KR20040087094A - High expression method of the b subunit of e. coli heat-labile enterotoxin in the chloroplast of plants - Google Patents

Abstract

PURPOSE: A high expression method of the B subunit of E. coli heat-labile enterotoxin in the chloroplast of plants is provided, thereby easily and efficiently forming immune response of the animal by introducing plants expressing animal disease inducing antigen into animals. CONSTITUTION: An antigen expression cassette for transforming the chloroplast of plants comprises (a) chloroplast rRNA operon promoter(Prrn); (b) ribosome binding nucleotide sequence; (c) antibiotic resistance gene; (d) polynucleotide containing the nucleotide sequence encoding an animal disease inducing antigen; and (e) 3'-untranslated region of chloroplast psbA gene, wherein the animal disease inducing antigen is B subunit of E. coli enterotoxin. A vector pMY-CH-LTB comprises the antigen expression cassette. A transgenic plant containing the antigen expression cassette in the chromosomal genome by homologous recombination, wherein the plant is animal edible plant; and the plant is Nicotiana tabacum/pMY-CH-LTB(KCTC 10447BP). The high expression method of the B subunit of E. coli heat-labile enterotoxin in the chloroplast of plants comprises inserting the antigen expression cassette into the chromosomal genome of a plant and expressing the animal disease inducing antigen from the plant chromosome.

Description

HIGH EXPRESSION METHOD OF THE B SUBUNIT OF E. COLI HEAT-LABILE ENTEROTOXIN IN THE CHLOROPLAST OF PLANTS}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a high expression method of Escherichia coli enterotoxin Ｂsubunit in plant chloroplasts, and more specifically, to an expression cassette, a vector, a transgenic plant, a recombinant protein and the above which can express an animal disease-causing antigen in chloroplasts It relates to an oral vaccine comprising a transgenic plant or recombinant protein.

[Private Technology]

Recombinant proteins of antigen genes are used as oral vaccines. Recombinant proteins have many advantages that can be used as oral vaccines in terms of low production, ease of administration and storage (Giddings G, Allison G, Brooks D and Carter A (2000) Nat Biotechnol 18: 1151-. 1155).

Oral vaccines increase the likelihood of forming mucosal immunity against infectious agents entering the body across mucosal surfaces (Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM and Arntzen CJ (1998) Nat Med 4: 607-609). However, subunit vaccines used for oral delivery are limited in their use due to proteolytic degradation in the intestine.

Recently, studies have been underway to use transgenic plants as an alternative means of producing and delivering vaccines. For example, Norwalk virus capsid protein, Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM and Arntzen CJ (2000)J Infect Dis182: 302-305), transmissible gastroenteritis coronavirus glycoprotein, Tuboly T, Yu W, Bailey A, Degrandis S, Du S, Erickson L and Nagy E (2000)Vaccine18: 2023-2028), Hepatitis B virus surface protein (Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS and Thanavala Y (2001)Proc Natl Acad Sci USA98: 11539-11544) and foot-and-mouth virus VPl (Dus Santos MJ, Wigdorovitz A, Trono K, Rios RD, Franzone PM, Gil F, Moreno J, Carrillo C, Escribano JM and Borca MV (2002)Vaccine20: 1141-1147) has been successfully expressed in plants.

In order to satisfy the vaccine dose, the amount of plant ingested should be of a practically practicable amount. However, in previous studies, protein expression in plants was mostly 0.01 to 0.1% of total water soluble protein, rarely exceeding 0.40% (Haq TA, Mason HS, Clements JD and Arntzen CJ ( 1995)Science268: 714-716; Gomez N, Carrillo C, Salinas J, Parra F, Borca MV and Escribano JM (1998) Virology249: 352-358; Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS and Thanavala Y (2001)Proc Natl Acad Sci USA98: 11539-11544; Mason HS, Warzecha H, Mor T and Arntzen CJ (2002)Trends Mol Med8: 324-329) Therefore, high expression of recombinant proteins that can be used as oral vaccines in plants is of great importance (Stoger E, Sack M, Fischer R and Christou P (2002)).Curr Opin Biotechnol13: 161-166)

There are about 60 chloroplasts per cell in a plant, and one chloroplast contains about 60 copies of DNA. Thus, plant chloroplast transformation can significantly increase the production of recombinant proteins. Indeed, Bt Cry2 Aa2 operon expressed 45.3% of the total water soluble protein in chloroplasts (De Cosa B, Moar W, Lee SB, Miller M and Danielle H (2001) Nat Biotechnol 19: 71-74). (Daniell H, Lee SB, Panchal T and Wiebe PO (2001) J Mol Biol 311: 1001-1009). In tomato chloroplasts, the foreign gene aadA is expressed in about 1% of the total water soluble protein. (Ruf S, Hermann M, Berger IJ, Carrer H and Bock R (2001) Nat Biotechnol 19: 870-875) GFP was expressed at 5% in potato chloroplasts (Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz). PTJ, Staub JM and Nehra NS (1999) Plant J 19: 209-216)

