Targeted protein expression platform using viral nucleocapsids

Abstract

The target protein expression vector of the present invention includes a polynucleotide encoding a viral nucleocapsid as a fusion partner of the target protein. Also, the target protein production method of the present invention includes the steps of: constructing a target protein expression vector including 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 a host cell to produce a transformant; and culturing the transformant.

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 and expressed in E. coli, in a host cell, particularly E. coli, using a viral nucleocapsid as a fusion partner. [Background technology]

[0002] The core of modern biotechnology is the production of recombinant proteins, and the most important thing is to produce them in large quantities while maintaining their inherent activity. Recombinant proteins produced in this way are very important for the production and recovery of foreign proteins, crystallization for functional research, industrialization, etc.

[0003] To date, many recombinant protein production studies have been conducted using Escherichia coli, because it has the advantages of being easy to manipulate, having a short growth time, stable expression, low cost, and easy scale-up. However, it has been pointed out that the recombinant proteins of foreign origin produced in Escherichia coli have the disadvantages of low expression levels and the formation of insoluble protein aggregates (inclusion bodies) of the produced recombinant proteins due to the lack of appropriate "post-translational chaperons" and "post-translational processing" (Francois Baneyx, Recombinant protein expression in Escherichia coli, Current Opinion in Biotechnology (1999), 10: 411-421).

[0004] To solve these problems, 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 own technical limitations, and the development of new expression technologies is required. Summary of the Invention [Problem to be solved by the invention]

[0005] A main object of the present invention is to provide an expression vector capable of efficiently expressing a target protein, particularly a difficult-to-express protein that is expressed in an insoluble form in E. coli, in a host cell, particularly E. coli.

[0006] Another object of the present invention is to provide a host cell that is transformed with the expression vector and is capable of efficiently expressing the target protein.

[0007] It is yet another object of the present invention 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 comprising a polynucleotide encoding a viral nucleocapsid as a fusion partner for a target protein.

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

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

[0011] According to the present invention, a target protein, particularly a difficult-to-express protein that is expressed in an insoluble form in E. coli, can be efficiently expressed in a host cell, particularly E. coli. [Brief description of the drawings]

[0012] FIG. 1 is a diagram for investigating the possibility of using viral nucleocapsid (NC) as a fusion partner, and shows the results of expressing NC together with a His-tag in Escherichia coli and confirming the results with SDS-PAGE. FIG. 2 is a diagram for investigating the possibility of using NC as a fusion partner, and shows the results of expressing NC in E. coli (expressed without a His-tag) and confirming the results with SDS-PAGE. FIG. 3 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. FIG. 4 shows the results of SDS-PAGE analysis of the expression of the target protein GFP when an expression vector according to one embodiment of the present invention was introduced into E. coli and treated with different IPTG concentrations. FIG. 5 shows the results of measuring the fluorescent signal of GFP, an expressed target protein, using a microplate reader when an expression vector according to one embodiment of the present invention is introduced into E. coli and treated with different IPTG concentrations. FIG. 6 shows the results of confirming the expression of the target protein, IFN-β, by SDS-PAGE after introducing an expression vector according to one embodiment of the present invention into E. coli. FIG. 7 shows the results of expressing IFNβ in E. coli without a fusion partner and checking by SDS-PAGE. FIG. 8 shows the results of an IFNβ reaction assay experiment in which the target protein from the experiment in FIG. 6, IFNβ, was purified by Ni-affinity chromatography and then serially diluted. FIG. 9 shows the amino acid sequences of the mutant NCs used in the experiment along with the wild type NC, and their interactions with zinc ions. FIG. 10 shows the results of SDS-PAGE confirming the expression of the fusion protein when each of the NCs shown in FIG. 9 was applied. FIG. 11 shows the results of introducing an expression vector according to one embodiment of the present invention into E. coli, inducing expression of the target protein GFP, purifying the protein by zinc affinity chromatography, and analyzing the protein by SDS-PAGE and measuring the fluorescent signal of GFP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

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

[0015] In the present invention, the target protein may be one or more selected from the group consisting of antigens, antibodies, cell receptors, enzymes, structural proteins, serum and cell proteins, but is not limited thereto.

