Modified polyhedrin promoter

Abstract

This invention provides a polyhedrin promoter that enables efficient expression of target proteins in baculovirus protein expression systems. [Solution] A method for producing a protein is provided, comprising: a modified polyhedrin promoter for a baculovirus protein expression system in which one or more A's in the A-rich sequence region of a burst sequence are replaced with T's; a DNA construct containing the modified polyhedrin promoter; and the preparation of the DNA construct containing a DNA sequence encoding a target protein.

Description

Technical Field

[0001] The present invention relates to a gene expression system using a baculovirus vector. In particular, the present invention relates to the modification of a polyhedrin promoter for increasing the gene expression level by a baculovirus vector.

Background Art

[0002] Baculovirus is a general term for viruses belonging to the family Baculoviridae, and is a large DNA virus that infects insects. The family Baculoviridae is roughly divided into two types: nucleopolyhedrovirus (NPV) and granulovirus (GV). In particular, research on NPV has advanced, and the baculovirus expression vector used for the expression of foreign genes is a vector developed based on NPV. When expressing a foreign gene, when cells derived from insects of the family Bombycidae (such as the BmN-4 cell line) are used as the host Bombyx mori a nuclear polyhedrosis virus (BmNPV) expression vector is used, and when cells derived from insects of the family Noctuidae (such as the Sf-9 cell line, High Five cell line, etc.) are used as host cells Autographa californica a nuclear polyhedrosis virus (AcMNPV) expression vector is used.

[0003] When a nuclear polyhedrosis virus infects a host cell, a proteinaceous crystal called a "polyhedron" in which hundreds of virus particles are encapsulated is produced in large quantities in the final stage of infection. The main component of the polyhedron is a protein called polyhedrin synthesized by the virus, and it comes to account for several tens of percent of the total protein in the infected cells. The polyhedrin gene ( polhBecause the promoter is very potent, a system of baculovirus expression vectors utilizing this promoter has been developed. Within the polyhedrin promoter sequence, there is a cis-element called a "burst sequence" in the approximately 50 bp region between the transcription start site and the translation start site, to which very late expression factor 1 (VLF-1) binds. This burst sequence is in the terminal phase. polh It is essential for the transcription burst (Non-Patent Literature 1). Modified polyhedrin promoters containing multiple burst sequences have been reported to date as polyhedrin promoters that highly express target proteins (Patent Literature 1), but there is a growing expectation for the development of even more potent modified polyhedrin promoters. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2008-301792 [Non-patent literature]

[0005] [Non-Patent Document 1] Rankin et al., Gene 70:39-49, 1988. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In view of the above circumstances, the present invention aims to provide a polyhedrin promoter that enables efficient expression of a target protein in a baculovirus protein expression system. [Means for solving the problem]

[0007] The burst sequence in the polyhedrin promoter, which is essential for protein expression by baculovirus expression vectors, is highly conserved; for example, its sequence is identical in BmNPV and AcMNPV (see A in Figure 1, SEQ ID NO: 1). Therefore, it is generally known that introducing mutations into the burst sequence reduces protein expression. However, the inventors have found that changing the 5'-AATAAAAAAA-3' (SEQ ID NO: 2) sequence, which is located within approximately 20 bp upstream from the translation start site in the wild-type polyhedrin promoter sequence, specifically the 5'-AATAAAAAAA-3' (SEQ ID NO: 2) sequence located between -19 and -10 when the polyhedrin translation start site is set to +1, to 5'-AATTTTAAAA-3' (SEQ ID NO: 3) increases gene expression by the modified polyhedrin promoter by approximately 6 to 7 times compared to the wild type. Furthermore, it was confirmed that gene expression increased to approximately 2 to 6 times the wild type when 5'-AATAAAAAAA-3' (sequence number 2) was changed to 5'-AATTTAAAAA-3' (sequence number 4), 5'-AATTATAAAA-3' (sequence number 5), or 5'-AATATTAAAA-3' (sequence number 6). This invention was completed based on the above findings.

