KR102032509B1 - Manufacturing method of Chinese cabbage producing immunoglobulin polymer or immunoglobulin Fc region recombinant protein polymer - Google Patents

Abstract

The present invention relates to a method for producing a multimeric producing cabbage of a recombinant protein containing an immunoglobulin or an immunoglobulin Fc site, specifically, a first type by introducing a gene encoding a recombinant protein containing an immunoglobulin or an immunoglobulin Fc site Preparing the converted cabbage; Preparing a second transgenic cabbage by introducing a gene encoding the J chain; And a step of crossing the first transformed cabbage and the second transformed cabbage. The method relates to a method for producing multimeric produced cabbage of a recombinant protein comprising an immunoglobulin or immunoglobulin Fc site.
According to the present invention, it is possible to prepare a cabbage that efficiently produces an immunoglobulin-derived recombinant protein including an immunoglobulin or an Fc region. In particular, when the antibody gene for the cancer cell specific antigen is applied to the present invention, it is possible to produce cabbage that produces the antibody, and to prevent or treat cancer by simply ingesting cabbage without medical administration. have.

Description

Manufacturing method of Chinese cabbage producing immunoglobulin polymer or immunoglobulin Fc region recombinant protein polymer

The present invention relates to a method for producing a multimeric producing cabbage of a recombinant protein comprising an immunoglobulin or an immunoglobulin Fc site, specifically, a first type by introducing a gene encoding a recombinant protein containing an immunoglobulin or an immunoglobulin Fc site Preparing the converted cabbage; Preparing a second transgenic cabbage by introducing a gene encoding the J chain; And a step of crossing the first transformed cabbage with the second transformed cabbage. The method relates to a method for producing multimeric produced cabbage of a recombinant protein comprising an immunoglobulin or immunoglobulin Fc site.

The production of industrially useful recombinant proteins, for example diagnostic molecules, vaccines, therapeutic antibodies and the like, has until now been mainly used systems using microbial fermentation or animal cells. However, these systems have major problems in terms of cost, scalability, and stability, and there is a continuous demand for new systems that can be improved or replaced.

As an alternative to this, a plant-based protein expression system can be considered. There are still limitations in terms of low protein yield, difficulty in separation and purification process, and it is a problem in the production of pharmaceuticals, but mass production is low at low cost and low risk of contamination of pathogens that are fatal to humans. The advantage is that it is a very attractive system for the production of proteins for the treatment of human diseases. Another advantage is that if a protein such as a recombinant vaccine is expressed in the edible plant tissue, the effect of the vaccine can be expected simply by edible without a separate medical administration.

Since the expression mechanism of the protein is different depending on the type of plant, various studies have to be conducted for each plant in order to develop a successful system, but related studies are still insignificant.

Therefore, the present inventors have studied the protein expression system for the Chinese cabbage, which is a representative plant used for edible worldwide as well as in Korea, and in particular, to efficiently produce immunoglobulins that can be very useful for biological diagnosis or disease treatment. To develop a cabbage that can be.

Ilchan Song, Do Sun Kim, Mi Kyung Kim, Arshad Jamal, Kyung-A Hwang, and Kisung Ko. 2015.Comparison of Total Soluble Protein in Various Horticultural Crops and Evaluation of Its Quantification Methods. Hort. Environ. Biotechnol. 56 (1): 123-129. 2015.

Therefore, the main object of the present invention is to provide a method for producing cabbage that can efficiently produce proteins such as immunoglobulins.

According to one aspect of the invention, the present invention comprises the steps of preparing a first transgenic cabbage by introducing a gene encoding a recombinant protein comprising an immunoglobulin or immunoglobulin Fc site; Preparing a second transgenic cabbage by introducing a gene encoding the J chain; It provides a method for producing multimeric produced cabbage of recombinant protein comprising an immunoglobulin or immunoglobulin Fc site comprising the step of crossing the first transformed cabbage and the second transformed cabbage.

In the cabbage production method of the present invention, it is preferable that the first transformed cabbage is used as a mother and the second transformed cabbage is used as a parent.

In the cabbage production method of the present invention, the immunoglobulin is preferably IgM.

In the cabbage production method of the present invention, the immunoglobulin is preferably an antibody against a cancer cell specific antigen.

In the cabbage production method of the present invention, it is preferable that the gene encoding the J chain is a gene encoding a protein having a form in which a KDEL motif is linked to the J chain.

In the cabbage production method of the present invention, it is preferable to prepare the first transgenic cabbage so that the expression of the gene encoding the recombinant protein comprising the immunoglobulin or immunoglobulin Fc region is controlled by the 35S promoter and 35S terminator.

In the cabbage production method of the present invention, it is preferable to prepare a second transformed cabbage so that the expression of the gene encoding the J chain is controlled by the 35S promoter and 35S terminator.

According to the present invention can be produced cabbage to efficiently produce immunoglobulin-derived recombinant protein containing an immunoglobulin or Fc region (Fc region). In particular, when the antibody gene for the cancer cell specific antigen is applied to the present invention, it is possible to prepare a cabbage that produces the antibody, and can be expected to prevent or treat cancer by simply ingesting cabbage without medical administration. have.