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

[0017] The expression vector of the present invention may be a plasmid, viral vector, phage particle or genomic insert and may, after introduction into a host cell, replicate independently of the host cell genome or may be integrated into the host cell genome.

[0018] The expression vector of the present invention may contain a polynucleotide encoding a target protein, or may be used by subsequently containing a polynucleotide encoding a target protein. In particular, in the latter case, the expression vector preferably contains a restriction enzyme recognition site, which is a component that can easily contain a polynucleotide encoding a target protein and is usually called a multiple cloning site (MCS).

[0019] The present invention is based on the new findings that when a viral nucleocapsid is applied as a fusion partner for a target protein, the target protein can be efficiently expressed in a soluble form, for example, a protein that is expressed in a soluble form without using a fusion partner or with other existing fusion partners.

[0020] In particular, the fusion partners used in the present invention have the advantage that they are very small in size (short peptide length) and therefore can minimize interference with the target protein.

[0021] In addition, the present invention has the advantage that it can use a zinc binding domain inherent to viral nucleocapsids, for example, by using the zinc binding domain of viral nucleocapsids, the fusion protein can be easily purified without a separate component 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 previously known viral nucleocapsid, or may be a fragment or mutant of the viral nucleocapsid, so long as the activity of the present invention, i.e., the activity of acting as a fusion partner for a target protein to enable the target protein to be expressed in a soluble form, particularly in E. coli, is maintained.

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

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

[0025] In the present invention, the viral nucleocapsid preferably comprises 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, more preferably 99% or more sequence identity to the amino acid sequence of SEQ ID NO:1. Preferably, the amino acid sequence satisfies the above sequence identity while maintaining the sequences of positions 14, 17, 22 and 27 of the amino acid sequence of SEQ ID NO: 1, more preferably while maintaining the sequences of positions 14, 17, 22, 27, 35, 38, 43 and 48, and more preferably while maintaining the sequences of positions 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, a polynucleotide encoding a viral nucleocapsid preferably comprises 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, more preferably 99% or more sequence identity with the base sequence of SEQ ID NO:2 or SEQ ID NO:3. Preferably, the base sequence satisfies the above sequence identity while encoding an amino acid sequence that maintains the sequences of positions 14, 17, 22, and 27 of the amino acid sequence of SEQ ID NO:1, more preferably, the sequences of positions 14, 17, 22, 27, 35, 38, 43, and 48, and more preferably, the sequences of positions 6, 9, 10, 13, 14, 17, 19, 22, 25, 27, 28, 31, 32, 33, 35, 37, 38, 40, 43, 46, 48, and 51.

[0027] As used herein, sequence identity refers to the 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 a position in the compared 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 the position shared by the amino acid or nucleotide sequences, respectively. For example, a variety of alignment algorithms and / or programs, including FASTA or BLAST, can be used to calculate the identity between two sequences.

[0028] The expression vector of the present invention may be for expressing a target protein in a cell selected from among any organism, including prokaryotes and eukaryotes, but is preferably for expressing a target protein in a microorganism of the genus Escherichia, 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-encoding polynucleotide, e.g., a restriction enzyme recognition site, e.g., a multiple cloning site (MCS); a linker sequence for providing spacing between the target protein and the fusion partner; a tag sequence, e.g., a histidine tag sequence; and / or a protease recognition site coding sequence for excising a portion of the expressed fusion protein. Each of these may be arranged in various positions and included in various numbers as required.

[0030] In a specific embodiment, the expression vector of the present invention is configured so that the viral nucleocapsid is linked to the N-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 viral nucleocapsid coding region, a target protein coding region or a cloning site for inserting a target protein coding polynucleotide (e.g., a restriction enzyme recognition site, e.g., a multiple cloning site (MCS)) and a terminator are operably linked 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 is linked to the viral nucleocapsid at the 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 coding region or a cloning site for inserting a target protein coding polynucleotide (e.g., a restriction enzyme recognition site, e.g., a multiple cloning site (MCS)), a viral nucleocapsid coding region, and a terminator are operably linked in this order from 5' to 3'.

