KR20150117645A - Method for producing recombinant human basic fibroblast growth factor rice seeds - Google Patents

Abstract

The present invention relates to a rice genetic codon-optimized OsrbFGF Gene, an associated vector, and a method for producing transformed rice seeds containing OsrbFGF using this vector, and a method for isolating and purifying OsrbFGF . Transgenic rice seeds containing OsrbFGF are prepared using OsrbFGF expression vectors specifically expressed in rice oil cells, and OsrbFGF is isolated and purified from rice seeds. The purity of OsrbFGF obtained is 95%, and OsrbFGF exhibits activity promoting in vivo NIH / 3T3 cell proliferation and promoting in vitro wound healing.

Description

METHOD FOR PRODUCING RECOMBINANT HUMAN BASIC FIBROBLAST GROWTH FACTOR FROM RICE SEEDS BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to genetic engineering, and more particularly, to a method of producing human cell growth factors in plants through genetic engineering techniques.

Fibroblast Growth Factor (FGF) is a group of proteins that have a very high affinity for heparin and similar biological activities. To date, 23 different fibroblast growth factors have been isolated and identified. A prototype of fibroblast growth factor, the basic fibroblast growth factor (bFGF or FGF-2), was first found in the pituitary and brain in the 1970s. It is a single-stranded protein with a molecular weight of 17 kDa and an isoelectric point of 9.6. This promotes the proliferation of NIH / 3T3 cells and inhibits stem cell differentiation. This can play an important role in the treatment of asthma, as it can promote the proliferation and migration of human airway smooth muscle cells, alter the contractile phenotype in vitro and thus cause attenuation of the airway remodeling. The basic fibroblast growth factor was also found to promote rat mammary epithelial cell lineage. It is also widely used in clinical applications for the treatment of burns and wounds because it can significantly increase wound healing rates. Compared to simple surgical treatments, this is more effective in the treatment of redness, swelling and burn healing, and the surface hardness of the skin is relatively small. Thus, fibroblast growth factors are commonly used for tissue repair and wound healing of cardiovascular and neurodegenerative diseases.

The basic fibroblast growth factor was originally isolated from the brain and pituitary gland and its origin is very limited. It is difficult to meet the wide demand for basic fibroblast growth in vivo and ex vivo. Over the past several decades, scientists have used recombinant basic fibroblast growth factors in Escherichia coli .) , yeast ( Pichia pastoris ), soybean seeds and silkworms. However, due to low yields, complicated processes and insolubility, neither satisfied the market demand, and it was particularly difficult to meet the increasing application requirements for basic fibroblast growth factor in cell therapy and mediastinology.

Recently, it has been reported that rice starch is used for the expression of a variety of recombinant pharmaceutical proteins including human lactoferrin, human lysozyme, rhIGF-1 fusion protein, human granulocyte colony-stimulating factor and human serum albumin. These results indicate that the rice starvation expression system is advantageous in that it is cost-effective, safe and easy to mass-produce scalable compared to other animal and plant cell expression systems. Therefore, rice starvation is regarded as the preferred expression platform for the mass production of pharmacological proteins, and its benefits are of great interest in academic research and industrial applications.

The expression of basic fibroblast growth factor in the rice endosperm system has not been reported yet.

It is an object of the present invention to provide a rice genetic codon-optimized gene expressing a basic fibroblast growth factor ( OsrbFGF gene) having a sequence of SEQ ID NO.

The present invention also The present invention provides a vector containing the OsrbFGF gene, preferably a rice starch-specific expression vector, and more preferably a vector having the structure of Fig.

Another object of the invention to provide a use of a vector containing a OsrbFGF the production of transgenic rice seeds containing OsrbFGF.

It is still another object of the present invention to provide a method for producing transformed rice seeds containing OsrbFGF .

Another object of the present invention is to provide a method for separating and purifying OsrbFGF from transformed rice stocks containing OsrbFGF .

The method for producing transformed rice seeds containing OsrbFGF comprises the following steps:

(1) preparing an OsrbFGF gene having a sequence of ID NO. 1;

(2) constructing an OsrbFGF expression vector and a selective marker gene vector that are specifically expressed in rice endosymbionts ;

(3) transforming the vectors obtained in step (2) together into rice union tissues;

(4) culturing said rice union tissue to obtain transgenic rice plants containing OsrbFGF , followed by screening and induction;

(5), culturing the transformed rice plant comprising the OsrbFGF to obtain the transgenic rice seeds containing OsrbFGF.