[0008] In other words, the present invention is as follows (1) to (13). (1) A modified polyhedrin promoter for baculovirus protein expression systems, wherein one or more A's in the A-rich sequence region of the burst sequence are replaced with T's. (2) The modified polyhedrin promoter described in (1) above, wherein the burst sequence is the base sequence represented by SEQ ID NO: 1, and the A-rich sequence region is 5'-AATAAAAAAA-3' (SEQ ID NO: 2), which is located in the region from the 34th to the 43rd base sequence represented by SEQ ID NO: 1. (3) The modified polyhedrin promoter described in (2) above, wherein one, two, or three of the fourth, fifth, and sixth A atoms in the A-rich sequence region represented by Sequence ID No. 2 are replaced with T. (4) The modified polyhedrin promoter described in (3) above, wherein the fourth, fifth, and sixth A in the A-rich sequence region represented by Sequence ID No. 2 are replaced with T. (5) The modified polyhedrin promoter described in (3) above, wherein the fourth and fifth A in the A-rich sequence region represented by Sequence ID No. 2 is replaced with T. (6) The modified polyhedrin promoter described in (3) above, wherein the fourth and sixth A in the A-rich sequence region represented by Sequence ID No. 2 are replaced with T. (7) The modified polyhedrin promoter described in (3) above, wherein the nucleotide sequence of the A-rich sequence region represented by Sequence ID No. 2 has the 5th and 6th A substituted with T. (8) A DNA construct comprising the modified polyhedrin promoter described in any of (1) through (7) above. (9) The DNA construct described in (8) above, which is plasmid DNA, transient expression plasmid, recombinant buckmid DNA, or recombinant baculovirus DNA. (10) A protein expression kit comprising the DNA construct described in (8) above. (11) A method for producing a protein, comprising preparing the DNA construct described in (8) above, which includes a DNA sequence encoding a target protein. (12) The method for producing the protein according to (11) above, wherein the DNA construct is recombinant baculovirus DNA. (13) The method for producing the protein according to (12) above, wherein the baculovirus DNA is AcMNPV DNA or BmNPV DNA. In this specification, the symbol "~" indicates a numerical range that includes the values ​​to its left and right. [Effects of the Invention]

[0009] The modified polyhedrin promoter provided by this invention can strongly induce the expression of a target gene linked downstream thereof. Therefore, in a protein expression system using a baculovirus expression vector, it becomes possible to express a significantly larger amount of protein compared to when using conventional vectors. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the results of measuring the activity of a modified polyhedrin promoter in which a mutation (substitution of A with T) was introduced into the A-rich region (SEQ ID NO: 2) of the burst sequence. A shows the nucleotide sequences of the burst sequences of BmNPV and AcMNPV. The nucleotide sequences shown here are listed in the sequence listing as SEQ ID NO: 1. TSS indicates the transcription start site. B shows the nucleotide sequences of the A-rich region of mutant viruses (Mut1, Mut2, Mut3, and Mut4) in which A in the A-rich region was substituted with T. C shows the results of measuring luciferase activity using each of the viruses shown in B. [Figure 2] Figure 2 shows the results of measuring the activity of modified polyhedrin promoters with varying numbers of Mut2 T mutations. A shows the nucleotide sequence of the A-rich region of the modified polyhedrin promoter with varying numbers of Mut2 T mutations. B shows the results of measuring luciferase activity using each virus shown in A. C shows the results of measuring luc mRNA and polh UTR mRNA levels using each virus shown in A by RT-qPCR. [Figure 3]Figure 3 shows the results of promoter activity analysis when a TTT substitution is introduced into the polyhedrin promoter of a high-expression vector containing a polh coding region (cds). A shows the nucleotide sequences of the A-rich sequence region of Luc30 used as a control and the A-rich sequence region of Luc30-TTT with the TTT mutation introduced. B shows the results of measuring luciferase activity using each virus shown in A. C shows the results of measuring luc mRNA levels by RT-qPCR using each virus shown in A. Results using the luc sequence or polh UTR sequence as PCR primers are shown. [Figure 4] Figure 4 shows the analysis results of AcMNPV with the TTT mutation introduced into the A-rich sequence region. A shows the nucleotide sequences of the A-rich sequence regions of pFB1-GFP and pFB1-Luc used as controls, and the A-rich sequence regions of pFB1-TTT-GFP and pFB1-TTT-Luc with the TTT mutation introduced. B shows the results of measuring GFP expression levels using each virus shown in A. C shows the results of measuring luciferase activity using each virus shown in A. [Figure 5] Figure 5 shows the results of investigating the effect of the TTT mutation on the expression of wild-type virus polh. A shows the nucleotide sequences of the A-rich sequence region of T3-WT used as a control and the A-rich sequence region of T3-WT-TTT with the TTT mutation introduced. B shows the results of measuring the amount of polh mRNA using each virus shown in A by RT-qPCR. Results using the polh cds sequence or polh UTR sequence as PCR primers are shown.