[0032] In yet another specific embodiment, the expression vector of the present invention is configured so that the user can select whether to link 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, in the 5' to 3' direction, a promoter, a first cloning site for inserting a polynucleotide encoding a target protein, a viral nucleocapsid encoding site, a second cloning site for inserting a polynucleotide encoding a target protein, and a terminator are operably linked.

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

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

[0035] According to another embodiment of the present invention, there is provided a host cell transformed with a target protein expression vector of the present invention.

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

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

[0038] According to yet another embodiment of the present invention, there is provided a method for efficiently producing a target protein in a heterologous or homologous host cell, the method comprising the steps of: constructing a target protein expression vector comprising a polynucleotide encoding a target protein and a viral nucleocapsid as a fusion partner of the target protein; introducing the target protein expression vector into a host cell to produce a transformant; and culturing the transformant.

[0039] In this regard, for specific matters regarding the target protein expression vector and matters regarding the introduction of the target protein expression vector, please refer to the matters described above.

[0040] For culturing the transformant, a culturing method known to be suitable for the host cell can be used. For example, when the host cell is Escherichia coli, a method of culturing in LB (Luria-Bertani) medium at a temperature of 16 to 37°C can be used. When the expression vector has an antibiotic selection marker (e.g., an ampicillin resistance gene), the growth of Escherichia coli containing the expression vector can be preferentially induced by adding the antibiotic (e.g., ampicillin) to the medium and culturing. In addition, when the expression vector has a component that regulates the transcription and / or translation of the target protein-encoding DNA, for example, a lac operon-related component, the expression of the target protein can be induced by adding a related substance, for example, IPTG.

[0041] The following examples are presented to aid in understanding the invention, and are not intended to limit the scope of the invention.

[0042] [Example] Manufacturing Example 1 To investigate the possibility of using the viral nucleocapsid (NC) as a fusion partner, we constructed a vector for expressing the HIV NC (Fig. 1), with six histidine residues added for purification.

[0043] The vector was constructed based on pGEMEX-1 (Promega), which is designed so that expression of the target protein is regulated by the T7 promoter, and the sequence of SEQ ID NO: 2 was used as the sequence encoding NC.

[0044] Manufacturing Example 2 A vector was constructed to express the gene in a form in which six histidines were omitted compared to Preparation 1 (FIG. 2).

[0045] Example 1 A vector was constructed to express the target protein, green fluorescent protein (GFP), in a form in which NC was linked to the N-terminus as a fusion partner (Figure 3).

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

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

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

[0049] The same method as in Example 1 was used, but the sequence encoding NC was the E. coli codon-optimized sequence shown in SEQ ID NO: 3.

[0050] In the constructed vector, the NC and IFNβ coding sequence sites are the same as SEQ ID NO:6, and the amino acid sequence of the NC+IFNβ fusion protein predicted to be expressed is the same as SEQ ID NO:7.

[0051] Comparative Example 1 The same method as in Example 2 was used, but a vector was constructed to express the target protein, human-derived IFNβ, in a form in which 6 histidines were linked to the N-terminus without NC as a fusion partner (FIG. 7).

[0052] Experimental Example 1 The vectors of Preparation Example 1, Preparation 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 Vitrogen TMThe pLysS plasmid was extracted from competent E. coli using Mini-prep, and pLysS was introduced into SHuffle T7 (NEB) competent E. coli, followed by selection with 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 at 16 to 37°C. When the OD600 value of E. coli reached 0.5 or higher, IPTG was added at 0 μM to 1 mM levels to activate the T7 promoter, and the cells were cultured at 30°C for 3 hours or at 16 to 20°C for 16 hours to ensure sufficient protein production. The fully cultured E. coli was centrifuged to remove the supernatant and then stored. Next, 0.3 ml of PBS was added to the E. coli harvest equivalent to 5 ml of LB medium, and ultrasonic disruption was performed to prepare a lysate, which was then centrifuged to separate the soluble and pellet fractions, and the total lysate, soluble fraction, and pellet fraction were analyzed by SDS-PAGE.