Moreover, in the above method, the OsrbFGF expression vector has the structure of FIG.

Furthermore, in the above method, the selectable marker gene vector has the structure of FIG.

A method for isolating and purifying OsrbFGF from a transformed rice stock oil containing OsrbFGF of the present invention comprises the steps of:

(1) OsrbFGF extract is prepared as a raw material by using transformed rice bran containing OsrbFGF ;

(2) OsrbFGF filtered and the OsrbFGF extract was filtered using positive pressure filtration equipment in order to obtain the water;

(3) On the heparin 6 Fast Flow column,OsrbFGF Separating and extracting the filtrate, and removing the impurity protein from the OsrbFGF filtrate bound to the heparin 6 Fast Flow column through a two-step washing method;

(4) In order to obtain the OsrbFGF purified in high purity, the elution of the heparin binding OsrbFGF 6 Fast Flow column with elution buffer OsrbFGF.

In particular, the method comprises the steps of:

(1) OsrbFGF extract was prepared with extraction buffer containing 50 mM PB (pH 7.5), 1 mM EDTA, 1 mM L-reduced glutathione, and 250 mM NaCl using transformed rice seeds containing OsrbFGF ;

(2) in order to obtain the OsrbFGF extract, the extract OsrbFGF and filtered through a pressure filtration apparatus and 3㎛ 0.22㎛ amount sequentially;

(3) isolating and purifying the OsrbFGF extract on a heparin 6 Fast Flow column and equilibrating the column with 50 mM PB (pH 7.5) containing 250 mM NaCl and 1 mM L-reduced glutathione; Through the two-step cleaning process to remove impurities from the protein extract OsrbFGF, wherein the washing buffer 1 50mM PB (pH 7.5), 600mM NaCl, 1mM L- reduction and containing glutathione, washing buffer 2 is 50mM PB (pH 7.5 ), 900 mM NaCl, 1 mM L-reduced glutathione;

(4) In order to obtain the OsrbFGF purified in high purity, and eluting the OsrbFGF coupled to the heparin 6 Fast Flow column with elution buffer OsrbFGF; The OsrbFGF elution buffer contained 10 mM Tris-HCl (pH 7.5), 1.7 mM NaCl, 1 mM L-reduced glutathione.

The present invention constitutes and successfully expresses a human bFGF gene that is advantageously expressed in rice oil and isolates purified OsrbFGF protein. The obtained OsrbFGF protein exhibits human basic fibroblast growth factor activity and lay down the basis for the production of human cell growth factor proteins using plant genetic engineering techniques.

1 is a schematic view of the structure of the plasmid pOsPMP2.
2 is a schematic view of the structure of the plasmid pOsPMP276.
3 is a schematic view of the structure of the plasmid pOsPMP277.
4 is a schematic view of the structure of the plasmid pOsPMP05.
Figure 5 is a schematic diagram of the results of PCR amplification and identification of positive-trait-transformed plants, from left to right lanes 1-8: positive transformed plants; Nc: Negative control, that is, using rice genomic DNA of non-transgenic rice TP309 as a template; Pc: Positive control, i.e., plasmid pOsPMP276 used as a template.
FIG. 6 is a schematic diagram showing the expression results of OsrbFGF in transgenic rice identified by Western blotting, wherein lanes 1-8: positive trait modified plants from left to right; Nc: rice seed extract of non-transgenic rice TP309; Pc: bFGF standard.
Figure 7 shows the expression levels of OsrbFGF.
Figure 8 shows the isolation and identification results of OsrbFGF from transformed rice seeds, where a shows the recovery of OsrbFGF ; b shows the detection results of SDS-PAGE during purification; c shows the results of identification of western blotting during purification, and from left to right in FIGS. b and c , lane M: molecular label; UF-5: 5 kDa ultrafiltration desalting; Elu: affinity elution; W2: impurity cleaning for the second time, W1: impurity cleaning for the first time; Extra: bFGF crude extract.
Figure 9 shows the results of the OsrbFGF activity assay in vitro and in vivo; a shows the effect of OsrbFGF on the proliferation of NIH / 3T3 cells; b shows wound healing rates at OsrbFGF treatment days 2, 4, 6, 8, 10, 12, 14 days; c and e show positive numbers of CD68 and PCNA at OsrbFGF treatment days 3, 7 and 14, respectively; d and f show the results of autoimmune radiography of CD68 and PCNA.