In addition, transformation with chloroplasts has several advantages over nuclear transformation. First, the chloroplasts of most major crops are maternally inherited to allow for preservation, and they do not pollute with other plants, thus facilitating crop management without contaminating the environment (Daniell et al., 1998; Scott and Wilkinson, 1999; Lutz KA, Knapp JE and Maliga P (2001) Plant Physiol 125: 1585-1590. Second, polycistronic operons can be used to express a number of genes in one chloroplast transformation (Staub JM). and Maliga P (1995) Plant J 7: 845-848. Third, the foreign gene is inserted into the chromosome by homologous recombination, so the insertion site can be expected. This does not cause the site effect caused by random insertion of foreign genes in nuclear transformation (Svab Z, Hajdukiewicz P and Maliga P (1990) Proc Natl Acad Sci USA 87: 8526-8530). Finally, chloroplast transformation Cases of no exogenous gene expression have yet been reported.

Enterotoxigenic E. coli (ETEC), on the other hand, produces at least one enteric toxin causing severe watery diarrhea. One of the enterotoxins is high molecular weight heat labile toxin (LT), which is similar to cholera toxin (CT). Clements JD and Finkelstein RA (1979) Infect Immun 24: 760-769; Clements JD , Yancey RJ and Finkelstein RA (1980) Infect Immun 29: 91-97). The dimeric toxin is a donut-like structure with a single 27-kDa A subunit that possesses toxic ADP ribosyl transferase activity. It consists of five 11.6-kDa B subunits that are very stably linked in a non-covalent bond. Dimeric toxins and cholera toxins bind by specific interactions of GM subganglia and B subunit pentamers on the epithelial cell surface of the intestine. E. coli enterotoxin B subunit and CTB are immunogenic (Clements JD and Finkelstein RA (1978) Infect immun 21: 1036-1039 .; Gilligan PH, Brown JC and Robertson DC (1983) Infection and Immunity 42 : 683-691) as well as adjuvant for co-administered antigen (Dickinson BL (1996) Mucosal Vaccines. (Pp. 73-87) San Diego, CA; Academic Press; Elson CO (1996) Mucosal vaccines (pp. 59- 72) Academic Press, New York, NY).

Recombinant E. coli toxin Ｂsubunit protein (rLTB), when expressed in Bacillus brevis and administered to rats' noses with diphtheria denatured toxin, substantially promotes the production of denatured toxin serum IgG antibodies, resulting in some detoxification in the nasal cavity and lungs. Specific mucosal IgG antibody response was induced. This indicates that rLTB can be used as an adjuvant for mucosal immunity (Kozuka S, Yasuda Y, Isaka M, Masaki N, Taniguchi T, Matano K, Moriyama A, Ohkuma K, Goto N, Udaka S and Tochikubo K). (2000) Vaccine 18: 1730-1737. The B subunit recombinant multimers of the dimeric toxin and the cholera toxin have been successfully produced in potatoes, which have been produced in animals (Mason HS, Haq TA, Clements JD and Arntzen CJ (1998) Vaccine 16: 1336-1343; Lauterslager TG, Florack DE, van der Wal TJ, Molthoff JW, Langeveld JP, Bosch D, Boersma WJ and Hilgers LA (2001) Vaccine 19: 2749-2755) and people (Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM and Arntzen CJ (1998) Nat Med 4: 607-609) has been confirmed to form an immune response by oral administration. However, the B subunit recombinant multimers of dimeric toxins and cholera toxins were found in tobacco (Haq TA, Mason HS, Clements JD and Arntzen CJ (1995) Science 268: 714-716) and potato (Mason HS, Warzecha H, Mor T and When produced by nuclear transformation of Arntzen CJ (2002) Trends Mol Med 8: 324-329), less than 0.01% and 0.19% of total water soluble proteins, respectively, need to establish a system and method capable of high expression. It is true.

In order to solve the problems of the prior art, an object of the present invention is to provide an expression cassette comprising an animal disease-causing antigen gene to be expressed in the chloroplast of the plant.

It is another object of the present invention to provide a transgenic plant in which an animal disease-causing antigen gene is expressed in chloroplasts.

In addition, an object of the present invention is to provide a method for producing a large amount of E. coli enterotoxin B subunit (heat labile enterotoxin B subunit) by plant chloroplast transformation method.

It is also an object of the present invention to provide a method for preventing and treating diseases caused by pathogenic microorganisms by transforming an animal edible plant to be capable of expressing an animal disease-causing antigen and orally administering the transformed plant to the animal. It is done.

