KR100861923B1 - Method for preparating Antigen of Hepatitis A Virus using Insect cells Tansformed with Recombinant Vector for Expressing an Antigen of Virus - Google Patents

Abstract

The present invention relates to a method for producing an insect cell transformed with a recombinant vector comprising an antigen gene of hepatitis A virus, and stably producing a VP1 or VP3 antigen protein of hepatitis A virus using these transformants. will be.

By using a transgenic insect cell expressing the antigenic gene of the hepatitis A virus of the present invention, a recombinant hepatitis A vaccine material can be produced more stably, and the transgenic plant expressing the hepatitis A virus antigen of the present invention There is an advantage that can be used as a functional food material and oral vaccine material.

Hepatitis A virus antigen gene, recombinant vector, transformed cell

Description

Method for preparating Antigen of Hepatitis A Virus using Insect cells Tansformed with Recombinant Vector for Expressing an Antigen of Virus}

1 is a schematic diagram of an expression vector pMT / BiP / V5-His used to transform Drosophila melanogaster S2 cells to express an antigen of hepatitis A virus used in the present invention.

2 is a schematic diagram of pMT / BiP / VP3-V5-His, pMT / BiP / VP1-S-V5-His and pMT / BiP / VP1-L-V5-His of the present invention, respectively.

3 is a schematic diagram of BCTV / CsVMV / VP1-S and BCTV / CsVMV / VP1-L of the present invention.

Figure 4 shows the results of investigating the antigens of His-labeled hepatitis A virus purified from the media portion by culturing transformed Drosophila melanogasster S2 cells by SDS-PAGE (A) and Western blot analysis (B). It is shown. (M is the molecular weight marker; lane 1 is the cell fraction of the untransformed cell line; lanes 2, 3 and 4 are media portions of transformed S2 cells expressing recombinant VP3, VP1-S and VP1-L, respectively; arrows are Recombinant VP3, VP1-S and VP1-L proteins)

Figure 5 shows the result of Western blot the recombinant protein of transgenic tobacco. (Lane 1: normal leaf segment; lane 2: leaf segment of tobacco transformed with BCTV / CaMV / VP1-S; lane 3: leaf segment of tobacco transformed with BCTV / CaMV / VP1-L)

Figure 6 shows the results of Western blot the recombinant protein of the lettuce transformant. (Lane 1: normal leaf segment; lanes 2-6: lettuce transformants transformed with BCTV / CaMV / VP1-L)

The present invention relates to a method for producing an insect cell transformed with a recombinant vector comprising an antigen gene of hepatitis A virus, and stably producing a VP1 or VP3 antigen protein of hepatitis A virus using these transformants. will be.

Vaccines are biological materials used to inactivate certain pathogens when they are reunited with antigens that have been remembered by stimulating the immune system through several mechanisms to ensure that specific pathogens are remembered as antigens before they enter the body. Vaccine materials are divided into attenuated live vaccine and inactivated germ vaccine.

The global vaccine market is expected to reach $ 17 billion in 2010, growing at an average annual rate of 13%. Accordingly, the domestic market is currently forming a market of about 150 billion won. The age-specific vaccine material market had the highest share of pediatric vaccines, with revenues of $ 2.5 billion in 2001, but the government's recent vaccinations for older people and travelers are increasing the demand for adult vaccines. Therefore, this fact suggests that the development of new vaccine material development strategy is very important in the future.

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

Therefore, dietary vaccines are considered to be the most desirable vaccines in view of the manpower, cost, and pain used for vaccination. However, all of the dietary vaccines developed to date are attenuated live bacteria vaccines for typhoid fever and polio. It's being stopped. Therefore, recently, the pharmaceutical functional food material expressing the antigen gene in plants has emerged as the next generation bio health food by combining the safety of recombinant vaccine material with the ease and utility of dietary vaccine material.

Hepatitis A virus is a 27-nm RNA virus belonging to the family Picornavirdae. Acute liver disease characterized by high fever, malaise, anorexia, nausea, abdominal pain, cancer and jaundice after an average of 28 days of incubation. It is a disease causing. Hepatitis A virus, like rotavirus, has no encapsulation, is a icosahedron of 27 nm in diameter, 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 hepatitis A endemic regions, people with blood clotting disorders, homosexuals, people with chronic liver disease, and type A Vaccination is recommended in pediatric groups living in areas of high hepatitis, especially when more than 10 people per 100,000 population occur, and widespread vaccination is necessary, especially for hepatitis B carriers, the elderly, and chronic kidney failure. Hepatitis A vaccination is essential for adolescents living in groups and living in groups.