The features and advantages of the present invention will be described in detail with reference to the accompanying drawings. The embodiments are provided merely for the purpose of describing the present invention and are not intended to limit the other contents disclosed by the present invention in any way.

Unless otherwise specified, the materials and reagents used in the examples are generally commercially available.

(Example 1) Construction of a vector specifically expressing recombinant human basic fibroblast growth factor in rice and preparation of transformed rice

Human bFGF The gene (gene bank accession number: NM002006) was synthesized by the Heron Blue Biotech company and the rice preferred genetic codon was bFGF 61.54% of the genetic codons of the genes were optimized, and human bFGF The nucleic acid of the gene was changed by 21.65%. Optimized human bFGF The gene sequence was as shown in SEQ ID NO. 1, and the amino acid sequence before and after codon optimization was not changed. This example demonstrates that recombinant human bFGF < RTI ID = 0.0 > In order to mediate the expression of the gene, a rice specific promoter Gt13a and its signal peptide were employed. To construct a rice starch-specific expression cassette, the plasmid pOsPMP2 shown in Fig. 1 was used. The synthesized codon-optimized human bFGF The gene was digested with SchI and XhoI and cloned into pOsPMP2 to construct the plasmid pOsPMP276 as shown in Fig. Subsequently, Agrobacterium To construct a tumefaciens -mediated plasmid, pOsPMP276 was digested with HindIII and EcoRI, and a 1983 bp expression cassette containing the promoter gene Gt13a and its signal peptide, codon-optimized bFGF gene and Nos The termination was ligated to the BINARY vector JH2600. The obtained Binalari plasmid was designated pOsPMP277 as shown in Fig. The Bynari plasmid pOsPMP277 was transformed into Agrobacterium tumefaciens strain EHA105 (Invitrogen Company, USA). As shown in Fig. 4, the plasmid pOsPMP277 and the plasmid pOsPMP05 contained Agrobacterium Through tumefaciens - mediated transformation, in particular, Daley , M .; Knauf , VC; Summerfelt , KR; Turner, JC Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants. Plant Cell Rep.1998, 17, through the method of 489-496, was transfected with the conversion in the callus (callus tissue) derived from a rice product TP309. They were then incubated together, screened and induced to produce seedlings. A total of 58 transgenic plants were obtained. Positive transformed plants were identified by PCR amplification. For PCR amplification, reverse primer sequences were used, starting from a forward primer starting from (5'-GAGGGTGTGGAGGCTC TTGT-3 ') signal peptide and (5'-GCCAGTGAATTCCCGA TCTAGTAAC-3') Nos termination gene. The PCR result is shown in Fig.

(Example 2) Analysis and identification of OsrbFGF

In this example, Western blotting was used to detect whether bFGF expressed in transformed rice (referred to herein as OsrbFGF) was harvested in oil-bearing cells. Approximately 100 mg of T1 transgenic rice seeds positive for PCT were extracted with 1 ml of extraction buffer (50 mM PB, pH 7.5, 1 mM EDTA, 1 mM L-reduced glutathione, 250 mM NaCl) at 4 ° C for about 50 minutes After being crushed, it was centrifuged at 10620 xg for 10 minutes to obtain a crude protein extract. 40㎕ of the crude protein extract with 350ng Ecoli - derived recombinant bFGF was separated by 15% SDS-PAGE gel, the gel was inhalation (blotting) to a nitrocellulose membrane. Nitrotetrazolium Blue chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP, BIOSHARP, China) The anti-basic fibroblast growth factor rabbit polyclonal antibody (Abcam, UK) and alkaline phosphatase goat anti-rabbit IgG (ZSGB-BIO, China) were used for the detection of the target protein . As a result, as shown in FIG. 6, OsrbFGF expressed in 35 transgenic rice strains was accumulated in rice oil. The results of western blotting were analyzed with Image-pro Plus software. The results showed that one kilogram of brown rice could produce 99.11-185.66 mg / gram OsrbFGF, and OsrbFGF expression levels are shown in FIG.