Figure 1 shows the structure of the production vector (pH2-GA733-IgM) prepared to transform the gene encoding IgM for the GA733 antigen according to an embodiment of the present invention.
Figure 2 shows the structure of the production vector (pH2GW-Jchain) produced to transform the gene encoding the J chain (J-chain) according to an embodiment of the present invention.
Figure 3 shows the structure of the production vector (pH2GW-Jchain-KDEL) produced to transform the gene encoding the protein of the type KDEL motif is linked to the J chain (J-chain) according to an embodiment of the present invention It is shown.
Figure 4 shows the transformation process of the cabbage used in one embodiment of the present invention.
Figure 5 is a PCR result of confirming the gene introduced into the Agrobacterium culture photograph and Agrobacterium for cabbage transformation used in one embodiment of the present invention.
Figure 6 shows the process of selecting the Chinese cabbage transformant T ₁ seed in hygromycin medium according to an embodiment of the present invention.
Figure 7 shows the hybridization method of the Chinese cabbage transformant according to an embodiment of the present invention, and thus immunoglobulin multimeric molecules expected to be produced.
Figure 8 shows the results of obtaining the transformants T ₀ and T ₁ individuals through transformation in accordance with an embodiment of the present invention and PCR and immunoblot results of assaying the transgene.
9 is a photograph of a spring-treated transformant T ₁ subject according to one embodiment of the present invention.
Figure 10 shows the PCR assay results (A) and immunoblot assay results (B) of the T ₁ subject according to an embodiment of the present invention.
Figure 11 shows the PCR assay results (A) and immunoblot assay results (B) of the hybrids obtained through the crossing of the transgenic cabbage according to an embodiment of the present invention.
Figure 12 is a protein of hybridized cabbage (EpCAM-IgMFC (♀) x J-chain K (♂)) and non-hybridized cabbage (EpCAM-IgM Fc) obtained through the crossing of the transgenic cabbage according to an embodiment of the present invention Expression is compared by SDS-PAGE (left) and immunoblot (right). 2nd mAb, Goat Anti-human IgM Fc, HRP conjugated, 1: 5000; +, Positive control (Chinese cabbage GA733-IgM Fc W plant); Negative control (Chinese cabbage NT plant); F, Purification fraction; CT: Column through. Grind 200 g of cabbage and refine.

Cabbage production method of the present invention comprises the steps of preparing a first transgenic cabbage by introducing a gene encoding a recombinant protein comprising an immunoglobulin or immunoglobulin Fc site; Preparing a second transgenic cabbage by introducing a gene encoding the J chain; And crossing the first transformed cabbage and the second transformed cabbage.

In this case, a gene encoding IgG, IgA, IgM, IgD, or IgE may be used as a gene used to prepare the first transgenic cabbage, and also includes a recombinant protein such as a form including an Fc region and linking other enzymes. You can also use a gene that encodes. According to the invention it is preferred to use a gene encoding IgM. The variable region of the immunoglobulin is not particularly limited, but in order not to adversely affect the normal physiological activity of the Chinese cabbage, it is preferable to use the variable region acting only on antigens derived from animals, especially humans. In particular, using the antibody gene for cancer cell-specific antigen can be obtained cabbage having an anticancer effect.

In the present invention, the J chain is used to connect the immunoglobulin molecules produced in the cabbage or the recombinant protein molecules including the immunoglobulin Fc sites to form a multimer, so that they can be more stably maintained in the cabbage. According to the present invention, the expression of the J chain gene enables stable expression of the recombinant protein including the immunoglobulin or immunoglobulin Fc region. In addition, when a gene encoding a protein having a form linked to a KDEL motif in the J chain, a cabbage in which the recombinant protein including the immunoglobulin or immunoglobulin Fc region is more stably expressed can be obtained.

Introducing the gene into the cabbage in the present invention can be achieved by using the transformation method of conventional cabbage known in the art. For example, Agrobacterium mediated transformation can be used. In addition, for the stable introduction of the gene it is preferable to produce and use a recombinant vector configured to express the gene. Various promoters and terminators that operate in cabbage can be used to configure the gene to be expressed in cabbage, and in particular, it is desirable to ensure that the expression of each gene is regulated by 35S promoter and 35S terminator for stable expression of the genes. Do.

In the present invention, a method of crossing a first transgenic cabbage into which a gene encoding a recombinant protein including an immunoglobulin or an immunoglobulin Fc region is introduced and a second transgenic cabbage into which a gene encoding a J chain are introduced are used. Through this, both genes can be introduced to produce cabbage that can be expressed. According to the present invention, when mating with the first transgenic cabbage and the second transgenic cabbage as the parent, the recombinant protein containing the immunoglobulin or immunoglobulin Fc region is more stably expressed than the opposite case. Cabbage can be obtained.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Transformed Chinese Cabbage Preparation

1-1. Materials and methods

Chinese cabbage for transformation (Seoul cabbage, Syngenta): about 1000 ~ 2000 grains

Transgene: J chain KDEL, EpCAM-IgM, EpCAM-IgG, EpCAM-IgA

HPT2 gene primers and each transgene specific primers for PCR

Transformation material: 1/2 Murashige & Skoog selection and regeneration medium, Hygromycine, Agrobacterium transfected (strain LBA4404)

1-1-1. Recombinant Vector for Transformation

In order to transform the gene encoding IgM for GA733 antigen, a cell surface glycoprotein that is highly expressed in colorectal carcinoma cells of human, the IgM gene was cloned into the pH2GW7 vector (using the LR reaction) to construct the structure as shown in FIG. 1. The preparation vector (pH2-GA733-IgM) was produced. The base sequence of this recombinant vector is shown in SEQ ID NO: 1.

In SEQ ID NO: 1, LB (left border) is 239 ~ 265th sequence, Tnos (NOS terminator) is 337 ~ 618th sequence, Hyg-R (hygromycin resistance gene) is 806 ~ 1828th sequence, Pnos (NOS promoter) is 1838-2120 sequence, P35S (35S protomer) sequence 2154-3171, attR1 sequence 3209-3233, GA733_IgM (IgM gene for GA733 antigen) sequence 3234-5132, attR2 sequence 5139-5163, T35S (35S terminator) is the 5174 ~ 5398 sequence, RB (right border) is the 5502 ~ 5527 sequence.

In order to transform the gene encoding the J-chain (J-chain), a recombinant vector (pH2GW-Jchain) having a structure as shown in Figure 2 was produced by cloning the J-chain gene in the pH2GW7 vector (using the LR reaction). The base sequence of this recombinant vector is shown in SEQ ID NO: 2.

