KR20120128730A - Method for Enhancing the Expression of Foreign Recombinant Protein Using Fusion Expression with Partial Polyhedrin of Baculovirus - Google Patents

Abstract

The present invention relates to a baculovirus transfer vector, a host cell comprising the transfer vector, and a method for mass production of the target protein using the transfer vector, which can greatly increase the expression efficiency of the foreign expression target protein. . The transfer vector of the present invention fuses a specific portion of the polyhedral protein of the baculovirus to the expression target protein and expresses it under a powerful polyhedral protein promoter, thereby significantly increasing the expression amount of the expression target protein in the baculovirus expression vector system. You can.

Description

Method for Enhancing the Expression of Foreign Recombinant Protein Using Fusion Expression with Partial Polyhedrin of Baculovirus}

The present invention relates to a baculovirus transfer vector system for increasing the expression level of a foreign recombinant protein, a host cell comprising the same, and a method for mass production of a foreign recombinant protein using the same. More specifically, the present invention relates to a baculovirus transfer vector system for expressing a foreign recombinant protein in the form of a fusion protein fused with some peptide or variant thereof of the polyhedral protein of baculovirus.

The Baculovirus Expression Vector System (BEVS) is an expression vector system that uses a powerful promoter of the Nucleopolyhedrovirus (NPV) gene. Because of the excellent biological and immunological activity of foreign proteins expressed by using cells, there are many aspects compared to other expression vector systems using E. coli, yeast, Mammal cells, etc. It has gained much attention because of its advantages (O'Reilly et al., Baculovirus Expression Vectors-A laboratory manual, WH Freeman and Company, New York, 1992). Currently, two expression vector systems using Autographa californica NPV (AcNPV) and Silkworm nuclear polymorphism virus (BmNPV) are widely used (Smith et al., Mol). Cell. Biol. 3: 2156-2165, 1983; Maeda et al., Nature 315: 592-594, 1985). When expressing foreign proteins in insect cells, the baculovirus expression vector has several advantages: it can mass-produce proteins to a degree similar to that of E. coli, and post-translational modifications such as glycosylation. Unlike the case where E. coli and yeast are used as host cells because of the effective post-translational modification, a large amount of foreign protein having a biological activity similar to that of a natural protein can be obtained in a large amount. In addition, the method of using mammalian cells with accurate post-translational modifications can obtain relatively high therapeutic value proteins compared to other systems.However, in the culture and maintenance of mammalian cells, various hormones and growth regulators derived from mammals are obtained. This is required in large quantities, so a significant cost is required. Therefore, the insect baculovirus expression vector system, which is a new production system capable of producing recombinant proteins at low cost, with similar post-translational modifications similar to mammalian cells, has attracted attention for the production of protein drugs.

However, in spite of these advantages, when the foreign protein is expressed in insect host cells by the baculovirus expression vector system using only the polyhedral protein promoter, the amount of the expressed protein may be significantly low. The reason why the foreign protein is expressed at low levels in insect cells is mainly due to the inefficient recognition of the signal sequence of the foreign gene by the host cell or the decrease of the host cell's protein processing ability following infection of the virus. have. In addition, the foreign protein is expressed using a strong promoter of the polyhedral protein, but has a limitation that the expression level of the foreign protein is less than that of the original polyhedral protein. In order to solve this problem, recently, the protein expressed by manipulating the signal sequence of an external gene is secreted to the outside of the host cell to increase its expression, or the expression amount is increased by fusion of a gene sequence serving as an enhancer. Many ways to increase are being developed. For example, when a fusion with the gene of melittin, an important factor for making bee venom, foreign protein was secreted extracellularly, resulting in a successful increase in expression (Tessier, DC, et al., gene, 1991, 98, 177-183). In addition, the expression of ubiquitin genes was improved by fusion with foreign genes, resulting in increased expression, and are currently marketed as Small Ubiquitin-like MOdifer (SUMO) (LifeSensors Inc.). .

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to effectively increase the expression level of the expression target protein in the baculovirus expression vector system. As a result, the fusion of a specific portion of the polymorphic protein of the baculovirus to the expression target protein and expression under a strong polymorphic protein promoter The present invention was completed by confirming that the foreign protein of the purpose of expression can be expressed with high efficiency.

Accordingly, it is an object of the present invention to provide a baculovirus transfer vector capable of greatly increasing the expression efficiency of a foreign expression target protein.

Another object of the present invention is to provide a host cell comprising the baculovirus transfer vector.

Another object of the present invention to provide a method for producing a large amount of the protein of interest using the baculovirus transfer vector.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a promoter of (v) a baculovirus polymeric protein; (Ii) a nucleotide sequence encoding a portion or variant thereof of a baculovirus polymorphic protein operably linked to said promoter; And (iii) one or more restriction enzyme recognition nucleotide sequences from which the nucleotide sequence encoding the protein of interest can be cloned, wherein a portion of the baculovirus polymorphic protein is a peptide consisting of the amino acid sequence of SEQ ID NO: 23 sequence , Wherein the variant is an amino acid substitution variant or truncation variant of the peptide, wherein the expression target protein is expressed in the form of a fusion protein with the peptide or a variant thereof, baculovirus for use in expressing the expression target protein. Provide a transition vector.

The present invention is based on the discovery that when a part or a variant thereof of a polyhedral protein in a baculovirus transfer vector is designed to be expressed by being fused to a front part of an expression target protein, the expression efficiency of the expression target protein is greatly increased. That is, in the baculovirus transfer vector of the present invention, the expression efficiency of the target protein can be successfully improved by expressing a part or a variant thereof of the baculovirus polymorphic protein in a fused form with the expression target protein.

As used herein, the term "baculovirus" refers to an insect pathogenic virus that is not pathogenic to humans or vertebrates, and has pathogenicity only to insects. Baculovirus is largely divided into Nucleopolyhedrovirus (NPV) and granulovirus (GV), the baculovirus in the present invention means the nuclear polyhedral virus.

The term "polymorphic protein of baculovirus" is used herein as a structural protein of baculovirus polymorphs synthesized in large quantities from the nucleus of insect cells at the end of infection (Smith et al., 1993, J. Virol., 46, 584-593), for example, the polyhedral protein of Autographa californica Nucleopolyhedrovirus (AcNPV), the polyhedral protein of Spodoptera exigua nuclear polyhedral virus (Elisabeth et al., 1992, J. gen. Viral., 13, 2813-2821), polyhedral proteins of the Numb polysomal virus (Iatrou et al., 1985, J. Virol., 54, 436-445) In the present invention, the polyhedral protein of the autografpa californica nuclear polyhedron virus (AcNPV) is preferably used.