[0011] The following describes embodiments for carrying out the present invention. When referring to "this embodiment," unless otherwise specified, it refers to all embodiments described herein. The first embodiment is a modified polyhedrin promoter for baculovirus protein expression systems (hereinafter also referred to as "the modified promoter according to this embodiment") in which one or more A's in the A-rich sequence region of the burst sequence are replaced with T's. The "burst array" according to this embodiment is not particularly limited as long as it is a sequence included in the polyhedrin promoter sequence existing on the genomic DNA of baculovirus. For example, the sequence represented by SEQ ID NO: 1, which is the burst array of BmNPV and AcMNPV, is particularly preferred. SEQ ID NO: 1: (Burst array of BmNPV and AcMNPV) 5’-ATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAAT-3’ The "A-rich sequence region" in the burst array refers to a region where at least 7 or more A's are continuously present. More specifically, for example, in the case of the burst array consisting of the base sequence represented by SEQ ID NO: 1, it is the sequence region from the 34th to the 43rd of the base sequence, which is the region consisting of the base sequence represented by 5’-AATAAAAAAA-3’ (SEQ ID NO: 2). In addition, the "burst array" according to this embodiment includes a base sequence having a sequence identity of preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more, most preferably 99% or more, with the base sequence represented by SEQ ID NO: 1, or a base sequence in which preferably about 1 to 5, about 1 to 3, most preferably about 1 or 2 nucleotides are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1, where the above A-rich sequence region is conserved, and the activity of the polyhedrin promoter containing the burst array is the same as or higher than the activity of the wild-type polyhedrin promoter. Here, the promoter activity can be evaluated using the amount of the transcription product from the gene transcriptionally regulated by the promoter as an index. Specifically, for example, the activity of the promoter can be evaluated by the reporter assay described in the examples.

[0012] The inventors of the present invention have found for the first time that when one, two or three A's present in the A-rich sequence region included in the burst sequence of the polyhedrin promoter are replaced with T, the activity of the polyhedrin promoter becomes about 2 to 6 times stronger compared to the wild-type polyhedrin promoter. Examples of the base sequences of the A-rich sequence regions that increase the activity of the polyhedrin promoter containing the burst sequence consisting of the base sequence represented by SEQ ID NO: 1 include the base sequences represented by the following SEQ ID NOs: 3 to SEQ ID NO: 9. A-rich array with 3 A's replaced by T's SEQ ID NO: 3 (TTT); 5'-AATTTTAAAA-3' (sequence obtained by replacing the 4th, 5th and 6th A's of SEQ ID NO: 2 with T) A-rich array with two A's replaced by T's SEQ ID NO: 4 (T12); 5'-AATTTAAAAA-3' (sequence obtained by replacing the 4th and 5th A's of SEQ ID NO: 2 with T) SEQ ID NO: 5 (T13); 5'-AATTATAAAA-3' (sequence obtained by replacing the 4th and 6th A's of SEQ ID NO: 2 with T) SEQ ID NO: 6 (T23); 5'-AATATTAAAA-3' (sequence obtained by replacing the 5th and 6th A's of SEQ ID NO: 2 with T) A-rich array with one A replaced by a T SEQ ID NO: 7 (T1); 5'-AATTAAAAAA-3' (sequence obtained by replacing the 4th A of SEQ ID NO: 2 with T) SEQ ID NO: 8 (T2); 5'-AATATAAAAA-3' (sequence obtained by replacing the 5th A of SEQ ID NO: 2 with T) SEQ ID NO: 9 (T3); 5'-AATAATAAAA-3' (sequence obtained by replacing the 6th A of SEQ ID NO: 2 with T)

[0013] The second embodiment is a DNA construct (DNA construct) containing the modified promoter according to the present embodiment. The DNA construct according to this embodiment includes a construct containing the modified promoter according to this embodiment, and encompasses all DNA constructs used for the purpose of expressing proteins using baculovirus systems and insect cell transient expression systems. Examples of DNA constructs according to this embodiment include plasmid DNA such as transfer vectors, donor plasmids, and transient expression plasmids, as well as baculovirus DNA and baculovirus DNA.