In SEQ ID NO: 2, LB (left border) is 194-220, Tnos (NOS terminator) is 292-573, Hyg-R (hygromycin resistance gene) is 761-1786, and Pnos (NOS promoter) is 1796-2078 sequence, P35S (35S protomer) is 2112-3129, attB1 is 3167-3191, J-chain (J chain gene) is 3192-3722, attB2 is 3723-3747, T35S (35S terminator) is the 3758 ~ 3982th sequence, RB (right border) is the 4086 ~ 4111th sequence.

Also, in order to transform a gene encoding a protein having a KDEL motif linked to a J-chain, a J2 + KDEL gene was cloned into the pH2GW7 vector (using an LR reaction) to obtain a recombinant vector having a structure as shown in FIG. 3. (pH2GW-Jchain-KDEL) was produced. The base sequence of this recombinant vector is shown in SEQ ID NO: 3.

In SEQ ID NO: 3, LB (left border) is 194-220, Tnos (NOS terminator) is 292-573, Hyg-R (hygromycin resistance gene) is 761-1786, and Pnos (NOS promoter) is 1796 ~ 2078th sequence, P35S (35S protomer) is 2112 ~ 3129th sequence, attB1 is 3167 ~ 3191th sequence, Jchain-K (gene chain with KDEL motif in J chain) is 3192 ~ 3734th sequence, attB2 is 3735 ~ The 3759th sequence, T35S (35S terminator) is 3770-3994, and the RB (right border) is 4098-4123th sequence.

KDEL motif is an amino acid sequence consisting of Lys-Asp-Glu-Leu and is known to play a role in preventing the secretion of proteins from endoplasmic reticulum (ER, endoplasmic reticulum). This was applied to investigate whether it would be helpful to produce J chain protein in Chinese cabbage.

1-1-2. Chinese cabbage transformation

Cabbage was transformed using the recombinant vector by the following method.

1-1-2-1. badge

The medium of Chinese cabbage for transformation was used in Table 1 and the medium of Agrobacterium strains in Table 2.

1-1-2-2. Inoculation explant collection and pretreatment

1) Peel off the germinated, seedlings to light for 3-4 hours (hardening process)

2) Grab the roots of the seedlings with tweezers and take out one or two and put them on a paper petri dish.

3) Cut the chlorophyll invisible (bottom of cotyledon) in the length of 0.7 ~ 1cm and cut it horizontally on the pretreatment medium (PM) and cut out the medium a little in the area to be damaged. cut)

4) Wrapped in foil and incubated for 2 days at 23 ℃

Changed pH from pre-culture medium to pH 5.8

1-1-2-3. Strain Culture and Inoculation

1) Strain culture (YEP medium + antibiotics) 100µl culture of strain stock (OD value: 0.8) on 50 days before the date of inoculation (about 5 pm)

2) Shake culture at 28 ℃ overnight at 220rpm

3) Incubate until the OD value grows to 0.8 on the day of inoculation

* Strains are transferred to a micro cuvette in a clean bench, and the entrance of the Erlenmeyer flask must be disinfected.

4) Transfer the strain with OD value of 0.8 to 50 ml tube and centrifuge for 20 min at 4 ℃, 4000rpm.

5) Discard the supernatant and place the pellet in 50 ml of MS medium (MS + glucose 36g / L, pH 5.2).

6) Shake incubation at 23 ° C. at 150 rpm for 1 hour

7) Transfer the pretreated hypocotyls to a high plate or 50ml tube and slowly inoculate with the strain

8) Shake for 10 minutes at 80rpm

9) Wipe the inoculated hypocotyl on the sterilized filter paper and transfer to the coculture medium (CM).

10) Wrapped in foil and incubated for 2 days at 23 ℃

-Reduced inoculation time by 10 minutes compared to the existing protocol

The inoculated hypocotyls were transferred to co-culture medium (pH 5.2) previously transferred to pre-culture medium.

1-1-2-4. Transformant Selection

1) Two days after inoculation, cancer cultivation

2) Transfer lower hypocotyls to selection medium (SI) and grow light (within 2 weeks after vaccination)

3) Subculture at intervals of 2 to 3 weeks

4) When callus is made at both ends, it is passaged as it is without cutting.

5) When the shoot can be seen with the naked eye, it will be moved to a high plate SI badge to secure a space for growth.

6) Subculture at intervals of 2 to 3 weeks (must be well shaped)

7) Once complete leaf formation is transplanted into RIM

8) After rooting, soak in water to separate the medium and purify (1 ~ 2 days)

9) Spring treatment at 4 ℃ for more than 6 weeks after purifying

In the existing protocol, which was transplanted to selection medium immediately after cancer culture, transplanted to selection medium after about 1 week of light culture after 2 days of cancer culture.

-When the shoot is visually confirmed, the transplantation to the regeneration medium is sufficiently transferred from the selection medium to the rooting medium without passing through the regeneration medium.

-If you look at the incidence of callus, 880 ~ 979 occurrences per 1000 hypocotyls occur, showing a maximum rate of 98%, shoot shoot rate is about 3%

1-1-2-5. Chinese cabbage transformant assay

Hygromycine 15 mg / ml antibiotic medium (1/2 Murashige & Skoog medium) was selected and seeded with about 20 seeds per medium.

Seed disinfection was disinfected using 70% EtOH and 4% sodium hypochlorite and rinsed again with distilled water three or more times.

About 5 to 10 individuals were selected per plate, and the selected individuals were transplanted into a commercial soil as the main leaves and roots developed (see FIG. 6).

PCR analysis was performed by HPT2 (hygromycin resistance gene) and transgene assay. Each transgene specific primer was prepared and used, and Takara rTaq (Takara Shuzo Co., Ltd., Shiga, Japan) was used. Positive control was performed using transformed Agrobacterium.

Pornographic through treatment (60 day treatment pornographic) was attempted to induce the mating T ₁ blooming and complex protein expression (see Fig. 7).