The term "promoter" as used herein refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Promoter sites are readily recognized by those skilled in the art. That is, an estimated start codon including an ATG motif has been identified, and an upstream site is an estimated promoter site from this start codon.

As used herein, the term "promoter of baculovirus polyhedron protein" refers to a DNA sequence essential for transcription and functional expression of the polyhedral protein, and preferably about upstream of the structural gene of nuclear polyhedron virus (AcNPV) polyhedral protein. 100 bp sequence site, more preferably upstream -1 bp to -52 bp site.

In the present invention, the "part of the baculovirus polymorphic protein" is a peptide consisting of the amino acid sequence of SEQ ID NO: 23 sequence. The peptide of SEQ ID NO: 23 is a peptide consisting of amino acid sequences of 19th to 110th amino acids of the polyhedral protein of Autographa californica nuclear polyhedron virus (AcNPV) (see FIG. 1 (a)). .

According to a preferred embodiment of the present invention, the variant of the polyhedral protein portion is an amino acid substitution variant or truncated mutant of the peptide consisting of the SEQ ID NO: 23 sequence, more preferably, SEQ ID NO: 24 SEQ ID NO: Any one selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31 It is a peptide consisting of the sequence of.

As used herein, the term “baculovirus transfer vector” serves to transfer a DNA fragment encoding an expression target protein fused with a portion or variant thereof to a DNA genome of baculovirus. A vector, which is generally used in the same sense as the expression vector of baculovirus.

The nucleotide sequence encoding a portion of the baculovirus polymorphic protein or variant thereof in the transfer vector of the invention is operatively linked to the promoter of the baculovirus. As used herein, the term “operably linked” means a functional linkage between a nucleic acid expression control sequence (eg, a promoter sequence) and another nucleic acid sequence, whereby the regulatory sequence is capable of transcription and / or transduction of the other nucleic acid sequence. You will control the translation.

The transition vector of the present invention includes one or more restriction enzyme recognition nucleotide sequences, ie, multiple cloning site (MCS) sequences, from which the nucleotide sequence encoding the protein of interest can be cloned. As the restriction enzyme, for example, Eag I, EcoR I, EcoR II, BamH I, Bgl II, BstB I, Hind III, Taq I, Not I, Hinf I, Sau3A, Pac I, Pov II, Sma I, Hae III, Hga I, Alu I, EcoR V, EcoP15 I, Kpn I, Pst I, Sac I, Sal I, Sca I, Spe I, Sph I, Stu I, Xba I, Xho I, etc. It doesn't work.

In the baculovirus transfer vectors of the present invention, the expression protein of interest is designed to be expressed in the form of a fusion protein with the peptide or variant thereof. That is, at least one restriction enzyme recognition nucleotide sequence capable of cloning the nucleotide sequence encoding the protein of interest (iii) is downstream of the nucleotide sequence encoding a part of (ii) the baculovirus polymorphic protein or a variant thereof. Linked downstream, the expression protein of interest is positioned to be expressed in the form of a fusion protein with a portion of the polyhedral protein or variants thereof when expressed.

In a fusion protein of a part or variant of a baculovirus polymorphic protein expressed from a baculovirus transfer vector of the present invention and an expression target protein, a proteolytic enzyme recognition site for cleaving and isolating the expression target protein from the polygonal protein is encoded. A nucleotide sequence may be additionally included, and such a sequence may be located between the (ii) sequence and the (iii) sequence. The protease recognition site may include, for example, Asp-Asp-Asp-Asp-Lys amino acid sequence recognized by Enterokinase (EK), but is not limited thereto.

The protein of interest to be expressed in the present invention is, for example, hormones, hormone analogs, enzymes, inhibitors, signaling proteins or parts thereof, antibodies or parts thereof, short chain antibodies, binding proteins or binding domains, antigens, adhesion proteins, Structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulators, blood coagulation factors or vaccine proteins.

The baculovirus transfer vector of the present invention may include a polyadenylation sequence as a transcription termination sequence, and preferably includes a polyadenylation sequence derived from a nuclear polyhedral virus (AcNPV) polyhedral protein.

The baculovirus transfer vector of the present invention may include a selection marker for selection, and the selection marker may include an antibiotic resistance gene commonly used in the art. Examples include resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin (G418), neomycin or tetracycline.

The baculovirus transfer vector of the present invention includes an origin of replication for replication of the vector, and may include, for example, a replication origin of a bacterium such as colE1 or p15A, and a bacterio such as f1 origin. It may include a replication start point of the pie.

The baculovirus transfer vectors of the invention can be further fused with other sequences to facilitate purification of the recombinant protein of interest expressed therefrom, such as, for example, glutathione S-transferase (GST). , Sequences encoding maltose binding protein (MBP), FLAG, 6x His (hexahistidine), Nus A (N utilization substance A), and Trx (thioredoxin). Because of the additional sequence for this purification, the protein expressed in the host is quickly and easily purified via affinity chromatography.

According to another aspect of the present invention, there is provided a host cell comprising the baculovirus transfer vector of the present invention. In the present invention, the host cell may be a prokaryotic or eukaryotic cell, and host cells capable of continuously and cloning and replicating the baculovirus transfer vector of the present invention are known in the art, for example, E. coli JM109. , Bacillus sp. Strains such as E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhi Enterobacteria and strains, such as Murium, Serratia Marcesons and various Pseudomonas species. In addition, the host cell in the case of producing a recombinant baculovirus using the baculovirus transfer vector of the present invention is preferably an insect cell, for example, the insect cell line Spodoptera frugiperda (Sf 21). Can be used.

According to yet another aspect of the present invention, there is provided a method for mass production of an expression target protein using the baculovirus transfer vector comprising the following steps: (a) preparing the baculovirus transfer vector step; (b) cloning an expression target protein coding nucleotide sequence into the baculovirus transfer vector; (c) cotransfection of the recombinant baculovirus transfer vector prepared in step (b) with a baculovirus DNA to a host cell; (d) selecting and isolating recombinant baculovirus from the host cell prepared in step (c); And (e) infecting the host cell with the recombinant baculovirus selected and isolated in step (d) to produce a large amount of the protein of interest.

In the following, the method of the present invention is explained according to each step.

