connected to pOET1.1 except... polhThe region outside the transcription unit serves as a second promoter.

[0037] Preferably, the nucleic acid construct further comprises two copies of a recombinant protein-coding gene, wherein one copy of the recombinant protein-coding gene is linked downstream of the first promoter, and the other copy is linked downstream of the second promoter.

[0038] Preferably, the recombinant protein is RSV F recombinant protein.

[0039] More preferably, the amino acid sequence of the RSV F recombinant protein is shown in SEQ ID NO: 1.

[0040] More preferably, the sequence of the RSV F recombinant protein encoding gene is shown in SEQ ID NO: 2.

[0041] Thirdly, the present invention provides an insect cell-baculovirus expression system, wherein the expression system is obtained by co-transfecting insect cells with the nucleic acid construct described in the first or second aspect (the nucleic acid construct contains a recombinant protein encoding gene) and the baculovirus genome.

[0042] Preferably, the baculovirus is the alfalfa silver-striped noctuid moth nucleopolyhedrovirus.

[0043] Preferably, the insect cells are Sf9 insect cells.

[0044] Fourthly, the present invention provides a method for preparing recombinant proteins, characterized in that the insect cell-baculovirus expression system described in the third aspect is used to culture the expression system and harvest the recombinant protein from the culture supernatant.

[0045] Terminology and related explanations:

[0046] 1. Chimeric promoters: These are chimeric regulatory structures formed by the directed assembly of two or more DNA elements with independent transcriptional regulatory functions through molecular biology techniques. They exhibit synergistic or synergistic transcriptional activation effects. Their core characteristic lies in the precise regulation of the expression intensity, specificity, or timing of a single target gene through the design of the element combination pattern, spatial arrangement, and orientation. Among them:

[0047] (1) Oriented assembly: For example, components are directly connected in series or connected by artificially designed connectors (to optimize the spatial distance between components or eliminate reading frame interference).

[0048] (2) Combination mode: For example, connecting at least one enhancer element in series with at least one promoter element, or connecting two or more promoter elements in series.

[0049] (3) Spatial arrangement: refers to the relative positional relationship of each element on the DNA strand.

[0050] (4) Direction: refers to the physical orientation of the element's DNA sequence relative to the transcription direction of the target gene, and is divided into: ① forward: the 5'→3' direction of the element is consistent with the transcription direction of the target gene; ② reverse: the 5'→3' direction of the element is opposite to the transcription direction of the target gene.

[0051] 2. Dual promoter system: refers to a system containing two independent promoter units, each with an insertion site downstream (towards the transcription direction) of the promoter unit, allowing a first target gene to be operatively linked to the first promoter unit and a second target gene to be operatively linked to the second promoter unit, forming two independent transcription units; independence means that each promoter can independently drive the transcription of its downstream target gene without depending on the action of the other promoter. In the dual promoter system used in this application, the first promoter is a single promoter ( polh The second promoter is a chimeric promoter (such as Rhr1+R). polh + HSP70 )

[0052] 3. Enhancer: A DNA cis-acting element whose core function is to significantly enhance (or activate) the transcription efficiency of its associated genes through mechanisms such as recruiting transcription factors or altering chromatin structure, thereby ultimately enhancing protein expression; in this application, the enhancer is hr1.

[0053] 4. Linker: refers to a sequence located between two functional elements (such as enhancer and promoter elements, or promoter and promoter elements) that serves to connect, space, or introduce a specific functional role (such as providing an enzyme cleavage site) without negatively affecting the function of the functional elements. In this application, it specifically refers to a linker at the DNA level (a non-coding DNA sequence that does not undergo transcription and translation).

[0054] 5. Insertion site: refers to a specific region in the vector or genome that allows the integration of foreign DNA, such as, but not limited to, multiple cloning sites, homologous arms, and other forms.

[0055] 6. In this application, "above" and "at least" are both understood to include the number itself. For example, "two or more" is equivalent to "≥2". The terms "connect" and "connect in series" include direct connection, connection through spacer sequences, connection through enzyme cleavage sites, and other conventional methods in the art. The term "comprising" is an open-ended term, and its semantic scope and legal protection scope cover the situation of "composed of" (the latter is a subset of the former).

[0056] The beneficial effects of this invention are:

[0057] (1) The specific dual-start subsystems (BV56, BV58) provided by this invention exhibit significant synergistic effects. Experiments have confirmed that, compared with traditional single-start subsystems... polh The promoter subsystem, the BV56 expression system, can increase the yield of the target recombinant protein (RSV F) by more than 3 times, and the BV58 expression system by more than 12 times, achieving highly efficient expression of the target protein. This reduces costs for large-scale industrial production and provides a foundation for commercial application.