According to the recent 10-year survey, hepatitis A is increasing in the western United States, the Middle East, and some parts of Asia, and there is concern about the spread of hepatitis A worldwide. This is starting to increase rapidly. The incidence of hepatitis A is 5-14 years, with approximately 30% of patients under 15 years old. In the United States, 1982 to 1993, hepatitis A was 47%, more than 37% of hepatitis B. Serological tests showed that 33% of the US population had had hepatitis A in the past. It is identified.

About 11-22% of patients with hepatitis A 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 those under 18 years of age. More than $ 200 million is reported to be about $ 300 million in 2000 currency value.

Havrix was developed in 1995 by Smith Klein B.B.  Is the world's first hepatitis A vaccine material, produced by inactivating HM175 strain in MRC5 cells, which are human diploid cells, inactivating them, and adsorbing them to aluminum hydroxide. The method requires three doses for adolescents 2-18 years old and two doses for adults, and because they are given by muscle, they are not perfect for the formation of intestinal mucosal immunity, which is the path of viral penetration. .

Existing hepatitis A virus antigens have been attempted using an expression system through Escherichia coli or baculovirus, but their expression levels are insignificant. I can't. In addition, the transformed insect cell method of the present invention enables higher and stable production of the expression level of the antigenic protein than the case of using baculovirus.

Accordingly, the present inventors have made efforts to develop a hepatitis A virus vaccine that is more stable and easy to be orally administered. As a result, the VP1 and VP3 genes are cloned into insect cell expression vectors and plant expression vectors, and thus, to insect cells or plant cells. As a result of the transformation, it was confirmed that the VP1 and VP3 genes were stably expressed in insect cells and plant cells, thereby completing the present invention.

The main object of the present invention is to prepare a recombinant hepatitis A virus antigen using insect cells transformed with a recombinant vector containing the hepatitis A virus antigen gene, the vaccine material comprising the recombinant hepatitis A virus antigen as an active ingredient To provide.

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In order to achieve the above object, the present invention comprises a metallothionein promoter (PMT), BiP signal sequence (BiP SS), V5 epitope (V5), poly histidine (His6 tag) and restriction enzyme cleavage site, In the group consisting of VP1-S represented by SEQ ID NO: 1, VP1-L represented by SEQ ID NO: 2 and VP3 represented by SEQ ID NO: 3 in an expression vector (pMT / BiP / V5-His) having a series map of 1 The recombinant hepatitis A virus antigen, characterized by culturing the drosophila melanogasster S2 transformed with the recombinant vector for insect cell expression of the hepatitis A virus antigen, into which the selected hepatitis A virus antigen gene is inserted. It provides a manufacturing method.

Preferably, the expression vector includes restriction enzyme cleavage sites Bgl II and Apa I, and the hepatitis A virus antigen gene is inserted between the Bgl II and Apa I.
In addition, to achieve the above object, an expression comprising two BCTV replicons between a T-DNA border and a CsVMV promoter, North (NOS) and a replication initiation protein (REP) between the two replicons. In the vector, a hepatitis A virus antigen gene selected from the group consisting of VP1-S represented by SEQ ID NO: 1, VP1-L represented by SEQ ID NO: 2, and VP3 represented by SEQ ID NO: 3 is inserted. 3 provides a recombinant vector for plant expression of hepatitis A virus antigen gene having a cleavage map of FIG. 3.

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Preferably, in the expression vector, restriction enzyme cleavage site isSmaI AndSpeIncluding I and aboveSmaI AndSpeProvided between I, a recombinant vector for plant expression of the hepatitis A virus antigen gene, characterized in that the hepatitis A virus antigen gene is inserted.
And it provides a plant cell transformed with the recombinant vector, a transformed plant, and provides a method for producing a recombinant hepatitis A virus antigen, characterized in that the cultured transformed plant cells or cultivated transformed plants.

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Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing an antigen of recombinant hepatitis A virus by culturing the drosophila melanogasster S2 transformed with a recombinant vector expressing the VP1 or VP3 gene, which is the antigen gene of hepatitis A virus.

At this time, the VP1 gene is a gene corresponding to the total length of 1480-2298 bp or 1480-2379 bp 819bp, 900bp, respectively, VP1-S represented by SEQ ID NO: 1 for each VP1 represented by SEQ ID NO: 2 It is represented by -L. And the VP3 gene is a gene with a total length of 738 bp located between 742-1479 bp.

In the present invention, as a vector used for expressing the hepatitis A virus antigen gene VP1-S, VP1-L or VP3 gene, preferably, insect insect expression vector pMT / BiP / V5-His can be used. , But not necessarily limited thereto.