Step (a): preparing the baculovirus transfer vector

The baculovirus transfer vectors of the present invention described above may be prepared by various methods known in the art, and specific methods thereof may be prepared by Sambrook et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). Which is hereby incorporated by reference. For example, a DNA fragment encoding a promoter region of a baculovirus polymorphic protein and a portion or variant thereof of the baculovirus polymorphic protein can be prepared and then cloned into a conventional plasmid vector.

Step (b): cloning the expression target protein coding nucleotide sequence into the baculovirus transfer vector.

Nucleotide sequence encoding the target protein to be expressed in the present invention can be obtained in a large amount through a variety of methods known in the art, for example, it can be obtained through a polymerase chain reaction (polymerase chain reaction). The nucleotide sequence encoding the resulting expression protein of interest is cloned into one or more restriction enzyme recognition nucleotide sequences present in the prepared transition vector. The cloning method may be carried out through DNA molecule manipulation using restriction enzymes and ligase known in the art, and sequencing method is used to determine whether the nucleotide sequence finally corrected for the baculovirus transfer vector has been cloned. You can check it through

Step (c): cotransfection of the recombinant baculovirus transfer vector prepared in step (b) with the baculovirus DNA to the host cell

To produce a recombinant baculovirus, the recombinant baculovirus transfer vector is cotransfected into a host cell along with the baculovirus whole genome DNA. Expression of the Transfer Vector Since the original baculovirus DNA fragment region existing to both sides of the nucleotide sequence encoding the foreign protein and the region corresponding to the fragment region in the entire baculovirus genome DNA are composed of the same nucleotide sequence, Homologous recombination occurs between them, resulting in a recombinant baculovirus. In the present invention, a virus which is co-transfected with a transfer vector to produce a recombinant baculovirus is not particularly limited as long as it is a wild type virus. The co-transfer method in the present invention can be carried out using a method for transferring genes into eukaryotic cells known in the art. For example, micro-injection (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology , 52: 456 (1973)), electroporation (Neumann, E et al., EMBO J. , 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene , 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol. , 5: 1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568-9572 (1990)), and the like. Most preferably, lipofection using liposomes (lipofection, liposometransfection) is used. The host cell used for the co-transfer preferably uses an insect cell line, for example, Sf21 (Spodoptera frugiperda 21; Invitrogen), Sf9 (Spodoptera frugiperda 9; Invitrogen), Tn5 (Trichoplusia ni 5; Invitrogen) and the like can be used. And Sf21 cell line is most preferred.

Step (d): selecting and separating recombinant baculovirus from the host cell prepared in step (c)

In general, since the production rate of recombinant baculovirus is very low, a process of selecting and separating only recombinant baculovirus having a DNA molecule encoding a protein of interest is performed. In the screening method, compared to the wild-type virus which forms a polyhedron, the recombinant virus does not form a polyhedron because the polyhedral protein is mutated, so only the virus that does not form a polyhedron is selected. For example, by observing the culture medium using a phase contrast microscope, the cells and nuclei are larger than normal cells, and the infection is confirmed from the cells that do not show the virus inclusions in the nucleus (occlusion negative, occ-). The solution is separated to obtain recombinant baculo viruses. On the other hand, a variety of methods for increasing the selection efficiency can be applied without selecting whether the polymorph is generated as a criterion, for example, a recombinant virus by inserting a selection marker gene on a transfer vector and expressing it. A method of screening may be used. In the present invention, in particular, to facilitate the selection of the recombinant baculovirus produced, the recombinant virus bAcGOZA disclosed in Korean Patent Publication No. 2003-0006477 can be used.

Subsequently, the isolated recombinant baculoviruses were isolated purely by the Plaque assay, and re-inoculated in, for example, a T-25 flask (manufactured by SPL) in order to multiply the amount of the purely isolated viruses. Get the viruses. For the quantification of the obtained virus, it is quantified by end-point dilution (King and Possee, 1992, The Baculovirus Expression System.A Laboratory Guide).

Step (e): infecting the host cell with the recombinant baculovirus selected and isolated in step (d) and generating a large amount of the protein of interest.

The purely isolated recombinant baculo virus primarily confirms whether the target protein is produced and whether the nucleotide sequence encoding the target protein exists on the viral genome through small-scale cultivation. Through proliferation, the target protein is expressed in large quantities and produced. Mass production of the protein of interest is carried out by infecting the host cell with the recombinant baculovirus prepared above, followed by culturing the infected host cell in large quantities. The host cell preferably uses an insect cell line, for example Sf21 (Spodoptera frugiperda 21; Invitrogen), Sf9 (Spodoptera frugiperda 9; Invitrogen), Tn5 (Trichoplusia ni 5; Invitrogen), and most preferably Sf21 cell line. . Expression and purification of recombinant proteins is carried out using methods of culturing insect cells and purification of proteins known in the art. For example, the target protein expressed in the present invention can be purified quickly and easily through affinity chromatography using the additional sequence introduced for the purification.

The present invention relates to a baculovirus transfer vector, a host cell comprising the transfer vector, and a method for mass production of the target protein using the transfer vector, which can greatly increase the expression efficiency of the foreign expression target protein. . The transfer vector of the present invention fuses a specific portion of the polyhedral protein of the baculovirus to the expression target protein and expresses it under a powerful polyhedral protein promoter, thereby significantly increasing the expression amount of the expression target protein in the baculovirus expression vector system. You can.

1A is a diagram schematically showing the structure of a baculovirus transfer vector of the present invention.
FIG. 1B shows various structures of a promoter of a baculovirus polyhedrin protein, a fluorescent protein (eGFP) that is a foreign expression target protein, and a portion or variant of a baculovirus polymorphic protein that is fused and expressed in front of the protein. It is a schematic diagram.
Figure 1c is a vector map of the transition vector pB9-AcPol19-110-EK made by inserting 19 to 110 of the amino acid sequence of the polyhedral protein into the transition vector pBacPAK9.
FIG. 2 shows the results of electrophoresis of fusion proteins produced in Sf21 host cells by recombinant baculo virus of the present invention expressing a fluorescent protein (eGFP) fused with some or various variants of the baculovirus polyhedral protein.
Figure 3 is a result of measuring the fluorescence of the fluorescent protein (eGFP) expressed by fusion with some or various variants of the baculovirus polyhedral protein in the present invention using a fluorescence photometer.
4A and 4B are photographs observed by confocal microscopy of cultured Sf21 cells infected with the recombinant baculo virus of the present invention expressing a fluorescent protein (eGFP) fused with a portion or various variants of the polyhedral protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1 Construction of Recombinant Baculovirus Transfer Vector

1-1. Preparation of Transfer Vector pAc-eGFP

The eGFP of the plasmid pEGFP (Clontech Co., Ltd.) containing eGFP (green fluorescence protein) derived from the North American jellyfish Aequorea victoria was cut with BamH I and EcoR I, and BamH I of pBacPAK9 (Clontech) It was inserted into the EcoR I cleavage site to prepare a transition vector pAc-eGFP (see (a) of Figure 1b). In this vector the eGFP gene is located downstream of the polyhedrin promoter (PPH).