Also, Southern blotting was used to identify transgenic rice plants in this example. Approximately 200 mg of each of the leaves of the transformed rice was pulverized with liquid nitrogen, and then extracted using a rapid-type plant genome DNA system (Tianson Biotech Co., Ltd., China) to obtain genomic DNA. Genomic DNA was digested with EcoRI , HindIII , and EcoRI and HindIII (New England BioLabs) overnight at 37 degrees Celsius, followed by 0.8% sepharose gel. The gel was then inhaled on MILLIPORE NY + membranes and hybridized according to the instructions of the DIG High Prime DNA Label and High Prime DNA Labeling and Detection Starter Kit I (Roche). A 824 bp probe derived from the bFGF coding region was amplified using primers (5'-GCATCCATAAATCGCCCCATAG-3 'and 5'-GCCAGTGAATT CCCGATCTAGTAAC-3'). The results showed that the two fragments could be detected after cleavage with EcoRI or HindIII , consistent with the results of the genetic analysis. A single band obtained from the double enzyme digestion with EcoRI and HindIII was compared with the entire expression cassette, confirming that the Agrobacterium bidirectional vector was present in the entire expression cassette.

(Example 3) Isolation and purification of OsrbFGF

Transgenic rice seeds were pulverized and extracted with extraction buffer (50 mM PB, pH 7.5, 1 mM EDTA, 1 mM L-reduced glutathione, 250 mM NaCl) at room temperature for approximately 1 hour. The extracts obtained were filtered sequentially through pressure filtration apparatus of 3 [mu] m and 0.22 [mu] m quantities, and then the filtrate was loaded on a Heparin 6 Fast Flow column (GE Healthcare, www.gelifesciences.com). The column was equilibrated with 50 mM PB containing 250 mM NaCl and 1 mM L-reduced glutathione. To remove the impurity protein and increase the recovery of OsrbFGF, a two step rinse method was used wherein rinse buffer 1 contained 50 mM PB (pH 7.5), 600 mM NaCl, 1 mM L-reduced glutathione, 2 contained 50 mM PB (pH 7.5), 900 mM NaCl, and 1 mM L-reduced glutathione. After washing the impurities, OsrbFGF bound to heparin 6 Fast Flow column was eluted with elution buffer (10 mM Tris-HCl (pH 7.5), 1.7 mM NaCl, 1 mM L-reduced glutathione) to obtain highly purified OsrbFGF protein Lt; / RTI > Here, the OsrbFGF recovery rate is shown in FIG. 8A, and the results of the determination of each step and the analysis results of Western blotting are shown in FIGS. 8B and 8C, respectively. Prior to lyophilization, OsrbFGF eluted fractions were concentrated and partially desalted using a 5kD ultrafilter from Pellicon XL (Millipore Corporation) and then diluted with Viva to remove endotoxin Flow 50 (Sartorius) 100 kD ultrafiltration device. The final OsrbFGF yielded more than 95% purity and the endotoxin content was less than 1 EU / ㎍. Finally, OsrbFGF was lyophilized (1:50, w / w) under the protection of OsrHSA.

(Example 4) Biological activity of OsrbFGF on the proliferation of NIH / 3T3 cells

NIH / 3T3 cells were cultured in 96 well plates in medium containing DMEM and supplemented with 1.5% FBS and 1% penicillin and streptomycin (P / S). The initial concentration of NIH / 3T3 cells, 1x10 ⁵ - were 5x10 ⁵ / ml. The cells were incubated at 37 ° C and 5% CO ₂ for approximately 24 hours. The medium was then removed and the cells were washed with PBS and treated with 0.25% trypsin for 3 min at 37 ° C. The treated cells were resuspended in DMEM medium, supplemented with 1.5% FBS and 1% P / S, and then centrifuged at 800 rpm for 3 minutes. The supernatant was removed and cells were resuspended in DMEM medium containing 1.5% FBS and 1% P / S. NIH / 3T3 cells were seeded in 96 well plates at a concentration of 4000 / ml and incubated again for 24 hours at 37 ° C and 5% CO ₂ . Finally, the cells were cultured for 48 hours in DMEM medium containing different concentrations of OsrbFGF (200 [mu] L / well) and then 20 [mu] l MTT (Thiazolyl Blue Tetrazolium Bromide) Lt; / RTI > for 4 hours. A microtiter plate was placed at 490 nm for reading on a VersaMax (Molecular Devices, USA). All data were analyzed using GraphPad Prism 5 software, and all experiments were repeated three times. The results showed that the proliferation of NIH / 3T3 cells was dose-dependent when the OsrbFGF concentration was 6 pg-5 ng / ml and could reach 1.15 x 10 ⁶ IU / mg as shown in Figure 9a; OsrbFGF has an activity of promoting in vitro NIH / 3T3 cell proliferation. This demonstrates that the OsrbFGF of the present invention has the same biological activity as commercially available rbFGF.

