Target protein expression platform using viral nucleocapsids

Abstract

To provide an expression vector that can efficiently express target proteins, particularly those that are poorly expressed insolublely in E. coli, in host cells, especially E. coli. [Solution] A method for producing a target protein is provided, comprising the steps of: constructing a target protein expression vector containing a polynucleotide encoding a viral nucleocapsid as a fusion partner of the target protein, and constructing a target protein expression vector containing a target protein and a polynucleotide encoding a viral nucleocapsid as a fusion partner of the target protein; introducing the target protein expression vector into host cells to produce a transformant; and culturing the transformant, thereby enabling the efficient expression of a target protein, particularly a poorly expressed protein that is insoluble in E. coli, in host cells, especially E. coli.

Description

Technical Field

[0001] The present invention relates to a target protein expression platform using a viral nucleocapsid. More specifically, the present invention relates to an expression vector, a transformed host cell, and a method for producing a target protein, which can efficiently express a target protein, particularly a difficult-to-express protein that is insoluble in Escherichia coli, in a host cell, particularly Escherichia coli, by using a viral nucleocapsid as a fusion partner.

Background Art

[0002] The core of modern biotechnology is the production of recombinant proteins. Among them, it is particularly important to produce a large amount while maintaining the intrinsic activity of the recombinant protein. The recombinant proteins produced in this way are very important for the production and recovery of foreign proteins, crystallization for functional research, industrialization, and the like.

[0003] To date, many studies on the production of recombinant proteins using Escherichia coli have been conducted because Escherichia coli is easy to manipulate, has a short growth time, stable expression, low cost, and can easily change the scale. However, recombinant proteins of foreign origin produced from Escherichia coli have been pointed out to have the disadvantage of forming insoluble protein aggregates (inclusion bodies) of the produced recombinant proteins because of low expression levels or the absence of appropriate "post-translational chaperons" and "posttranslational processing" (Francois Baneyx, Recombinant protein expression in Escherichia coli, Current Opinion in Biotechnology (1999), 10:411-421).

[0004] To address these issues, methods have been developed to express recombinant proteins by fusing them with highly water-soluble proteins such as MBP, NusA, SUMO, Thioredoxin, or GST (Dominic Esposito and Deb K Chatterjee, Enhancement of soluble protein expression through the use of fusion tags, Current Opinion in Biotechnology (2006), 17:353-358). However, these methods also have their technical limitations, and the development of new expression techniques is needed. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The main objective of the present invention is to provide an expression vector that can efficiently express target proteins, particularly poorly expressed proteins that are insoluble in E. coli, in host cells, especially E. coli.

[0006] Another object of the present invention is to provide a host cell that can be transformed with the expression vector and achieve efficient expression of the target protein.

[0007] A further object of the present invention is to provide a method for efficiently producing the target protein using the expression vector and / or the host cell. [Means for solving the problem]

[0008] The present invention provides a target protein expression vector containing a polynucleotide encoding a viral nucleocapsid as a fusion partner for the target protein.

[0009] The present invention also provides host cells transformed with the target protein expression vector.

[0010] The present invention also provides a method for producing a target protein, comprising the steps of: preparing a target protein expression vector containing a target protein and a polynucleotide encoding a viral nucleocapsid as a fusion partner of the target protein; introducing the target protein expression vector into host cells to produce a transformant; and culturing the transformant. [Effects of the Invention]

