WO2009131423A2 - Novel hepatitis a virus antigen gene and transformed plants with the gene - Google Patents

Abstract

The present invention relates to a novel hepatitis A virus antigen gene and transformed plants with the gene, more particularly to a novel hepatitis A virus antigen gene synthesized by optimization of a genetic code, a recombinant vector containing the gene and plants transformed with the recombinant vector.  According to the invention, the use of a synthesized antigen gene of hepatitis A virus makes it possible to prepare recombinant hepatitis A virus vaccine materials at a high efficiency, and transformed plants expressing a hepatitis A virus antigen are useful as functional food materials and orally administrated vaccine materials.

Description

Antigen Genes of Novel Hepatitis A Virus and Plants Transformed with the Genes

The present invention relates to an antigen gene of a novel hepatitis A virus and a plant transformed with the gene, and more specifically, to an antigen gene of a novel hepatitis A virus synthesized by gene codon optimization, and a recombinant containing the gene. It relates to a vector and a plant transformed with the recombinant vector.

Vaccines are biological materials used to inactivate certain pathogens when they are reunited with antigens that have once been remembered by stimulating the immune system through several mechanisms to ensure that a particular pathogen is remembered as an antigen before it enters the body. The vaccine material is divided into attenuated live vaccine material and inactivated bacterium vaccine material.

The global vaccine material market is expected to reach $ 17 billion in 2010, growing at an annual average of about 13%. As a result, Korea is currently forming a market of about 150 billion won. The age-specific vaccine material market had the highest share in the pediatric vaccine sector, with sales of approximately $ 2.5 billion in 2001, but the demand for adult vaccines has recently been boosted by the government's vaccinations for older people and travelers. Is also increasing.

However, currently available vaccine materials have many disadvantages in terms of efficacy, ease, manufacture and distribution. For example, the bacteriophage vaccine material is safe, but has the disadvantage of having to be inoculated several times, and the viable vaccine material is excellent in immunogenicity, but cannot be administered to pregnant women and those with reduced immune system, and is expensive to produce and requires refrigeration There is a disadvantage. In addition, these vaccine materials induce a systemic immune response, but do not elicit an immune response on the mucosal cell surface, which is the path of penetration of most bacteria and viruses.

Therefore, the dietary vaccine material is considered to be the most desirable type of vaccine in view of the manpower, cost, and pain used for vaccination. However, all of the dietary vaccine materials developed to date are attenuated live vaccines against typhoid fever and polio. It's being stopped. Therefore, recently, a pharmaceutical functional food material that expresses and uses an antigen gene in a plant is emerging as a next-generation bio health food by combining safety of recombinant vaccine material and excellence and utility of dietary vaccine material.

Hepatitis A (hepatitis A) virus is an RNA virus of 27nm size belonging to the Picornavirdae, acute liver characterized by symptoms such as fever, malaise, anorexia, nausea, abdominal pain, amnyo, jaundice after incubating an average of 28 days Cause disease. Hepatitis A virus, like rotavirus, has no encapsulation, is a icosahedron with a diameter of 27 nm, has a 7.5 kb single-stranded RNA genome, and has one long ORF (P1-3). The main route of transmission of hepatitis A is fecal-oral passage, like rotavirus, and is transmitted by contaminated food or drinking water.

Since the recent incidence of hepatitis A has been increasing globally, ACIP has been associated with people traveling to areas with hepatitis A endemic, people with blood clotting disorders, homosexuals, people with chronic liver disease, and type A Vaccination is recommended for the pediatric population living in areas with high hepatitis frequency, especially when more than 10 people per 100,000 population occur, and extensive vaccination is needed, and hemodialysis is performed due to chronic hepatitis B carriers, the elderly, and chronic kidney failure. Hepatitis A vaccination is mandatory for adolescents living in groups with other patients.

According to the recent 10-year survey, hepatitis A is increasing in the western United States, the Middle East, and some Asian countries, and there is concern about the spread of hepatitis A worldwide. Hepatitis A is growing rapidly in younger people. The prevalence of hepatitis A is 5-14 years, with approximately 30% of patients under 15 years old. In the United States, 1982 to 1993, the incidence of hepatitis A was about 47%, more than about 37% of hepatitis B, and serological tests showed that 33% of the US population in the past had hepatitis A. It has been found that there is a history of illness.