Figure 1 shows the LTB expression cassette contained in the pMY-CH-LTB vector,

2 is a structural diagram of a pLD-CtV2 vector,

3 is a structural diagram of a pMY-CH-LTB vector.

Figure 4 shows the process of homologous recombination of the LTB expression cassette of the pMY-CH-LTB vector on the chloroplast genome of the plant,

Figure 5 briefly shows the amplification site for each PCR primer set type for the chloroplast genome of the transgenic plant,

Figure 6a and 6b is a PCR analysis of the chloroplast DNA of wild type tobacco leaves and transgenic tobacco leaves,

Figure 7 shows the LTB Southern blot results of the transgenic plant,

8a and 8b confirm the expression of LTB protein in transgenic plants by Western blot,

9 is a quantification of recombinant LTB protein expressed in a transgenic plant,

Figure 10 is a graph showing the strong binding capacity of GM1-gangliosides of recombinant LTB protein expressed from the transgenic plants of the present invention.

In order to achieve the above object, the present invention includes (a) a chloroplast rRNA operon promoter (Prrn), (b) ribosomal binding sequence, (c) antibiotic resistance gene, and (d) a base sequence encoding an animal disease-causing antigen. To provide an antigen expression cassette for plant chloroplast transformation comprising a polynucleotide and (e) 3'-untranslated region of the chloroplast psbA gene.

The present invention also provides a vector comprising the above antigen expression cassette.

The present invention also provides a transgenic plant wherein the antigen expression cassette is inserted by homologous recombination on the chloroplast genome.

In another aspect, the present invention provides a recombinant protein of the animal disease-causing antigen produced from the transgenic plant.

In another aspect, the present invention provides a method for inserting the above-mentioned antigen expression cassette on the chloroplast genome of a plant for mass production of an animal disease-causing antigen from the plant chloroplast.

The present invention also provides an anti-E. Coli enterotoxin vaccine composition comprising the transformed plant or E. coli enterotoxin B subunit recombinant protein.

The present invention also provides an expression cassette comprising (a) a nucleotide comprising a nucleotide sequence encoding the animal disease-causing antigen to be expressed on the chloroplast of the plant, and (b) the expression cassette on the chloroplast genome of the plant To insert a transgenic plant, (c) cultivate the transgenic plant to produce a recombinant antigen protein, and (d) administer the animal to the animal disease-causing antigen by administering the produced recombinant antigen protein or a plant comprising the same to the animal. It provides an immunogenic method comprising forming an antibody against.

Hereinafter, the present invention will be described in detail.

The present inventors have identified a method for producing a large amount of animal disease-causing antigens using plant chloroplast transformation, and administering the antibody by administering to the animal a plant expressing the animal disease-causing antigen or a recombinant antigenic protein isolated from the plant. We have established a system that can be formed.

Animal disease-causing antigens referred to in the present invention are proteins produced from pathogenic microorganisms, ie viruses, bacteria or fungi. A representative example is E. coli Heat-labile enterotoxin B subunit (GeneBank No. 3062900).

The present invention provides an antigen expression cassette comprising a nucleotide comprising a nucleotide sequence encoding the animal disease-causing antigen to be expressed in the plant chloroplast.

In one embodiment, the invention encodes (a) a chloroplast rRNA operon promoter (Prrn), (b) a ribosomal binding sequence, (c) an antibiotic resistance gene, and (d) an E. coli enterotoxin B subunit protein. A polynucleotide comprising a nucleotide sequence and (e) an antigen expression cassette for plant chloroplast transformation comprising the 3'-untranslated region of the chloroplast psbA gene are prepared. The chloroplast rRNA operon promoter is a promoter expressing the chloroplast rRNA (GeneBank No. 11799), and the ribosome binding base sequence may be a conventional base sequence to which plant ribosomes can bind, preferably GGAGG. The ribosomal binding base sequence may be located at the 3 'end of the promoter. The antibiotic resistance gene is a gene not observed in wild-type plant cells, and may be an antibiotic resistance gene that is commonly used. For example, the kanamycin resistance gene, the streptomycin resistance gene, the ampicillin resistance gene, the spectinomycin resistance gene, and the aminoglycoside adenylyl transferase ( aadA ) gene, which is preferably resistant to streptomycin or spectinomycin. (GeneBank No. 23476452). The 3'-untranslated region of the chloroplast psbA gene is preferably Gene bank No. It contains the base sequence described in 11465934.

In addition, the antigen expression cassette of the present invention may further comprise a chloroplast genomic sequence at both ends of the cassette to be homologously recombined into a specific position on the chloroplast genome. Preferably 16S rDNA Gene Bank No. 2924257), trnI (GeneBank No. 2924257) and trnA (GeneBank No. 2924257) nucleotide sequences are those containing some (16S rDNA, 158 bp) or all (trnI, 779 bp; trnA, 782 bp), respectively.

