KR20210003472A - Vector for foreign protein expression using temperature sensitive protein - Google Patents

Abstract

The present invention relates to a vector used in a method for expressing a foreign protein using a temperature-sensitive protein. The present invention can easily overexpress a target foreign protein to produce a protein therapeutic in a simple and economical way.

Description

Vector for foreign protein expression using temperature sensitive protein

The present invention relates to a vector used in a foreign protein expression method using a temperature-sensitive protein.

As the patent period of synthetic drugs expired, it became possible to produce and sell a generic drug called “generic” that has the same chemical structure as the synthetic drug and can function biologically in the body.

In addition, the patent period for protein therapeutics that can be produced in human tissues or cells using genetic recombination technology is also expired. “Biosimilar”, which can be called a “generic” of protein therapy, is attracting attention as a new growth engine of the Korean pharmaceutical industry in that it can produce higher added value than generics.

The protein therapy market includes protein drug types such as peptides, enzymes, antibodies, cytokines, hormones, and various types of drugs such as anti-cancer, anti-diabetic and anti-angiogenic. Protein therapeutics targeting disease treatment have been released. Generics require large-scale plant facilities for chemical synthesis. In addition, since protein therapeutics are made in animal cells through methods such as genetic manipulation, facilities for culturing cells are required, and DNA containing therapeutic genes must be put into animal cells through molecular biology experiments to induce expression. Many domestic conglomerates and pharmaceutical companies are interested in the biosimilar business, and the development of protein therapeutics and the development of brackish water are in progress. Among protein therapeutics, peptides and hormones form a large market, followed by the cytokine market, and the antibody production market is growing rapidly.

Biosimilars are also aimed at protein therapeutics, but as they have high utility value as a functional Medicare for disease prevention, they have the potential to be applied to the development of various health and health products. As for the market size of health functional foods and antioxidant/anti-aging health foods, in the case of Korea, the domestic health and health functional food market in 2007 is estimated to be 2.5 trillion won, showing a growth of 10-15% annually. Therefore, the development of protein therapy and functional Medicare technology can steadily grow as it can lead to expansion of the pharmaceutical market with the concept of increasing patient compliance with drugs and various types of preventive treatments.

In the 1980s, gene recombination technology was applied and protein therapeutics drugs have been released through various research and development. In October 2005, Europe prepared guidelines for biosimilar approval through EMEA (European Medicines Agency) for the first time in the world, and in 2006, Genotropin of Pfizer was first approved as a biosimilar. The global protein therapy market was $86.8 billion in 2007, and is expected to more than double to $160.1 billion in 2013 through rapid growth of more than 10.9% per year (BCC Research 2008). As of 2011, the global sales of biosimilars are $3.2 billion, but as the patent expiration approaches, sufficient conditions are being created for the growth of the protein biosimilar market.

Domestically and internationally, the development of biosimilars targets protein drugs that have already been released on the market and have been verified for safety and effectiveness, so low R&D costs and shortening of development period are of major interest. However, in order not to lose the medicinal efficacy, enormous facility management costs are consumed to extract protein therapeutics through various processes in a cell culture facility under conditions that maintain complete aseptic conditions and low temperatures. In addition, high cost is required for maintaining cell culture, such as FBS, medium, and other nutrients that enter the culture medium. Therefore, although biosimilars are in the spotlight as a method of developing protein therapeutics, biosimilars require a strategic approach to lower the cost of protein therapeutics, so the development of technology that can mass-produce stable therapeutic proteins while reducing costs. I need this.

Korean Patent Laid-Open Publication No. 10-2010-132516 is a method for preparing a human protein in a plant, especially a recombinant human lysosomal enzyme in plant endosperm, obtaining the protein and confining the protein in endosperm that is not ultimately absorbed by the embryo. , A first step of transforming the plant so that the presence of a large amount of the protein in the endosperm does not negatively affect seed vitality and germination rate; And a second step of accumulating the protein in the endosperm of the seed of the plant, wherein in the first step of the plant transformation, an endosperm-specific promoter upstream of the gene encoding the protein is used, as well as tissue specificity of the protein. It relates to a method characterized in that a signal peptide is used for simultaneous translational delivery of the newly synthesized protein into the lumen of the endoplasmic reticulum of endoderm cells for accumulation.

However, this method uses plant endosperm, differs from the present invention, which uses brown rice with intact rice, and requires the use of an endosperm specific promoter and the like, so the method is complicated.

The present invention aims to provide a vector capable of producing a large amount of protein therapeutic agents without side effects by using brown rice in a simple and economical way.

The present invention relates to a vector used in a method of overexpressing a foreign protein using intact brown rice, that is, a method for producing a large amount of foreign protein by inducing germination by introducing foreign gene DNA into brown rice with intact rice.

In the present invention, after cloning a target gene in a plant plasmid, the cloned target gene is cut to prepare a vector with a CaMV promoter and an NOS terminator, and then the water content of brown rice is lowered to less than 5%, and the vector is added to the brown rice with lower water. And a swelling buffer are mixed and added to treat the brown rice so that water and vectors are absorbed, the treated brown rice is treated with a chlorine disinfectant and washed, and then the brown rice containing the target gene-containing vector is immersed in water and cultured (germination). And, it relates to the structure of a vector used in a foreign gene introduction and protein expression method using brown rice for extracting a target protein in which a target gene is expressed from cultured brown rice.

The swelling buffer may include saline-sodium citrate (SSC) buffer.

As a promoter in the vector, a CaMV 35S promoter may be used.

The vector may contain a marker gene. When a marker gene is included, it is easy to determine whether the protein is expressed after expression. As the marker gene, it is preferable to select a gene that is easy to detect, such as a β-glucuronidase gene and a green fluorescent protein gene.