PMT / BiP / V5-His (3.6 kb) used for the preparation of the recombinant vector in the present invention can be easily obtained from Invitrogen Co., CA, a metallothionein promoter , BiP signal sequence, V5 epitope tag and polyhistidine site.

In addition, Drosophila melanogaster Schneider 2, (Invitrogen, USA) cells, a host cell used for expressing hepatitis A virus antigen in the present invention, is a genus Drosophila widely used in the field of genetic engineering. (Drosophila) is a cell line known as one of the cell lines belonging.

The VP1-S, VP1-L or VP3 genes were cloned into the BglII-ApaI sites of the restriction enzyme cleavage site of pMT / BiP / V5-His, respectively, introduced into Drosophila melanogasster S2 cells, transformed, and then cultured. Insect cells are produced to produce hepatitis A virus antigen.

The insect cell can be used in any of the conventional methods in the field of genetic engineering, so long as it can effectively express the desired hepatitis A virus antigen in high yield for such transformation and culture. Especially preferably, the transformed insect cell is cultured using a Nunc T-flask. The present invention also provides a method for producing a recombinant hepatitis A virus antigen, which is characterized by culturing the transformed insect cells.

In addition, the same method can be applied to Spodoptera, Trichoflugia, silkworm cells and other insect cells in addition to Drosophila insect cells in connection with the present invention.

In still another aspect of the present invention, a vector for use in expressing the hepatitis A virus antigen gene VP1-S, VP1-L or VP3 gene in the present invention, a plant expression vector comprising a geminivirus replicon Can be used.

Preferably, it has two BCTV (beet curly top virus) replicas between two T-DNA borders, and a promoter, NOS and replication initiator protein (REP) between the two replicas. ), And a recombinant vector for plant expression, characterized in that the hepatitis A virus antigen gene VP1-S, VP1-L or VP3 can be inserted, but is not necessarily 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 Application Publication No. 10-2006-0013000. In other words, the replicas of BCTV (using ATCC # PVMC-6) belonging to the Gemini virus were cloned, and REP (replication initiator protein) gene of BCTV was put into cis to reconstruct the skeleton of the expression system. In addition, PCR replicon was cloned into the CaMV 35S promoter + GFP + Nos poly-A by PCR using primers having Hi n dIII and Sal I restriction enzyme sites, and the REP gene and BCTV replicon of BCTV Was cloned by PCR using a primer having a Sac I restriction enzyme site. The CsVMV promoter was cloned by PCR using primers containing restriction enzymes of Sal I and Xba I 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. Restriction Enzyme Cleavage Sma I And Spe Clones the VP1-S, VP1-L or VP3 gene between I.

Plant cells and plants producing hepatitis A virus antigens are prepared by introducing the gene into Agrobacterium using expression plasmids for plant cell transformation comprising the VP1-S, VP1-L or VP3 genes. As the Agrobacterium, for example, Agrobacterium tumefaciens can be used. In the present invention, Agrobacterium tumefacien used a GV3101 line containing a hygromycin resistance gene pCAMBIA 1300 plasmid.

The plant cell can be used in any of the conventional methods in the field of genetic engineering, so long as it can effectively express the desired hepatitis A virus antigen in high yield for such transformation and culture. The present invention also provides a method for producing a recombinant hepatitis A virus antigen, comprising culturing the transformed plant cells.

In particular, in the present invention, tobacco and lettuce were used as a transgenic plant, but any plant capable of economically producing recombinant proteins such as tomatoes, peppers, beans, rice, and corn may 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.

The VP1-S, VP1-L or VP3 genes express the proteins that make up the hepatitis virus antigen. Therefore, as established with the transformed insect cells and the plant cells express hepatitis A virus antigen during the regeneration process, expression of such recombinant protein can be confirmed by Western blot analysis.

The present invention clones the hepatitis A virus gene VP1-S, VP1-L or VP3 gene and introduces it into insect cells or plants to greatly enhance the expression efficiency of the gene of hepatitis A virus antigen. Therefore, the transformant producing the hepatitis A virus antigen prepared according to the present invention can be usefully used as a vaccine for preventing or treating hepatitis. In particular, it provides an oral hepatitis preventive vaccine composition containing the transformed plant as an active ingredient.

The composition may be used as it is, or after drying the transformed plant in powder form, it may also be 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 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. It is certain.

In the present invention, "vaccine" refers to a composition used for the prevention or treatment of an infectious disease. Does the vaccine contain an antigen or can express an antigen, thereby inducing an immune response to the antigen. In order to prevent or treat infection, transmission, and epidemic of hepatitis A virus, the vaccine comprising the recombinant vector of the present invention can be used in a desired form.