[0014] The third embodiment is a protein expression kit for use in a baculovirus protein expression system or an insect cell transient expression system, comprising one or more DNA constructs containing the modified promoter according to this embodiment (hereinafter also referred to as "the kit according to this embodiment"). The kit according to this embodiment includes, as essential components, one or more DNA constructs containing the modified promoter according to this embodiment, such as a transfer vector, a donor plasmid, a transient expression plasmid, buckmid DNA (recombinant buckmid DNA), or baculovirus DNA (recombinant baculovirus DNA) (recombinant buckmid DNA is included in recombinant baculovirus DNA). Furthermore, the kit may also include insect cells for protein expression (e.g., BmN-4 cell line, Sf-9 cell line, High Five cell line, etc.), reagents for introducing the DNA construct into cells, etc.

[0015] The fourth embodiment is a method for producing a protein, which includes a step of preparing a DNA construct (i.e., the DNA construct according to the second embodiment) that includes a DNA sequence encoding a target protein (the protein to be produced) and a modified promoter according to this embodiment. (This is also referred to as the "method for producing a protein according to this embodiment"). The protein production method according to this embodiment includes expressing the target protein in a baculovirus protein expression system, and includes at least the step of constructing a DNA construct that includes the DNA sequence encoding the target protein and the modified promoter according to this embodiment. Recombinant baculoviruses for protein expression can be easily produced by those skilled in the art, for example, by producing recombinant baculoviruses in insect cells by producing recombinant baculoviruses in E. coli, transfecting insect cells with the recombinant baculoviruses in insect cells, or by co-transfecting insect cells with a transfer vector containing the DNA encoding the target protein and linearized baculovirus genomic DNA. However, the method is not limited to these methods. For more details on baculovirus protein expression systems, see, for example, Irons et al., Curr Protoc Protein Sci. 2018 Feb 21:91:5.5.1-5.5.22. doi: 10.1002 / cpps.45. Protein Production Using the Baculovirus Expression System; Chambers et al., Curr Protoc Protein Sci. 2018 Feb 21:91:5.4.1-5.4.6. doi: 10.1002 / cpps.47. Overview of the Baculovirus Expression System; and van Oers et al., Review J Gen Virol. 2015 Jan;96(Pt 1):6-23. doi: 10.1099 / vir.0.067108-0. Epub 2014 Sep 22. Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology.

[0016] The recombinant baculovirus created is, in the case of AcMNPV, the fall armyworm ( Spodoptera frugiperda ) derived from Sf-9 and Sf-21, and nettle moth ( Trichoplusia ni The target protein may be expressed by infecting cultured cells such as High Five derived from ) or other organisms. In the case of cultured cells, BmNPV may be expressed by infecting silkworm ovary-derived BmN-4 cells, but if expression in cultured cells is difficult, or if expression occurs but the activity is low, it may be expressed using silkworm larvae or pupae. The expressed proteins can be purified using various methods, such as purification with tags, gel filtration, and desalting. Secreted proteins can be purified from the infected cell culture medium, while other proteins can be purified from the infected cell lysate. Furthermore, methods for producing virus-like particle (VLP) vaccines, which involve highly expressing the outer skin protein of the virus and forming virus-like particles within insect cells, may also be used for vaccine antigen production.