[0058] (2) The expression system provided by this invention can maintain high expression even after three generations of viral amplification, showing good genetic stability. Therefore, this expression system can be used to enhance the expression of trimeric macromolecular proteins such as RSV F protein, and has the potential to produce high-difficulty proteins (such as complex membrane proteins, multi-subunit complexes, etc.). Attached Figure Description

[0059] Figure 1 Example 2: ELISA results of target protein expression levels in all embodiments;

[0060] Figure 2 In Example 2, it involves polh ELISA results of target protein expression levels in all embodiments;

[0061] Figure 3 In Example 2, it involves p10 ELISA results of target protein expression levels in all embodiments;

[0062] Figure 4 In Example 2, it involves p6.9 ELISA results of target protein expression levels in all embodiments;

[0063] Figure 5 In Example 2, it involves HSP70 ELISA results of target protein expression levels in all embodiments;

[0064] Figure 6 Example 3: ELISA results of target protein expression levels in all embodiments;

[0065] Figure 7 In Example 4, the expression level of the target protein after three generations of amplification of baculovirus containing a dual promoter system in Sf9 insect cells is detected, including the results of SDS-PAGE detection of cell supernatant (left) and Western blot detection (right).

[0066] Figure 8 : Structural diagram of the initial transfer vector pOET1.1;

[0067] Figure 9 : Structural diagram of the recombinant transfer vector pOET1-BV56;

[0068] Figure 10 : Structural diagram of the recombinant transfer vector pOET1-BV58. Detailed Implementation

[0069] To more accurately and clearly understand and grasp the purpose, technical solution, and advantages of this invention, the invention will be described in detail below based on specific embodiments and related experimental data.

[0070] Example 1: Novel Promoter Design and Screening Strategies for Baculoviruses

[0071] Step 1: Promoter sequence design and gene synthesis:

[0072] To obtain novel promoters suitable for baculovirus expression systems that can enhance protein expression, this invention creates chimeric promoters and evaluates the efficacy of different promoters by comparing the expression levels of the target protein in the model. The specific process is as follows:

[0073] (1) Chimeric promoter design: First, through literature review, enhancers and promoter elements suitable for baculovirus expression systems were initially selected; then, based on this, chimeric promoters were designed: at least one enhancer element was connected in series with at least one core promoter element (to jointly drive the expression of a single target protein), or two or more core promoter elements were connected in series (to jointly drive the expression of a single target protein), and the combination mode, spatial arrangement, and orientation of the elements were designed. The elements were directly connected in series or selectively connected through linkers to obtain chimeric promoters;

[0074] (2) Model target gene design: The respiratory syncytial virus fusion protein (RSV F protein) suitable for production by baculovirus expression system was used as the model target protein. Based on previous research results, the RSV F recombinant protein with the research code name BR58 was specifically selected (its amino acid sequence is shown in SEQ ID NO: 1, and an 8×His tag was introduced at the C-terminus for easy purification). According to the codon preference of the host Sf9 insect cells, the coding sequence of the target protein (i.e. the target gene) was optimized to obtain the optimized nucleic acid sequence (shown in SEQ ID NO: 2).

[0075] (3) Construction of transcriptional enhancement units: Different promoter sequences are connected to the 5' end of the optimized target gene to construct a series of transcriptional enhancement units; then, the complete DNA fragments containing different promoters and the target gene are synthesized into a whole gene.

[0076] Step 2: Construction of recombinant transfer plasmid and expression of target protein:

[0077] The synthesized DNA fragments were cloned into the baculovirus transfer vector pOET1.1 (its structure is shown below). Figure 8 As shown in the sequence (SEQ ID NO: 29), the corresponding recombinant transfer vector (recombinant transfer plasmid) was constructed. Then, using flashBAC... TM Baculovirus expression system kit, which combines various recombinant transfer plasmids with flashBAC TM Baculovirus genomic DNA was co-transfected into Sf9 insect cells, strictly following the kit instructions. Seven days after transfection, the cell supernatant was collected; this was the P0 generation virus solution containing recombinant baculovirus. The P0 generation virus solution was used to infect new Sf9 cells to obtain the P1 generation virus. Six to seven days after P1 generation virus infection of Sf9 cells, the cell culture supernatant was collected for subsequent detection of the target protein expression level.

[0078] Step 3: Detection of target protein expression level and evaluation of promoter efficacy:

[0079] (1) Detection of target protein expression: The expression level of the target protein was detected by ELISA. The specific steps are as follows: 100 μL of Motavizumab (Mota, which can bind to the Pre-F and Post-F conformations of RSV F protein) or AM14 (specifically binds to the Pre-F conformation) or other antibodies were coated into a 96-well plate at 4°C overnight. The next day, after washing three times with PBST and patting dry, 200 μL of 5% skim milk prepared with PBST was added to each well, and the plate was blocked at room temperature for 1 hour. After blocking, the plate was washed three times and patted dry. 100 μL of diluted cell supernatant sample was added to each well, and the plate was incubated at room temperature for 1-2 hours. After washing again, 100 μL of Rabbit anti-His-tag antibody was added to each well, and the plate was incubated at room temperature for 1 hour. After washing, 100 μL of horseradish peroxidase-labeled secondary antibody was added, and the plate was incubated at room temperature for 1 hour. After thoroughly washing five times and patting dry, add 100 μL of freshly prepared TMB chromogenic solution to each well and incubate for 10-15 minutes. Then, add stop solution to terminate the reaction. Read the absorbance values ​​(OD values) at 450 nm and 570 nm wavelengths using a microplate reader. The final OD value is expressed as the difference between A450nm and A570nm.