"Vaccination means the vaccination of the vaccine to actively create immunity (humidity immunity, cellular immunity, or both) in the body or in the culture of the organism. Charges and / or epidemic can be prevented.

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 can be used as one epitope, one epitope in a protein usually contains about 7 to 15 amino acids, for example, 8, 9, 10, 12 or 14 amino acids. Antigens are also called immunogens. In the case of using an antigen to express 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.

"Gene" refers to genetic material and includes nucleic acids such as RNA and DNA. The gene may be one having a naturally derived or artificially designed sequence.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Preparation of Recombinant Vector

(1) Hepatitis A Virus Antigen Gene Amplification

Hepatitis A virus was extracted from a fecal sample from a Korean male patient diagnosed with hepatitis A using TRI Reagent (Molecular Research Center, Cincinnati, USA) and a series of nested RT-PCR methods. The P1 gene of the virus was obtained. First, cDNA was synthesized using 20 ng of hepatitis A virus RNA extracted using random hexamer (Promega, USA), and then a forward primer (5'-AGTGGCCTTGACCACATTCTGT-3 ') and a reverse primer (5'-ACTCCAAGTCTCCAGCTGCAATT-3') were used. Amplifying the cDNA flanking site containing the P1 gene of hepatitis A virus, and then using the forward primer (5'-GCTCTAGAATGAATATGTCCAAACAAGG-3 ') and the reverse primer (5'-GACTAGTCTCAAATCTTTTATCTTCCTC-3') Genetic amplification. The PCR conditions were all hepatitis A by PCR for 30 cycles at 94 ° C, 3 minutes, denaturation 94 ° C, 1 minute, annealing 55 ° C, 1 minute, height 72 ° C, 1 minute, extension 72 ° C, and 7 minutes. Viral P1 gene was amplified. PCR results were ligated into pCR 2.1-TOPO vector (Invitrogen, USA) and transformed with E. coli TOP10. The E. coli strain containing the PI gene of hepatitis A virus (KCTC10987BP: E. coli TOP10 / pCR2.1-TOPO (HAV P1) has been deposited with KCTC. Wherein the sequence located between 1480-2298 bp is VP1-S of SEQ ID NO: 1, the sequence located between 1480-2379 bp is VP1-L of SEQ ID NO: 2, and between 742-1479 bp The sequence located in was designated VP3 of SEQ ID NO: 3.

(2) Preparation of Expression Vector

1) Construction of pMT / BiP / VP1-V5-His and pMT / BiP / VP3-V5-His Expression Vectors of VP1 and VP3 Genes

 pMT / BiP / V5-His (3.6 kb) was obtained from Invitrogen Co., CA. pMT / BiP / V5-His was digested with restriction enzymes BglII and Apa I at 37 ° C. for 2 hours. In addition, HAV-VP3 and HAV-VP1 obtained above were digested with restriction enzymes BglII and ApaI also at 37 ° C. for 2 hours to obtain a BglII-ApaI fragment containing the gene of hepatitis A virus antigen. The BglII-ApaI fragments of HAV-VP3 and HAV-VP1 thus obtained were ligated between the BglII and ApaI cleavage sites of pMT / BiP / V5-His at 25 ° C. for 2 hours with T4 DNA ligation. Hepatitis A virus antigen expression vectors pMT / BiP / VP1-V5-His and pMT / BiP / VP3-V5-His were obtained. The structure is shown in FIG. Since the VP1 gene corresponds to a total length of 819 and 900 bp of 1480-2298 bp or 1480-2379 bp, two types of cloning, HAV-V1-S and HAV-V1-L, are shown in FIG. Was performed.

In the expression vector pMT / BiP / VP1 (VP3) -V5-His the gene insertion was in the proper direction and the read frame was confirmed by restriction enzyme mapping and DNA sequencing.

2) Construction of BCTV-CsVMV- VP1-S / L, a Plant Expression Vector Including BCTV Replicon

BCTV vectors containing CsVMV promoters are prepared by the method described in Korean Patent Application Publication No. 10-2006-0013000. Replicon of BCTV (using ATCC # PVMC-6) belonging to the Gemini virus was cloned, and the replication system of the expression system was reconstituted by adding the replication initiator protein (REP) gene of BCTV to cis. In addition, PCR replicon was cloned into the CaMV 35S promoter + GFP + Nos poly-A by PCR using primers having Hi n dIII and Sal I restriction enzyme sites, and the REP gene and BCTV replicon of BCTV of Cloning was performed by PCR using primers with Sac I restriction enzyme sites. The CsVMV promoter was cloned by PCR using primers containing restriction enzymes of Sal I and Xba I in BCTV vectors, respectively.