About 11-22% of hepatitis A patients will be hospitalized, and if they are hospitalized, they will be absent for about 27 days, and when the illness occurs, an average of 11 contacts per patient should be administered. . In the United States, the direct and indirect costs per hepatitis A patient are reported to be $ 1,817-2,459 for adults and $ 433-1,492 for children under 18 years of age. The cost of medical care is about $ 200 million or more, which is reported to be about $ 300 million in 2000.

Havrix, developed in 1995 by Smith Klein BK, is the world's first hepatitis A vaccine, incubated with HM175 strain in MRC5 cells, a human diploid cell, inactivated, and inactivated with aluminum hydroxide. As a bacterium vaccine material produced by adsorption, it is required to inoculate three times for adolescents 2-18 years old and two times for adults, and it is not perfect to form intestinal mucosal immunity, which is the path of virus penetration because of inoculation into muscles. Persistence is also a problem.

Conventional hepatitis A virus antigen production has been attempted using an expression system through Escherichia coli or baculovirus. However, the expression level of the hepatitis A virus antigen has not been accurately reported.

Accordingly, the present inventors have made intensive efforts to develop a hepatitis A virus vaccine that is more stable and easy to administer orally, and when using a plant transformed with a novel hepatitis A virus antigen gene synthesized by gene codon optimization, The present invention was completed by confirming that hepatitis A virus antigen is efficiently expressed.

Summary of the Invention

The main object of the present invention is to provide a novel gene encoding a hepatitis A virus antigen and a recombinant vector comprising the gene.

Another object of the present invention is to provide a novel gene encoding the hepatitis A virus antigen or a plant transformed with the recombinant vector.

Still another object of the present invention is to provide a method for preparing hepatitis A virus antigen and a hepatitis A virus antigen.

In order to achieve the above object, the present invention provides a gene encoding a hepatitis A virus antigen represented by the amino acid sequence of SEQ ID NO: 1 and a recombinant vector comprising the gene.

The present invention also provides a hepatitis A virus antigen having the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a plant transformed with the gene or the recombinant vector.

The present invention also comprises the steps of culturing the transformed plant; And it provides a method for producing a hepatitis A virus antigen comprising the step of recovering the hepatitis A virus antigen.

Other features and embodiments of the present invention will become more apparent from the following detailed description and the appended claims.

1 compares the nucleotide sequences of the native hepatitis A virus antigen gene and the hepatitis A virus antigen gene synthesized by gene codon optimization.

2 shows a schematic diagram of BCTV / CsVMV / synthesis VP1 in accordance with the present invention.

Figure 3 shows the recombinant protein expressed in the tomato cell system by Western blot (lane M: molecular weight marker; lane 1: protein extracted from the leaves of plants transformed with native VP1 gene; lane 2: codon optimization according to the invention Protein extracted from the leaves of plants transformed with the synthetic VP1 gene; lane 3: protein from E. coli transformed with the native VP1 gene (positive control); lane 4: negative control (from normal plants not transformed) Protein (negative control).

Detailed Description of the Invention and Specific Embodiments

The present invention relates to a novel hepatitis A virus antigen gene synthesized by gene codon optimization to be suitable for a plant, a recombinant vector comprising the gene, and a plant transformed with the gene or the recombinant vector.

In the present invention, the recombinant vector has two BCTV replicons between the T-DNA border and an expression vector comprising a CsVMV promoter, North (NOS) and a replication initiation protein (REP) between the two replicons. Hepatitis A virus antigen gene represented by the amino acid sequence of SEQ ID NO: 1 is inserted, and may have a cleavage map of FIG. 1, wherein the plant is tomato, lettuce, pepper, soybean, rice and corn. It may be characterized in that selected from the group consisting of.

In one embodiment of the present invention, hepatitis A virus antigen gene was synthesized by gene codon optimization. The synthetic gene was synthesized using a codon optimization program developed by GENEART, which was commissioned by Takara Korea.

In another embodiment of the present invention, a recombinant vector comprising the synthesized hepatitis A virus antigen gene was prepared, and the recombinant vector was introduced into a plant.

In the present invention, in order to produce a recombinant vector containing a hepatitis A virus antigen gene, a plant expression vector comprising a geminivirus replicon was used, and preferably, two BCTVs between two T-DNA borders. Hepatitis A having a beet curly top virus replicon, comprising a promoter, a NOS and a replication initiator protein (REP) between the two replicons and synthesized by the codon optimization Recombinant vectors for plant expression may be used, wherein viral antigen genes can be inserted, but are not limited thereto.