Various types of restriction enzyme sites may be placed in the vector to facilitate the insertion of foreign genes.

The present invention relates to a vector used for foreign gene introduction and protein expression using brown rice, including a promoter, a gene encoding a target protein, an intein, an elastin-like peptide repeat gene, and a terminator.

In addition, the present invention relates to a vector used for the introduction of foreign genes and protein expression using brown rice, characterized in that the promoter is a CaMV promoter.

In addition, the present invention relates to a vector used for foreign gene introduction and protein expression using brown rice, characterized in that the vector further includes a marker gene for confirming protein expression.

In addition, the present invention relates to a vector used for foreign gene introduction and protein expression using brown rice, characterized in that the marker gene is at least one of β-glucuronidase gene and green fluorescent protein gene. The marker gene is not particularly limited as long as it does not affect the expression or function of the target protein when expressed, but preferably, at least one of the easy-to-detect β-glucuronidase gene and the green fluorescent protein gene is used.

In addition, the present invention relates to a vector used for introduction of foreign genes and protein expression using brown rice, characterized in that two or more restriction enzyme sites are present in the vector.

When the vector of the present invention is used, a protein therapeutic agent without side effects can be prepared in a simple and economical way by expressing a foreign gene through germination of rice.

According to the present invention, in a method in which foreign genes are easily introduced into brown rice by processing water and DNA together in brown rice with minimal moisture, the foreign gene is effectively introduced into brown rice and cultured by culturing brown rice to easily overexpress the target foreign protein. Protein therapeutics can be produced in an economical way.

1 is a schematic diagram of a vector for foreign gene introduction and protein expression using brown rice.
2 is a detailed schematic diagram of a vector according to an embodiment for introduction of foreign genes and protein expression using brown rice.

Hereinafter, the configuration of the present invention will be described in more detail through specific examples. However, it is apparent to those of ordinary skill in the art that the scope of the present invention is not limited only to the description of the examples.

1. A method of introducing foreign genes into brown rice with a complete form of rice

The present inventors have invented a system that can be expressed and purified by introducing foreign genes useful in plants (brown rice). First, a foreign gene is introduced using a vector gene that has been previously developed.

Vector DNA composition: pGEM vector (promega) + CaMV promoter + GFP + NOS terminator

(A) Introducing foreign genes into the rice buds of brown rice

1) Minimize the moisture content of rice to less than 5% by using a vacuum pump. Specifically, brown rice is put in a desiccator and a vacuum is maintained for about 2 hours.

2) Mix 10ug/ml DNA, 0.5x SSC, and 20% DMSO in brown rice with minimal moisture, and treat in a refrigerator for 12 hours. At this time, moisture is absorbed by the brown rice, and the volume of the brown rice increases by 2-3 times, and DNA is absorbed into the rice with the moisture.

3) Soak the treated brown rice in 20% chlorax for 3 minutes, wash it 5 times with distilled water, and sterilize it.

(B) Culture of brown rice with foreign genes introduced

Brown rice into which the foreign gene was introduced is immersed in sterilized distilled water and incubated in a 28°C incubator for 5-10 days.

2. Vector construction for protein expression

A vector using ELP (Elistin-like polypeptide) was constructed to purify the protein obtained by expressing the foreign gene introduced into the rice bud of brown rice.

Specifically, a vector using an intein protein was constructed to separate the ELP protein and the target protein.

In addition, three frames of multicloning sites were formed in the vector for commercialization of the fabricated vector DNA.

3. Insertion of marker protein into vector

Various marker proteins can be inserted into the vector. The marker protein is not particularly limited as long as it is a gene that does not affect the expression or function of the foreign protein, and in the embodiment of the present invention, green fluorescent protein was selected as a marker protein and inserted into a vector.

4. Protein expression verification

Protein purification method

1) Grind 1-2g of rice grown for 5-7 days in a mortar using liquid nitrogen.

2) After transferring to a centrifuge tube, add 20 ml of 1x PBS + 2M NaCl buffer and stir vigorously for 1 minute.

3) Freeze in the freezer for 1-2 hours and thaw at 37℃. Repeat this process twice.

4) Centrifuge at 4℃ for 20 minutes at 13,000rpm.

5) Transfer the supernatant to a new tube and leave it at 37℃ for 30 minutes.

6) After preheating the centrifuge to 37°C, centrifuge at 15,000 rpm for 30 minutes.

7) Discard the supernatant, add 20 ml of cold 1x PBS +2M NaCl buffer to the pellet, stir vigorously for 3 minutes, and store at 4°C for 1 hour.

8) Centrifuge for 20 minutes at 13000rpm in a centrifuge cooled to 4℃.

9) Transfer the supernatant to a new tube and store at 37°C for 30 minutes.

10) Repeat the above steps 6), 7), and 8) twice.

11) Transfer the supernatant to a new tube and use it for concentration and purity analysis.

Claims (5)

A vector used for foreign gene introduction and protein expression using brown rice, including a promoter, a gene encoding a target protein, an intein, an elastin-like peptide repeat gene, and a terminator.
The method according to claim 1,
The promoter is a vector used for introduction of foreign genes and protein expression using brown rice, characterized in that the promoter is a CaMV promoter.
The method according to claim 1,
The vector used for foreign gene introduction and protein expression using brown rice, characterized in that the vector further includes a marker gene for confirming protein expression.
The method of claim 3,
The marker gene is a vector used for introduction of foreign genes and protein expression using brown rice, characterized in that at least one of β-glucuronidase gene and green fluorescent protein gene.
The method according to claim 1,
A vector used for foreign gene introduction and protein expression using brown rice, characterized in that at least two restriction enzyme sites are present in the vector.

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