PCR amplification of hepatitis A virus VP1 gene was provided with SmaI and SpeI restriction enzyme sites for easy cloning. PCR conditions were 30 cycles at a total denaturation 94 ℃, 5 minutes, denaturation 94 ℃, 1 minute, annealing 55 ℃, 1 minute, elongation 72 ℃, 1 minute, expansion 72 ℃, 7 minutes. PCR result was ligated to the pGEM-T vector (Promega, USA), transformed into E. coli DH5α, plasmid was isolated and cut with SalI and SpeI to confirm the band of about 2.0kb, to determine whether the gene insertion Could. Hepatitis A virus antigen expression vector was produced recombinant BCTV-CsVMV-VP1-S / L which is a plant expression binary vector inserted BCTV replicon (Fig. 3).

In the recombinant expression vector BCTV-CsVMV-HAV (VP3, VP1-S, VP1-L), the gene insertion was in the proper direction and the read frame was confirmed by restriction enzyme mapping and DNA sequencing.

Example 2: Construction of Stable Transformants

(1) Transformation of Insect Cells Using Drosophila Melanogasster S2 Cells

Drosophila melanogasster S2 cells, a host cell used to express the hepatitis A virus gene, are non-cracked with M3 insect medium (Sigma) containing 10% IMS (insect medium supplement, Sigma). Incubated at 27 ° C using a Nunc) (T-25, Denmark) flask.

Hepatitis A virus antigen expression vector pMT / BiP / HAV (VP3, VP1-S, VP1-L) -V5-His and the selected plasmid pCoHygro (1: 1) using lipofectin Ratio of exponentially growing Drosophila melanogasster S2 cells were transformed according to the same method as proposed in Korean Patent 10-0557343. Subsequently, antibiotics, hygromycin (300 µg / ml), were added to the culture medium to establish a transformed cell line stably after a transgenic cell line selection process for about 4 weeks.

(2) Transformation of Tobacco Using Agrobacterium

Agrobacterium tumefaciens with recombinant BCTV-CsVMV-HAV plasmid inserted into sterile LB liquid medium and antibiotics (Kanamycin 50mg / l) was added to control shaking at 28 ° C and 150 rpm for 36 hours. The cells were incubated in a incubator, centrifuged at 2500 rpm for 10 minutes, and the centrifuged bacteria were suspended in liquid MSO medium and used for inoculation of tobacco slices.

Precultured Sections Agrobacterium After soaking in solution for 15 minutes, wipe excess Agrobacterium on the sections from sterilized filter paper and then 0.2 mg / l 3- in redistribution medium (MS salt, 0.3% phytagel, 30 g / L sucrose) without antibiotics. Inoculated on indoleacetic acid (IAA) and 1.0 mg / l zeatine added to the medium, and cultured for 2 days at 25 ℃. Two days later, transformants were selected by transfer to regeneration medium with antibiotics (150 mg / l thimentin, 30 mg / l hygromycin).

When the transformants were selected for 21 days, when the shoots were induced, roots were induced by cutting the stems grown in the medium from which MSO (MS salt, 0.3% phytagel, 30 g / L sucrose) hormone was removed. After two months of regeneration, the transgenic plants were transferred to pollen and subjected to soil purification.

(3) Transformation of Lettuce Using Agrobacterium

Agrobacterium tumefaciens with recombinant BCTV-CsVMV-HAV plasmid inserted into sterile LB liquid medium and antibiotics (Kanamycin 50mg / l) was added to control shaking at 28 ° C and 150 rpm for 36 hours. Incubated in the incubator and centrifuged at 2500 rpm for 10 minutes, the centrifuged bacteria were suspended in liquid MSO medium and used for inoculation of lettuce (Hanbat Blue Skirt, Non-bio Bio) slices.

Precultured Sections Agrobacterium After soaking in solution for 15 minutes, wipe off excess Agrobacterium on the sections from sterile filter paper, and then 0.1 mg / l 3- in regeneration medium (MS salt, 0.3% phytagel, 30 g / L sucrose) without antibiotics. Incubated at 25 ° C. for 2 days by incubation in a medium to which indole acetic acid (IAA) and 0.5 mg / L 6-benzylaminopurine (BA) were added. Two days later, transformants were selected by transfer to regeneration medium with antibiotics (150 mg / l thimentin, 10 mg / l hygromycin).

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, 30 g / L sucrose) hormone was removed. After the regeneration, the transformed plants were transferred to pollen and subjected to soil purification.