The present invention also provides a vector comprising the above antigen expression cassette. The vector includes an antigen expression cassette in a conventional plant expression vector, and the present invention provides, for example, a pMY-CH-LTB vector.

The pMY-CH-LTB vector is a vector pLD-CtV2 shown in Figure 2 (Prrn, Ribosomal RNA Promoter; aadA , aminoglycoside adenylyltransferase; TpsbA, 3'PsbA; 16S, trnI, and trnA, border sequences) Eco RI and Xba the The antigen-expressing gene is inserted at the restriction site I, and the structural diagram of the vector is shown in FIG. 3 (SD-LT-B, LTB antigen gene containing Shine-Dalgarno sequence (GGAGG)).

The present invention also provides a transgenic plant wherein the antigen expression cassette is inserted by homologous recombination on the chloroplast genome. The transgenic plant may introduce a vector containing an antigen expression cassette into plant cells, and the antigen expression cassette in the vector may be inserted by homologous recombination at a specific position on the chloroplast genome and then selected using antibiotic selection medium.

The transgenic plant of the present invention may be any kind of plant that can be eaten by animals, and may also be whole plants, plant tissues or plant cells, and plants regenerated from the plant tissues and plant cells may also correspond thereto. . Available transgenic plants include potatoes, sweet potatoes, pineapples, bananas, tomatoes, lettuce, lettuce, cabbages, cabbage, carrots or avocados.

As an example in the present invention, transgenic sikmulreul manufacturing was, this Republic of Korea on the Life Sciences Institute gene bank located in Yuseong Nico tiahna other Vacuum / pMY-CH-LTB (Nicotiana tabacum / pMY-CH-LTB) to March 2003 21 It was deposited on a date and received accession number KCTC 10447BP.

The present invention also provides a recombinant protein of an animal disease-causing antigen produced from a transgenic plant. Recombinant proteins of animal disease-causing antigens can be isolated by conventional protein purification methods. In one embodiment of the present invention, E. coli Heat-lablid enterotoxin B subunit was transformed into plant chloroplasts to obtain a recombinant protein. In this case, the E. coli enterotoxin B subunit recombinant protein expressed in plant cells was expressed at a rate of 2.5% to 3.0% of the total water soluble protein, and showed about 250-fold higher expression titer than that obtained by nuclear transformation. The E. coli enterotoxin B subunit recombinant protein of the present invention can bind gangliosides by maintaining the same activity as the wild type.

The present invention also provides a method for inserting an antigen expression cassette onto the chloroplast genome of a plant to mass produce an animal disease-causing antigen from the plant chloroplast. More specifically, (a) preparing an antigen expression cassette to express an animal disease-causing antigen gene in plant chloroplasts, (b) inserting the antigen expression cassette into a vector, and then inserting the inserted vector into plant cells, (c) selecting the transformed plants with the antigen expression cassette, and (d) culturing the transformed plants. It is obvious that the above-described methods can be carried out by methods known in the art.

Animal disease-causing antigen-recombinant protein prepared by the above-mentioned methods or a plant, plant tissue, or plant cell expressing the same can be used for forming immunity to pathogenic microorganisms.

That is, the present invention provides an anti-E. Coli enterotoxin vaccine composition comprising a transgenic plant or E. coli toxin B subunit recombinant protein as an active ingredient. The anti-E. Coli toxin vaccine composition may comprise a transgenic plant or E. coli toxin B subunit recombinant protein alone, and may further include a pharmaceutically acceptable carrier. When the vaccine composition further comprises a carrier, the content of the transgenic plant or E. coli enterotoxin B subunit recombinant protein is preferably 0.001 to 50% by weight.

The anti-E. Coli enterotoxin vaccine composition of the present invention may be prepared in oral or parenteral form, and preferably, the vaccine composition containing a transgenic plant as an active ingredient is preferably used orally, and the E. coli enterotoxin B subunit recombinant protein Vaccine compositions comprising as an active ingredient can be used orally or parenterally. In addition, the transformed plants may be whole plants, plant tissues or plant cells, preferably plant parts containing a large amount of chloroplasts.

The dose of the anti-E. Coli enterotoxin vaccine composition of the present invention is preferably adjusted in consideration of the purpose, condition and age of the patient.