1-2. Preparation of Transfer Vector pAc-19-110-eGFP

Amino acid sequences 19 to 110 of the AcNPV polyhedral protein were subjected to polymerase chain reaction (PCR) using the wild-type baculovirus AcNPV genome DNA as a template and primers of the first and second sequences listed below. DNA fragments encoding up to have been obtained [SEQ ID NO: 1'-CCCGGGATAATGAAGTACTACAAAAATTTAGGTG-3 '; SEQ ID NO: 2 SEQ ID NO: 5'-GAGCTCCCATGGTAACAATGGGGAAGCTGTCTTC-3 ']. The DNA fragment encoding the polyhedral protein amino acid sequence Nos. 19 to 110 of the AcNPV was cloned into pMD18 (manufactured by TaKaRa), cut with Sma I and Nco I, and then cut with Sma I and Nco I using inserts. It was inserted into the obtained pAc-eGFP to prepare a transition vector pAc-19-110-eGFP (see (b) of Figure 1b). In this vector, the fusion protein coding DNA fragment of the polyhedral protein and the eGFP (green fluorescent protein) is located downstream of the polyhedrin promoter (PPH).

1-3. Preparation of Transfer Vector pAc-NGNN19-110-eGFP

The polymerase chain reaction (PCR) was carried out using the primers of the following SEQ ID NOs 3 and 4 from the transition vector pAc-19-110-eGFP prepared above to obtain the polymorphic protein amino acid sequence 19 to 110 Point mutations 32 to 35 of the sequence were performed to obtain a mutant polyhedral protein and eGFP encoding lysine, arginine, lysine, and lysine amino acids substituted with asparagine, glycine, asparagine, and asparagine, respectively.

SEQ ID NO: 3'-AGATCTATAATGAAGTACTACAAAAATTTAGG TGCCGTTATCAAGAACGCTAATGGCAATAATCACTTC-3 '; SEQ ID NO: 4 SEQ ID NO: 5'-CTGCAGTTACTTGTACAGCTCGTCCAT-3 ']. The DNA fragment encoding the fusion protein fused with the amino acid-substituted variant peptide and the eGFP was point-mutated from 32 to 35 of the AcNPV polyhedral protein amino acid sequences 19 to 110 prepared above. Cloned into RBC) and cleaved with Bgl II and Pst I and inserted into the cleavage sites of Bgl II and Pst I of pBacPAK9 (Clontech) to prepare a transition vector pAc-NGNN19-110-eGFP (Fig. c)). In this vector the fusion gene is also located downstream of the polyhedrin promoter (PPH).

1-4. Preparation of Transfer Vector pAc-19-85-eGFP

From the polypeptide vector AcAc-19-110-eGFP, SOE PCR was performed using primers of the first and fourth sequences, and the fifth and sixth sequences, respectively, DNA fragments encoding fusion proteins in which peptides up to 85 and eGFP were fused were obtained. [SEQ ID NO: 5'-GCTCACCATGGTAAGCTTCATCGTGTCGG-3 '; SEQ ID NO: 6 SEQ ID NO: 5'-ACGATGAAGCTTACCATGGTGAGCAAGGG-3 ']. The DNA fragment encoding the peptide of the above-described polyhedral protein amino acid sequence Nos. 19-85 of AcNPV and the protein fused with eGFP was cloned into a T & A vector (manufactured by RBC), cut with EcoR I and Pst I and pBacPAK9 ( It was inserted into the cleavage site of EcoR I and Pst I of Clontech Co., Ltd. to prepare a transition vector pAc-19-85-eGFP (see (d) of Figure 1b). In this vector the fusion gene is located downstream of the polyhedrin promoter (PPH).

1-5. Transition vector pAc-32-110-eGFP

The polymerase chain reaction (PCR) was carried out using the primers of SEQ ID NO: 4 and SEQ ID NO: 7 from the transition vector pAc-19-110-eGFP to form a polyhedral protein amino acid sequence from 32 to 110. DNA fragments encoding the fusion proteins fused with peptide and eGFP were obtained (SEQ ID NO: 7'-GAATTCATAATGAAGCGCAAGAAGCACTTCGC-3 '). The DNA fragment encoding the peptide of the above-described polyhedral protein amino acid sequence 32 to 110 of AcNPV and a protein fused with eGFP was cloned into a T & A vector (produced by RBC) and cut with EcoR I and Pst I to pBacPAK9 (Clontech And a transfection vector pAc-32-110-eGFP was prepared by inserting them into the cleavage sites of EcoR I and Pst I (see FIG. 1B). In this vector the fusion gene is located downstream of the polyhedrin promoter (PPH).

1-6. Transition vector pAc-32-85-eGFP

Polyhedral protein amino acid sequence Nos. 32 to 85 by SOE PCR using the primers of SEQ ID NO: 4 and 7 and SEQ ID NO: 8 and 9 from the transition vector pAc-32-110-eGFP A DNA fragment encoding a fusion protein fused with a peptide consisting of a peptide and an eGFP was obtained. [SEQ ID NO: 8'-GCTCACCATGGTAAGCTTCATCGTGTCGG-3 '; SEQ ID NO: 9 SEQ ID NO: 5'-ACGATGAAGCTTACCATGGTGAGCAAGGG-3 ']. The DNA fragment encoding the peptide of the above-mentioned polyhedral protein amino acid sequence of AcNPV peptides 32 to 85 and the protein fused with eGFP was cloned into a T & A vector (produced by RBC) and cut with EcoR I and Pst I to pBacPAK9 (Clontech And a transfection vector pAc-32-85-eGFP was prepared by inserting them into the cleavage site of EcoR I and Pst I (see FIG. 1B). In this vector the fusion gene is located downstream of the polyhedrin promoter (PPH).