[0017] Where this specification is translated into English and contains the singular forms "a," "an," and "the," they shall be considered to include both singular and plural forms unless the context clearly indicates otherwise. Furthermore, in this specification, "approximately" or "to what extent" means a numerical range of ±10%. The present invention will be further explained below with reference to examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Examples]

[0018] 1. Materials and Methods 1-1. Preparation of transfer vectors (donor vectors) pBm31 was used as the transfer vector for the creation of recombinant BmNPV (Ko et al., J. Virol. 74, 11240-11246 2000). pFastBac1 (Thermo Fisher Scientific) was used as the donor vector for the creation of recombinant AcMNPV. The KOD-Plus Mutagenesis Kit (Toyobo) was used for introducing mutations into each vector, and the In-Fusion HD Cloning Kit (TaKaRa) was used for inserting foreign genes.

[0019] 1-2. Creation of recombinant viruses Recombinant BmNPV was prepared by co-transfection of BmN-4 cells with a linearized viral genome (Zhou et al., Virology 240:282-292, 1998) and a transfer vector using X-tremeGENE HP DNA Transfection Reagent (Roche Applied Science), followed by homologous recombination. Clones were isolated from the viral suspension by the plaque assay, and viruses containing the target mutation were identified and used by PCR and DNA sequencing. The viral infectivity titer was quantified by the plaque assay. The Bac-to-Bac system (Thermo Fisher Scientific) was used to produce recombinant AcMNPV. Recombinant donor vectors were introduced into E. coli DH10Bac to create recombinant buckmid within the E. coli. PCR was used to identify E. coli cells exhibiting the expected translocation, and buckmid DNA was purified from these cells. The buckmid DNA was transfected into cultured Sf-9 cells to create recombinant viruses. The viral infectivity titer was determined using the TCID. 50 Quantitative analysis was performed using the method described below.

[0020] 1-3. Luciferase assay BmN-4 cells were infected with recombinant BmNPV, or Sf-9 cells were infected with recombinant AcMNPV at a multiple infection degree (MOI) of 5, and the cells were harvested after 3 days. Luciferase activity was measured using the Luciferase Assay System (Promega).

[0021] 1-4. Quantitative RT-PCR (RT-qPCR) RNA was extracted from BmN-4 or Sf-9 cells two days after viral infection using TRI Reagent (Sigma-Aldrich). First-strand cDNA was extracted using Reverse Transcriptase XL (AMV) (TaKaRa), and RT-qPCR was performed using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems) and the StepOne Real-Time PCR System (Applied Biosystems). The primers used are as follows: polh coding T3COpolh-qF:5'-T(G / T)GGCATGA ACAACGAATAC-3'(Sequence ID 10) T3COpolh-qR:5'-TGTAGAAGTTCTCCCATATG-3'(Sequence ID 11) polh UTR polhUTR-F:5'-TCTGTGCGTTGTTGATTTAC-3'(Sequence ID 12) polhUTR-R:5'-AGCTAGATTGCTGCTACAAG-3'(Sequence ID 13) luc luc-qF:5'-TCACTTACGCTGAGTACTTC-3'(SEQ ID NO: 14) luc-qR:5'-CTGTTGAGCAATTCACGTTC-3'(Sequence ID 15)

[0022] 1-5. Determination of GFP and POLH (polyhedrin) BmN-4 cells were infected with recombinant BmNPV, or Sf-9 cells with recombinant AcMNPV, at a multiple infection degree (MOI) of 5. Cells were collected after 3 days. POLH and GFP expression were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie brilliant blue (CBB) staining, and images were acquired using the ChemiDoc XRS+ imaging system (Bio-Rad). The amount of the corresponding bands was quantified using Image Lab software (Bio-Rad).

[0023] 1-6. Measurement of Polygonal Numbers BmN-4 cells were infected with recombinant BmNPV at a multiple infection degree (MOI) of 5 and collected after 3 days. The cell pellet was lysed with 0.1% SDS, and the number of polyhedrons was measured using a hemocytometer.