The present invention also provides an immunogenic method for an animal disease-causing antigen. More specifically, the immunogenic method of the present invention prepares an expression cassette comprising (a) a nucleotide comprising a nucleotide sequence encoding an animal disease-causing antigen on the chloroplast of a plant, and (b) the expression cassette. Is inserted into the chloroplast genome of the plant to prepare a transgenic plant, (c) culturing the transgenic plant to produce a recombinant antigen protein, and (d) producing the recombinant antigen protein or plant tissue comprising the same. To form an antibody against the antigen. Each procedure for the above method can be easily implemented by those skilled in the art.

E. coli enterotoxin B subunit high expression method in the chloroplast of the present invention and the immunogenic method using a transgenic plant or recombinant antigen protein can easily produce a large amount of recombinant protein compared to the nuclear transformation method, Immunization can be induced simply. Therefore, the transgenic plant or recombinant antigen protein of the present invention can be used as an additive in livestock feed or beverages in addition to vaccine compositions.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example

1. Plant Materials

Tobacco ( Nicotiana tabacum cv TI560) seeds were surface sterilized in 70% alcohol for 3 minutes, followed by 15 minutes in 10% sodium hypochlorite. The seeds were washed repeatedly with distilled water five times and sown in a medium (pH 5.7) containing MS (4.6 g / L) added with sucrose (30 g / L) and bactoagar (7.5 g / L) for 16 hours. / 8 hr dark under light conditions, grown at 25 ℃.

2. Preparation of Chloroplast Expression Vector

The plasmid pLD-CtV2 was constructed for stable chloroplast transformation in tobacco (De Cosa B, Moar W, Lee SB, Miller M and Danielle H (2001) Nat Biotechnol 19: 71-74).

E. coli enterotoxin toxin B subunit (hereinafter referred to as "LTB") was prepared from genomic DNA of E. coli strain No. 1032. PCR primers were prepared by introducing a ribosome binding site (GGAGG) into the 8 bases upstream from the initiation codon in E. coli LTB from which the signal peptide was removed. The forward primer contains SEQ ID NO: 1, the back primer contains SEQ ID NO: 2, and each primer contains the Eco RI and Xba I restriction enzyme cleavage sites.

PCR conditions were repeated 30 times with denaturation (94 ° C., 1 min)-> heat cooling (55 ° C., 1 min)-> elongation (72 ° C., 1 min), and the reaction solution was performed in a total of 50 µl. The PCR product was cloned into the pGemTeasy vector of Promega, and the pMY-CH-LTB vector was prepared by conjugating the fragments of the restriction enzymes Eco RI and Xba I to the same restriction site of the pLD-CtV2 vector.

The pMY-CH-LTB vector was transformed into M15 cells of Escherichia coli, and the expressed recombinant LTB protein was purified by Qiagen's QIAexpress System.

Figure 1 shows the LTB expression cassette contained in the pMY-CH-LTB vector, LTB expression cassette has a total length of 1.7 kb and chloroplast rRNA operon promoter (Prrn), ribosomal binding site (GGAGG), antibiotic spectinomycin or 3'-untranslated region of the streptomycin resistance gene, aminoglycoside adenylyltransferase ( aadA ) gene, LTB gene and chloroplast psbA gene for stabilization of mRNA.

3. Chloroplast transformation

The transformed plants were selected by homologous recombination of the LTB expression cassette contained in the pMY-CH-LTB vector on the chloroplast genome of tobacco.

Figure 4 shows the process of homologous recombination of the LTB expression cassette of the pMY-CH-LTB vector on the chloroplast genome of the plant, transformed with the LTB expression cassette, wild-type plant (WT-CtDNA) of the pMY-CH-LTB vector The plant (T-CtDNA) is shown. The arrow indicates the transfer direction. The 1.7 kb LTB expression cassette was inserted into a homologous recombination by trnI and trnA on the 4.47 kb wild-type chloroplast genome, totaling 6.17 kb. In addition, the probe shown in Figure 4 is a probe, it is then used as a means for confirming the insertion and the number of insertion of LTB expression cassette in transgenic plants.

In the following method, a transgenic plant was prepared in which the LTB expression cassette was inserted on the chloroplast genome.

Tobacco leaves grown sterile in hormone-free MS agar medium were placed on filter paper on top of MS agar medium. Tobacco leaves were bombarded using a Biolistic PDS-1000 / Helium Particle Delivery System (Bio Rad) with gold (1 μm) or tungsten (0.7 μm) particles.

1) Mix the microcarriers (50 mg / ml of gold particles per ml of glycerol) in glycerol to release the pellets.

2) Mixing (Mix every step below)

end. 5 μg DNA is added to the microcarrier tube and mixed for 1 minute.

I. Add 50 M of 2.5 M CaCl ₂ and mix for 1 minute.

All. Add 0.1 M spermidine in 20 μl and mix for 1 minute.

3) Centrifuge the mixture for 2 seconds.

