KR101158755B1 - Vector for expressing NC protein of HIV and method for producing NC protein using the same - Google Patents

Abstract

The present invention relates to a vector for NC protein expression of HIV and a method for producing NC protein using the same. More specifically, the present invention relates to a vector for NC protein expression of HIV and a method for producing NC protein using the same, characterized in that the mRNA stabilization factor (mRNA stability element) is sequentially connected to the lower portion of the intron sequence and NC gene will be.

The NC protein expression vector of HIV of the present invention can express a wild type NC protein in an animal cell, and its expression efficiency is improved as compared with the prior art, thereby developing an antiviral agent effective against HIV. It can be used to.

HIV, nucleocapsid (NC), expression, vector, mRNA stability element

Description

Vector for expressing NC protein of HIV and method for producing NC protein using the same}

1 is a schematic diagram showing the manufacturing process of the vector pCMV (-HA) NC / RRE for HIV NC protein expression according to the present invention.

Figure 2 is a result of Western blot to compare the amount of NC protein expression in cells transformed with the vector for expressing the NC NC protein according to the present invention.

The present invention relates to a vector for NC protein expression of HIV and a method for producing NC protein using the same. More specifically, the present invention relates to a vector for NC protein expression of HIV and a method for producing NC protein using the same, characterized in that the mRNA stabilization factor (mRNA stability element) is sequentially connected to the lower portion of the intron sequence and NC gene will be.

Nucleocapsid proteins of human immunodeficiency virus (hereinafter referred to as 'NC') are not only structural roles for viral individualization, but also functionally important for the viral life cycle. The function is as follows. First, NC proteins are involved in the genetic encapsidation of viruses. This function is derived from two zincfinger domains consisting of the unique Cys-X2-Cys-X4-His-X4-Cys motif (CCHC motif), which exhibits high conservedness in all retroviruses, and HIV It is known to be essential for RNA packaging and infectious virus production. Second, NC proteins are known to promote tRNA primer annealing and strand transfer during virus reverse transcription (RT), suggesting that NC proteins play an important role in viral replication. It can be seen that. Third, NC proteins have the nucleic acid chaperone activity required for the viral life cycle. Recently, NC proteins have been reported to play a role even when viral DNA is inserted into host cell chromosomes. Therefore, the study of these NC proteins will be very important not only to reveal the biological functions of NC proteins in the HIV life cycle, but also to develop effective antiviral agents for key HIV proteins.

An essential prerequisite for understanding the biological function of NC proteins in vivo is to develop methods for effectively expressing NC proteins in animal cells. However, in the case of other structural proteins of HIV, animal cell expression is restricted because the codon usage of the virus is different from the genetic code usage of the animal cell or because it contains an inhibitory sequence (INS) in the gene. There is a case. In the case of NC protein, unlike other structural proteins of HIV, despite the absence of INS, there was a problem that the expression in animal cells is significantly low. Therefore, most studies on NC proteins have been carried out using recombinant NC proteins expressed in E. coli, or using genetic analysis. The inventors have filed a patent application (Korean Patent Registration No. 0553154) for producing HIV NC protein in animal cells through previous studies. The method described above is characterized by linking the intron sequence on top of the NC gene or further linking the FLAG gene on top of the NC gene. However, when only the intron sequence is linked to the upper NC gene, the expression efficiency is low, and when additionally linked to the FLAG gene, there is a disadvantage in that it is expressed in a modified NC protein form rather than a wild type.

The inventors of the present invention, while studying the production method of the HIV NC protein that can overcome the problems presented above when the additional expression of mRNA stability factor (mRNA stability element) in the intron sequence and the lower part of the NC gene The present invention has been completed by confirming that a natural type NC protein can be expressed and that the expression efficiency is improved.

Accordingly, it is an object of the present invention to provide an NC protein expression vector for HIV and a method for producing NC protein using the same, wherein an mRNA stability element is further connected to the intron sequence and the lower part of the NC gene. .

In order to achieve the above object, the present invention provides a vector for NC protein expression of HIV characterized in that the mRNA stabilization factor (mRNA stability element) is further connected to the intron sequence and the lower part of the NC gene.

The present invention also provides a transformant transformed with the vector.

The present invention also provides a method for producing NC protein of HIV using the vector.

Hereinafter, the present invention will be described in detail.

The production of HIV Nucleocapsid (NC) proteins will be very important for the development of effective antiviral agents, but to date the expression of HIV NC proteins in animal cells is very low. In order to improve this, the inventors of the present invention have been using the NC protein expression vector for HIV, which is characterized by additionally expressing an mRNA stability element (mRNA stability element) at the bottom of the intron sequence and the NC gene. It was confirmed that there is an effect that the expression of NC protein is significantly improved.