[0024] 2.Results 2-1. Analysis of viruses in which mutations have been introduced into the burst sequence In the two viruses commonly used as baculovirus vectors (BEVS: Baculovirus Expression Vector Systems), BmNPV and AcMNPV... polh The burst sequences are exactly the same (Figure 1A, Sequence ID 1). From previous studies, the burst sequences are polh It is important for expression levels, and it is known that introducing mutations reduces its expression (Ooi et al., J Mol Biol. 210:721-36, 1989). Using the luciferase (Luc) reporter expression BmNPV, the A-rich sequences (-19 to -10; translation start site is +1) within the burst sequence are polhThe effect on promoter-dependent reporter expression was investigated. For the experimental method, Luc-SP (Katsuma and Matsuda-Imai, Biochem. Biophys. Res. Commun. 679:1-5, 2023), which contains 46 base pairs derived from the pGL-3 vector, was used as the wild-type (WT) strain. Four recombinant viruses, Mut1 (all 7 A's from -16 to -10 replaced with T), Mut2 (all 3 A's from -16 to -14 replaced with T), Mut3 (all 4 A's from -13 to -10 replaced with T), and Mut4 (all 2 A's from -19 to -18 replaced with T), were created by cotransfecting cultured cells BmN-4 with a linearized viral genome and a transfer vector into which the mutations were introduced, and then performing homologous recombination. As shown in the figure, the recombinant viral genome also contains 46 base pairs derived from the pGL-3 vector and a linker sequence derived from the transfer vector pBm31 (Figure 1B).

[0025] Each virus shown in Figure 1B was used to infect silkworm BmN-4 cells at an MOI of 5. The cells were collected after 3 days, and luciferase activity was quantified using the Luciferase Assay System (Promega). As a result, Mut1 and Mut2 showed a 4-5 fold increase in activity. Mut3 showed no change, and Mut4's activity was reduced by approximately half. This indicates that the TTT mutation in Mut2 contributes to the increased activity. From these results, it was found that replacing all three A cells from -16 to -14 with T cells unexpectedly increased reporter expression. On the other hand, replacing the four A cells from -13 to -10 had little effect, and replacing the two A cells from -19 to -18 with T cells decreased expression.

[0026] 2-2. Detailed analysis of Mut2 TTT mutations Mutant viruses were created by altering the number of T mutations in Mut2 (TTT). Viruses were created with only one A substitution (T1, -16A replaced with T; T2, -15A replaced with T; T3, -14A replaced with T), and viruses with two A substitutions (T12, -16~-15 A replaced with T; T13, -16 and -14 A replaced with T; T23, -15~-14 A replaced with T) (Figure 2A). Recombinant viruses were created in the same manner as shown in Figure 1B. Luciferase activity was measured using each virus shown in Figure 2A. When only one A was replaced with T, T1 showed almost the same activity as WT, while T2 and T3 showed approximately twice the activity of WT. When two substitutions were made, T12 and T13 showed approximately five times the activity of WT, and T23 showed approximately 2.5 times the activity of WT, but the rate of increase was lower compared to Mut2, in which all were replaced with T (Figure 2B). Next, using each virus shown in Figure 2A, luc mRNA and polh The transcription level of UTR mRNA was evaluated by RT-qPCR. Each virus shown in Figure 2A infected silkworm BmN-4 cells at MOI=5, and the cells were harvested after 2 days. RNA was extracted using TRI Reagent (Sigma-Aldrich). Reverse Transcriptase XL (AMV) (TaKaRa) was used to prepare first-strand cDNA, and RT-qPCR was performed using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems) and the StepOne Real-Time PCR System (Applied Biosystems). PCR primers were selected based on the reporter luc sequence and the downstream sequences of luc. polh The UTR sequence was used. The RT-qPCR results showed a similar trend to the luciferase assay regardless of which primer was used. Specifically, T1 showed almost no change from WT, T2 and T3 showed a slight increase, T12 and T13 showed approximately 3-4 times the activity of WT, and T23 showed approximately 2 times the activity. However, the rate of increase was lower compared to Mut2, in which all T cells were substituted. From these results, it was revealed that a slight increase in expression was observed with one nucleotide substitution, a significant increase with two nucleotide substitutions, and the increase in expression was maximized with three nucleotide substitutions. Furthermore, it was found that the increase in reporter activity was due to an increase at the mRNA level.