4) Remove the supernatant, add 140 μl of 100% EtOH, then centrifuge for 2 seconds and remove the solution quickly (leave the pellet intact). Repeat once.

5) Add 40-60 μl of 100% EtOH and tap to release the pellets.

6) Mix 2-3 seconds before impact.

7) Dispense 6 to 8 µl into the microcarrier and apply an impact.

8) After the impact, co-culture in a co-culture medium for 3-5 days (cancer condition).

2-3 days after the impact, the leaves were cut into 0.5 cm ² sections and RMOP selection medium (0.43 g MS salt, 1.0 mg / L 6-BAP, 0.1 mg / L) containing 500 mg / L of antibiotic spectinomycin. NAA, 100 mg / L inositol, 30 g / L sucrose, 6 g / L phytagar, 1 mg / L thiamine-HCl, pH 5.8). After 5-6 weeks, the shoots produced in the selection medium were cut into small sections (~ 0.2 cm ² ) and placed on the selection medium and incubated.

4. PCR and Southern Blot Analysis

Total genomic DNA was extracted as follows.

The leaf tissue was ground with liquid nitrogen, and suspended therein with 0.5 ml of extract buffer (100 mM Tris / HCl, 50 mM EDTA, 500 mM NaCl, pH 7.5). 45 μl of 20% SDS was added thereto and then left at 65 ° C. for 10 minutes. The same amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added thereto, followed by centrifugation at 12,000 rpm for 3 minutes, and then the supernatant was extracted and transferred to a new tube, followed by addition of the same amount of chloroform. Was repeated. The extracted supernatant was transferred to a tube, added with 0.8 volume of isopropyl alcohol, left at -20 ° C. for 30 minutes, and then centrifuged (12,000 rpm, 15 minutes) to obtain pellets. The pellet was purified by 1 ml of 70% ethanol. Washed. The DNA pellet was then dried and dissolved in sterile distilled water.

PCR was performed to amplify the LTB fragment from the extracted genomic DNA. Primers used were 1F of SEQ ID NO: 3 / 1R primer set of SEQ ID NO: 4, 2F of SEQ ID NO: 5 / 2R primer set of SEQ ID NO: 6, 3F of SEQ ID NO: 7 / 3R primer set of SEQ ID NO: 8, and 4F of SEQ ID NO: 9 / 4R primer set of SEQ ID NO: 10. PCR was performed under the same conditions as described in the preparation of chloroplast expression vectors using DNA isolated from the transformant and wild-type leaves.

Figure 5 briefly shows the amplification sites for each PCR primer set for the chloroplast genome containing the LTB expression cassette of the transgenic plant.

The 1F / 1R primer set binds to wild-type chloroplast DNA and aadA to amplify a 1.65 kb DNA fragment, the 2F / 2R primer set binds to aadA and trnA to amplify a 1.9 kb DNA fragment, and the 3F / 3R primer set 2.8 kb of DNA fragments are amplified by binding to wild-type chloroplast DNA and LTB, and the 4F / 4R primer set binds to amplification of 1.7 kb of DNA fragments by binding to LTB and wild-type chloroplast DNA.

6 is a PCR analysis of chloroplast DNA of wild type tobacco leaves and transformed tobacco leaves. WT is the result of PCR using DNA extracted from wild tobacco leaf as a template, lanes 1 to 5 are the results of PCR with each 1F / 1R to 4F / 4R primer set as a template using DNA extracted from chloroplasts of transformed tobacco leaves, M Is a 1 kb DNA size marker.

In FIG. 6, it was confirmed that LTB expression cassette was successfully introduced into the target site of the chloroplast genome by homologous recombination in all transgenic plants. The primer sets used for PCR are each paired with primers that bind to chloroplast DNA adjacent to the LTB expression cassette and primers that bind within the LTB expression cassette. When the LTB expression cassette is not inserted by homologous recombination at the target position, the primers This is because PCR products are not amplified by PCR in sets. In addition, the 2F / 2R primer set is intended to confirm that the LTB gene is stably inserted alongside the aadA gene.

On the other hand, no PCR product was found in wild-type tobacco.

5. Sudden blot analysis

Sudden blots were performed to verify the specific site insertion of the LTB expression cassette in the transgenic plants and to confirm the number of copies of the inserted LTB expression cassette.

10 ug of total genomic DNA isolated from the leaves of the transgenic and wild type plants was cut with restriction enzyme Bgl II and electrophoresed on a 0.8% agarose gel. DNA on an agarose gel was transferred to Highbond-N + (Amersham), reacted with trnI-trnA (0.81 kb) probe labeled with ² P-dCTP, washed and exposed to X-ray film.