[0011] According to the present invention, target proteins, particularly those that are poorly expressed insolublely in E. coli, can be efficiently expressed in host cells, especially E. coli. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a diagram used to investigate the potential use of viral nucleocapsids (NCs) as fusion partners. It shows the results of expressing NCs together with His-tags in E. coli and confirming this using SDS-PAGE. [Figure 2] Figure 2 is a diagram used to investigate the potential of NC as a fusion partner, showing the results of expressing NC in E. coli (without His-tag) and confirming it by SDS-PAGE. [Figure 3] Figure 3 shows the results of introducing an expression vector according to one embodiment of the present invention into E. coli and confirming the expression of the target protein, GFP, by SDS-PAGE. [Figure 4] Figure 4 shows the results of confirming the expression of the target protein, GFP, by SDS-PAGE after introducing an expression vector according to one embodiment of the present invention into E. coli and treating it with different IPTG concentrations. [Figure 5] Figure 5 shows the results of measuring the fluorescence signal of the expressed target protein, GFP, using a microplate reader when an expression vector according to one embodiment of the present invention was introduced into E. coli and treated with different IPTG concentrations. [Figure 6]Figure 6 shows the results of introducing an expression vector according to one embodiment of the present invention into E. coli and confirming the expression of the target protein, IFNβ, by SDS-PAGE. [Figure 7] Figure 7 shows the results of expressing IFNβ in E. coli without a fusion partner and confirming this by SDS-PAGE. [Figure 8] Figure 8 shows the results of IFNβ reaction analysis experiments performed after purifying the target protein IFNβ by Ni-affinity chromatography using the method described in Figure 6, followed by serial dilution. [Figure 9] Figure 9 shows the amino acid sequences of the mutant NC used in the experiment along with wild-type NC, and their interactions with zinc ions. [Figure 10] Figure 10 shows the results of SDS-PAGE confirmation of fusion protein expression when each NC in Figure 9 was applied. [Figure 11] Figure 11 shows the results of introducing an expression vector according to one embodiment of the present invention into E. coli, inducing the expression of the target protein GFP, followed by purification by zinc affinity chromatography and analysis by SDS-PAGE and fluorescence signal measurement of GFP. [Modes for carrying out the invention]

[0013] According to one embodiment of the present invention, a vector is provided that enables the efficient expression of a target protein in heterogeneous or homogeneous host cells, and is a target protein expression vector comprising a polynucleotide encoding a viral nucleocapsid as a fusion partner for the target protein.

[0014] As used herein, the term "target protein" refers to any protein that a person skilled in the art would like to produce in large quantities, and which can be expressed in a host cell by inserting a polynucleotide encoding the said protein into a recombinant vector.

[0015] In the present invention, the target protein may be selected from one or more of the group consisting of an antigen, an antibody, a cell receptor, an enzyme, a structural protein, a serum, and a cell protein, but is not limited thereto.

[0016] As used herein, the term "expression vector" is a vector for introducing and expressing a polynucleotide encoding a polypeptide of interest into a heterologous or homologous host cell, and means a circular or linear polynucleotide, for example, a DNA or RNA molecule. The expression vector may contain a promoter and / or a terminator sequence, and may also contain components necessary for use in a vector such as an origin of replication, a selection marker, a polyadenylation signal, and the like.

[0017] The expression vector of the present invention may be a plasmid, a viral vector, a phage particle, or a genomic insert, and after being introduced into a host cell, it may be replicated without relation to the genome of the host cell or integrated into the genome of the host cell.

[0018] The expression vector of the present invention may contain a polynucleotide encoding a target protein, or may be for later use by including a polynucleotide encoding a target protein. Particularly in the latter case, it is preferably a component that can easily include a polynucleotide encoding a target protein, and usually may contain a restriction enzyme recognition site called a multiple cloning site (MCS).

[0019] The present invention is based on a new research result that when a viral nucleocapsid is applied as a fusion partner of a target protein, the target protein can be efficiently expressed in a water-soluble form, for example, when no fusion partner or other existing fusion partners are used, while the protein is expressed in an insoluble form.

[0020] In particular, the fusion partner used in this invention has the advantage of being extremely small in size (a short-length peptide), which minimizes interference with the target protein.

[0021] Furthermore, the present invention has the advantage of being able to use the zinc-binding domain inherent to the viral nucleocapsid. For example, by using the zinc-binding domain of the viral nucleocapsid, the fusion protein can be easily purified without other components for purification, such as a histidine tag, for example, by using a zinc affinity purification method.

[0022] In the present invention, the viral nucleocapsid may be a publicly known viral nucleocapsid, or it may be a fragment or mutant of the viral nucleocapsid, as long as the activity of the present invention, i.e., the activity of acting as a fusion partner of the target protein to enable the target protein to be expressed in water-soluble form, particularly in E. coli, is maintained.