[0053] First, the vectors of Preparation Example 1 and Preparation Example 2 were introduced into E. coli to induce the expression of NC. As a result, it was shown that NC was expressed normally, i.e., in large quantities, with the expected size and water solubility, at both 16°C and 20°C, as shown in Figures 1 and 2. In particular, as shown in Figure 2, it was shown that NC was expressed almost 100% in water solubility even in the absence of a His-tag. This indicates that NC may be used as a fusion partner for the purpose of increasing the expression efficiency of target proteins, especially for the purpose of expressing NC in a water soluble form.

[0054] The vector of Example 1 was introduced into E. coli to induce expression of the target protein, GFP, and as a result, it was shown that it was normally expressed at both 16°C and 20°C, as shown in Figure 3. GFP is known to be expressed in an insoluble form in E. coli. Therefore, these results show that according to the method of the present invention, a protein that is expressed in an insoluble form in E. coli can be expressed in a soluble form.

[0055] Furthermore, when the vector of Example 1 was introduced into E. coli and treated with different IPTG concentrations, GFP expression was investigated, and it was shown that even at low IPTG concentrations, GFP was efficiently expressed in proportion to the IPTG concentration, as shown in Figure 4, and the GFP fluorescent signal of the expressed protein was measured with a microplate reader, and it was shown that the GFP fluorescent signal increased in proportion to the IPTG concentration, as shown in Figure 5. This shows that the method of the present invention can efficiently express a target protein while maintaining its original function.

[0056] The vectors of Example 2 and Comparative Example 1 were introduced into E. coli to induce expression of the target protein IFNβ. As a result, as shown in Figures 6 and 7, in the case of Example 2, the proportion of proteins expressed insoluble at 30°C was relatively high, but a large amount of protein was expressed normally, especially in soluble form, at both 16°C and 30°C, whereas in the case of Comparative Example 1, many proteins were expressed insoluble. This indicates that according to the method of the present invention, proteins that are expressed insoluble in E. coli can be expressed in soluble form.

[0057] Furthermore, the vector of Example 2 was introduced into E. coli to induce expression of the target protein IFNβ, which was purified by Ni-affinity chromatography, and then an IFNβ reaction assay (Invivogen) was performed by serially diluting the sample at 1 / 2 dilution from the initial concentration of 200 nM. As a result, as shown in Figure 8, an IFNβ reaction was observed in the purified product, and the IFNβ reaction decreased as the dilution ratio increased. In this case, culture medium was used as the control group (Con). These results indicate that the method of the present invention can efficiently express a target protein while maintaining its original function.

[0058] Example 3 A vector was constructed to express the target protein, E. coli heat-labile enterotoxin B subunit (LTB), in the form in which NC is linked to the N-terminus as a fusion partner, using the same method as in Example 1, with the addition of six histidines for purification.

[0059] Example 4 A vector was constructed in the same manner as in Example 3, except that cysteine ​​(C) at positions 14, 17, and 27 in the NC amino acid sequence of SEQ ID NO: 1 was replaced with serine (S) (ΔZF1 in FIG. 9). These residues, together with histidine (H) at position 22, are the residues that interact with zinc ions in the first zinc-binding domain of NC.

[0060] Example 5 A vector was constructed in the same manner as in Example 3, except that the cysteines at positions 35, 38, and 48 in the NC amino acid sequence of SEQ ID NO: 1 were replaced with serine (ΔZF2 in FIG. 9 ). These residues, together with histidine (H) at position 43, are the residues that interact with zinc ions in the second zinc-binding domain of NC.

[0061] Example 6 A vector was constructed in the same manner as in Example 3, except that the cysteines at positions 14, 17, 27, 35, 38, and 48 in the NC amino acid sequence of SEQ ID NO: 1 were expressed in a form in which they were replaced with serine (ΔZF1+2 in Figure 9).

[0062] Experimental Example 2 The vectors of Examples 3 to 6 were transformed into E. coli Shuffle / T7 / pLysS in the same manner as in Experimental Example 1, and then the production of fusion proteins was induced. Each of the produced fusion proteins was analyzed by SDS-PAGE.