Mating combination

1.IgA (parent) x J chain KDEL (parent), J chain KDEL (parent) x IgA (parent)

2.IgM (parent) x J chain KDEL (parent), J chain KDEL (parent) x IgM (parent)

PCR assay of cabbage plants (T ₁ XT ₁ ) resulting from the hybridization was performed and protein expression was confirmed. Hybridization was expected to induce the production of large vaccine proteins with greater vaccine effectiveness in cabbage plants.

1-2. result

As shown in Figure 8 to secure the T ₀ and T ₁ through the Chinese cabbage transformation.

Homozygous T ₁ seed was obtained through self fertilization of T ₀ transgenic plants (see FIG. 9).

Only the individuals whose transgene amplification was confirmed through PCR assay of each transformant T ₁ into which J chain KDEL and IgM were introduced were selected, and the expression of two target proteins was confirmed in the selected individuals (see FIG. 10).

In IgM-introduced individuals, both T ₀ and T ₁ generations confirmed protein expression, and in J-introduced individuals, the protein size was small, so it was difficult to confirm by immunoblot. See FIG. 10).

Immunoblotting experiments were conducted under two antibody conditions. For IgM, the positive control group was Chinese cabbage EpCAM-IgM Fc, and the first condition was obtained using Goat Anti-human IgM Fc μ-chain, HRP conjugated, 1: 5000 secondary antibody. Mouse anti-human EpCAM (1: 2500) primary antibody, Goat Anti-mouse IgG 2a heavy chain, HRP conjugated (1: 5000) secondary antibody were used (see FIG. 10).

PCR assay and protein expression were also confirmed in hybrid plants in the same manner (crossing combination: IgM (parent) x J chain KDEL (parent), J chain KDEL (parent) x IgM (parent)] (see FIG. 11).

Target protein expression was analyzed by SDS-PAGE (Experimental conditions: 10% Acrylamide gel, 20 μl aliquots) and protein immunoblot experiments.

As a result, it was confirmed that IgM protein expression was more stably expressed in hybridized plants of IgM and J chain KDEL (see FIG. 11).

It was confirmed that there is a difference in the expression level of the complex protein by changing the combination of the parents, it was confirmed that more stable expression is made when the transformant is introduced to the IgM introduced mother (see Figure 11).

The target protein was purified from hybridized plants and the purified protein was confirmed by immunoblot (see FIG. 12).

In the case of IgM without KDEL, the target protein was not confirmed on SDS-PAGE, but IgM X J-chain KDEL was confirmed on SDS-PAGE, and the immunoblotting result showed a large difference in expression rate when compared with the same quantitative control group. (See FIG. 12).