[0023] In the present invention, the viral nucleocapsid may be a retrovirus, such as a retrovirus nucleocapsid of HIV-1, SIV, MuLV, etc.

[0024] In the present invention, the viral nucleocapsid is preferably a human immunodeficiency virus (HIV) nucleocapsid.

[0025] In the present invention, the viral nucleocapsid preferably contains an amino acid sequence having 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. Preferably, the amino acid sequence satisfies the sequence identity while maintaining the sequences of amino acids 14, 17, 22, and 27 of the amino acid sequence of Sequence ID No. 1; more preferably, while maintaining the sequences of amino acids 14, 17, 22, 27, 35, 38, 43, and 48; and more preferably, while maintaining the sequences of amino acids 6, 9, 10, 13, 14, 17, 19, 22, 25, 27, 28, 31, 32, 33, 35, 37, 38, 40, 43, 46, 48, and 51.

[0026] In the present invention, the polynucleotide encoding the viral nucleocapsid preferably contains a base sequence having 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more sequence identity with the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3. Preferably, the sequence maintains the sequences of amino acids 14, 17, 22, and 27 of the amino acid sequence of Sequence ID No. 1; more preferably, the sequences of amino acids 14, 17, 22, 27, 35, 38, 43, and 48; and more preferably, the sequences of amino acids 6, 9, 10, 13, 14, 17, 19, 22, 25, 27, 28, 31, 32, 33, 35, 37, 38, 40, 43, 46, 48, and 51, while including a base sequence that satisfies the sequence identity requirement.

[0027] In this specification, sequence identity means sequence identity between an amino acid sequence and a reference amino acid sequence, or between a nucleotide sequence and a reference nucleotide sequence. Sequence identity can be determined by comparing the positions of each sequence that can be aligned for comparison purposes. If the same amino acid or base occupies the same position in the comparison sequence, then the molecules are identical at that position. The degree of identity between amino acid or nucleotide sequences is a function of the number of identical amino acids or nucleotides at positions shared by each amino acid or nucleotide sequence. For example, the identity between two sequences can be calculated using a variety of alignment algorithms and / or programs, including FASTA or BLAST.

[0028] The expression vector of the present invention may be for expressing a target protein in cells selected from all organisms, including prokaryotes and eukaryotes, but is preferably for expressing a target protein in Escherichia microorganisms, and more preferably for expressing a target protein in Escherichia coli.

[0029] The expression vector of the present invention may further include other components, such as a cloning site for inserting a target protein-coding polynucleotide, such as a restriction enzyme recognition site, such as a multiple cloning site (MCS); a linker sequence for providing a spacing between the target protein and its fusion partner; a tag sequence, such as a histidine tag sequence; and / or a protease recognition site-coding sequence for cleaving a portion of the expressed fusion protein. Each of these may be arranged in various ways as needed and may be included in various numbers.

[0030] In one specific embodiment, the expression vector of the present invention is configured such that a viral nucleocapsid is ligated to the N-terminus of a target protein. As a specific example of such an embodiment, the expression vector of the present invention includes a construct in which a promoter, a viral nucleocapsid encoding site, a target protein encoding site or a cloning site for inserting a target protein encoding polynucleotide (e.g., a restriction enzyme recognition site, e.g., a multiple cloning site (MCS)), and a terminator are operably ligated in the 5' to 3' direction.

[0031] In another specific embodiment, the expression vector of the present invention is configured such that the target protein has a viral nucleocapsid ligated to its C-terminus. As a specific example of such an embodiment, the expression vector of the present invention includes a construct in which a promoter, a target protein encoding site or a cloning site for inserting a target protein encoding polynucleotide (e.g., restriction enzyme recognition site, e.g., multiple cloning site (MCS)), a viral nucleocapsid encoding site, and a terminator are operably ligated in the 5' to 3' direction.