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

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

[0065] As a result, it was shown that the fusion protein could be purified with a fairly high recovery rate through zinc affinity chromatography (Figure 11), indicating that efficient purification is possible using the zinc-binding domain of NC without the need for a separate purification tag such as a histidine tag.

[0066] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than restrictive sense. The scope of the present invention is set forth in the claims, not in the above description, and all differences within the scope of the equivalents thereto should be understood to be included in the present invention.

[0067] <Array list> SEQ ID NO:1 Sequence name: Amino acid sequence of HIV nucleocapsid Array type: AA Organism name: Human immunodeficiency virus array: QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQAN

[0068] SEQ ID NO:2 Sequence Name: Nucleotide sequence encoding the HIV nucleocapsid Sequence type: DNA Organism name: Human immunodeficiency virus array: CAGCGGGGAAACTTCAGGAACCAGAGAAAAACTGTGAAGTGCTTCAATTGCGGAAAGGAGGGCCACATCGCTAAGAACTGCCGCGCCCCCAGAAAGAAAGGCTGCTGGAGATGCGGCAGAGAGGGCCACCAGATGAAGGACTGCACAGAGAGACAGGCAAAC

[0069] SEQ ID NO:3 Sequence Name: Nucleotide sequence encoding the HIV nucleocapsid Sequence type: DNA Organism name: synthetic construct array: CAGCGTGGTAACTTCCGTAACCAGCGTAAAACCGTTAAATGCTTCAACTGCGGCAAAGAAGGCCACATCGCGAAAAACTGCCGTGCGCCGCGTAAAAAAGGCTGCTGGCGTTGCGGCCGTGAAGGCCACCAGATGAAAGATTGCACCGAACGTCAGGCGAAC

[0070] SEQ ID NO:4 Sequence Name: Nucleotide sequence encoding the fusion protein of NC and GFP Sequence type: DNA Organism name: synthetic construct array: AGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTT GTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTG TTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGC AGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA

[0071] SEQ ID NO:5 Sequence name: Amino acid sequence of fusion protein of NC and GFP Array type: AA Organism name: synthetic construct array: SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0072] SEQ ID NO:6 Sequence Name: Nucleotide sequence encoding the fusion protein of NC and IFNβ Sequence type: DNA Organism name: synthetic construct array: AAATCTTACAACCTGCTGGGCTTCCTGCAGCGTTCCTCCAACTTCCAGTGCCAGAAGCTGCTGTGGCAGCTGAACGGTCGCCTGGAATACTGCCTGAAAGATCGTATGAACTTCGATATCCCGG AAGAAATCAAACAGTTGCAGCAGTTCCAGAAAGAAGATGCGGCGTTAACCATCTACGAAATGCTGCAAAACATTTTCGCTATCTTCCGTCAGGATTCCAGCTCCACCGGCTGGAACGAAACCATC GTTGAAAACCTTCTGGCGAACGTTATCACCAGATCAATCATCTGAAAACCGTGCTGGAAGAGAAACTGGAAAAAGAAGACTTCACCCGTGGTAAACTGATGTCCTCGCTGCACCTGAAGCGTTACTACGGTCGTATCCTGCATTATCTGAAAGCGAAAGAGTACAGCCACTGCGCTTGGACCATTGTTCGTGTTGAAATTCTGCGCAATTTCTACTTCATCAACCGTCTGACCGGTTACCTGCGTAAC

[0073] SEQ ID NO:7 Sequence name: Amino acid sequence of fusion protein of NC and IFNβ Array type: AA Organism name: synthetic construct array: KSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN

Claims

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

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

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

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

1.

5. The target protein expression vector of claim 1 , wherein the vector is for expressing a target protein in E. coli.

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

7. constructing a target protein expression vector comprising a polynucleotide encoding a target protein and a viral nucleocapsid as a fusion partner of the target protein; introducing the target protein expression vector into a host cell to produce a transformant; and culturing the transformant.

8. The method for producing a target protein according to claim 7 , wherein the target protein is at least one selected from the group consisting of antigens, antibodies, cellular 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 human immunodeficiency virus nucleocapsid.

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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