Figure 7 shows the Southern blot results, WT is a wild type plants, 1 to 5 are transformed plants. As a result of Southern blot, 4.47 kb of DNA fragment was detected in wild type, and the transformed plant was identified as 6.17 kb including LTB expression cassette (1.7 kb). Therefore, in all five transgenic plants of the present invention, the LTB expression cassette is inserted between tranI and trnA of the chloroplast genome.

6. Weston Blot Analysis

Expression of recombinant LTB protein in transgenic plants was confirmed.

In a greenhouse, transformed plants (plants 2 and 5) and wild-type plants (~ 0.5 g) were added with liquid nitrogen, and ground in a mortar and 1 ml of protein extract buffer (50 mM HEPES, pH 7.5, 10 mM). Potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 1 mM DTT, 2 mM phenylmethanesulfonyl fluoride). The suspensions were quantified by Bradford assay and then electrophoresed on 100 μg total water soluble protein on 15% SDS-PAGE gel. In addition, the recombinant LTB expressed in E. coli M15 cells as a positive control was electrophoresed at 0.5 to 4 ㎍. The developed protein bands were transferred to high bond C membranes (Promega) using a Mini Trans Blot ^™ electrophoretic Transfer Cell (BioRad). The membrane was soaked in TBST buffer containing 5% nonfat powdered milk (TBS containing 0.05% Tween-20) and then in a 1: 2000 ratio containing TBST buffer containing 2.5% nonfat powdered milk and rabbit LTB antiserum. The mixture was placed in an antibody mixture and allowed to stand at room temperature with stirring. Membranes were washed three times with TBST buffer, alkaline phosphatase conjugated anti-rabbit IgG diluted 1: 7000 and reacted for 2 hours, then washed three times with TBST buffer and once with TMN buffer. After washing, the membrane was reacted with BCIP / NBT (USB) to induce color development.

Figure 8 confirms the expression of the LTB protein in transgenic plants by Weston blot, 6a is the electrophoresis of the total water-soluble protein of wild-type and transgenic plants without thermal denaturation, 6b is electrophoresis after thermal denaturation. LTB is the electrophoresis of the recombinant LTB protein purified as a positive control, Transnasplastomic plants are the electrophoresis of the total water soluble protein (100 ug) of the transgenic plants (2 and 5), WT is the total water soluble protein of wild type plants. "P" means pentamer, "M" means monomer, "Te" means tetramer and "Tr" means trimer.

In FIG. 8A, expression of the oligomeric LTB protein of about 45 kDa was observed in the transgenic plants without heat denaturation. In FIG. 8B, which was thermally denatured, 5 polymers, 4 polymers, 3 polymers, and 12 kDa molecular weight monomers similar to the LTB size derived from bacteria were separated. This is in line with previous reports on expressed LTB in nuclear transfection of maize (Chikwamba R, Cunnick J, Hathaway D, McMurray J, Mason Hand Wang K). (2002) Transgenic res11: 479-493). In addition, when comparing the two transgenic plants, it was confirmed that the LTB protein expression level between the two species can be produced a stable transgenic plant.

7. LTB Protein Quantitation by ELISA

ELISA analysis was performed to estimate LTB protein levels in leaf tissues of transgenic plants 2 and 5.

Leaves of transformed and wild type plants were left in liquid nitrogen and then ground, and homogenized by addition of bicarbonate buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ ) at pH 9.6. At this time, the concentration of total water soluble protein was determined by the Bradford reaction reagent (Sigma).

Total soluble protein extracted from the transgenic and wild-type plants was placed in a microtiter plate at 100 μl per well and left overnight at 4 ° C. Plates were washed three times with TBST buffer, 300 μl of PBS containing 1% BSA (bovine serum albumin) was added to each well, and left at 37 ° C. for 2 hours. Again, 100 μl of rabbit-LTB antibody diluted 1: 5000 in 0.01 M PBS containing 0.5% BSA was added to each well, left at 37 ° C. for 2 hours, and washed four times with PBST. Each well was added 0.01 M PBS containing 0.5% BSA diluted 1: 0000 of goat anti-rabbit IgG conjugated with horseradish-derived peroxidase to react at 37 ° C. for 2 hours, and then washed four times with PBST buffer. It was. The plate was placed in 100 μl of each well with TMB substrate (Pharmingen) for 30 minutes at room temperature and room temperature to maximize the reaction rate, and the absorbance was measured at 405 nm using an ELISA reader.

The amount of LTB protein in each transgenic plant was measured three times and confirmed by the ratio to the total water soluble protein. 9 is a graph quantifying the recombinant LTB protein expressed in the transgenic plant, the recombinant LTB protein was expressed in 2-3% in the total water soluble protein.

8. LTB-GM1 binding assay

The ganglioside binding capacity of the recombinant LTB protein was measured.