[0027] 2-3. polh Analysis of promoter activity when TTT substitutions are introduced into A-rich sequences of high-expression vectors containing coding regions (cds). It is affected by BEVS polh Promoter-based transfer vectors, or donor vectors, can be broadly divided into two types. polh These are either cds-containing or cds-free. Luckow and Summers modified the AcMNPV transfer vector by inserting or deleting at various sites, resulting in 34-base or 35-base sequences. polh Results have shown that vectors containing cds exhibit high expression (Luckow and Summers, Virology 170:31-39, 1989). Based on this sequence, donor vectors for the Bac-to-Bac system (such as pFastBac1) have been created and are now widely used. The present inventors used BmNPV polh Random mutations were introduced into the cds, and this virus also polh We have shown that the expression of a polyhedrin promoter-dependent reporter increases when cds contains 30 base pairs (Katsuma and Matsuda-Imai, J Mol Biol. 436:168595, 2024). The results shown in Figures 1 and 2 are: polh Since the results were obtained based on a vector that does not contain cds, polh We investigated whether the TTT mutation contributes to increased expression even when a high-expression vector containing 30 cds is used as the base. The control used was: polhThis is a Luciferase reporter virus containing 30 csd bases (Katsuma and Matsuda-Imai, J Mol Biol. 436:168595, 2024), and a Luciferase reporter virus called Luc30-TTT was created by introducing the TTT mutation based on this (Figure 3A).

[0028] Luciferase activity was measured using the virus shown in Figure 3A. Introducing the TTT mutation resulted in an approximately 1.4-fold increase in activity compared to Luc30 (Figure 3B). Next, each virus shown in Figure 3A was used to infect silkworm BmN-4 cells with an MOI of 5, and the cells were collected after 2 days and subjected to RT-qPCR. The PCR primers were based on the sequence of the reporter luc and the downstream sequences of luc. polh The UTR sequence was used. The RT-qPCR results showed a similar trend to the luciferase assay regardless of which primer was used. In other words, it was found that the introduction of the TTT mutation increased the reporter mRNA level by approximately 1.4 to 1.5 times. Based on the above results, polh In cds-addition vectors, the TTT mutation was found to increase reporter expression at the mRNA level.

[0029] 2-4. Construction of AcMNPV by introducing the TTT mutation into the A-rich sequence Up to this point, we have presented the results of our investigation into the effects of TTT mutations using BmNPV, but globally, BEVS systems based on AcMNPV are more commonly used. Therefore, we investigated the effects of TTT mutations in AcMNPV using a Bac-to-Bac system. pFastBac1 was used as the donor vector, but this is polhThis type contains cds. Luciferase and green fluorescent protein (GFP) were used as reporters. For recombinant AcMNPV production, recombinant donor vectors were introduced into E. coli DH10Bac to produce recombinant buckmid within the E. coli. E. coli cells exhibiting the expected translocation were identified by PCR, and buckmid DNA was purified from these cells. The buckmid DNA was transfected into cultured Sf-9 cells to produce recombinant viruses. The viral infectivity titer was determined using TCID. 50 Quantitative analysis was performed using the following method. pFB1-TTT-GFP or pFB1-TTT-Luc reporter viruses were created by introducing the TTT mutation into the A-rich sequence of the pFB1-GFP or pFB1-Luc reporter virus used as a control (Figure 4A).

[0030] GFP expression levels were investigated using the virus shown in Figure 4A by SDS-PAGE. Sf-9 cells were infected with each recombinant AcMNPV at a multiple infection degree (MOI) of 5 and harvested after 3 days. GFP expression was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie brilliant blue (CBB) staining, and images were acquired using the ChemiDoc XRS+ imaging system (Bio-Rad). The amount of the corresponding band was quantified using Image Lab software (Bio-Rad). As a result, the introduction of the TTT mutation increased GFP expression by approximately 1.5 times. Next, luciferase activity was measured using the virus shown in Figure 4A. Sf-9 cells were infected with each recombinant AcMNPV at a multiple infection degree (MOI) of 5 and harvested after 3 days. The assay results showed that introducing the TTT mutation increased activity by approximately twofold compared to the control. These results indicate that, in the AcMNPV system, TTT mutations further increase expression.