[0032] In yet another specific embodiment, the expression vector of the present invention is configured to allow the user to choose to apply the fusion partner to the N-terminus of the target protein or, conversely, to the C-terminus of the target protein. As a specific example of such an embodiment, the expression vector of the present invention includes a construct in which a promoter, a first cloning site for inserting a target protein-coding polynucleotide, a viral nucleocapsid-coding site, a second cloning site for inserting a target protein-coding polynucleotide, and a terminator are operably linked in the 5' to 3' direction.

[0033] The expression vector of the present invention may include a T7 promoter and a T7 terminator so that the transcription of the target protein and fusion partner encoding DNA is regulated by the bacteriophage T7 system. This allows for more effective production of the target protein in E. coli.

[0034] The expression vector of the present invention described above can be fabricated, for example, using a conventional recombinant method based on an existing expression vector.

[0035] According to another embodiment of the present invention, host cells transformed with the target protein expression vector of the present invention are provided.

[0036] In the present invention, the host cell may be selected from cells of any organism, including prokaryotes and eukaryotes, but is preferably a cell of a microorganism of the genus Escherichia, and more preferably Escherichia coli.

[0037] As used herein, the terms "transformation" or "introduction" mean introducing a polynucleotide into a host so that the polynucleotide can be replicated as an extrachromosomal factor or by completion of chromosome integration. Methods for transformation with the expression vector according to the present invention may include, but are not limited to, electroporation, calcium phosphate (CaPO4), calcium chloride (CaCl2), microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposome, or lithium acetate-DMSO.

[0038] A method for efficiently producing a target protein in heterologous or homogeneous host cells is provided, comprising the steps of: preparing a target protein expression vector containing the target protein and a polynucleotide encoding a viral nucleocapsid as a fusion partner of the target protein; introducing the target protein expression vector into host cells to produce a transformant; and culturing the transformant.

[0039] In this regard, specific details regarding target protein expression vectors and their introduction can be found in the information described above.

[0040] For culturing transformants, culture methods known to be suitable for the host cell can be used. For example, if the host cell is E. coli, a culture method using LB (Luria-Bertani) medium at a temperature of 16-37°C can be used. If the expression vector contains components of an antibiotic selection marker (e.g., an ampicillin resistance gene), the growth of E. coli containing the expression vector can be preferentially induced by adding the antibiotic (e.g., ampicillin) to the culture medium. Furthermore, if the expression vector contains components that regulate the transcription and / or translation of target protein-coding DNA, such as lac operon-related components, the expression of the target protein can be induced by adding related substances, such as IPTG.

[0041] The following examples are provided to aid in understanding the invention and do not limit the scope of protection of the invention.

[0042] [Examples] Manufacturing Example 1 To investigate the potential of using viral nucleocapsids (NCs) as fusion partners, a vector expressing HIV virus NCs was constructed (Figure 1). Six histidine molecules were also added for purification.

[0043] The pGEMEX-1(Promega) vector, designed to regulate the expression of the target protein by the T7 promoter, was used as the sequence for Sequence ID No. 2 to encode NC.

[0044] Manufacturing Example 2 A vector was prepared to express the product in a form in which six histidine molecules were excluded compared to the above-mentioned production example 1 (Figure 2).

[0045] Example 1 We constructed a vector for expressing the target protein, GFP (green fluorescent protein), in a form in which an NC (non-cylinder nucleotide) is linked to the N-terminus as a fusion partner (Figure 3).

[0046] The same method as in Manufacturing Example 2 was used, but a sequence encoding NC (SEQ ID NO: 2) and a target protein encoding sequence were inserted into the MCS of the pGEMEX-1(Promega) vector.

[0047] In the constructed vector, the NC and GFP encoding sequences are the same as those in SEQ ID NO: 4, and the amino acid sequence of the NC+GFP fusion protein expected to be expressed is the same as that of SEQ ID NO: 5.

[0048] Example 2 A vector was constructed to express the target protein, human-derived IFNβ (interferon-beta), in a form in which a NC (noncortical cell) was linked to the N-terminus as a fusion partner (Figure 6). Six histidine molecules were also added for purification.