1-7. Transition vector pAc-32-59-eGFP

Polyhedral protein amino acid sequence Nos. 32 to 59 by SOE PCR using the primers of SEQ ID NO: 4 and 7 and the following SEQ ID NO: 10 and 11 from the transition vector pAc-32-110-eGFP A DNA fragment encoding a fusion protein fused to a peptide consisting of a peptide and an eGFP was obtained. [SEQ ID NO: 10'-GCTCACCATGGTAGGATCCTCAGCCACTAGG-3 '; SEQ ID NO: 11 SEQ ID NO: 5'-GCTGCGGATCCTACCATGGTGAGCAAGGG-3 ']. The DNA fragment encoding the peptide of the above-described polyhedral protein amino acid sequence of AcNPV amino acids 32 to 59 and the protein fused with eGFP was cloned into a T & A vector (produced by RBC), and digested with EcoR I and Pst I to pBacPAK9 ( The transition vector pAc-32-85-eGFP was prepared by inserting the cleavage site of EcoR I and Pst I of Clontech Co., Ltd. (see (g) of FIG. 1B). In this vector the fusion gene is located downstream of the polyhedrin promoter (PPH).

1-8. Transition vector pAc-32-59 / 90-110-eGFP

Polyhedral protein amino acid sequence Nos. 32 to 59 by SOE PCR using the primers of SEQ ID NO: 4 and 7 and the following SEQ ID NO: 12 and 13 from the transition vector pAc-32-110-eGFP DNA fragments encoding the fusion proteins in which the peptide consisting of No. 90 and No. 110 and the eGFP were fused were obtained. [SEQ ID NO: 12'-CTCTTTTCCTTTAGGATCCTCAGCCACTAGG-3 '; SEQ ID NO: 13 Sequence: 5'-GCTGAGGATCCTAAAGGAAAAGAGTTCTACAGG-3 ']. The DNA fragments encoding the peptides of the above-described polyhedral protein amino acid sequences 32-59 and 90-110 of the ENPFP fused to the EGFP were cloned into a T & A vector (manufactured by RBC), and EcoR I and Pst. Cutting with I was inserted into the cutting sites of EcoR I and Pst I of pBacPAK9 (manufactured by Clontech) to prepare a transition vector pAc-32-59 / 90-110-eGFP (see (h) of FIG. 1b). In this vector the fusion gene is located downstream of the polyhedrin promoter (PPH).

1-9. Transition vector pAc-KRKK77-110-eGFP

PCR was performed using the primers of SEQ ID NO: 4 and SEQ ID NO: 14 from the transition vector pAc-19-110-eGFP to form a polyhedral protein amino acid sequence from 32 to 35 and from 77 to 110 A DNA fragment encoding a fusion protein fused with peptide and eGFP was obtained (SEQ ID NO: 14 SEQ ID NO: 5'-GAATTCATAATGAAGCGCAAGAAGAATGTTAAACCCGA-3 '). DNA fragments encoding the above-described polyhedral protein amino acid sequences 32 to 35 and 77 to 110 peptides of AcNPV and proteins fused with eGFP were cloned into T & A vectors (manufactured by RBC), and EcoR I and Pst I It was cut into and inserted into the cleavage sites of EcoR I and Pst I of pBacPAK9 (manufactured by Clontech) to prepare a transition vector pAc-KRKK77-110-eGFP (see (i) of Figure 1b). In this vector the fusion gene is located downstream of the polyhedrin promoter (PPH).

1-10. Transition vector pAc-KRKK90-110-eGFP

PCR was carried out using the primers of SEQ ID NO: 4 and SEQ ID NO: 15 from the transition vector pAc-19-110-eGFP to form a polyhedral protein amino acid sequence from 32 to 35 and from 90 to 110 DNA fragments encoding the fusion proteins fused with peptide and eGFP were obtained (SEQ ID NO: 15 SEQ ID NO: 5'-GAATTCATAATGAAGCGCAAGAAGAAAGGAAAAGAGTTC-3 '). The DNA fragments encoding the above-described polyhedral protein amino acid sequence of AcNPV amino acid sequence 32 to 35 and 90 to 110 peptide and a protein fused with eGFP were cloned into a T & A vector (manufactured by RBC) and EcoR I and Pst I Was cut into the EcoR I and Pst I cleavage site of pBacPAK9 (Clontech) to prepare a transition vector pAc-KRKK90-110-eGFP (see (j) of Figure 1b). In this vector the fusion gene is located downstream of the polyhedrin promoter (PPH).

Example 2 Preparation of Recombinant Virus

Each of the transfer vectors prepared in Example 1 and the genome DNA of the recombinant virus bAcGOZA (published patent No. 2003-0006477) were mixed in 100 μl of SF900 II medium (manufactured by Gibco), and the cellfectin II (cellfectin II, 5 μl of Invitrogen) was mixed with 100 μl of SF900 II medium, and each of them was allowed to stand at 27 ° C. for 15 minutes, and then the two mixtures were mixed and left at 27 ° C. for 15 minutes. This mixture was dispensed into an insect cell line, Sf21 (Spodoptera frugiperda 21, manufactured by Invitrogen) at 2.0 X 10 ⁶ per 25 mm ² culture flask, inoculated into the culture medium, and further cultured to form a recombinant baculo virus. Observation of the culture medium with a phase contrast microscope (x100) confirmed the presence of infection from cells with a larger cell and nucleus cell size than normal cells and no virus inclusions in the nucleus (occlusion negative, occ-). After centrifugation for minutes, the supernatant was separated to isolate recombinant baculo viruses. Subsequently, the isolated recombinant baculoviruses were purely separated by a Plaque assay and re-inoculated into a T-25 flask (SPL) to obtain the virus by propagating the purely isolated viruses. The obtained viruses were quantified by the dilution terminal method (King and Possee, 1992, The Baculovirus Expression System. A Laboratory Guide).