Accordingly, the present invention provides a vector for HIV NC protein expression, characterized in that the intron sequence, HIV NC (nucleocapsid) gene and mRNA stability element (mRNA stability element) is sequentially connected.

More specifically, a) any one sequence selected from the group consisting of an SV40 19s mRNA intron sequence, a modified SV40 16S mRNA intron sequence, and a β-globin intron sequence b) an HIV NC (nucleocapsid) gene; And c) an mRNA vector for HIV NC protein expression, characterized in that mRNA stability elements are sequentially connected.

The "NC protein of HIV" means a nucleocapsid protein of human immunodeficiency virus (HIV), a pathogen causing AIDS (acquired Immunodeficiency Syndrome), which protein binds strongly to viral genomic RNA A ribocleoprotein core complex is formed The HIV NC protein sequence expressed in one embodiment of the present invention is derived from the ARV2 / SF line and is disclosed as the 380th to 434rd amino acid sequence of GenBank No. P03349. Meanwhile, the NC gene sequence used in one embodiment of the present invention was derived from a pJC1 vector (HIV Nucleocapsid Protein; Expression in E. coli, Purification and Characterization. J. Biol. Chem. 268, 16519-16527, 1993).

As used herein, the term "expression" refers to the production of a protein or nucleic acid in a cell.

As used herein, the term “expression vector” refers to a gene construct that is capable of expressing a protein of interest or RNA of interest in a suitable host cell, and includes a gene construct comprising essential regulatory elements operably linked to express the gene insert.

As used herein, the term “operably linked” refers to the functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the encoding nucleic acid sequence.Operative linkage with recombinant vectors may be accomplished using genetic recombination techniques well known in the art. And site-specific DNA cleavage and ligation using enzymes generally known in the art.

Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors can include expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers, and the like, and can be prepared in a variety of ways, and the promoters of the vectors may be constitutive or inducible. Can be.

In the present invention, the intron sequence may be one known in the art, and examples thereof include an SV40 19s mRNA intron sequence, a modified SV40 16S mRNA intron sequence, and a β-globin intron sequence. Preferably, an SV40 19s mRNA intron sequence represented by SEQ ID NO: 1, a modified SV40 16S mRNA intron sequence represented by SEQ ID NO: 2, and a β-globin intron sequence represented by SEQ ID NO: 7 may be used. .

The intron sequence used in one embodiment of the present invention is derived from the intron sequence of pCMV-HA (Clontech Laboratories, Inc) vector (SV40 splice donor / splice acceptor; hereinafter referred to as 'SV40 SD / SA'), Are the 672-702th SV40 19S mRNA intron sequence, and the 672-768 are the modified SV40 16S mRNA intron sequence.

The "mRNA stability element" (mRNA stability element) refers to the element (stability element) to increase the stability by binding to the 3 'end of the mRNA, preferably RRE (Rev response element), WPRE (woodchuck post-transcriptional regulatory element) ), Beta-actin 3'-UTR (β-actin 3'-untranslated regions) and RSV stabilizing factor (Rous Sarcoma Virus stability element) can be any one selected from the group.

Among the mRNA stability elements, “RRE” refers to Rev responsive element (RRE), which is a cis-acting element present in the env gene of HIV-1 RNA. Binds directly to Rev protein. “WPRE (woodchuck hepatitis virus post-transcriptional regulatory element)” refers to the “post-transcriptional regulatory element (PRE)” derived from the Woodchuck hepatitis virus, and has a sheath at post-transcriptional level. refers to viral sequences that act as (cis) to regulate transport from the nucleus to the cytoplasm, thereby increasing the accumulation of gene transcripts in the cytoplasm. Finally, the beta-actin 3'-untranslated regions (RSV) and the RSV stabilizing factor (RSV) stabilizer (Rous Sarcoma Virus stability elements) both regulate the transport of cytoplasm from the nucleus to the cytoplasm, or to prevent degradation. Is a sequence that increases the accumulation of gene transcripts within. In one embodiment of the present invention, the RRE sequence was used as an mRNA stability element, and the RRE is derived from the HIV HXB2 strain, and GenBank accession No. An RRE sequence known as K03455 was used, and SEQ ID NOs: 7769 to 8002, which correspond to RRE, are represented by SEQ ID NO. On the other hand, WPRE may use a sequence derived from the Wood Chuck hepatitis virus WHV8 strain (Journal of Virology Apr. 1999, p.2886-2892), the 1093bp ~ 1684bp portion corresponding to the WPRE of the sequence disclosed in SEQ ID NO: 4 Indicated. In addition, "beta-actin 3'-untranslated regions" and "Rous Sarcoma Virus stability element" may use the sequences described in the following documents (The 3 ' -end of the human beta-actin gene enhances activity of the beta-actin expression vector system: construction of improved vector., Journal of Biochemical & Biophysical Methods. 36 (1): 63-72, 1997; A 3 'UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus., RNA-A Publication of the RNA Society., 12 (1): 102-10, 2006.).