Example 3: Confirmation of Hepatitis A Virus Antigen Gene Introduction by Western Blot Analysis

In the examples below, Western blot analysis was performed on protein samples using SDS-PAGE according to the Lamley method (Laemmli, UK, Nature 227: 680-685, 1970). That is, the proteins electrophoresed on the gel were transferred onto nitrocellulose and reacted with a mouse anti-V5 (Invitrogen Co.) multicellular antibody followed by rabbit anti-mouse IgG alkaline phosphatase conjugate (1: 1000, v / v). Was labeled. After washing, BCIP / NBT solution (Amersco Co., OH) was added and the reaction was stopped with distilled water.

(1) Confirmation of Recombinant Protein Expression in Drosophila Melanogasster S2 Cells

To determine whether the drosophila melanogasster S2 cells transformed with pMT / BiP / HAV (VP3, VP1-S, VP1-L) -V5-His stably express the recombinant hepatitis A virus antigen It was confirmed by Western blot analysis using anti-V5 multi-cell antibody (Invitrogen) (Fig. 4).

As a result, the transformed Drosophila melanogasster S2 cells successfully expressed the VP1 and VP3 antigens, and the recombinant VP3 was about 7 kDa larger than the predetermined molecular weight of 27 kDa by 33-34 kDa. Recombinant VP1-S was about 38-39 kDa, about 8 kDa than the expected molecular weight of 30 kDa, and recombinant VP1-L was about 39-40 kDa, which was about 6 kDa larger than the expected molecular weight of 33 kDa.

Recombinant hepatitis A virus antigen is present inside and outside (medium) of transformed Drosophila melanogasster S2 cells, and according to densitometric scanning, secreted hepatitis A virus antigen (i.e., media portion) Hepatitis A virus antigens present in E. coli) account for about 85% of the total hepatitis A virus antigen production. That is, stably transformed S2 cells secrete the expressed hepatitis A virus antigen effectively extracellularly (in medium). No recombinant hepatitis A virus antigen was detected inside or outside untransformed S2 cells. This result shows that the expression of recombinant hepatitis A virus antigen in transformed S2 cells contains pMT / BiP / HAV (VP3, VP1-S, VP1-L)-containing the hepatitis A virus antigen gene introduced into the cell. Suggests that it is due to V5-His. In addition, the amount of recombinant hepatitis A virus antigen produced from transformed S2 cells is about 4 mg / l.

(2) Confirmation of Recombinant Protein Expression in Tobacco Transgenic Plants

To confirm the expression of the hepatitis A virus antigen protein from transgenic tobacco, 105 mg of acetone-concentrated protein was analyzed by 12% SDS-PAGE and western blot (Western blot). 5).

As a result, the transformed tobacco fragment successfully expressed the VP1 antigen, and the recombinant VP1-S showed a molecular weight of about 30 kDa and the recombinant VP1-L of about 33 kDa.

(3) Transformation of lettuce plants Recombinant protein  Expression confirmation

In order to confirm the expression of hepatitis A virus antigen protein from the transformants of lettuce, the protein samples extracted from the same amount of each transformant were analyzed by 12% SDS-PAGE and Western blot. (FIG. 6).

As a result, the transformants of lettuce successfully expressed the VP1 antigen and recombinant VP1-L showed a molecular weight of about 33 kDa.

As described above, the recombinant hepatitis A virus antigen is introduced by inducing and transforming the hepatitis A virus antigen genes VP1 and VP3 genes, which cause hepatitis A in the human body, into S2 cells or Agrobacterium and cultured or re-differentiated. It can be obtained and a vaccine material can be prepared as an active ingredient. Since the present invention is a stable expression system for the production of hepatitis A virus envelope proteins VP1 and VP3, it does not require complicated processes such as baculovirus infection, and has a high expression level and a continuous process configuration. In particular, the transgenic plant of the present invention can be used as a food material and oral dose vaccine material because it can show the efficacy of the hepatitis vaccine by ingesting as food.