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Manufacturing method of Chinese cabbage producing immunoglobulin          polymer or immunoglobulin Fc region recombinant protein polymer <130> PA-D17331 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 11798 <212> DNA <213> Artificial Sequence <220> PH 2 -GA733-IgM <400> 1 tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc 60 atccgtgttt caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga 120 gcaaagtctg ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc 180 ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg 240 gcaggatata ttgtggtgta aacaaattga cgcttagaca acttaataac acattgcgga 300 cgtttttaat gtactgaatt aacgccgaat tgaattatca gcttgcatgc cggtcgatct 360 agtaacatag atgacaccgc gcgcgataat ttatcctagt ttgcgcgcta tattttgttt 420 tctatcgcgt attaaatgta taattgcggg actctaatca taaaaaccca tctcataaat 480 aacgtcatgc attacatgtt aattattaca tgcttaacgt aattcaacag aaattatatg 540 ataatcatcg caagaccggc aacaggattc aatcttaaga aactttattg ccaaatgttt 600 gaacgatctg cttgactcta gggaattaat tcctgaatca ctgcgaccgg ccctcccgcg 660 acccagccga gcgagcttag cgaactgtgg acgagaactg tgccaccaag cgtaaggccg 720 ttctctcgca tttgccttgc taggctcgcg cgagttgctg gctgaggcgt tctcgaaatc 780 agctcttgtt cggtcggcat ctactctatt cctttgccct cggacgagtg ctggggcgtc 840 ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc gcgcttctgc 900 gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg tcgcatcgac 960 cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa 1020 gaccaatgcg gagcatatac gcccggagcc ttggcgatcc tgcaagctcc ggatgcctcc 1080 gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag aagaagatgt 1140 tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg accgctgtta 1200 tgcggccatt gtccgtcagg acattgttgg agccgaatcc gcgtgcacga gtgccggact 1260 tcggggcagt cctcgcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga 1320 cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga 1380 aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg 1440 tctggctaag atcggccgca gcgatagcat ccatagcctc cgcgaccggc tgaagaacag 1500 cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc 1560 aataggtcag gctctcgctg aactccccaa tgtcaagcac ttccggaatc gggagcgcgg 1620 ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta 1680 cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct 1740 ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag 1800 acgtcgcggt gagttcaggc tttttcatag gggggattcg agttgagagt gaatatgaga 1860 ctctaattgg ataccgaggg gaatttatgg aacgtcagtg gagcattttt gacaagaaat 1920 atttgctagc tgatagtgac cttaggcgac ttttgaacgc gcaataatgg tttctgacgt 1980 atgtgcttag ctcattaaac tccagaaacc cgcggctcag tggctccttc aacgttgcgg 2040 ttctgtcagt tccaaacgta aaacggcttg tcccgcgtca tcggcggggg tcataacgtg 2100 actcccttaa ttctccgctc atgatcgata attcgagctc tcccatatgg tcgactagag 2160 ccaagctgat ctcctttgcc ccggagatca ccatggacga ctttctctat ctctacgatc 2220 taggaagaaa gttcgacgga gaaggtgacg ataccatgtt caccaccgat aatgagaaga 2280 ttagcctctt caatttcaga aagaatgctg acccacagat ggttagagag gcctacgcgg 2340 caggtctcat caagacgatc tacccgagta ataatctcca ggagatcaaa taccttccca 2400 agaaggttaa agatgcagtc aaaagattca ggactaactg catcaagaac acagagaaag 2460 atatatttct caagatcaga agtactattc cagtatggac gattcaaggc ttgcttcata 2520 aaccaaggca agtaatagag attggagtct ctaagaaagt agttcctact gaatcaaagg 2580 ccatggagtc aaaaattcag atcgaggatc taacagaact cgccgtgaag actggcgaac 2640 agttcataca gagtctttta cgactcaatg acaagaagaa aatcttcgtc aacatggtgg 2700 agcacgacac tctcgtctac tccaagaata tcaaagatac agtctcagaa gaccaaaggg 2760 ctattgagac ttttcaacaa agggtaatat cgggaaacct cctcggattc cattgcccag 2820 ctatctgtca cttcatcaaa aggacagtag aaaaggaagg tggcacctac aaatgccatc 2880 attgcgataa aggaaaggct atcgttcaag atgcccctgc cgacagtggt cccaaagatg 2940 gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 3000 aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt 3060 cgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggact ccggtatttt 3120 tacaacaatt accacaacaa aacaaacaac aaacaacatt acaatttact attctagtcg 3180 acctgcaggc ggccgcacta gtgatatcac aagtttgtac aaaaaagcag gctatggcta 3240 ctcaacgaag ggcaaaccct agctctctcc atctaattac tgtattctct ctgctagctg 3300 ctgtcgtctc cgctgaggtc gacacggcca cttttgccgc agctcaggaa gaatgtgtct 3360 gtgaaaacta caagctggcc gtaaactgct ttgtgaataa taatcgtcaa tgccagtgta 3420 cttcagttgg tgcacaaaat actgtcattt gctcaaagct ggctgccaaa tgtttggtga 3480 tgaaggcaga aatgaatggc tcaaaacttg ggagaagagc aaaacctgaa ggggccctcc 3540 agaacaatga tgggctttat gatcctgact gcgatgagag cgggctcttt aaggccaagc 3600 agtgcaacgg cacctccacg tgctggtgtg tgaacactgc tggggtcaga agaacagaca 3660 aggacactga aataacctgc tctgagcgag tgagaaccta ctggatcatc attgaactaa 3720 aacacaaagc aagagaaaaa ccttatgata gtaaaagttt gcggactgca cttcagaagg 3780 agatcacaac gcgttatcaa ctggatccaa aatttatcac gagtattttg tatgagaata 3840 atgttatcac tattgatctg gttcaaaatt cttctcaaaa aactcagaat gatgtggaca 3900 tagctgatgt ggcttattat tttgaaaaag atgttaaagg tgaatccttg tttcattcta 3960 agaaaatgga cctgacagta aatggggaac aactggatct ggatcctggt caaactttaa 4020 tttattatgt tgatgaaaaa gcacctgaat tctcaatgca gggtctaaaa ctcgagcttc 4080 cagtgattgc tgagctgcct cccaaagtga gcgtcttcgt cccaccccgc gacggcttct 4140 tcggcaaccc ccgcaagtcc aagctcatct gccaggccac gggtttcagt ccccggcaga 4200 ttcaggtgtc ctggctgcgc gaggggaagc aggtggggtc tggcgtcacc acggaccagg 4260 tgcaggctga ggccaaagag tctgggccca cgacctacaa ggtgaccagc acactgacca 4320 tcaaagagag cgactggctc ggccagagca tgttcacctg ccgcgtggat cacaggggcc 4380 tgaccttcca gcagaatgcg tcctccatgt gtgtccccga tcaagacata gccatccggg 4440 tcttcgccat ccccccatcc tttgccagca tcttcctcac caagtccacc aagttgacct 4500 gcctggtcac agacctgacc acctatgaca gcgtgaccat ctcctggacc cgccagaatg 4560 gcgaagctgt gaaaacccac accaacatct ccgagagcca ccccaatgcc actttcagcg 4620 ccgtgggtga ggccagcatc tgcgaggatg actggaactc cggggagagg ttcacgtgca 4680 ccgtgaccca cacagacctg ccctcgccac tgaagcagac catctcccgg cccaaggggg 4740 tggccctgca caggcccgat gtctacttgc tgccaccagc ccgggagcag ctgaacctgc 4800 gggagtcggc caccatcacg tgcctggtga cgggcttctc tcccgcggac gtcttcgtgc 4860 agtggatgca gagggggcag cccttgtccc cggagaagta tgtgaccagc gccccaatgc 4920 ctgagcccca ggccccaggc cggtacttcg cccacagcat cctgaccgtg tccgaagagg 4980 aatggaacac gggggagacc tacacctgcg tggtggccca tgaggccctg cccaacaggg 5040 tcaccgagag gaccgtggac aagtccaccg gtaaacccac cctgtacaac gtgtccctgg 5100 tcatgtccga cacagctggc acctgctact gactcgagac ccagctttct tgtacaaagt 5160 ggtgatatcc cgcggccatg ctagagtccg caaaaatcac cagtctctct ctacaaatct 5220 atctctctct atttttctcc agaataatgt gtgagtagtt cccagataag ggaattaggg 5280 ttcttatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat gtatttgtat 5340 ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc agtgacctgc 5400 aggcatgcga cgtcgggccc aagcttagct tgagcttgga tcagattgtc gtttcccgcc 5460 ttcagtttaa actatcagtg tttgacagga tatattggcg ggtaaaccta agagaaaaga 5520 gcgtttatta gaataatcgg atatttaaaa gggcgtgaaa aggtttatcc gttcgtccat 5580 ttgtatgtgc atgccaacca cagggttccc ctcgggatca aagtacttta aagtacttta 5640 aagtactgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc 5700 cgtcttctga aaacgacatg tcgcacaagt cctaagttac gcgacaggct gccgccctgc 5760 ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact 5820 agaaccggag acattacgcc atgaacaaga gcgccgccgc tggcctgctg ggctatgccc 5880 gcgtcagcac cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct 5940 gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg gagctggcca 6000 ggatgcttga ccacctacgc cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg 6060 cccgcagcac ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc 6120 tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc ggccggccgc atggtgttga 6180 ccgtgttcgc cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg 6240 ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc ctcaccccgg 6300 cacagatcgc gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg 6360 ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag 6420 tgacgcccac cgaggccagg cggcgcggtg ccttccgtga ggacgcattg accgaggccg 6480 acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga 6540 cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg 6600 gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg 6660 tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg 6720 ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc 6780 gtatatgatg cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct 6840 gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc 6900 ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc 6960 gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg 7020 attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc 7080 caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg 7140 cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc 7200 attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc 7260 acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag 7320 tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt 7380 gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa 7440 tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa 7500 gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca 7560 cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga 7620 tgtacgcggt acgccaaggc aagaccatta ccgagctgct atctgaatac atcgcgcagc 7680 taccagagta aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc 7740 ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga 7800 acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga 7860 acccccaagc ccgaggaatc ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg 7920 cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca 7980 acgcatcgag gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg 8040 caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg 8100 cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg 8160 cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt 8220 gatccgctac gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc 8280 cagtgtgtgg gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa 8340 ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga 8400 cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac 8460 ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg 8520 ccgcctggtg acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag 8580 cgaaaccggg cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat 8640 cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc 8700 cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag 8760 atggttgttc aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg 8820 tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga 8880 ggcggggcag gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc 8940 atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa 9000 aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat 9060 tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat 9120 gtaagtgact gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact 9180 tattaaaact cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga 9240 agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg 9300 tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc 9360 agggcgcgga caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct 9420 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg 9480 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 9540 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 9600 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 9660 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 9720 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 9780 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 9840 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 9900 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 9960 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 10020 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 10080 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 10140 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 10200 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 10260 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 10320 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 10380 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 10440 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 10500 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc atgatatatc 10560 tcccaatttg tgtagggctt attatgcacg cttaaaaata ataaaagcag acttgacctg 10620 atagtttggc tgtgagcaat tatgtgctta gtgcatctaa tcgcttgagt taacgccggc 10680 gaagcggcgt cggcttgaac gaatttctag ctagacatta tttgccgact accttggtga 10740 tctcgccttt cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag gccaagcgat 10800 cttcttcttg tccaagataa gcctgtctag cttcaagtat gacgggctga tactgggccg 10860 gcaggcgctc cattgcccag tcggcagcga catccttcgg cgcgattttg ccggttactg 10920 cgctgtacca aatgcgggac aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg 10980 gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa 11040 ccggatcaaa gagttcctcc gccgctggac ctaccaaggc aacgctatgt tctcttgctt 11100 ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa 11160 tgtcattgcg ctgccattct ccaaattgca gttcgcgctt agctggataa cgccacggaa 11220 tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg gagaatctcg ctctctccag 11280 gggaagccga agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc 11340 ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg ccatccactg 11400 cggagccgta caaatgtacg gccagcaacg tcggttcgag atggcgctcg atgacgccaa 11460 ctacctctga tagttgagtc gatacttcgg cgatcaccgc ttcccccatg atgtttaact 11520 ttgttttagg gcgactgccc tgctgcgtaa catcgttgct gctccataac atcaaacatc 11580 gacccacggc gtaacgcgct tgctgcttgg atgcccgagg catagactgt accccaaaaa 11640 aacatgtcat aacaagaagc catgaaaacc gccactgcgc cgttaccacc gctgcgttcg 11700 gtcaaggttc tggaccagtt gcgtgacggc agttacgcta cttgcattac agcttacgaa 11760 ccgaacgagg cttatgtcca ctgggttcgt gcccgaat 11798 <210> 2 <211> 4193 <212> DNA <213> Artificial Sequence <220> <223> pH2GW-Jchain <400> 2 accctccgcg agatcatccg tgtttcaaac ccggcagctt agttgccgtt cttccgaata 60 gcatcggtaa catgagcaaa gtctgccgcc ttacaacggc tctcccgctg acgccgtccc 120 ggactgatgg gctgcctgta tcgagtggtg attttgtgcc gagctgccgg tcggggagct 180 gttggctggc tggtggcagg atatattgtg gtgtaaacaa attgacgctt agacaactta 240 ataacacatt gcggacgttt ttaatgtact gaattaacgc cgaattgaat tatcagcttg 300 catgccggtc gatctagtaa catagatgac accgcgcgcg ataatttatc ctagtttgcg 360 cgctatattt tgttttctat cgcgtattaa atgtataatt gcgggactct aatcataaaa 420 acccatctca taaataacgt catgcattac atgttaatta ttacatgctt aacgtaattc 480 aacagaaatt atatgataat catcgcaaga ccggcaacag gattcaatct taagaaactt 540 tattgccaaa tgtttgaacg atctgcttga ctctagggaa ttaattcctg aatcactgcg 600 accggccctc ccgcgaccca gccgagcgag cttagcgaac tgtggacgag aactgtgcca 660 ccaagcgtaa ggccgttctc tcgcatttgc cttgctaggc tcgcgcgagt tgctggctga 720 ggcgttctcg aaatcagctc ttgttcggtc ggcatctact ctattccttt gccctcggac 780 gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga 840 cggccgcgct tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga 900 ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct 960 gatagagttg gtcaagacca atgcggagca tatacgcccg gagccttggc gatcctgcaa 1020 gctccggatg cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc 1080 tccagaagaa gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt 1140 caatgaccgc tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt 1200 gcacgaggtg ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct 1260 gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat 1320 cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga 1380 atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatagcatcc atagcctccg 1440 cgaccggctg aagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct 1500 gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa ctccccaatg tcaagcactt 1560 ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac 1620 catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag 1680 cacgagattc ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga 1740 tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttcataggg gggattcgag 1800 ttgagagtga atatgagact ctaattggat accgagggga atttatggaa cgtcagtgga 1860 gcatttttga caagaaatat ttgctagctg atagtgacct taggcgactt ttgaacgcgc 1920 aataatggtt tctgacgtat gtgcttagct cattaaactc cagaaacccg cggctcagtg 1980 gctccttcaa cgttgcggtt ctgtcagttc caaacgtaaa acggcttgtc ccgcgtcatc 2040 ggcgggggtc ataacgtgac tcccttaatt ctccgctcat gatcgataat tcgagctctc 2100 ccatatggtc gactagagcc aagctgatct cctttgcccc ggagatcacc atggacgact 2160 ttctctatct ctacgatcta ggaagaaagt tcgacggaga aggtgacgat accatgttca 2220 ccaccgataa tgagaagatt agcctcttca atttcagaaa gaatgctgac ccacagatgg 2280 ttagagaggc ctacgcggca ggtctcatca agacgatcta cccgagtaat aatctccagg 2340 agatcaaata ccttcccaag aaggttaaag atgcagtcaa aagattcagg actaactgca 2400 tcaagaacac agagaaagat atatttctca agatcagaag tactattcca gtatggacga 2460 ttcaaggctt gcttcataaa ccaaggcaag taatagagat tggagtctct aagaaagtag 2520 ttcctactga atcaaaggcc atggagtcaa aaattcagat cgaggatcta acagaactcg 2580 ccgtgaagac tggcgaacag ttcatacaga gtcttttacg actcaatgac aagaagaaaa 2640 tcttcgtcaa catggtggag cacgacactc tcgtctactc caagaatatc aaagatacag 2700 tctcagaaga ccaaagggct attgagactt ttcaacaaag ggtaatatcg ggaaacctcc 2760 tcggattcca ttgcccagct atctgtcact tcatcaaaag gacagtagaa aaggaaggtg 2820 gcacctacaa atgccatcat tgcgataaag gaaaggctat cgttcaagat gcccctgccg 2880 acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc 2940 caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg 3000 cacaatccca ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg 3060 agaggactcc ggtattttta caacaattac cacaacaaaa caaacaacaa acaacattac 3120 aatttactat tctagtcgac ctgcaggcgg ccgcactagt gatatcacaa gtttgtacaa 3180 aaaagcaggc tatggccaag aaccatttgc ttttctgggg agtcctggcg gtttttatta 3240 aggctgttca tgtgaaaggt tcaagagatt cttctgcctc agcctcccga gtagctggga 3300 ttacagccca agaagatgaa aggattgttc ttgttgacaa caaatgtaag tgtgcccgga 3360 ttacttccag gatcatccgt tcttccgaag atcctaatga ggacattgtg gagagaaaca 3420 tccgaattat tgttcctctg aacaacaggg agaatatctc tgatcccacc tcaccattga 3480 gaaccagatt tgtgtaccat ttgtctgacc tctgtaaaaa atgtgatcct acagaagtgg 3540 agctggataa tcagatagtt actgctaccc agagcaatat ctgtgatgaa gacagtgcta 3600 cagagacctg ctacacttat gacagaaaca agtgctacac agctgtggtc ccactcgtat 3660 atggtggtga gaccaaaatg gtggaaacag ccttaacccc agatgcctgc tatcctgact 3720 gaacccagct ttcttgtaca aagtggtgat atcccgcggc catgctagag tccgcaaaaa 3780 tcaccagtct ctctctacaa atctatctct ctctattttt ctccagaata atgtgtgagt 3840 agttcccaga taagggaatt agggttctta tagggtttcg ctcatgtgtt gagcatataa 3900 gaaaccctta gtatgtattt gtatttgtaa aatacttcta tcaataaaat ttctaattcc 3960 taaaaccaaa atccagtgac ctgcaggcat gcgacgtcgg gcccaagctt agcttgagct 4020 tggatcagat tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt 4080 ggcgggtaaa cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt 4140 gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca accacagggt tcc 4193 <210> 3 <211> 4205 <212> DNA <213> Artificial Sequence <220> <223> pH 2 GW-Jchain-K <400> 3 accctccgcg agatcatccg tgtttcaaac ccggcagctt agttgccgtt cttccgaata 60 gcatcggtaa catgagcaaa gtctgccgcc ttacaacggc tctcccgctg acgccgtccc 120 ggactgatgg gctgcctgta tcgagtggtg attttgtgcc gagctgccgg tcggggagct 180 gttggctggc tggtggcagg atatattgtg gtgtaaacaa attgacgctt agacaactta 240 ataacacatt gcggacgttt ttaatgtact gaattaacgc cgaattgaat tatcagcttg 300 catgccggtc gatctagtaa catagatgac accgcgcgcg ataatttatc ctagtttgcg 360 cgctatattt tgttttctat cgcgtattaa atgtataatt gcgggactct aatcataaaa 420 acccatctca taaataacgt catgcattac atgttaatta ttacatgctt aacgtaattc 480 aacagaaatt atatgataat catcgcaaga ccggcaacag gattcaatct taagaaactt 540 tattgccaaa tgtttgaacg atctgcttga ctctagggaa ttaattcctg aatcactgcg 600 accggccctc ccgcgaccca gccgagcgag cttagcgaac tgtggacgag aactgtgcca 660 ccaagcgtaa ggccgttctc tcgcatttgc cttgctaggc tcgcgcgagt tgctggctga 720 ggcgttctcg aaatcagctc ttgttcggtc ggcatctact ctattccttt gccctcggac 780 gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga 840 cggccgcgct tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga 900 ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct 960 gatagagttg gtcaagacca atgcggagca tatacgcccg gagccttggc gatcctgcaa 1020 gctccggatg cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc 1080 tccagaagaa gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt 1140 caatgaccgc tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt 1200 gcacgaggtg ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct 1260 gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat 1320 cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga 1380 atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatagcatcc atagcctccg 1440 cgaccggctg aagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct 1500 gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa ctccccaatg tcaagcactt 1560 ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac 1620 catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag 1680 cacgagattc ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga 1740 tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttcataggg gggattcgag 1800 ttgagagtga atatgagact ctaattggat accgagggga atttatggaa cgtcagtgga 1860 gcatttttga caagaaatat ttgctagctg atagtgacct taggcgactt ttgaacgcgc 1920 aataatggtt tctgacgtat gtgcttagct cattaaactc cagaaacccg cggctcagtg 1980 gctccttcaa cgttgcggtt ctgtcagttc caaacgtaaa acggcttgtc ccgcgtcatc 2040 ggcgggggtc ataacgtgac tcccttaatt ctccgctcat gatcgataat tcgagctctc 2100 ccatatggtc gactagagcc aagctgatct cctttgcccc ggagatcacc atggacgact 2160 ttctctatct ctacgatcta ggaagaaagt tcgacggaga aggtgacgat accatgttca 2220 ccaccgataa tgagaagatt agcctcttca atttcagaaa gaatgctgac ccacagatgg 2280 ttagagaggc ctacgcggca ggtctcatca agacgatcta cccgagtaat aatctccagg 2340 agatcaaata ccttcccaag aaggttaaag atgcagtcaa aagattcagg actaactgca 2400 tcaagaacac agagaaagat atatttctca agatcagaag tactattcca gtatggacga 2460 ttcaaggctt gcttcataaa ccaaggcaag taatagagat tggagtctct aagaaagtag 2520 ttcctactga atcaaaggcc atggagtcaa aaattcagat cgaggatcta acagaactcg 2580 ccgtgaagac tggcgaacag ttcatacaga gtcttttacg actcaatgac aagaagaaaa 2640 tcttcgtcaa catggtggag cacgacactc tcgtctactc caagaatatc aaagatacag 2700 tctcagaaga ccaaagggct attgagactt ttcaacaaag ggtaatatcg ggaaacctcc 2760 tcggattcca ttgcccagct atctgtcact tcatcaaaag gacagtagaa aaggaaggtg 2820 gcacctacaa atgccatcat tgcgataaag gaaaggctat cgttcaagat gcccctgccg 2880 acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc 2940 caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg 3000 cacaatccca ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg 3060 agaggactcc ggtattttta caacaattac cacaacaaaa caaacaacaa acaacattac 3120 aatttactat tctagtcgac ctgcaggcgg ccgcactagt gatatcacaa gtttgtacaa 3180 aaaagcaggc tatggccaag aaccatttgc ttttctgggg agtcctggcg gtttttatta 3240 aggctgttca tgtgaaaggt tcaagagatt cttctgcctc agcctcccga gtagctggga 3300 ttacagccca agaagatgaa aggattgttc ttgttgacaa caaatgtaag tgtgcccgga 3360 ttacttccag gatcatccgt tcttccgaag atcctaatga ggacattgtg gagagaaaca 3420 tccgaattat tgttcctctg aacaacaggg agaatatctc tgatcccacc tcaccattga 3480 gaaccagatt tgtgtaccat ttgtctgacc tctgtaaaaa atgtgatcct acagaagtgg 3540 agctggataa tcagatagtt actgctaccc agagcaatat ctgtgatgaa gacagtgcta 3600 cagagacctg ctacacttat gacagaaaca agtgctacac agctgtggtc ccactcgtat 3660 atggtggtga gaccaaaatg gtggaaacag ccttaacccc agatgcctgc tatcctgaca 3720 aagatgaact ctgaacccag ctttcttgta caaagtggtg atatcccgcg gccatgctag 3780 agtccgcaaa aatcaccagt ctctctctac aaatctatct ctctctattt ttctccagaa 3840 taatgtgtga gtagttccca gataagggaa ttagggttct tatagggttt cgctcatgtg 3900 ttgagcatat aagaaaccct tagtatgtat ttgtatttgt aaaatacttc tatcaataaa 3960 atttctaatt cctaaaacca aaatccagtg acctgcaggc atgcgacgtc gggcccaagc 4020 ttagcttgag cttggatcag attgtcgttt cccgccttca gtttaaacta tcagtgtttg 4080 acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat aatcggatat 4140 ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg 4200 gttcc 4205