The present inventors attempted to express NC proteins in animal cells by introducing NC genes into eukaryotic expression vectors containing a CMV (cytomegalovirus) promoter in a previous study (Korean Patent Registration No. 0553154), but this expression was not expressed. It was confirmed that the increase in the number of copies of NC mRNA is required because it is related to the stability of mRNA. In order to solve this problem, the inventors introduced an intron sequence on top of the NC gene to increase the stability of the NC mRNA or inserted FLAG at the N-terminal expression. It was confirmed that the protein is expressed, and when FLAG is inserted into the N-terminal, it was confirmed that it is expressed in the form of the N-terminal FLAG fusion NC protein. However, the method has a disadvantage in that the expression efficiency of NC protein is not high or it is not in the form of natural NC protein. Therefore, the inventors confirmed that the addition of mRNA stabilizing factors to sequentially linked intron sequences and HIV NC (nucleocapsid) genes, while increasing the expression of HIV NC protein can produce a natural NC protein. Conventionally, no report has been disclosed that the mRNA stabilizing factor increases HIV NC protein expression.

Specifically, in an embodiment of the present invention, a vector for constructing an HIV NC protein expression sequence, in which an SV40 SD / SA intron sequence, an HIV NC (nucleocapsid) gene, and a RRE (Rev response element) are sequentially connected, was constructed and transformed with the vector. It was confirmed that NC protein was highly expressed in the transformant (see Example <1-2>).

Meanwhile, standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and described in the following literature (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual) , 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984) and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

The present invention also provides a cell transformed with the vector for NC protein expression of HIV.

Vectors for the expression of NC proteins of HIV are known in the art such as conventional transfection methods such as DEAE-dextran, calcium phosphate method, microinjection method, DNA-containing liposome method, lipofectamine-DNA complex method, etc. As such, it may be performed by selecting appropriate standard techniques depending on the host cell (Molecular Cloning, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press (1989)).

Cells that can be transformed with the vector of the present invention are not particularly limited, but COS-7 cells, 293T cells, HEK293T, CHO and HeLa, etc. are preferably used, and more preferably, COS-7 cells and 293T cells.

The present invention also provides a method for producing HIV NC protein comprising culturing a cell transformed with the vector.

In one embodiment of the present invention was cultured by a method known in the art 293T cells transformed with the vector for expressing the NC protein of HIV of the present invention, it was confirmed that the native type of NC NC protein (Example < 1-2>).

Proteins expressed in the transformants can be purified in a conventional manner, for example, salting out (eg ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fraction precipitation with acetone, ethanol, etc.), dialysis HIV NC proteins of the present invention can be purified, alone or in combination, by techniques such as gel filtration, ion exchange, column chromatography such as reversed phase column chromatography, and ultrafiltration (Maniatis et al, Molecular Cloning: A Laboratory). Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990).

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

pCMV (- HA NC / RRE  Vector NC  Production of protein

<1-1> pCMV (- HA NC / RRE  Vector building

A vector for HIV NC protein expression was constructed by sequentially connecting the SV40 SD / SA intron sequence, the HIV nucleocapsid (NC) gene, and the Rev response element (RRE).

The construction of the vector is shown in FIG. First, in order to amplify RRE, a pLP1 vector (Invitrogen) was used as a template using a forward primer (5'-GCGCTCGAGAGGAGCTTTGTTCCTTGGG-3 '; SEQ ID NO: 5) and a reverse primer (5'-TAAGGTACCAGGAGCTGTTGATCCTTTA-3'; SEQ ID NO: 6). PCR was performed. In the primer sequence, a restriction enzyme site of Xho I and Kpn I was produced. After amplification of RRE, Xho I and Kpn I were treated, and the restriction enzyme-treated fragment was cloned into pCMV (-HA) NC vector (Korea Registration No. 0553154) treated with Xho I and Kpn I prepared first. It was named pCMV (-HA) NC / RRE.

<1-2> pCMV (- HA NC / RRE  Vector NC  Expression of protein

293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) culture containing 1% streptomycin / penicillin and 10% (v / v) fetal bovine serum (FBS). One day before the transduction, the cultured cells were inoculated in a 12-well plate at a density of about 1 × 10 6 per well and cultured to show 60-80% confluency at the time of transduction. Meanwhile, 1 µg of pCMV (-HA) NC / RRE prepared in Example <1-1> was mixed with 2 µl of JetPEI (Polyplus-transfection Inc), followed by 200 µl of serum-free culture at room temperature. The reaction was carried out for 20 minutes. Subsequently, the culture medium of the cells to be transduced was exchanged with 1 ml of serum-free culture medium, and the reaction solution was added to the serum-free culture medium, and further cultured in a carbon dioxide cell incubator for 4 hours. After further incubation, the cell cultures were exchanged for DMEM cultures containing 10% (v / v) FBS and then incubated again for 2 days to obtain cells.