Example 3: Protein Electrophoresis Analysis of Recombinant Proteins

Recombinant baculoviruses prepared in Example 2 were aliquoted to 1.0 x 10 ⁶ per 60 mm x 15 mm culture plates to 5 virus particles per cell in insect cell line Sf21 cells, and allowed to stand for 1 hour to infect Sf21 cells. At 4 days after inoculation, the cells were observed by fluorescence microscopy (MCXI600 inverted microscopy, manufactured by Micros, 100-fold magnification) and examined for infection by showing fluorescence in infected cells. At this time, a wild type baculovirus AcNPV (Clontech, Cat. No. K1601-D) was used as a control. The recombinant viruses were harvested from the plate and the culture was centrifuged at 1,000 × g for 5 minutes. To the precipitate was added a cytosolic solution (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5% glycerol, 0.1% Triton schnol, 00) and an ultrasonic digester (Ultrasonic processor, 30% Amp., Pulse 10 sec stop 5 sec; Sonics Company), and left for 30 minutes on ice. 5X sample buffer [0.6 ml 1 M Tris-HCl (pH 6.8), 5 ml 50% glycerol, 2 ml 10% SDS, 0.5 ml 2-mercaptoethanol, 1 ml 1% bromophenol blue, 0.9 ml H ₂ O) was added and mixed, heated in a bath at 00 ° C. for 10 minutes, allowed to stand on ice for a while, and then centrifuged at 13000 rpm for 10 minutes. The supernatant was subjected to 12% SDS Sorbent GE. After electrophoresis, gels were stained with coomasie brillant blue to confirm protein expression. Among the nine polyhedral protein subcombinations, a total of nine polyhedral amino acid sequences were obtained. RAc-19-110-eGFP with 1% Tan fusion of amino acid sequences from 1 to 0 was present at the expected size of 37.89 kDa, while other polyhedral protein combinations were also present at the expected position, but rAc-19 The -85-eGFP and rAc-32-85-eGFP were also present at the 26.95 kDa position, the expected position of the fusion with the polyhedral protein portion and the position of the eGFP. Expression of a total of nine polyhedral protein fragments or variants thereof by fusion to fluorescent protein (eGFP) is expressed more than expression of fluorescence protein (eGFP) alone without fusion of a portion or variant thereof of polyhedral protein. It was confirmed that the amount was significantly increased.

Example  4: Quantitative Comparison of Recombinant Proteins

Recombinant baculoviruses prepared in Example 2 were aliquoted at 1.0 × 10 ⁶ per 60 mm × 15 mm culture plate so as to have 5 virus particles per cell in the insect cell line Sf21 and allowed to stand for 1 hour to infect Sf21 cells. At 4 days after inoculation, the cells were observed with a fluorescence microscope (MCXI600 inverted microscopy, manufactured by Micros, 100-fold magnification), and the infection was confirmed by fluorescence in the infected cells. At this time, a wild type baculovirus AcNPV (Clontech, Cat. No. K1601-D) was used as a control. Recombinant viruses confirmed infection were harvested from the plate and the culture was centrifuged at 1,000 × g for 5 minutes. Add 160 µl of cell lysis solution (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5% glycerol, 0.1% Triton X-100) to the precipitate and place an ultrasonic digester (Ultrasonic processor, 30% Amp., Pulse 10 sec stop 5 sec). , Sonics) and left on ice for 30 minutes. 740 μl of phosphate buffered saline was added to the reaction solution, and 100 μl of 1M sodium carbonate was added and reacted at 37 ° C. for 1 hour. The lysed cell lysate was analyzed with a fluorescence photometer (K2TM, manufactured by ISS) to determine whether the emission wavelength of the fluorescent protein was detected at 510 nm (FIG. 3). As a result, the fluorescence intensity values of rAc-32-85-eGFP and rAc-32-59 / 90-110-eGFP were the highest compared to other polyhedral protein partial combinations with 162098.5 ㅁ 6049.1 and 158564.6 ㅁ 1957.0 intensity, respectively. The figure was higher than that of rAc-eGFP. The fluorescence intensity value of the recombinant protein rAc-19-110-eGFP, which is a fusion of the polyhedral protein amino acid sequence Nos. 19 to 110, which is a site including all of the polyhedral protein partial combinations, is 124621 ㅁ 2628.4 intensity Is shown (Table 1).

Recombinant virus Measures Sf21 cells 4225.2 ± 765.0 AcNPV 5297.9 ± 238.4 rAc-eGFP 28847.1 ± 3184.1 rAc-19-110-eGFP 124621 ± 2628.4 rAc-NGNN19-110-eGFP 45020.6 ± 2723.3 rAc-19-85-eGFP 106175.6 ± 5467.0 rAc-32-110-eGFP 113241.0 ± 5102.1 rAc-32-85-eGFP 162098.5 ± 6049.1 rAc-32-59-eGFP 43367.8 ± 865.2 rAc-32-59 / 90-110-eGFP 158564.6 ± 1957.0 rAc-KRKK77-110-eGFP 87026.5 ± 6349.5 rAc-KRKK90-110-eGFP 118503.2 ± 6408.9

The same result as in the protein electrophoresis analysis, it was confirmed that the expression amount when fusion of a total of nine polyhedral protein portion combinations with eGFP, significantly increased than eGFP unfused with the combination of the polyhedral protein portion Is the result.

Example 5 Confocal Electron Microscopy of Recombinant Baculovirus

The recombinant baculovirus rAc-19-110-eGFP prepared in Example 2 was dispensed at 1.0 x 10 ⁶ per 60 mm x 15 mm culture plate to 5 virus particles per cell in insect cell line Sf21 cells, and left for 1 hour. Infected with Sf21 cells. Three days after the inoculation, a portion of the cells were collected, the cells were fixed with methanol on a slide glass, the nuclei were stained with propidium iodide (PI), and then mounted and observed with a confocal laser scanning microscope (FIG. 4). As a result, the recombinant protein produced using rAc-19-110-eGFP, rAc-32-110-eGFP and rAc-KRKK77-110-eGFP was found to exist in the nucleus, and the remaining rAc-eGFP, rAc- NGNN19-110-eGFP, rAc-19-85-eGFP, rAc-32-85-eGFP, rAc-32-59-eGFP, rAc-32-59 / 90-110-eGFP and rAc-KRKK90-110-eGFP The recombinant protein produced was found to be present in the cytoplasm.

Example 6: Preparation of the transition vector pB9-AcPol19-110-EK comprising the polyhedral protein amino acid sequence 19 to 110

PCR was performed from the transfer vector pAc-19-110-eGFP using primers of SEQ ID NO: 16 and 17, to encode the polyhedral protein amino acid sequences 19 to 110 and enterokinase (EK) sites. DNA fragments were obtained. [SEQ ID NO: 16'-ACTAGTATAATGAAGTACTACAAAAAT-3 '; SEQ ID NO: 17 Sequence: 5'-GAATTCCTTGTCGTCGTCATCGGTAACAATGGG -3 ']. The gene was cloned into a T & A vector (RBC), cleaved with Bgl II and EcoR I, and inserted into the cleavage sites of BamH I and EcoR I of pBacPAK9 (Clontech) to transfer the transition vector pB9-AcPol19-110-EK. Produced. In this vector the fusion gene is located downstream of the polyhedrin promoter (PPH).