Claims (7)

Preparing a first transgenic cabbage by introducing a gene encoding a recombinant protein comprising an immunoglobulin or an immunoglobulin Fc site;
Preparing a second transgenic cabbage by introducing a gene encoding the J chain; And
And crossing the first transformed cabbage and the second transformed cabbage.
The first transformed Chinese cabbage as a mother, and the second transformed Chinese cabbage as a parent, characterized in that the mating
A method for producing a multimeric producing cabbage of a recombinant protein comprising an immunoglobulin or an immunoglobulin Fc region.
delete The method of claim 1,
The immunoglobulin is IgM, characterized in that the immunoglobulin or immunoglobulin Fc region containing a multi-protein production cabbage production method comprising the Fc region. The method of claim 1,
The immunoglobulin is an antibody against a cancer cell specific antigen, characterized in that the immunoglobulin or immunoglobulin Fc region containing a recombinant protein multi-protein production cabbage production method. The method of claim 1,
The gene encoding the J chain is a multimeric production cabbage production method comprising a recombinant protein comprising an immunoglobulin or immunoglobulin Fc region, characterized in that the gene encoding a protein of the form KDEL motif is linked to the J chain. The method of claim 1,
The immunoglobulin or immunoglobulin Fc region is characterized in that the first transgenic cabbage is prepared such that the expression of the gene encoding the recombinant protein including the immunoglobulin or immunoglobulin Fc region is controlled by the 35S promoter and the 35S terminator. Method for producing multimeric produced cabbage of recombinant protein. The method of claim 1,
Method for producing a multimeric produced cabbage of recombinant protein comprising an immunoglobulin or immunoglobulin Fc site, characterized in that the second transgenic cabbage is prepared so that the expression of the gene encoding the J chain is regulated by the 35S promoter and 35S terminator .

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