[0080] (2) Evaluation of promoter expression efficacy: Based on the expression level of the target protein, promoters with high expression intensity are screened out.

[0081] Example 2: Design and Verification of Chimeric Promoters

[0082] The main purpose of this embodiment is to screen and traditional polhPromoters with comparable or better expression levels. Following the procedures described in Example 1, chimeric promoter design and expression efficacy evaluation were performed.

[0083] (a) Promoter design

[0084] Three hybrid promoter design strategies are employed: connecting one enhancer element in series with one promoter element; connecting two promoter elements in series; and connecting one enhancer element in series with two promoter elements. Simultaneously, a single promoter is used as a reference to compare the effects with the hybrid promoter approach. Specific implementation methods include:

[0085] Single promoter: polh , p10 , p6.9 or HSP70 ;

[0086] Chimeric promoter I (enhancer + promoter): hr1+ polh Rhr1+ polh hr1+ HSP70 or Rhr1+ HSP70 ;

[0087] Chimeric promoter II (promoter X + promoter Y): polh + HSP70 Or R polh + HSP70 ;

[0088] Chimeric promoter III (enhancer + promoter X + promoter Y): hr1+ polh + HSP70 hr1+R polh + HSP70 Rhr1+ polh + HSP70 Rhr1+R polh + HSP70 hr1+ p10 + HSP70 hr1+R p10 + HSP70 Rhr1+ p10 + HSP70 Rhr1+R p10 + HSP70 hr1+ p6.9 + HSP70 hr1+R p6.9 + HSP70 Rhr1+ p6.9 + HSP70 Or Rhr1+R p6.9 + HSP70 .

[0089] Regarding the specific implementation methods described above, it should be noted that:

[0090] 1. Regarding the "R" prefix: (1) The absence of the "R" prefix before the element name indicates that the element is inserted in the forward orientation in the transcriptional enhancement unit (its natural 5'→3' direction is consistent with the transcription direction of the target gene; the sequence read along the transcription direction is the original 5'→3' sequence of the element); (2) The presence of the "R" prefix before the element name indicates that the element is inserted in the reverse orientation in the transcriptional enhancement unit (its natural 5'→3' direction is opposite to the transcription direction of the target gene; the sequence read along the transcription direction is the reverse complementary sequence of the original sequence of the element).

[0091] 2. Regarding the nucleic acid sequences of each element: (1) The sequence of the enhancer element hr1 is shown in SEQ ID NO: 3, and the promoter element polh , p10 , p6.9 , HSP70 The sequences are shown as SEQ ID NO: 4, 5, 6, 7 respectively; (2) Elements with the prefix "R", namely Rhr1, R polh R p10 R p6.9 The sequences are the reverse complementary sequences of SEQ ID NO: 3, 4, 5, and 6, respectively.

[0092] 3. Regarding the specific structure and nucleic acid sequence of the chimeric promoter: (1) Different elements in the chimeric promoter are linked by linkers, specifically linker 1, linker 2, linker 3 or their reverse complementary sequences (indicated by underlining, e.g. linker1 , linker2 , respectively representing the inverse complementary sequences of linker 1 and linker 2), the sequences of linker 1, linker 2 and linker 3 are shown as SEQ ID NO: 8, 9 and 10 respectively. (2) The “Promoter Sequence” column in Table 1 marks the specific sequences of each chimeric promoter used in this embodiment.

[0093] (II) Construction of Transcription Enhancement Units

[0094] After determining the promoter sequence, the target gene was linked downstream of the promoter to construct a transcriptional enhancement unit. Since the initial vector pOET1.1 itself contains a... polh Promoter (see) Figure 8 Therefore, the following approach is adopted in this embodiment:

[0095] (1) Targeting polh As an example of a single promoter (BV33), the target gene (BR58 protein coding sequence) is directly inserted into pOET1.1. polh Start the downstream of the sub-initiator;

[0096] (2) For those using other single promoters ( p10 , p6.9 or HSP70 Examples of chimeric promoters (BV37~BV54) are used to synthesize nucleic acid sequences containing the corresponding promoter and target gene, which are then linked to pOET1.1 through an insertion site.

[0097] In this embodiment, the detection results of the target protein expression levels corresponding to different promoters are shown in the last three columns of Table 1.

[0098] Table 1. Results of different promoter designs and target protein expression levels

[0099]

[0100] Notes: (1) In the chimeric promoter structure, the hyphen “-” indicates covalent linkage (i.e., phosphodiester bond linkage); in the specific implementation of this embodiment, the elements before and after each “-” are directly covalently linked; however, as another optional implementation, a restriction endonuclease recognition sequence (such as GAGCTC) can be introduced between adjacent elements for ligation, so that each element can be flexibly manipulated (inserted, deleted, replaced, etc.) through enzyme digestion in subsequent studies. (2) Pre-F protein refers to the conformational form of RSV F protein before fusion, and the percentage of Pre-F protein (%) = (OD AM14 / OD Mota ×100% (the same below); The names in the form of "BV + number" correspond to a promoter design strategy, and also to the recombinant transfer vector and recombinant baculovirus constructed using the strategy (the same below).