Example 7 Preparation of Transfer Vectors Using Peptides in Polyhedral Protein Amino Acid Sequences 19-110

PCR was performed from the transfer vector pAc-NGNN19-110-eGFP using the primers of SEQ ID NO: 16 and SEQ ID NO: 17 to obtain a DNA fragment encoding a polyhedral protein partial sequence and an enterokinase (EK) site. .

DNA encoding a polyhedral protein partial sequence and an enterokinase (EK) site by PCR using the primers of SEQ ID NO: 16 and SEQ ID NO: 18 from the transfer vector pAc-19-85-eGFP Fragments were obtained (SEQ ID NO: 18 SEQ ID NO: 5'-GAATTCCTTGTCGTCGTCATCAAGCTTCATCGTGTCGG-3 ').

DNA encoding a polyhedral protein partial sequence and an enterokinase (EK) site by PCR using the primers of SEQ ID NO: 19 and SEQ ID NO: 17 from the transfer vector pAc-32-110-eGFP A fragment was obtained (SEQ ID NO: 19 SEQ ID NO: 5'-AGATCTACTAGTATAATGAAGCGCAAGAAGCACTTC-3 ').

PCR was performed using the primers of SEQ ID NO: 19 and 18 from the transfer vector pAc-32-85-eGFP to obtain a DNA fragment encoding a polyhedral protein partial sequence and an enterokinase (EK) site. .

DNA encoding a polyhedral protein partial sequence and an enterokinase (EK) site by PCR using the primers of SEQ ID NO: 19 and SEQ ID NO: 20 from the transfer vector pAc-32-59-eGFp Fragments were obtained (SEQ ID NO: 20 ': 5'-GAATTCCTTGTCGTCGTCATCAGGATCCTCAGCCACTAGG-3').

PCR was performed using the primers of SEQ ID NO: 19 and 17 from the transfer vector pAc-32-59 / 90-110-eGFP to encode a polyhedral protein partial sequence and an enterokinase (EK) site. DNA fragments were obtained.

PCR was performed from the transfer vector pAc-KRKK77-110-eGFP using primers of SEQ ID NO: 21 and 17 to obtain a DNA fragment encoding a polyhedral protein partial sequence and an enterokinase (EK) site. [SEQ ID NO: 21 SEQ ID NO: 5'-AGATCTACTAGTATAATGAAGCGCAAGAAGAATGTTAAACCCGA-3 '].