[0049] The same method as in Example 1 was used, but the sequence of Sequence ID No. 3, which is an E. coli codon-optimized sequence, was used as the sequence to encode NC.

[0050] In the constructed vector, the NC and IFNβ encoding sequences are the same as those in SEQ ID NO: 6, and the amino acid sequence of the NC+IFNβ fusion protein expected to be expressed is the same as that of SEQ ID NO: 7.

[0051] Comparative Example 1 Using the same method as in Example 2, a vector was prepared to express the target protein, human-derived IFNβ, in a form in which the NC was not linked as a fusion partner, but rather six histidines were linked to the N-terminus (Figure 7).

[0052] Experimental Example 1 The vectors from Production Example 1, Production Example 2, Example 1, Example 2, and Comparative Example 1 were transformed into E. coli Shuffle / T7 / pLysS and cultured. Shuffle / T7 / pLysS was BL21 star(DE3)pLysS One Shot. TM vector TMThe pLysS plasmid was extracted from competent E. coli via Mini-prep, and pLysS was introduced into SHuffle T7(NEB) competent E. coli. The transformed cells were then sorted using chloramphenicol (CM), an antibiotic marker contained in pLysS. All transformed E. coli were cultured in LB medium containing 50 μg / ml ampicillin and 34 μg / ml chloramphenicol. The culture temperature was set to 16–37°C. When the OD600 value of the E. coli reached 0.5 or higher, IPTG was added at a level of 0 μM–1 mM to activate the T7 promoter. After adding IPTG, the cells were cultured for approximately 3 hours at 30°C or 16 hours at 16–20°C to ensure sufficient protein production. The fully cultured E. coli were then centrifuged, the supernatant was removed, and the cells were stored. Next, 0.3 ml of PBS was added to 5 ml of E. coli harvested in LB medium, and ultrasonic grinding was performed to prepare a lysate. The lysate was then centrifuged to separate it into a soluble fraction and a pellet fraction, and the total lysate, soluble fraction, and pellet fraction were separated and analyzed by SDS-PAGE.

[0053] First, the vectors from Production Example 1 and Production Example 2 were introduced into E. coli, respectively, to induce NC expression. As shown in Figures 1 and 2, the expression was normal at both 16°C and 20°C, i.e., in large quantities, with the expected size and water solubility. In particular, as shown in Figure 2, it was shown that expression was almost 100% water-soluble even without the His-tag. This suggests that NC may be useful as a fusion partner for increasing the expression efficiency of target proteins, especially for achieving water-soluble expression.

[0054] When the vector from Example 1 was introduced into E. coli, the expression of the target protein, GFP, was induced. As shown in Figure 3, it was shown that GFP was expressed normally at both 16°C and 20°C. GFP is known to be expressed insolublely in E. coli. Therefore, these results indicate that, according to the method of the present invention, proteins expressed insolublely in E. coli can be expressed in a water-soluble manner.

[0055] Furthermore, when the vector from Example 1 was introduced into E. coli and GFP expression was investigated under different IPTG concentrations, the results, as shown in Figure 4, showed that even at low IPTG concentrations, GFP expression was efficient in proportion to the IPTG concentration. The GFP fluorescence signal of the expressed protein was measured using a microplate reader, and as shown in Figure 5, the GFP fluorescence signal increased in proportion to the IPTG concentration. This indicates that, according to the method of the present invention, the target protein can be efficiently expressed while maintaining its original function.

[0056] When the vectors of Example 2 and Comparative Example 1 were introduced into E. coli, respectively, to induce the expression of the target protein, IFNβ, the results, as shown in Figures 6 and 7, were observed. In Example 2, the proportion of insoluble expression at 30°C was relatively high, but a large amount of protein was expressed normally, particularly in water-soluble, at both 16°C and 30°C. In contrast, in Comparative Example 1, many proteins were expressed insolublely. This indicates that, according to the method of the present invention, proteins that are expressed insolublely in E. coli can be expressed in water-soluble.