[0031] 2-5. Wild-type virus polhThe effect of TTT mutations on expression The burst sequence of wild-type viruses (strains found in nature) is polh Although it is expected that the expression is optimized, previous experiments have shown that introducing the TTT mutation in BEVS increases reporter expression. Therefore, we investigated how polh expression changes when the TTT mutation is introduced into wild-type viruses. As viruses, we used a control (disclosed in Katsuma and Matsuda-Imai, J Mol Biol. 436:168595, 2024) produced using a transfer vector containing wild-type polh, and a virus in which the TTT mutation was introduced into T3-WT ("T3-WT-TTT") (Figure 5A). The production method was the same as the method shown in "2-1", but in this experiment, both viruses (T3-WT and T3-WT-TTT) polh The gene product, POLH (polyhedrin), crystallizes, and a large number of polyhedra (inclusion bodies) are formed within the cell.

[0032] Each virus shown in Figure 5A was used to infect silkworm BmN-4 cells with an MOI of 5, and the cells were collected after 2 days and subjected to RT-qPCR. The PCR primers were polh The cds sequence and the downstream polh The UTR sequence was used. The RT-qPCR results showed that, regardless of which primer was used, the introduction of the TTT mutation was successful. polh It was found that the mRNA level increased by approximately 1.4 to 1.5 times (Figure 5B).

[0033] Using the virus shown in Figure 5A, the expression level of POLH was investigated by SDS-PAGE. As a result, the introduction of the TTT mutation increased POLH expression by approximately 1.1 to 1.2 times (Figure 5C). Polyhedron production was investigated using the virus shown in Figure 5A. BmN-4 cells were infected with recombinant BmNPV at a multiple infection degree (MOI) of 5 and collected after 3 days. The cell pellet was lysed with 0.1% SDS, and the number of polyhedrons was measured using a hemocytometer. The results showed that the TTT mutant virus tended to produce more polyhedrons than the wild type, but no statistically significant difference was observed. Based on these results, even in wild-type viruses, the introduction of the TTT mutation can lead to... polh It was revealed that this could induce an increase in the expression of [the gene]. [Industrial applicability]

[0034] This invention is a technology for improving the efficiency of protein expression using a baculovirus expression system. Therefore, this invention is expected to be useful in fields that perform protein expression using eukaryotic cells, such as agriculture and medicine.

Claims

1. A modified polyhedrin promoter for baculovirus protein expression systems, in which one or more A's in the A-rich sequence region of the burst sequence are replaced with T's.

2. The modified polyhedrin promoter according to claim 1, wherein the burst sequence is the nucleotide sequence represented by SEQ ID NO: 1, and the sequence of the A-rich sequence region is 5'-AATAAAAAAA-3' (SEQ ID NO: 2), which is located in the region from the 34th to the 43rd nucleotide sequence represented by SEQ ID NO:

1.

3. The modified polyhedrin promoter according to claim 2, wherein one, two, or three of the fourth, fifth, and sixth A atoms in the A-rich sequence region represented by Sequence ID No. 2 are replaced with T.

4. The modified polyhedrin promoter according to claim 3, wherein the fourth, fifth, and sixth A in the nucleotide sequence of the A-rich sequence region represented by Sequence ID No. 2 are replaced with T.

5. The modified polyhedrin promoter according to claim 3, wherein the fourth and fifth A in the nucleotide sequence of the A-rich sequence region represented by Sequence ID No. 2 are replaced with T.

6. The modified polyhedrin promoter according to claim 3, wherein the fourth and sixth A in the nucleotide sequence of the A-rich sequence region represented by Sequence ID No. 2 are replaced with T.

7. The modified polyhedrin promoter according to claim 3, wherein the nucleotide sequence of the A-rich sequence region represented by Sequence ID No. 2 has the 5th and 6th A substituted with T.

8. A DNA construct comprising a modified polyhedrin promoter according to any one of claims 1 to 7.

9. The DNA construct according to claim 8, which is a plasmid DNA, a transient expression plasmid, recombinant buckmid DNA, or recombinant baculovirus DNA.

10. A protein expression kit comprising the DNA construct described in claim 8.

11. A method for producing a protein, comprising preparing a DNA construct according to claim 8, which includes a DNA sequence encoding a target protein.

12. The method for producing a protein according to claim 11, wherein the DNA construct is recombinant baculovirus DNA.

13. The method for producing a protein according to claim 12, wherein the baculovirus DNA is AcMNPV DNA or BmNPV DNA.

Images