Meanwhile, western blot was performed to confirm whether NC protein is expressed in the transformed cells. First, in order to extract the protein from the cells obtained, each cell was lysed in lysis solution (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% TyitonX-100, 1% Sodium deoxycholate, 1% SDS, protase After dissolving with an inhibitor coktail, the supernatant was obtained by centrifugation at 15,000 rpm for 20 minutes, followed by 15% SDS-PAGE. Then, Western blot was performed using an Anti-NC monoclonal antibody (A of FIG. 2), and the expression amount thereof was numerically represented (B of FIG. 2).

As a result, as shown in FIG. 2, the expression level of NC protein in the cells transformed with the pCMV (-HA) NC / RRE vector was 116.28 IOD. This indicates that the RRE sequence is significantly improved compared to pCMV (-HA) NC without the RRE sequence inserted under the NC gene (see lane 2 and lane 3). I could see that.

As described above, the NC protein expression vector of HIV is a wild type NC protein in an animal cell, characterized by sequentially connecting an mRNA stability element to an intron sequence and a lower part of the NC gene. Can be expressed, the expression efficiency is improved compared to the prior art.

<110> YOU, JI CHANG <120> Vector for expressing NC protein of HIV and method for producing          NC protein using the same <160> 7 <170> Kopatentin 1.71 <210> 1 <211> 30 <212> DNA <213> SV40 late 19s mRNA intron <400> 1 gtaagtttag tctttttgtc ttttatttca 30 <210> 2 <211> 97 <212> DNA <213> Modified SV40 late 16S mRNA intron <400> 2 gtaagtttag tctttttgtc ttttatttca ggtcccggat ccggtggtgg tgcaaatcaa 60 agaactgctc ctcagtggat gttgccttta cttctag 97 <210> 3 <211> 234 <212> DNA <213> Rev response element (RRE): NL4-3 strain <400> 3 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat 60 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 120 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 180 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcct 234 <210> 4 <211> 592 <212> DNA <213> Woodchuck hepatitis B virus (WPRE region) <400> 4 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc 420 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tg 592 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> 5223 sense primer for amplification of RRE <400> 5 gcgctcgaga ggagctttgt tccttggg 28 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> 3223 anti-sense primer for amplification of RRE <400> 6 taaggtacca ggagctgttg atccttta 28 <210> 7 <211> 340 <212> DNA <213> Human beta-globin intron <400> 7 tacacatatt gaccaaatca gggtaatttt gcatttgtaa ttttaaaaaa tgctttcttc 60 ttttaatata cttttttgtt tatcttattt ctaatacttt ccctaatctc tttctttcag 120 ggcaataatg atacaatgta tcatgcctct ttgcaccatt ctaaagaata acagtgataa 180 tttctgggtt aaggcaatag caatatttct gcatataaat atttctgcat ataaattgta 240 actgatgtaa gaggtttcat attgctaata gcagctacaa tccagctacc attctgcttt 300 tattttatgg ttgggataag gctggattat tctgagtcca 340  

Claims (7)

a) any one sequence selected from the group consisting of an SV40 19s mRNA intron sequence, a modified SV40 16S mRNA intron sequence represented by SEQ ID NO: 2, and a β-globin intron sequence b) an HIV nucleocapsid (NC) gene; And c) an mRNA vector for HIV NC protein expression, characterized in that mRNA stability elements are sequentially linked. According to claim 1, wherein the SV40 19s mRNA intron sequence consists of SEQ ID NO: 1, β-globin intron sequence is HIV NC protein expression vector, characterized in that consisting of SEQ ID NO. The method of claim 1, wherein the mRNA stabilizing factor is RRE (rev response element), WPRE (woodchuck post-transcriptional regulatory element), beta-actin 3'-UTR (β-actin 3'-untranslated regions) and RSV stabilizing factor ( HIV NC protein expression vector, characterized in that any one selected from the group consisting of Rous Sarcoma Virus stability element. The vector according to claim 1, wherein the HIV NC protein expression vector has a cleavage map of pCMV (-HA) NC / RRE as shown in FIG. 1. Cells transformed with the vector of any one of claims 1 to 4. 6. The transformed cell of claim 5 wherein said cell is a COS-7 or 293T cell. A method for producing HIV NC protein comprising culturing the transformed cells of claim 5.

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