(Example 5) Effect of OsrbFGF on wound healing

In order to prepare a splinting model, we have used, in particular, ( Galiano , RD; Michaels , V .; Dobryansky , M .; Levine , JP; Gurtner , GC Quantitative and reproducible murine model of excisional wound healing. Regen . 2004, 12, 485-492 ) , 200-250 g female SD rats (Shanghai SLAC Laboratory Animal Co., Ltd.) were randomly divided into four groups. Four randomized model groups were used to test wound healing rates: three OsrbFGF dose groups, a group of 2000 ng / mo (n = 15), a group of 500 ng / ml (n = 15), a group of 125 ng / Physiological saline negative control group (n = 15). Wound closure was observed at 0, 2, 4, 6, 8, 10, 12 and 14 days, respectively. Wound healing rate {(initial wound area - unhealed area / initial wound area) x 100} was calculated and Wu , Y .; Chen, L .; Scott, PG; Tredget , EE Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis . Pathological and immunohistochemical staining observations were performed according to the method of Stem Cells 2007, 25, 2648-2659 . The results showed that the wound healing rate was OsrbFGF dose-dependent. Here, as shown in Figure 9b, during the first 8 days of treatment, low dose efficacy was significantly higher than high dose efficacy (e.g., 500ng vs. 2000ng). Usually, reconstruction of scarred skin can promote epithelial cell formation and CD68 expression as well as proliferation of cellular nuclear antigen (PCNA). Thus, this example also detected expression of PCNA and CD68 at days 3, 7 and 14 of treatment. The results showed that expression of PCNA and CD68 in the OsrbFGF dose groups was significantly increased in the OsrbFGF dose groups at 3 and 7 days of treatment compared to the negative control group, (Fig. 9C to Fig. 9F). In the early stages of treatment, OsrbFGF can accelerate wound closure by stimulating proliferation of PCNA and CD68 by stimulating cell proliferation and then decreasing the expression of cells, PCNA and CD68, and at the later stages of treatment, And prevent cell proliferation during wound closure.