The promoter may be characterized in that the CaMV35S promoter or CsVMV promoter, the REP gene may be characterized in that the REP gene of BCTV.

The BCTV vector containing the CsVMV promoter used in the present invention can be produced by the method described in Korean Patent No. 10-0625334. That is, the replicon of BCTV belonging to the gemini virus was cloned, and the REP gene of BCTV was put into cis to reconstruct the skeleton of the expression system. In addition, PCR replicon was cloned in front of CaMV 35S promoter + GFP + Nos poly-A by PCR using a primer having HindIII and SalI restriction enzyme sites, and BCTV REP gene and BCTV replicon were cloned into SacI. Cloning was performed by PCR using primers with restriction enzyme sites. The CsVMV promoter was cloned by PCR using primers containing restriction enzymes of SalI and XbaI in BCTV vectors, respectively.

In an expression vector prepared by the above method, which has two BCTV replicons between T-DNA borders, and includes a CsVMV promoter, North (NOS) and a replication initiation protein (REP) between the two replicons. The hepatitis A virus antigen gene synthesized above was cloned between restriction enzyme cleavage sites Sma I and Spe I.

Plant cells or plants producing hepatitis A virus antigens were prepared by introducing the synthetic genes into strains of the genus Agrobacterium using plant plasmid expression plasmids containing the synthesized hepatitis A virus antigen gene. Agrobacterium genus strain Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) can be used, the Agrobacterium tumefaciens used in the present invention GV3101 containing the hygromycin resistance gene pCAMBIA 1300 plasmid The strain was used.

The transformation can be used any method in the field of genetic engineering, as long as it can effectively express the hepatitis A virus antigen in high yield.

In particular, in the present invention, but the tomato was used as a transgenic plant, any plant that can economically produce recombinant proteins such as lettuce, pepper, soybean, rice, corn can be used without limitation. In addition, even if the plant used for transformation is a sexually propagating plant, it will be apparent to those skilled in the art that it can be repeatedly reproduced by tissue culture or the like.

In the present invention, "vector" means a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli DH5α, E. col JM101, E. coli TOP10, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL1-Blue (Stratagene), E. coli B, E. coli BL21 and the like. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the aforementioned E. coli , strains of the genus Agrobacterium, such as Agrobacterium A4, bacilli , such as Bacillus subtilis , Salmonella typhimurium or Serratia marghesen another variety of enteric bacteria and Pseudomonas (Pseudomonas) in strains such as marcescens) may be used as host cells.

Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al ., Supra . Alternatively, electroporation (Neumann et al. , EMBO J. , 1: 841, 1982) can also be used for transformation of these cells.

It is apparent to those skilled in the art that the genes used in the present invention may exist in host cells independently in plasmids or inserted into the host chromosomes.

In the present invention, when the foreign protein is expressed in a cell, the gene encoding the foreign protein is one or repeated two or more times, or the gene encoding the foreign protein repeated two or more times is the same or different from each other. This is possible and any combination of the above is possible.

On the other hand, it will be apparent to those skilled in the art that the induction of expression of a foreign protein can be expressed by a suitable other promoter which can induce expression in a growth arrest phase in a host cell.

In another aspect, the present invention relates to a method for producing a recombinant hepatitis A virus antigen using the transformant.

According to the present invention, since the synthesized hepatitis A virus antigen gene expresses the protein constituting the hepatitis A virus antigen, the transformed plant expresses the hepatitis A virus antigen during the regeneration process, Expression was confirmed by Western blot.

Therefore, the use of the novel hepatitis A virus gene according to the present invention greatly improves the expression efficiency of the hepatitis A virus antigen in tomato plants, and thus, the transformant can be usefully used as a vaccine for preventing or treating hepatitis. . In particular, it can provide an oral hepatitis preventive vaccine composition containing the transformed plant as an active ingredient.

The composition may be used as is or after drying the transgenic plant, in powder form, used with other food or food ingredients, and may be appropriately used according to conventional methods. The mixed amount of the plant as an active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment), and it is obvious that the plant as an active ingredient can be used without a specific maximum allowable range since there is usually no problem in terms of safety. Do.