[0101] Based on the results in Table 1, the corresponding attached figures are as follows: Figures 1-5 As shown. Among them, Figure 1 The results of all implementation methods (BV33~BV54) are presented for cross-sectional comparison; Figures 2-5 Results for all implementations including a specific promoter are presented separately for intra-group comparison. Figure 2 , Figure 3 , Figure 4 , Figure 5 Each contains polh , p10 , p6.9 , HSP70 The results of all implementations of the promoter are shown in the bar chart below, which indicates the elements used in the corresponding promoter design [“√” or “√(R)” indicates that the element is present, where “R” indicates orientation].

[0102] (a) Table 1 and Figure 1 The results show:

[0103] 1. The proportion of Pre-F protein expressed by all promoters is close to 100%, which proves that all promoters express the conformally correct RSV F protein.

[0104] 2. In a single-starter strategy: Utilize polh The promoter expression yields the highest production of the target protein. p10 , p6.9 Secondly, HSP70 The lowest value reflects the superiority of traditional very late promoters.

[0105] 3. In the chimeric promoter I (enhancer + promoter) strategy: BV41 (hr1 + HSP70 Best performing, BV37 (hr1+) polh Therefore, BV41 was selected to proceed to the next round of promoter screening.

[0106] 4. In the chimeric promoter II (promoter X + promoter Y) strategy: the tested BV39 ( polh + HSP70 ) and BV40 (R polh + HSP70 The protein yield of promoter combinations was lower than other strategies, and even worse than the worst-performing single promoter strategy. HSP70 The promoter does not select this strategy group to proceed to the next round of screening.

[0107] 5. In the chimeric promoter III (enhancer + promoter X + promoter Y) strategy:

[0108] (1) Promoter X is polh In the designs (BV43~BV46): the best performing is BV46 (Rhr1+R). polh + HSP70 BV43 (hr1+) polh + HSP70 ) and BV44 (hr1+R polh + HSP70 Slightly less, BV45 (Rhr1+) polh + HSP70 The results were poor, so BV46 was selected for the next round of screening.

[0109] (2) Promoter X is p10 In the design (BV47~BV50): the best performing is BV47 (hr1+). p10 + HSP70 The other three are BV48 (hr1+R) p10 + HSP70 BV49 (Rhr1+)p10 + HSP70 ) and BV50 (Rhr1+R p10 + HSP70 All of them were poor, so BV47 was selected for the next round of screening.

[0110] (3) Promoter X is p6.9 In the design (BV51~BV54): the best performing is BV51 (hr1+). p6.9 + HSP70 The other three are BV52 (hr1+R) p6.9 + HSP70 BV53 (Rhr1+) p6.9 + HSP70 ) and BV54 (Rhr1+R p6.9 + HSP70 All of them were poor, so BV51 was selected for the next round of screening.

[0111] (two) Figures 2-5 The results of the intragroup comparison showed that:

[0112] 1. Includes polh In the promoter design ( Figure 2 ):

[0113] (1) A comparison of BV33, BV37, and BV38 shows that: polh Adding an enhancing sub-element hr1 to the basic structure, regardless of the direction of hr1, did not enhance the expression.

[0114] (2) A comparison of BV33, BV39, and BV40 shows that: polh Add a promoter to the basics HSP70 The expression level of the target protein was significantly reduced, especially in polh In the reverse case (BV40);

[0115] (3) Comparison of BV33 with BV43~BV46 shows: ① In polh On the basis of adding hr1 and HSP70 However, BV43, BV44, and BV46 did not significantly increase the expression level of the target protein; however, they achieved similar results to BV33 (a single promoter). polh ① Similar expressive efficacy; ② It is worth mentioning that, i) BV44, compared to BV43, polh The reverse setting has little impact on expression efficiency; ii) compared to BV43, the reverse setting of hr1 in BV45 significantly reduces expression efficiency; iii) however, compared to BV43, the reverse setting of hr1 in BV45 significantly reduces expression efficiency. polh Reverse settings actually improve expressive efficiency.

[0116] therefore, Figure 2 The following implications are given: simply combining enhancers and promoters does not necessarily improve expressive efficacy; enhancer hr1, promoter X ( polh The direction of the transcription of the target gene does not necessarily need to be consistent with the direction of transcription of the target gene. The effect of reversing the transcription on expression efficiency cannot be generalized and needs to be verified through specific experiments.

[0117] 2. Includes p10 In the promoter design ( Figure 3 ):

[0118] (1) Comparison of BV34 with BV47~BV50 shows that: p10 On the basis of adding hr1 and HSP70 It failed to increase the expression level of the target protein; among them, BV47 achieved the same effect as BV34 (a single promoter). p10 The expression efficiency of BV48 to BV50 is basically the same, while the expression efficiency of BV48 to BV50 is significantly reduced.

[0119] (2) Comparison of BV47~BV50 shows that when all three components are set to positive (BV47), better performance can be achieved; while when hr1, p10 Setting one or both of these settings in reverse will result in a significant reduction in expressive efficiency.