<110> YAHOH Communication Co., Ltd. <120> Recombinant Vector for Expressing an Antigen of Virus, and Method          for preparating Antigen of Hepatitis A Virus using Tansformated          Insect cells and plant cells <130> P06-B214 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 819 <212> DNA <213> Hepatitis A Virus <400> 1 gttggagatg attcaggagg tttttcaaca acagtttcta cagagcagaa tgttcctgat 60 ccccaagttg gtataacaac catgagggac ctaaaaggaa aagctaatag gggaaagatg 120 gatgtttcag gagtgcaagc acctgtggga gctattacaa caattgagga tccagtttta 180 gcaaagaaag tgcctgaggc atttcctgaa ttgaagcctg gagagtccag acatacatca 240 gatcatatgt ctatttataa attcatggga aggtctcatt ttctgtgtac ttttaccttc 300 aattcaaata ataaagagta cacatttcca ataactctgt cttcgacttc taatcctcct 360 catggtttac catcaacatt aaggtggttt ttcaatctgt ttcagttgta tagaggacca 420 ttggacttga caattattat cacaggagcc actgatgtgg atggtatggc ctggttcact 480 ccagtaggcc ttgctgttga caccccttgg gttgaaaagg agtcagcctt atctattgat 540 tacaaaactg cccttggagc tgttagattt aacacaagaa gaacagggaa cattcagatt 600 agattgccat ggtattctta tttgtatgcc gtgtctggag cactggacgg tttgggagat 660 aagacagatt ctacatttgg attggtttct attcagattg caaattacaa tcattctgat 720 gaatatttgt cctttagttg ttatttgtct gtcacagaac aatcagagtt ttattttcct 780 agagctccat taaattcaaa tgctatgttg tccactgag 819 <210> 2 <211> 900 <212> DNA <213> Hepatitis A Virus <400> 2 gttggagatg attcaggagg tttttcaaca acagtttcta cagagcagaa tgttcctgat 60 ccccaagttg gtataacaac catgagggac ctaaaaggaa aagctaatag gggaaagatg 120 gatgtttcag gagtgcaagc acctgtggga gctattacaa caattgagga tccagtttta 180 gcaaagaaag tgcctgaggc atttcctgaa ttgaagcctg gagagtccag acatacatca 240 gatcatatgt ctatttataa attcatggga aggtctcatt ttctgtgtac ttttaccttc 300 aattcaaata ataaagagta cacatttcca ataactctgt cttcgacttc taatcctcct 360 catggtttac catcaacatt aaggtggttt ttcaatctgt ttcagttgta tagaggacca 420 ttggacttga caattattat cacaggagcc actgatgtgg atggtatggc ctggttcact 480 ccagtaggcc ttgctgttga caccccttgg gttgaaaagg agtcagcctt atctattgat 540 tacaaaactg cccttggagc tgttagattt aacacaagaa gaacagggaa cattcagatt 600 agattgccat ggtattctta tttgtatgcc gtgtctggag cactggacgg tttgggagat 660 aagacagatt ctacatttgg attggtttct attcagattg caaattacaa tcattctgat 720 gaatatttgt cctttagttg ttatttgtct gtcacagaac aatcagagtt ttattttcct 780 agagctccat taaattcaaa tgctatgttg tccactgagt ccatgatgag cagaattgca 840 gctggagact tggagtcatc agtggatgat cctagatcag aggaagataa aagatttgag 900                                                                          900 <210> 3 <211> 738 <212> DNA <213> Hepatitis A Virus <400> 3 atgatgagaa atgaatttag agttagcact actgaaaatg ttgtgaattt gtcaaattat 60 gaagatgcaa gggcaaaaat gtcttttgcc ttggatcagg aagattggaa gtctgatcct 120 tctcaaggtg gtggaattaa gatcactcat tttactacct ggacatccat tccaacttta 180 gctgcccagt ttccatttaa tgcttcagat tcagttgggc aacaaattaa agttattcca 240 gttgacccat attttttcca gatgacaaac actaatcctg atcaaaaatg tataactgcc 300 ctggcttcta tttgtcagat gttttgcttt tggagggggg atcttgtttt tgattttcag 360 gtttttccaa ccaaatacca ttcaggtaga ttattgtttt gttttgttac tgggaatgag 420 ttaatagatg ttactggaat tacattaaag caagcaacta ctgccccttg tgcagtaatg 480 gacattacag gagtgcaatc aaccttgaga tttcgtgttc cttggatttc tgatacaccc 540 tatcgagtaa ataggtacac gaagtcagca catcaaaaag gtgagtacac tgccattggg 600 aagcttattg tgtattgtta taatagactg acttctcctt ctaatgttgc ttctcatgtt 660 agagttaatg tttatctttc agcaattaat ttggaatgtt ttgctcctct ttatcatgct 720 atggatgtta ctacacag 738 <210> 4 <211> 2385 <212> DNA <213> Hepatitis A Virus <400> 4 tctagaatga atatgtccaa acaaggaatt ttccagactg