PCR was performed from the transfer vector pAc-KRKK90-110-eGFP using primers of SEQ ID NO: 22 and 17 to obtain a DNA fragment encoding a polyhedral protein partial sequence and an enterokinase (EK) site. [SEQ ID NO: 22 ': 5'-AGATCTACTAGTATAATGAAGCGCAAGAAGAAAGGAAAAGAGTTC-3']. Each DNA fragment obtained by PCR was cloned into a T & A vector (RBC Co., Ltd.), cut into Bgl II and EcoR I, and inserted into the cleavage sites of BamH I and EcoR I of pBacPAK9 (Clontech Co., Ltd.), respectively. Produced. In this vector the fusion gene is located downstream of the polyhedrin promoter (PPH).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Chungbuk National University Industry Academic Cooperation Foundation <120> Method for Enhancing the Expression of Foreign Recombinant          Protein Using Fusion Expression with Partial Polyhedrin of          Baculovirus <160> 31 <170> Kopatentin 1.71 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 1 cccgggataa tgaagtacta caaaaattta ggtg 34 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 2 gagctcccat ggtaacaatg gggaagctgt cttc 34 <210> 3 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 3 agatctataa tgaagtacta caaaaattta ggtgccgtta tcaagaacgc taatggcaat 60 aatcacttc 69 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 4 ctgcagttac ttgtacagct cgtccat 27 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 5 gctcaccatg gtaagcttca tcgtgtcgg 29 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 6 acgatgaagc ttaccatggt gagcaaggg 29 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 7 gaattcataa tgaagcgcaa gaagcacttc gc 32 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 8 gctcaccatg gtaagcttca tcgtgtcgg 29 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 9 acgatgaagc ttaccatggt gagcaaggg 29 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 10 gctcaccatg gtaggatcct cagccactag g 31 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 11 gctgcggatc ctaccatggt gagcaaggg 29 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 12 ctcttttcct ttaggatcct cagccactag g 31 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 13 gctgaggatc ctaaaggaaa agagttctac agg 33 <210> 14 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 14 gaattcataa tgaagcgcaa gaagaatgtt aaacccga 38 <210> 15 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 15 gaattcataa tgaagcgcaa gaagaaagga aaagagttc 39 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 16 actagtataa tgaagtacta caaaaat 27 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 17 gaattccttg tcgtcgtcat cggtaacaat ggg 33 <210> 18 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 18 gaattccttg tcgtcgtcat caagcttcat cgtgtcgg 38 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 19 agatctacta gtataatgaa gcgcaagaag cacttc 36 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 20 gaattccttg tcgtcgtcat caggatcctc agccactagg 40 <210> 21 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 21 agatctacta gtataatgaa gcgcaagaag aatgttaaac ccga 44 <210> 22 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 22 agatctacta gtataatgaa gcgcaagaag aaaggaaaag agttc 45 <210> 23 <211> 92 <212> PRT <213> Autographa californica nucleopolyhedrovirus <400> 23 Lys Tyr Tyr Lys Asn Leu Gly Ala Val Ile Lys Asn Ala Lys Arg Lys   1 5 10 15 Lys His Phe Ala Glu His Glu Ile Glu Glu Ala Thr Leu Asp Pro Leu              20 25 30 Asp Asn Tyr Leu Val Ala Glu Asp Pro Phe Leu Gly Pro Gly Lys Asn          35 40 45 Gln Lys Leu Thr Leu Phe Lys Glu Ile Arg Asn Val Lys Pro Asp Thr      50 55 60 Met Lys Leu Val Val Gly Trp Lys Gly Lys Glu Phe Tyr Arg Glu Thr  65 70 75 80 Trp Thr Arg Phe Met Glu Asp Ser Phe Pro Ile Val                  85 90 <210> 24 <211> 92 <212> PRT <213> Autographa californica nucleopolyhedrovirus <400> 24 Lys Tyr Tyr Lys Asn Leu Gly Ala Val Ile Lys Asn Ala Asn Gly Asn   1 5 10 15 Asn His Phe Ala Glu His Glu Ile Glu Glu Ala Thr Leu Asp Pro Leu              20 25 30 Asp Asn Tyr Leu Val Ala Glu Asp Pro Phe Leu Gly Pro Gly Lys Asn          35 40 45 Gln Lys Leu Thr Leu Phe Lys Glu Ile Arg Asn Val Lys Pro Asp Thr      50 55 60 Met Lys Leu Val Val Gly Trp Lys Gly Lys Glu Phe Tyr Arg Glu Thr  65 70 75 80 Trp Thr Arg Phe Met Glu Asp Ser Phe Pro Ile Val                  85 90 <210> 25 <211> 67 <212> PRT <213> Autographa californica nucleopolyhedrovirus <400> 25 Lys Tyr Tyr Lys Asn Leu Gly Ala Val Ile Lys Asn Ala Lys Arg Lys   1 5 10 15 Lys His Phe Ala Glu His Glu Ile Glu Glu Ala Thr Leu Asp Pro Leu              20 25 30 Asp Asn Tyr Leu Val Ala Glu Asp Pro Phe Leu Gly Pro Gly Lys Asn          35 40 45 Gln Lys Leu Thr Leu Phe Lys Glu Ile Arg Asn Val Lys Pro Asp Thr      50 55 60 Met lys leu  65 <210> 26 <211> 79 <212> PRT <213> Autographa californica nucleopolyhedrovirus <400> 26 Lys Arg Lys Lys His Phe Ala Glu His Glu Ile Glu Glu Ala Thr Leu   1 5 10 15 Asp Pro Leu Asp Asn Tyr Leu Val Ala Glu Asp Pro Phe Leu Gly Pro              20 25 30 Gly Lys Asn Gln Lys Leu Thr Leu Phe Lys Glu Ile Arg Asn Val Lys          35 40 45 Pro Asp Thr Met Lys Leu Val Val Gly Trp Lys Gly Lys Glu Phe Tyr      50 55 60 Arg Glu Thr Trp Thr Arg Phe Met Glu Asp Ser Phe Pro Ile Val  65 70 75 <210> 27 <211> 54 <212> PRT <213> Autographa californica nucleopolyhedrovirus <400> 27 Lys Arg Lys Lys His Phe Ala Glu His Glu Ile Glu Glu Ala Thr Leu   1 5 10 15 Asp Pro Leu Asp Asn Tyr Leu Val Ala Glu Asp Pro Phe Leu Gly Pro              20 25 30 Gly Lys Asn Gln Lys Leu Thr Leu Phe Lys Glu Ile Arg Asn Val Lys          35 40 45 Pro Asp Thr Met Lys Leu      50 <210> 28 <211> 28 <212> PRT <213> Autographa californica nucleopolyhedrovirus <400> 28 Lys Arg Lys Lys His Phe Ala Glu His Glu Ile Glu Glu Ala Thr Leu   1 5 10 15 Asp Pro Leu Asp Asn Tyr Leu Val Ala Glu Asp Pro              20 25 <210> 29 <211> 49 <212> PRT <213> Autographa californica nucleopolyhedrovirus <400> 29 Lys Arg Lys Lys His Phe Ala Glu His Glu Ile Glu Glu Ala Thr Leu   1 5 10 15 Asp Pro Leu Asp Asn Tyr Leu Val Ala Glu Asp Pro Lys Gly Lys Glu              20 25 30 Phe Tyr Arg Glu Thr Trp Thr Arg Phe Met Glu Asp Ser Phe Pro Ile          35 40 45 Val     <210> 30 <211> 38 <212> PRT <213> Autographa californica nucleopolyhedrovirus <400> 30 Lys Arg Lys Lys Asn Val Lys Pro Asp Thr Met Lys Leu Val Val Gly   1 5 10 15 Trp Lys Gly Lys Glu Phe Tyr Arg Glu Thr Trp Thr Arg Phe Met Glu              20 25 30 Asp Ser Phe Pro Ile Val          35 <210> 31 <211> 25 <212> PRT <213> Autographa californica nucleopolyhedrovirus <400> 31 Lys Arg Lys Lys Lys Gly Lys Glu Phe Tyr Arg Glu Thr Trp Thr Arg   1 5 10 15 Phe Met Glu Asp Ser Phe Pro Ile Val              20 25

Claims (6)

A baculovirus transfer vector for use in expressing a protein of interest characterized in that it comprises a nucleic acid sequence:
(a) a promoter of baculovirus polyhedral protein;
(b) a nucleotide sequence encoding a portion or variant thereof of a baculovirus polymorphic protein operably linked to said promoter; And
(c) one or more restriction enzyme recognition nucleotide sequences from which the nucleotide sequence encoding the protein of interest can be cloned:
However, part of the baculovirus polymorphic protein is a peptide consisting of the amino acid sequence of SEQ ID NO: 23 sequence, the variant is an amino acid substitution variant or truncation variant of the peptide, the expression protein of interest and the peptide or a variant thereof Is expressed in the form of a fusion protein.
The method of claim 1, wherein the variant is SEQ ID NO: 24 sequence, SEQ ID NO: 25 sequence, SEQ ID NO: 26 sequence, SEQ ID NO: 27 sequence, SEQ ID NO: 28 sequence, SEQ ID NO: 29 sequence, SEQ ID NO: 30 sequence And a peptide consisting of any one amino acid sequence selected from the group consisting of the thirty-first sequence.
The baculovirus transfer vector according to claim 1, further comprising a fusion protein cleavage site between the peptide or a variant thereof and the protein of interest.
4. The baculovirus transfer vector according to claim 3, wherein the fusion protein cleavage site is an Enterokinase cleavage site.
A host cell comprising the baculovirus transfer vector of any one of claims 1 to 4.
Mass production method of expression target protein comprising the following steps:
(a) preparing a baculovirus transfer vector of any one of claims 1 to 4;
(b) cloning an expression target protein coding nucleotide sequence into the baculovirus transfer vector;
(c) cotransfection of the recombinant transfer vector prepared in step (b) with a baculovirus DNA to a host cell;
(d) selecting and isolating recombinant baculovirus from the host cell prepared in step (c); And
(e) infecting the host cell with the recombinant baculovirus selected and isolated in step (e) and producing a large amount of the protein of interest.

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