Monosialoganglioside GM1 was diluted in bicarbonate buffer at pH 9.6 and placed in 100 μl in each well of a microtiter plate and coated at 4 ° C. overnight. As a control, each well was coated with 100 μl of BSA (3.0 μg ml ⁻¹ ). Plates were washed three times with PBST each and reacted with 1% BSA diluted with 0.01 M PBS to inhibit nonspecific reactions. The plate was washed three times with PBST, and then the total water-soluble protein isolated from the transformed or wild-type plants was added at various concentrations and reacted at 37 ° C for 2 hours. Since the primary antibody and secondary antibody reaction was carried out in the same manner as the quantification of LTB protein by ELISA.

10 is a graph showing the strong binding capacity of the recombinant LTB protein expressed from the transgenic plants of the present invention to GM1-ganglioside, it did not show the binding capacity for the negative control BSA. These results indicate that the plant-derived LTB subunit maintains its natural activity of cooperatively binding to GM1.

The transgenic plants expressing more recombinant LTB protein in the transgenic plants (No. 2), Nicotiana Tabacum / pMY-CH-LTB (Nicotiana tabacum / pMY-CH) to the Gene Bank of Life Science Research Institute located in Yuseong-gu, Daejeon, Korea -LTB), dated March 21, 2003, and received accession number KCTC 10447BP.

As mentioned above, the present invention introduced an E. coli enterotoxin B subunit, an animal disease-causing antigen into the chloroplast genome, and confirmed an expression system about 250-fold improvement compared to nuclear transformation. A plant expressing an animal disease-causing antigen can be dieted in an animal by the method of the present invention to form an animal's immune response simply and efficiently.

<110> YANG, Moon-Sik          KANG, Tae-Jin <120> HIGH EXPRESSION METHOD OF THE B SUBUNIT OF E. COLI HEAT-LABILE          ENTEROTOXIN IN THE CHLOROPLAST OF PLANTS <130> dpp20030302 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> sense primer for amplifying LTB <400> 1 ggggaattca ggaggttcag tcatggctcc ccagtctatt acagaac 47 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for amplifying LTB <400> 2 gggggtaccc tagttttcca tactgattgc 30 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 1F primer <400> 3 aaaacccgtc ctcagttcgg attgc 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 1R primer <400> 4 ccgcgttgtt tcatcaagcc ttacg 25 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 2F primer <400> 5 ctgtagaagt caccattgtt gtgc 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> 2223 primer <400> 6 tgactgccca cctgagagcg gaca 24 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 3F primer <400> 7 tcctcagttc ggattgcagg c 21 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 3R primer <400> 8 gggggtaccc tagttttcca tactgattgc 30 <210> 9 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> 4F primer <400> 9 ggggaattca ggaggttcag tcatggctcc ccagtctatt acagaac 47 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 4R primer <400> 10 tatgccatcc taaggtgctg c 21

Claims (12)

(a) chloroplast rRNA operon promoter (Prrn); (b) ribosomal binding sequences; (c) antibiotic resistance genes; (d) a polynucleotide comprising a nucleotide sequence encoding an animal disease causing antigen; And (e) 3'-untranslated region of chloroplast psbA gene An antigen expression cassette for plant chloroplast transformation comprising a. The antigen expression cassette of claim 1, wherein the animal disease-causing antigen is E. coli enterotoxin B subunit. A vector comprising the antigen expression cassette of claim 1. The vector of claim 3, wherein the vector is a pMY-CH-LTB vector. The transgenic plant of claim 1 or 2, wherein the antigen expression cassette is inserted by homologous recombination on the chloroplast genome. The plant of claim 5, wherein the plant is an animal edible plant. 6. The plant of claim 5, wherein the plant is a whole plant, plant tissue or plant cell. The method of claim 5, wherein the plant is Nikko tiahna other Vacuum / pMY-CH-LTB (Nicotiana tabacum / pMY-CH-LTB) (KCTC 10447BP) would plants. Animal disease-causing antigen protein produced from the transgenic plant of claim 5. A method of mass-producing an animal disease-causing antigen from a plant chloroplast by inserting the antigen expression cassette of claim 1 into the chloroplast genome of a plant. An anti-E. Coli enterotoxin vaccine composition comprising the transformed plant of claim 5 or the E. coli enterotoxin B subunit recombinant protein of claim 9. (a) preparing an expression cassette comprising a nucleotide comprising a nucleotide sequence encoding an animal disease-causing antigen on a chloroplast of a plant; (b) inserting said expression cassette onto the chloroplast genome of the plant to produce a transgenic plant; (c) culturing the transgenic plant to produce a recombinant antigenic protein; And (d) administering the produced recombinant antigen protein or a plant comprising the same to an animal to form an antibody against an animal disease-causing antigen Immunogenesis method comprising a.