[0057] Furthermore, the vector from Example 2 was introduced into E. coli to induce the expression of the target protein, IFNβ. After purification by Ni-affinity chromatography, the IFNβ reaction was analyzed (Invivogen) by serially diluting the solution by 1 / 2 from an initial concentration of 200 nM. As shown in Figure 8, the IFNβ reaction was confirmed in the purified product, and it was shown that this IFNβ reaction decreased as the dilution ratio increased. In this case, culture medium was used as the control group (Con). These results demonstrate that, according to the method of the present invention, the target protein can be efficiently expressed while maintaining its original function.

[0058] Example 3 A vector was prepared to express the target protein, the heat-labile enterotoxin B subunit (LTB) of E. coli, in a form in which a nucleotide chorionic molecule (NC) is linked to the N-terminus as a fusion partner. In this process, six histidine molecules for purification were added, and the same method as in Example 1 was used.

[0059] Example 4 A vector was prepared in the same manner as in Example 3, but in the NC amino acid sequence of Sequence ID No. 1, cysteine ​​(C) at positions 14, 17, and 27 was replaced with serine (S) to express the vector (ΔZF1 in Figure 9). These residues, along with histidine (H) at position 22, interact with zinc ions in the first zinc-binding domain of NC.

[0060] Example 5 The vector was prepared in the same manner as in Example 3, but in the NC amino acid sequence of Sequence ID No. 1, cysteine ​​residues at positions 35, 38, and 48 were replaced with serine residues to express the vector (ΔZF2 in Figure 9). These residues, along with histidine (H) at position 43, interact with zinc ions in the second zinc-binding domain of NC.

[0061] Example 6 The vector was prepared in the same manner as in Example 3, but in the NC amino acid sequence of Sequence ID No. 1, cysteine ​​at positions 14, 17, 27, 35, 38, and 48 was replaced with serine to express the vector (ΔZF1+2 in Figure 9).

[0062] Experimental Example 2 The vectors from Examples 3 to 6 were used to transform E. coli Shuffle / T7 / pLysS in the same manner as in Experimental Example 1. After inducing the production of fusion proteins, the produced fusion proteins were analyzed by SDS-PAGE.

[0063] As a result, as shown in Figure 10, in Examples 3 and 5, most of the fusion proteins were expressed normally in water, whereas in Examples 4 and 6, the proportion of fusion proteins expressed in water decreased to about 50%. This indicates that the residue that interacts with zinc ions in the first zinc-binding domain of NC is important for the water-soluble expression of the target protein.

[0064] Experimental Example 3 The vector from Example 1 was transformed into E. coli Shuffle / T7 / pLysS using the same method as in Experimental Example 1. After inducing the production of the fusion protein, it was purified by zinc affinity chromatography and then analyzed by SDS-PAGE and GFP fluorescence signal measurement (using a microplate reader).

[0065] As a result, as shown in Figure 11, the fusion protein could be purified with a considerably high recovery rate via zinc affinity chromatography. This indicates that efficient purification is possible using the zinc-binding domain of NC without the need for other purification tags such as histidine tags.

[0066] The present invention has been described above, focusing on its preferred embodiments. Those with ordinary skill in the art to which the present invention pertains will understand that the present invention may be embodied in modified forms that do not depart from its essential characteristics. Therefore, the disclosed embodiments should be considered in an explanatory rather than restrictive manner. The scope of the present invention is defined in the claims, not in the foregoing description, and all differences within an equivalent scope should be understood as being included within the present invention.

[0067] <array list> Sequence ID 1 Sequence name: Amino acid sequence of HIV nucleocapsid Array type: AA Organism name: Human immunodeficiency virus array: QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQAN

[0068] Sequence ID 2 Sequence name: Nucleotide sequence encoding the HIV nucleocapsid Sequence type: DNA Organism name: Human immunodeficiency virus array: CAGCGGGGAAACTTCAGGAACCAGAGAAAAACTGTGAAGTGCTTCAATTGCGGAAAGGAGGGCCACATCGCTAAGAACTGCCGCGCCCCCAGAAAGAAAGGCTGCTGGAGATGCGGCAGAGAGGGCCACCAGATGAAGGACTGCACAGAGAGACAGGCAAAC