[0120] therefore, Figure 3 The following insights are given regarding enhancers and promoter X ( p10 The orientation setting has a significant impact on expressive effectiveness; in comparison Figure 2 The insights provided further demonstrate that the impact of component orientation on expressive performance cannot be generalized and requires case-by-case analysis.

[0121] 3. Includes p6.9 In the promoter design ( Figure 4 ):

[0122] (1) A comparison of BV35 with BV51~BV54 shows that: p6.9 On the basis of adding hr1 and HSP70 The expression levels of the target protein were not improved. The expression efficacy was ranked from highest to lowest as follows: BV35 > BV51 > BV53 > BV52 > BV54.

[0123] (2) Comparison of BV51~BV54 shows that when all three components are set in the positive direction (BV51), relatively good expressive performance can be achieved; while when hr1, p6.9 Setting one or both of them in reverse will result in a slight decrease in expressive efficiency.

[0124] therefore, Figure 4The following insights are given regarding enhancers and promoter X ( p6.9 The direction setting has a certain impact on expressive effectiveness; comparison Figure 2 , Figure 3 The insights provided further demonstrate that the impact of component orientation on expressive performance cannot be generalized and requires case-by-case analysis.

[0125] 4. Includes HSP70 In the promoter design ( Figure 5 ):

[0126] (1) A comparison of BV36 with BV41 and BV42 shows that: HSP70 Adding hr1 to the basics can enhance expressive efficacy, and the positive setting of hr1 (BV41) is more effective than the negative setting (BV42).

[0127] (2) A comparison of BV36 with BV39 and BV40 shows that: HSP70 Based on (promoter Y), only one additional promoter is added. polh (Promoter X) can lead to reduced expressive efficiency, especially polh In the case of reverse settings.

[0128] (3) Comparison of BV36 and BV43~BV54 shows: ① In HSP70 Based on (promoter Y), add an enhancer hr1 and a promoter X ( polh , p10 or p6.9 The effects varied, with eight chimeric promoters (BV43, BV44, BV46, BV47, BV51~BV54) showing higher expression efficiency than single promoters. HSP70 The other four (BV45, BV48, BV49, and BV50) are lower than single promoters. HSP70 ; ② Adopt polh As an implementation of promoter X (BV43~BV46), its overall expression performance is superior to that of the implementation using... p6.9 (BV51~BV54) are implementation methods for promoter X, and employ... p10 (BV47~BV50) are implementations of promoter X, but there are still significant differences in individual cases.

[0129] therefore, Figure 5 The following insights are provided: When using the same... HSP70In the case of promoter Y being the immediate promoter of the target gene, (1) regarding the orientation of hr1: Although the chimeric promoter I design (enhancer + promoter) shows that hr1 is more effective when hr1 is in the forward direction than in the reverse direction (BV41 vs. BV42), the chimeric promoter III design (enhancer + promoter X + promoter Y) shows that in some embodiments, the hr1 orientation is slightly better (e.g., BV43 vs. BV45), while in other embodiments, the hr1 orientation is slightly better (e.g., BV44 vs. BV46); (2) regarding the orientation of promoter X: In some embodiments, the orientation of promoter X has little effect ( polh (BV43 vs. BV44), while in other embodiments, the orientation setting of the promoter X has a greater impact ( p10 (BV47 vs. BV48).

[0130] (III) Based on the above analysis, the following conclusions can be drawn:

[0131] The influence of the component combination mode, spatial arrangement, and orientation design of chimeric promoters on expression efficiency (enhancement or reduction) and its degree is not fixed and needs to be analyzed in conjunction with experimental data. Based on the experimental results, the chimeric promoter BV41(hr1+) with better performance was selected. HSP70 BV46 (Rhr1+R) polh + HSP70 BV47 (hr1+) p10 + HSP70 ) and BV51 (hr1+ p6.9 + HSP70 Proceed to the next round of experiments.

[0132] Example 3: Design and Verification of Dual-Boot Subsystem

[0133] Based on the results of Example 2, this example optimizes the promoter design strategy as follows: In addition to using the superior chimeric promoter strategies (BV41; BV46; BV47; BV51) selected in Example 2, an independent... polh The promoters form a dual promoter system (BV55; BV56; BV57; BV58), and this system is used to construct a tandem expression system containing two independent transcription units: the first transcription unit is generated by... polh The first transcription unit consists of a promoter and its driven target gene I, while the second transcription unit consists of the chimeric promoter and its driven target gene II. In this embodiment, the nucleic acid sequences of target gene I and target gene II are identical (both are coding sequences for BR58). The two transcription units function independently and do not interfere with each other, aiming to increase the total protein yield through the additive effect of the expressed products.