ttgggagtgg ccttgaccac 60 attctgtctt tggcagatat tgaggaagaa caaatgattc agtccgttga taggactgca 120 gtgactggtg cttcttattt tacttctgtt gaccaatctt cagttcacac tgctgaggtt 180 ggttcacatc agattgaacc tttgaaaact tctgttgata aacctggttc taagaagact 240 cagggggaga agtttttcct gattcattct gctgattggc tcactacaca tgctcttttt 300 catgaagttg caaaattgga tgtggtgaaa ttactgtaca atgagcagtt tgctgtccaa 360 ggtttgttga gataccatac atatgcaaga tttggcattg agattcaagt tcagataaac 420 cccacaccct ttcagcaagg gggactaatt tgtgccatgg ttcctggtga ccaaagttat 480 ggttcaatag catcctcgac tgtttatcca catggtctgt taaattgtaa catcaataat 540 gtagttagaa taaaggttcc atttatttat actagaggtg cttaccattt taaagatcca 600 caatacccag tttgggaatt aacaatcaga gtttggtcag agttgaatat tggaacggga 660 acttcagctt acacttcact taatgtttta gctaggttta cagatttgga gttgcatggg 720 ttaactcctc tttctacaca gatgatgaga aatgaattta gagttagcac tactgaaaat 780 gttgtgaatt tgtcaaatta tgaagatgca agggcaaaaa tgtcttttgc cttggatcag 840 gaagattgga agtctgatcc ttctcaaggt ggtggaatta agatcactca ttttactacc 900 tggacatcca ttccaacttt agctgcccag tttccattta atgcttcaga ttcagttggg 960 caacaaatta aagttattcc agttgaccca tattttttcc agatgacaaa cactaatcct 1020 gatcaaaaat gtataactgc cctggcttct atttgtcaga tgttttgctt ttggaggggg 1080 gatcttgttt ttgattttca ggtttttcca accaaatacc attcaggtag attattgttt 1140 tgttttgtta ctgggaatga gttaatagat gttactggaa ttacattaaa gcaagcaact 1200 actgcccctt gtgcagtaat ggacattaca ggagtgcaat caaccttgag atttcgtgtt 1260 ccttggattt ctgatacacc ctatcgagta aataggtaca cgaagtcagc acatcaaaaa 1320 ggtgagtaca ctgccattgg gaagcttatt gtgtattgtt ataatagact gacttctcct 1380 tctaatgttg cttctcatgt tagagttaat gtttatcttt cagcaattaa tttggaatgt 1440 tttgctcctc tttatcatgc tatggatgtt actacacagg ttggagatga ttcaggaggt 1500 ttttcaacaa cagtttctac agagcagaat gttcctgatc cccaagttgg tataacaacc 1560 atgagggacc taaaaggaaa agctaatagg ggaaagatgg atgtttcagg agtgcaagca 1620 cctgtgggag ctattacaac aattgaggat ccagttttag caaagaaagt gcctgaggca 1680 tttcctgaat tgaagcctgg agagtccaga catacatcag atcatatgtc tatttataaa 1740 ttcatgggaa ggtctcattt tctgtgtact tttaccttca attcaaataa taaagagtac 1800 acatttccaa taactctgtc ttcgacttct aatcctcctc atggtttacc atcaacatta 1860 aggtggtttt tcaatctgtt tcagttgtat agaggaccat tggacttgac aattattatc 1920 acaggagcca ctgatgtgga tggtatggcc tggttcactc cagtaggcct tgctgttgac 1980 accccttggg ttgaaaagga gtcagcctta tctattgatt acaaaactgc ccttggagct 2040 gttagattta acacaagaag aacagggaac attcagatta gattgccatg gtattcttat 2100 ttgtatgccg tgtctggagc actggacggt ttgggagata agacagattc tacatttgga 2160 ttggtttcta ttcagattgc aaattacaat cattctgatg aatatttgtc ctttagttgt 2220 tatttgtctg tcacagaaca atcagagttt tattttccta gagctccatt aaattcaaat 2280 gctatgttgt ccactgagtc catgatgagc agaattgcag ctggagactt ggagtcatca 2340 gtggatgatc ctagatcaga ggaagataaa agatttgaga ctagt 2385  

Claims (10)

Restriction cleavage site comprising a metallothionein promoter (PMT), BiP signal sequence (BiP SS), V5 epitope (V5), poly histidine (His6 tag) and Bgl II and Apa I, In the expression vector (pMT / BiP / V5-His) having a map, hepatitis A virus antigen gene of VP1-S represented by SEQ ID NO: 1 or VP1-L represented by SEQ ID NO: 2 is present between Bgl II and Apa I. A method for producing a recombinant hepatitis A virus antigen, comprising culturing the drosophila melanogasster S2 transformed with the recombinant vector for insect cell expression of the hepatitis A virus antigen inserted in the. delete delete delete delete delete delete delete delete delete

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