In the present invention, "vaccine" refers to a composition used for the prevention or treatment of an infectious disease. The vaccine may contain an antigen or may be capable of expressing an antigen to induce an immune response to the antigen. A vaccine comprising the recombinant vector of the present invention can be used for the prevention or treatment of infection, transmission and epidemic of hepatitis A virus.

By "vaccination" is meant that the vaccination of the vaccine actively produces immunity (humidary immunity, cellular immunity, or both) in the body or culture of the organism. This can prevent infection, proliferation, charge, and / or epidemic of pathogens.

An "antigen" refers to a molecule containing one or more epitopes that can induce an antigen-specific immune response by stimulating the host's immune system. The immune response may be a humoral immune response and / or a cellular immune response. Although three to several amino acids may be one epitope, one epitope of a protein usually contains about 7-15 amino acids, for example, 8, 9, 10, 12 or 14 amino acids. Antigens are also called immunogens. In addition, when an antigen is expressed using an antigen using a polynucleotide or vector encoding an antigen protein, in the present invention, such a polynucleotide and a vector are also referred to as an antigen, and these can also be used as vaccine components.

A "gene" refers to a genetic material and includes nucleic acids such as RNA and DNA, and may have a naturally derived or artificially designed sequence.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only to illustrate the invention, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1 Construction of Expression System of Hepatitis A Virus Antigen Gene

Example 1-1 Synthesis of Hepatitis A Virus Antigen Gene

Codon optimization was performed by Takara Korea Co., Ltd. to synthesize a hepatitis A virus antigen gene (SEQ ID NO: 1) of about 903 bp. The synthetic gene was commissioned by Takara Korea Co., Ltd., and was synthesized using a codon optimization program developed by GENEART. Codon optimization was performed by substituting codons with tomato codons and avoiding very high or very low GC content. (FIG. 1).

Example 1-2 Construction of BCTV-CsVMV-synthetic VP1 Expression Vector Containing BCTV Replicon

BCTV vectors containing CsVMV promoters were prepared by the method described in Korean Patent No. 10-0625334.

In other words, the clones of BCTV (beet curly to virus, ATCC PVMC-6) belonging to the Gemini virus were cloned, and the replication system of the expression system was reconstituted by inserting the replication initiator protein (REP) gene of BCTV into cis. In addition, the replicon of BCTV was cloned in front of CaMV 35S promoter + GFP + Nos poly-A by PCR using primers of SEQ ID NO: 2 and SEQ ID NO: 3 having HindI II and Sal I restriction enzyme sites, and BCTV The REP gene and BCTV replicon of were cloned by PCR using primers of SEQ ID NO: 4 and SEQ ID NO: 5 having Sac I restriction enzyme sites. The CsVMV promoter was cloned by PCR using primers of SEQ ID NO: 6 and SEQ ID NO: 7 having Sal I and Xba I restriction enzymes in BCTV vectors, respectively.

SEQ ID NO: 5'-AAGCTTCTGCAGATGCGAATATATTTTTATTC-3 '

SEQ ID NO: 5'-GTCGACCTTGCTTAGCAAGTTTCTTGTGGG-3 '

SEQ ID NO: 5'-GAGCTCATTTTTATTAATAAAAATATTATTTTTTTAATAC-3 '

SEQ ID NO: 5'-GAGCTCCTTGCTTAGCAAGTTTCTTGTGGG-3 '

SEQ ID NO: 5'-GGATCCCATGGAGTCAAAGATTCAAATAG-3 '

SEQ ID NO: 5'-GGTACCCCCGATCTAGTAACATAGATGAC-3 '

The hepatitis A virus VP1 gene synthesized above was designed to have Sma I and Spe I restriction enzyme sites for easy cloning during PCR amplification. PCR conditions were performed 30 times in total for 5 minutes at 94 DEG C, 1 minute at 94 DEG C, 1 minute at 55 DEG C, 1 minute at 72 DEG C, and 72 minutes at 72 DEG C ..

The PCR product was ligated into a pGEM-T vector (Promega, USA), and then transformed into E. coli DH5a, plasmids were isolated and cut with Sal I and Spe I to identify a band of about 2.0 kb. Insertion was confirmed. Hepatitis A virus antigen expression vector produced a recombinant vector BCTV-CsVMV-synthetic VP1 which is a binary vector for plant expression inserted with BCTV replicon (FIG. 2).