[0134] Regarding the specific implementation method, it should be noted that:

[0135] 1. Regarding the construction of the tandem expression system: Because the initial vector pOET1.1 itself contains a polh Therefore, in this embodiment, the dual promoter system is constructed as follows: the core structure is synthesized to contain "BR58 gene + chimeric promoter (such as Rhr1 + R..."). polh + HSP70 The nucleic acid construct of the "BR58 gene" [the above core structure is not a closed system; conventional structures such as restriction enzyme sites, spacer sequences (located between the promoter and the target gene), and transcription termination signal fragments (located at the 3' end of the target gene) can be added as needed]; then, the nucleic acid construct is ligated into the pOET1.1 vector through the insertion site. polh Downstream of the promoter, a recombinant transfer vector with a dual promoter system was obtained. Finally, the expression and expression level of the target protein were detected according to steps 2 and 3 of Example 1, and compared with the effect of the single promoter system BV33.

[0136] 2. Recombinant transfer vectors with dual promoter subsystems and their structural framework (where a set of square brackets [] corresponds to one transcription unit):

[0137] (1) pOET1-BV55 ( polh hr1+ HSP70 The structural framework of pOET1.1 plasmid backbone sequence is as follows: polh -Interval sequence-Target gene I] - [linker1-hr1-linker2-linker3- HSP70 [-spacer sequence-target gene II] - pOET1.1 plasmid backbone sequence;

[0138] (2) pOET1-BV56 ( polh ;Rhr1+R polh + HSP70 The structural framework of pOET1.1 plasmid is as follows: pOET1.1 plasmid backbone sequence - [ polh -spacer sequence-target gene I] - [ linker2 -Rhr1- linker1 -R polh -linker3- HSP70 - Spacer sequence - Target gene II] - pOET1.1 plasmid backbone sequence; the structure of the recombinant transfer vector pOET1-BV56 using BR58 as the target protein is as follows: Figure 9 As shown;

[0139] (3) pOET1-BV57 ( polhhr1+ p10 + HSP70 The structural framework of pOET1.1 plasmid backbone sequence is as follows: polh -spacer sequence-target gene I] - [linker1-hr1-linker2- p10 -linker3- HSP70 [-spacer sequence-target gene II] - pOET1.1 plasmid backbone sequence;

[0140] (4) pOET1-BV58 ( polh hr1+ p6.9 + HSP70 The structural framework of pOET1.1 plasmid backbone sequence is as follows: polh -spacer sequence-target gene I] - [linker1-hr1-linker2- p6.9 -linker3- HSP70 - Spacer sequence - Target gene II] - pOET1.1 plasmid backbone sequence; the structure of the recombinant transfer vector pOET1-BV58 using BR58 as the target protein is as follows Figure 10 As shown.

[0141] In this embodiment, the expression levels of the target protein corresponding to different promoters are shown in Table 2. Based on the results in Table 2, the corresponding figures are shown below. Figure 6 As shown.

[0142] Table 2. Promoter design and target protein expression level detection results

[0143]

[0144] Table 2 and Figure 6 The display shows that in the second round of promoter screening, only... polh promoter and hr1+ HSP70 The combination of promoters (BV55) failed to outperform a single promoter. polh The expression levels of the other dual-promoter subsystems (BV56, BV57, and BV58) were all superior to those of the single promoter. polh The promoter (BV33) was selected. Therefore, three dual-promoter subsystems, BV56, BV57, and BV58, were chosen for further research.

[0145] Example 4: Expression of target protein using a dual promoter system

[0146] Based on Example 3, three dual-promoter systems—BV56, BV57, and BV58—were selected for further viral amplification and protein expression to verify their genetic stability. Specifically, baculoviruses containing dual-promoter systems were amplified three times in Sf9 insect cells, and the content of the target protein in the supernatant was determined using SDS-PAGE and Western blot. The operating steps are as follows:

[0147] 1. Amplification of baculovirus and expression of target protein: The P1 generation virus harvested in Example 3 was transfected into new Sf9 cell well plates. Seven days after cell infection, the cell supernatant was collected to obtain the P2 generation virus. The P2 generation virus was then used to infect Sf9 cells in logarithmic growth phase (viable cell density 2 × 10⁶ cells / well) in shake flasks at a ratio of 1:60. 6 P3 generation virus was obtained by centrifuging at 27°C with a cell density of 120 rpm and a viability greater than 95% for 3-5 days. The P3 generation virus was cultured at 27°C with shaking at 120 rpm for 3-5 days. When the cell diameter significantly increased and the viable cell density and viability significantly decreased, the cell supernatant was collected by centrifugation at 4000 rpm for 20 min. The cell supernatant was then analyzed by SDS-PAGE and Western blot according to the following methods to determine the content of the target protein in the supernatant.

[0148] 2. SDS-PAGE detection: The cell supernatant and RSV F protein standard (purchased from Sinocare, 2 μg) obtained in step (1) above were mixed with 5× loading buffer at a ratio of 4:1 and heated at 95℃ for 10 min. Then, the processed sample was added to the gel loading wells, the gel was placed in the electrophoresis apparatus, electrophoresis buffer was added, and electrophoresis was performed at 120V for 60 min. The gel was removed and stained with rapid protein staining solution for 60 min, destained with deionized water, and then imaged using a chemiluminescence imaging system.

