In one embodiment of the present invention, the protein of interest may be aflibercept represented by SEQ ID NO: 19, but is not limited thereto.

The PolyA signal sequence used in the expression cassette of the present invention may be selected from the group consisting of a bovine growth hormone (BGH) Poly A signal sequence, a SV40-derived PolyA signal sequence, an HIV-1 poly A signal sequence, a β-globin poly A signal sequence, an HSV TK polyA signal sequence, and a polyomavirus polyA signal sequence. Among them, the SV40-derived polyA signal sequence or the BGH poly A signal sequence may be preferred, without being limited thereto. More preferably, it may be a poly A signal sequence represented by SEQ ID NO: 13 or 14.

In one embodiment of the present invention, the expression cassette for producing a protein of interest may, if necessary, comprise additional nucleotide sequences such as a linker, a restriction enzyme cleavage site and a Kozak nucleotide sequence within the range that does not affect the basic transcription and translation of the protein of interest. More specifically, for example, the expression cassette may comprise a linker represented by SEQ ID NO: 16, a SacII restriction enzyme cleavage site represented by SEQ ID NO: 17, an Hpal/Kozak nucleotide sequence represented by SEQ ID NO: 18, and the like.

The expression cassette for producing a protein of interest according to the present invention may regulate intron splicing, and may have any one or more effects selected from the group consisting of an effect of increasing transcription of the protein of interest, an effect of promoting mRNA export out of the nucleus, an effect of increasing the efficiency of translation of the protein of interest, an effect of reducing transcription-associated genome instability, and an effect of inducing high expression of the protein of interest.

In one example of the present invention, it can be confirmed that, when the intron sequence is introduced, the expression level is increased by as little as 1.90 times to as much as 2.25 times compared to a control group in which the intron sequence is not introduced (see FIGS. 2 to 3 and Tables 1 and 2).

The present invention provides a vector containing the expression cassette for producing a protein of interest. The expression vector that is used for transduction may be any vector, but may preferably be a MarEx vector.

In one example of the present invention, as the vector, a pCT-EEI1 vector was constructed by cloning via PacI/HpaI sites into an MarEx vector (Korean Patent No. 10-1076602, hereinafter referred to as pCT) having dihydrofolate reductase (DHFR) as a selection marker.

The present invention provides a transformant transformed with a vector containing an expression cassette for production of a protein of interest. In one embodiment of the present invention, the transformant is a eukaryotic cell. Preferably, the eukaryotic cell is an animal cell, and the animal cell may be any type of cell that allows expression of a target gene, such as a mammalian cell. For example, a mammalian cell suitable for the present invention may be one or more selected from the group consisting of mouse cells (e.g., COP, L, C127, Sp2/0, NS-0, NS-1, At20, NIH3T3, etc.), rat cells (e.g., PC12, PC12h, GH3, MtT, etc.), hamster cells (e.g., BHK, CHO, CHO-K1, GS defective CHO, CHO-DUXB11, CHO-DG44, etc.), monkey cells (e.g., COS1, COS3, COS7, CVI, Vero, etc.), human cells (e.g., HeLa, HEK 293 cells, HEK 293 EBNA cells, Namalwa cells, retina-derived PER-C6, cells derived from diploid fibroblasts, myeloma cells, HepG2, etc.), hybridoma cells, embryonic stem cells, and fertilized egg cells, without being limited thereto. More preferably, the transformant may be a Chinese hamster ovary (CHO) cell, a rat myeloma cell, a baby hamster kidney (BHK) cell, a hybridoma cell, a Namalwa cell, an embryonic stem cell, or a fertilized egg cell, and most preferably, may be a Chinese hamster ovary (CHO) cell.

The present invention also provides a method for producing a protein of interest comprising a step of culturing a transformant comprising an expression cassette for producing the protein of interest. More preferably, the method may comprise steps of: transfecting cells with a vector that expresses the intron sequence and the protein of interest by a single promoter; selecting stably transfected cells; culturing the transfected cells under conditions suitable for expression of the protein of interest; and recovering the protein of interest from the cells or the cell culture. Insertion of the gene of the protein of interest is preferably performed by a method known to those skilled in the art. For culturing, it is preferable to use a culture medium suitable for the host cell. In addition, purification of the protein of interest is preferably performed by a method known to those skilled in the art.

In one embodiment of the present invention, the present invention provides a method for transient transformation with an expression vector containing an expression cassette and production of a protein of interest. After transient transduction of the expression vector into host cells using polyethylenimine (PEI), the transduced cells were cultured in an incubator at 30 to 37° C. under 5 to 8% CO₂for 1 to 3 weeks to produce the protein of interest (see Example 1-2).

The present invention also provides a method for increasing the production of a protein of interest comprising steps of: a) preparing an expression-enhancing intron in which a splicing donor sequence, a branch sequence, a polyY sequence and a splicing acceptor sequence are combined with the expression-enhancing sequence represented by SEQ ID NO: 1: b) inserting the expression-enhancing intron, prepared in step a), into a vector; c) transforming a cell with the vector prepared in step b); and d) producing the protein of interest using the transformant prepared in step c).

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited by the examples.

The examples of the present invention are intended to prepare an intron sequence capable of strongly expressing the gene of the protein of interest in combination with a promoter/enhancer, and to construct an expression cassette using the same, and to test the effect thereof.

Example 1. Preparation of Expression-Enhancing Sequence and Evaluation of Effect
Example 1-1. Preparation of Expression-Enhancing Intron

Expression-enhancing intron 1 (hereinafter referred to as EEI1) was prepared by combining a cytomegalovirus immediate early gene-derived sequence (SEQ ID NO: 1) containing a splicing donor sequence, an elongation factor 1 alpha promoter sequence-derived branch (SEQ ID NO: 4), and a poly Y tract and splicing acceptor sequence (SEQ ID NO: 7) through a PCR method, and cloned via PacI/HpaI sites into a MarEx vector (Korean Patent No. 10-1076602, hereinafter referred to as pCT) having dihydrofolate reductase (DHFR) as a selection marker, thereby constructing a pCT-EEI1 vector. The cloning method is well known in the art and is described, for example, in Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). The aflibercept gene sequence, developed as a vascular endothelial growth factor inhibitor, was cloned into the expression cassette of the pCT-EEI1 vector via HpaI/ClaI sites, thereby constructing a pCT-EEI1-Aflibercept vector expressing aflibercept.