The gene insertion in the recombinant vector BCTV-CsVMV-HAV (synthetic VP1) and the reading frame were confirmed by restriction enzyme mapping and DNA sequencing.

Example 2: Transformation of Tomato Using Agrobacterium

Agrobacterium tumefaciens (GV3101) containing the recombinant vector BCTV-CsVMV-synthetic VP1 prepared above was added to sterile LB liquid medium and antibiotics (kanamycin 50mg / l) were added for 36 hours. After incubation in a shaker controlled at a speed of 150rpm at 28 ℃, put in a centrifuge and centrifuged at 2500rpm for 10 minutes, the centrifuged bacteria were suspended in liquid MSO medium and used for inoculation of tomato (MicroTom) slices. .

After immersing the precultured sections in Agrobacterium solution for 15 minutes, the excess Agrobacterium on the sections was wiped off the sterile filter paper, followed by regeneration medium (MS salt, 0.3% phytagel, sucrose 30) without antibiotics. g / l) was incubated at 25 ° C. for 2 days with a medium added with 0.1 mg / l 3-indolacetic acid (IAA) and 0.5 mg / l 6-benzylaminopurine (BA). After 2 days, transformants were selected by transfer to regeneration medium to which antibiotics (thymentin 150 mg / l, hygromycin 10 mg / l) were added.

In order to induce the roots when the shoots were induced while selecting the transformants, the roots were induced by cutting the stems grown in the medium from which the MSO (MS salt, 0.3% phytagel, sucrose 30 g / L) hormone was removed. The replanted transgenic plant was transferred to a pollen, and the soil was purified.

Example 3: Confirmation of Synthetic Hepatitis A Virus Antigen Expression

To confirm the expression of the synthetic hepatitis A virus antigen gene, Western blot was performed using SDS-PAGE by the Lamley method (Laemmli UK, Nature, 227: 680, 1970).

That is, in order to confirm the expression of hepatitis A virus antigen protein from the transformed tomato, after performing a western blot on the protein sample extracted from the transgenic leaf fragments, the electrophoresed proteins on the gel It was transferred to nitrocellulose membrane and reacted with rabbit anti-VP1 multicellular group antibody and then labeled with Goat anti-rabbit IgG alkaline phosphatase conjugate (1: 1000, v / v). After washing, BCIP / NBT solution (Amersco Co., OH) was added and the reaction was stopped with distilled water.

As a result, the synthetic VP1 antigen was expressed at high efficiency in the leaf slices of the transformed tomato, and the recombinant protein showed a molecular weight of about 40 kDa (FIG. 3).

In addition, in plants transformed with a BCTV / CsVMV / native VP1 recombinant vector containing a native VP1 gene (lane 1 of FIG. 3, Korean Patent Application No. 10-2006-0124041), the expression level of hepatitis A virus antigen protein is increased. Although very insignificant, hepatitis A virus antigen protein was expressed more than 10 times higher in tomato transformed with the synthetic VP1 gene (lane 2 in FIG. 3).

As described in detail above, according to the present invention, a recombinant hepatitis A virus antigen is obtained by culturing a plant transformed with a recombinant vector comprising a synthetic hepatitis A virus antigen gene produced by gene codon optimization to be suitable for a plant. It is possible to produce a vaccine material having this as an active ingredient. In particular, the transgenic plant according to the present invention shows the efficacy of the hepatitis vaccine by ingestion as food, it can be usefully used as a functional food material and oral dose vaccine material.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (7)

1. A gene encoding a hepatitis A virus antigen represented by the amino acid sequence of SEQ ID NO: 1.
2. Hepatitis A virus antigen having the amino acid sequence of SEQ ID NO: 1.
3. Recombinant vector comprising the gene of claim 1.
4. The expression vector of claim 3, wherein the recombinant vector has two BCTV replicons between T-DNA borders, and includes a CsVMV promoter, North (NOS), and a replication initiation protein (REP) between the two replicons. A recombinant vector having a hepatitis A virus antigen gene represented by the amino acid sequence of SEQ ID NO: 1 inserted into a vector and having a cleavage map of FIG.
5. Plant transformed with the gene of claim 1 or the recombinant vector of claim 3.
6. The transformed plant of claim 5, wherein the plant is selected from the group consisting of tomatoes, lettuce, peppers, beans, rice, and corn.
7. Culturing the transformed plant of claim 5; And recovering the hepatitis A virus antigen.

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