[0069] Sequence ID 3 Sequence name: Nucleotide sequence encoding the HIV nucleocapsid Sequence type: DNA Organism name: Synthetic construct array: CAGCGTGGTAACTTCCGTAACCAGCGTAAAACCGTTAAATGCTTCAACTGCGGCAAAGAAGGCCACATCGCGAAAAACTGCCGTGCGCCGCGTAAAAAAGGCTGCTGGCGTTGCGGCCGTGAAGGCCACCAGATGAAAGATTGCACCGAACGTCAGGCGAAC

[0070] Sequence ID 4 Sequence name: Nucleotide sequence encoding a fusion protein of NC and GFP Sequence type: DNA Organism name: Synthetic construct array: AGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTT GTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTG TTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGC AGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA

[0071] Sequence ID 5 Sequence name: Amino acid sequence of NC and GFP fusion protein Array type: AA Organism name: Synthetic construct array: SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0072] Sequence ID 6 Sequence name: Nucleotide sequence encoding a fusion protein of NC and IFNβ Sequence type: DNA Organism name: Synthetic construct array: AAATCTTACAACCTGCTGGGCTTCCTGCAGCGTTCCTCCAACTTCCAGTGCCAGAAGCTGCTGTGGCAGCTGAACGGTCGCCTGGAATACTGCCTGAAAGATCGTATGAACTTCGATATCCCGG AAGAAATCAAACAGTTGCAGCAGTTCCAGAAAGAAGATGCGGCGTTAACCATCTACGAAATGCTGCAAAACATTTTCGCTATCTTCCGTCAGGATTCCAGCTCCACCGGCTGGAACGAAACCATC GTTGAAAACCTTCTGGCGAACGTTATCACCAGATCAATCATCTGAAAACCGTGCTGGAAGAGAAACTGGAAAAAGAAGACTTCACCCGTGGTAAACTGATGTCCTCGCTGCACCTGAAGCGTTACTACGGTCGTATCCTGCATTATCTGAAAGCGAAAGAGTACAGCCACTGCGCTTGGACCATTGTTCGTGTTGAAATTCTGCGCAATTTCTACTTCATCAACCGTCTGACCGGTTACCTGCGTAAC

[0073] Sequence ID 7 Sequence name: Amino acid sequence of NC and IFNβ fusion protein Array type: AA Organism name: Synthetic construct array: KSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN

Claims

1. A target protein expression vector containing a polynucleotide encoding a viral nucleocapsid as a fusion partner for the target protein.

2. The target protein expression vector according to claim 1, wherein the target protein is selected from one or more of the group consisting of antigens, antibodies, cell receptors, enzymes, structural proteins, serum, and cellular proteins.

3. The target protein expression vector according to claim 1, wherein the viral nucleocapsid is a nucleocapsid of human immunodeficiency virus.

4. The target protein expression vector according to claim 1, wherein the viral nucleocapsid comprises the amino acid sequence of SEQ ID NO:

1.

5. The target protein expression vector according to claim 1, wherein the vector is for expressing a target protein in Escherichia coli.

6. A host cell transformed with a target protein expression vector according to any one of claims 1 to 5.

7. The steps include: preparing a target protein expression vector containing a target protein and a polynucleotide encoding a viral nucleocapsid as a fusion partner for the target protein; The steps include: introducing the target protein expression vector into host cells to produce a transformant; A method for producing a target protein, comprising the step of culturing the aforementioned transformant.

8. The method for producing a target protein according to claim 7, wherein the target protein is selected from one or more of the group consisting of antigens, antibodies, cell receptors, enzymes, structural proteins, serum, and cellular proteins.

9. The method for producing a target protein according to claim 7, wherein the viral nucleocapsid is a nucleocapsid of human immunodeficiency virus.

10. The method for producing a target protein according to claim 7, wherein the viral nucleocapsid comprises the amino acid sequence of SEQ ID NO:

1.

11. The method for producing a target protein according to claim 7, wherein the host cell is Escherichia coli.

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