[0149] 3. Western blot analysis: Place the gel from step (2) above, after electrophoresis, in a transfer clamp and wet it with transfer buffer. Cut a PVDF membrane to the same size as the gel, activate it with methanol, and cover it on the gel. Fix the transfer clamp and place it in the transfer apparatus. Transfer the membrane at 120V for 60min. After the transfer, remove the PVDF membrane and block it in 5% skim milk at room temperature for 1h or overnight at 4℃. After blocking, wash with PBST, add primary antibody (RSV F protein-specific antibody D25), and incubate at room temperature for 1h. After that, wash with PBST, add secondary antibody (HRP Donkey anti-human IgG Antibody), and incubate at room temperature for 1h. After that, wash with PBST again, add developing solution, react in the dark for 30-60s, and image using a chemiluminescence imaging system. The images were analyzed using ImageJ software, and the supernatant protein content of the cell was determined using a sample with a known concentration of RSV F protein.

[0150] SDS-PAGE test results are as follows: Figure 7 As shown on the left, the Western blot detection results are as follows: Figure 7 (Right) and as shown in Table 3.

[0151] Table 3. ImageJ analysis of protein content in cell supernatant

[0152]

[0153] Notes: (1) The ImageJ measurement area in the table is the peak area of ​​the protein band measured by the software in Western blot. (2) The cell supernatant protein content data is calculated from the known concentration of RSV F protein. Taking BV33 as an example, the calculation formula is: BV33 cell supernatant protein content = [(BV33 ImageJ measurement area × RSV F protein loading amount, i.e., 2 μg) / RSV F protein ImageJ measurement area] / BV33 loading volume × BV33 loading dilution factor.

[0154] Figure 7 The results in Table 3 show that:

[0155] 1. polh The protein yield of the single promoter system (BV33) was 5.69 μg / mL;

[0156] 2. polh With Rhr1+R polh + HSP70 The protein yield of the dual promoter system (BV56) was 22.09 μg / mL, which was 3.88 times that of BV33.

[0157] 3. polhWith hr1+ p6.9 + HSP70 The protein yield of the dual promoter system (BV58) was 70.09 μg / mL, which is 12.32 times that of BV33.

[0158] 4. polh With hr1+ p10 + HSP70 The protein yield of the dual promoter system (BV57) was 3.19 μg / mL, significantly lower than that of the single promoter system. polh The use of promoter baculovirus (BV33) demonstrated that not all dual-promoter systems consistently increase protein yield. This may be because both promoters are at a very late stage. polh and p10 Competition for transcriptional resources may exist, which may cause dual promoters to be less effective than single promoters.

[0159] The above data demonstrate that specific dual promoter expression systems (BV56, BV58) can significantly increase the yield of target proteins in insect cell-baculovirus expression systems, and exhibit good genetic stability after three amplifications in Sf9 cells. However, not all dual promoter systems can achieve the expected results, and specific experiments are needed to verify them. It is important to emphasize that the target protein used in the examples, namely the RSV F recombinant protein, belongs to the "complex protein that needs to be correctly assembled into a trimer and maintain a specific conformation" mentioned in the background art. After expression, it needs to self-assemble into a trimer, and the trimer has two conformations: Pre-F (before fusion) and Post-F (after fusion). Pre-F is the main conformation that induces highly efficient neutralizing antibodies and has significantly better immunogenicity than Post-F. However, the Pre-F conformation is inherently unstable and easily irreversibly converts to Post-F. Therefore, the expressed RSV F protein monomer needs to be able to assemble into a trimer and maintain a stable Pre-F conformation. This places extremely high demands on the expression system. As a result, expression systems that can express ordinary recombinant proteins usually have problems such as low expression levels, insufficient trimer assembly efficiency, and a high proportion of Post-F when facing complex proteins like RSV F with unstable conformations. This makes it difficult to meet the process and cost requirements of RSV recombinant protein vaccines. The experimental data provided in this embodiment show that the BV56 and BV58 dual promoter expression system solves the problem of high-level expression of complex proteins such as RSV F, which are conformationally unstable and require trimerization. This is beneficial for the subsequent development of RSV recombinant protein vaccines and therefore has important industrial value.

[0160] Explanation of the mechanism by which a specific dual-start subsystem exhibits higher expressive efficiency:

[0161] 1. A dual-promoter system uses two different promoters to drive the transcription of a target gene, compared to a single promoter system. polhThe promoter can produce more transcripts of the target gene, thus enhancing the expression of the target protein.

[0162] 2. Regarding the two promoters in a chimeric promoter: (1) Although the two promoters in a chimeric promoter may interfere with each other (which may be the reason why some chimeric promoters are not as effective as single promoters), the experimental data of the better-performing chimeric promoters (such as BV46 and BV51) have already demonstrated that there is no significant adverse interference between these promoters. (2) Among them, the reason why avoiding interference and bringing better results may be: early promoters HSP70 With late promoter p6.9 or very late promoter polh They can complement each other when forming, avoid competition between promoters, and prolong the expression window of the target gene, thereby enhancing the expression of the target protein.

[0163] 3. Regarding the orientation setting of hr1 in a chimeric promoter: (1) Chimeric promoters with reverse orientation (such as Rhr1+R) polh + HSP70 The good expression efficiency of hr1 indicates that its enhancing effect does not depend on direction but mainly on its physical structure (nucleic acid sequence). This may be because: hr1 mainly enhances transcription initiation by recruiting the host factor hr1-BP and alleviates chromatin-mediated transcriptional repression by changing chromatin structure (Venkaiah B, et al. Biochemistry. 2004Jun 29;43(25):8143-51). Therefore, hr1 set in the opposite direction of transcription can sometimes also enhance expression. (2) Since hr1 itself may be transcribed (Moldován N, et al. Sci Rep. 2018 Jun 5;8(1):8604), in the case of a promoter set upstream of hr1 (such as Rhr1+R), polh Situation, among which polh (equivalent to the promoter upstream of HR1 transcription), HR1 may be transcribed and enhanced in the vicinity through some still unknown pattern. HSP70 Promoter activity is enhanced, thereby increasing the transcription of the target gene; this is also the possible reason why reversed promoters in chimeric promoters play an active rather than disruptive role.

[0164] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Based on the above preferred embodiments, those skilled in the art are capable of making some conventional modifications to the technical solutions described in the embodiments, or making equivalent substitutions for some of the technical features, such as, but not limited to:

[0165] 1. In the dual promoter subsystem, the two promoter units can be arranged in series (located on one DNA strand) or in parallel (located on different DNA strands).

[0166] 2. In the dual promoter system, the two promoters can be the same or different target genes to meet different expression needs (when they are the same, they are used to increase the total protein yield through the superposition effect of the expression products; when they are different, they are used to realize the expression of different genes in the same expression system).

[0167] All conventional modifications and equivalent substitutions made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A nucleic acid construct, characterized in that, The nucleic acid construct includes a chimeric promoter, which includes an enhancer element, promoter X and promoter Y in tandem. The enhancer element, promoter X and promoter Y are hr1, p6.9 and hsp70, respectively, and the sequences of hr1, p6.9 and hsp70 are shown in SEQ ID NO: 3, 6 and 7, respectively. The structure of the chimeric promoter is as follows: connector I - enhancer element - connector II - promoter X - connector III - promoter Y; Specifically, connector I adopts the sequence shown in SEQ ID NO: 8, connector II adopts the sequence shown in SEQ ID NO: 9, and connector III adopts the sequence shown in SEQ ID NO:

10.

2. The nucleic acid construct according to claim 1, characterized in that, The sequence of the chimeric promoter is shown in SEQ ID NO:

25.

3. The nucleic acid construct according to claim 1 or 2, characterized in that, The nucleic acid construct also includes a recombinant protein-coding gene, which is linked downstream of the chimeric promoter.

4. The nucleic acid construct according to claim 3, characterized in that, The recombinant protein is RSV F recombinant protein.

5. The nucleic acid construct according to claim 4, characterized in that, The amino acid sequence of the RSV F recombinant protein is shown in SEQ ID NO:

1.

6. The nucleic acid construct according to claim 5, characterized in that, The sequence of the RSV F recombinant protein encoding gene is shown in SEQ ID NO:

2.

7. A nucleic acid construct, characterized in that, The nucleic acid construct includes a dual-promoter subsystem, which comprises a first promoter and a second promoter, wherein: i) The first promoter is a single polh promoter; ii) The second promoter is the chimeric promoter as described in claim 1 or 2; Furthermore, insertion sites are provided downstream of the first promoter and the second promoter, respectively.

8. The nucleic acid construct according to claim 7, characterized in that, The nucleic acid construct is a baculovirus transfer vector.

9. The nucleic acid construct according to claim 8, characterized in that, The baculovirus transfer vector is constructed using pOET1.1 as its backbone and includes the first promoter and the second promoter; wherein the second promoter is inserted into a region of pOET1.1 other than the transcription unit containing the first promoter.

10. The nucleic acid construct according to any one of claims 7-9, characterized in that, The nucleic acid construct also contains two copies of a recombinant protein-coding gene; wherein one copy of the recombinant protein-coding gene is linked downstream of the first promoter, and the other copy is linked downstream of the second promoter.

11. The nucleic acid construct according to claim 10, characterized in that, The recombinant protein is RSV F recombinant protein.

12. The nucleic acid construct according to claim 11, characterized in that, The amino acid sequence of the RSV F recombinant protein is shown in SEQ ID NO:

1.

13. The nucleic acid construct according to claim 12, characterized in that, The sequence of the RSV F recombinant protein encoding gene is shown in SEQ ID NO:

2.

14. An insect cell-baculovirus expression system, wherein the expression system is obtained by co-transfecting insect cells with the nucleic acid construct according to any one of claims 3-6 or any one of claims 10-13.

15. The expression system according to claim 14, characterized in that, The baculovirus is the alfalfa silver-striped noctuid moth nucleopolyhedrovirus.

16. The expression system according to claim 14 or 15, characterized in that, The insect cells were Sf9 insect cells.

17. A method for preparing recombinant protein, characterized in that, The insect cell-baculovirus expression system according to any one of claims 14-16 is used to culture the expression system and harvest the recombinant protein from the culture supernatant.

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