<110> HEALTHGEN BIOTECHNOLOGY CO., LTD. <120> METHOD FOR PRODUCING RECOMBINANT HUMAN BASIC FIBROBLAST GROWTH          FACTOR FROM RICE SEEDS <130> 13P420045 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 468 <212> DNA <213> Artificial Sequence <220> <223> rice genetic codon-optimized gene expressing human basic          fibroblast growth factor <400> 1 atggccgccg gctccatcac caccctcccg gccctcccgg aggacggcgg ctccggcgcc 60 ttcccgccgg gccacttcaa ggacccgaag cgcctctact gcaagaacgg cggcttcttc 120 ctccgcatcc acccggacgg ccgcgtggac ggcgtgcgcg agaagtccga cccgcacatc 180 aagctccagc tccaggcgga ggagcgcggc gtggtgtcca tcaagggcgt gtgcgccaac 240 cgctacctcg ccatgaagga ggacggccgc ctcctcgcca gcaagtgcgt gaccgacgag 300 tgcttcttct tcgagcgcct ggagagcaac aactacaaca cctaccgcag ccgcaagtac 360 accagctggt acgtggccct caagcgcacc ggccagtaca agctcggctc caagaccggc 420 ccgggccaga aggccatcct cttcctcccg atgagcgcca agagctga 468 <210> 2 <211> 1983 <212> DNA <213> Artificial Sequence <220> <223> Expression cassette sequence containing Gtl3a promoter and its          signal peptide, the codon-optimized bFGF gene and Nos terminator <400> 2 agcttcaacc tgctgagaag aacaactgac ggtcataagg agagggagct tttcgatagg 60 tgccgtgcag ttcaaagagt tagttagcag taggatgaag atttttgcac atggcaatga 120 gaagttaatt atggtgtagg caacccaaat gaaacaccaa aatatgcaca agacagtttg 180 ttgtattctg tagtacagaa taaactaaag taatgaaaga agatggtgtt agaaaatgaa 240 acaatattat gagtaatgtg tgagcattat gggaccacga aataaaaaaa gaacattttt 300 atgagcagtg tgttctcaat gagccttgaa tgttatcacc caggataaga aacccttaag 360 caatgaaaca tgcaagcgtt taatgtgcaa agttggcatt ctccacgaca taatgcaaaa 420 gaagatataa tctatgacat agcaagtcat gcatcatttc atgcctctgt caacctattc 480 atttctagtc atctaggtaa gtatcttaag ctaaagtgtt agaacttccc atacataagt 540 cataactgat gacaattggg tgtaacacat gacaaaccag agagtcaagc aagataaagc 600 aaaaggatgt gtacataaaa ctacagagct atatgtcatg ttgcgaaaag aggagagctt 660 ataagacaag ccatgactca aaaaaaattc acatgcctac tgtggcccat atatcatgca 720 acaatccaaa aactcacagg tctcggtgtt gatcgtgtca acatgtgacc accctaaaaa 780 ctcttcacta aatattaaag tattgctaga acagagcttc aagatataag tcatgatcac 840 caacaaccat gttcaaaaag aaatagaaag ctatggcaca gcaacaaaaa gcaaaagcat 900 gcatggatat aatctttaac atcatccatg tcatattgca aaagaaagaa agagagaaca 960 atacaaatga tgtgtcaatt acacatccat cattatccat ccaccttccg tgtaccacac 1020 ttcatatatc atgagtcact tcatgtctgg acattaacaa actctatctt aacattcaaa 1080 tgcatgagac tttatctcac tataaatgca caatgattta gcattgtttc tcacaaaacc 1140 attcaagttc attagtacta caacaacatg gcatccataa atcgccccat agttttcttc 1200 acagtttgct tgttcctctt gtgcaatggc tctctagcca tggccgccgg ctccatcacc 1260 accctcccgg ccctcccgga ggacggcggc tccggcgcct tcccgccggg ccacttcaag 1320 gacccgaagc gcctctactg caagaacggc ggcttcttcc tccgcatcca cccggacggc 1380 cgcgtggacg gcgtgcgcga gaagtccgac ccgcacatca agctccagct ccaggcggag 1440 gagcgcggcg tggtgtccat caagggcgtg tgcgccaacc gctacctcgc catgaaggag 1500 gacggccgcc tcctcgccag caagtgcgtg accgacgagt gcttcttctt cgagcgcctg 1560 gagagcaaca actacaacac ctaccgcagc cgcaagtaca ccagctggta cgtggccctc 1620 aagcgcaccg gccagtacaa gctcggctcc aagaccggcc cgggccagaa ggccatcctc 1680 ttcctcccga tgagcgccaa gagctgagct cgagctcgaa tttccccgat cgttcaaaca 1740 tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat 1800 aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg acgttattta 1860 tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg atagaaaaca 1920 aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagatc 1980 ggg 1983 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer 1 for identifying the positive transformed plants by PCR          amplification <400> 3 gagggtgtgg aggctcttgt 20 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer 2 for identifying the positive transformed plants by PCR          amplification <400> 4 gccagtgaat tcccgatcta gtaac 25 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer 1 for amplification a 824 bp probe derived from bFGF coding          region <400> 5 gcatccataa atcgccccat ag 22 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer 1 for amplification a 824 bp probe derived from bFGF coding          region <400> 6 gccagtgaat tcccgatcta gtaac 25

Claims (12)

A rice genetic codon-optimized gene encoding a human basic fibroblast growth factor having a sequence of SEQ ID NO: 1. An expression vector comprising the gene of claim 1. 3. The method of claim 2,
Wherein said vector is a rice oil cell-specific expression vector. The method of claim 3,
The vector is a vector having the structure of FIG. Use of a vector for use in a method for producing a transformed rice seed using the vector of any one of claims 2 to 4, wherein the transformed rice seed is a vector expressing a human basic fibroblast growth factor. Use of a gene to produce a transgenic rice seed using the gene of claim 1. A method for producing transformed rice seeds expressing human basic fibroblast growth factor,
(1) construct an OsrbFGF gene having a sequence of SEQ ID NO: 1 expressing human basic fibroblast growth factor;
(2) constructing an OsrbFGF expression vector and a selective marker gene vector that are specifically expressed in rice endosymbionts ;
(3) transforming the vectors obtained in step (2) together into rice union tissues;
(4) culturing the rice union tissue to obtain a transgenic rice plant containing OsrbFGF expressing human basic fibroblast growth factor, followed by screening and induction;
(5) 5) culturing the transgenic rice plants containing OsrbFGF to obtain transgenic rice seeds containing OsrbFGF . 8. The method of claim 7,
Wherein the OsrbFGF expression vector has the structure of Fig. 8. The method of claim 7,
Wherein said selectable marker gene vector has the structure of FIG. A method for separating and purifying human basic fibroblast growth factor from transformed rice seeds comprising OsrbFGF,
The method comprising:
(1) preparing an OsrbFGF extract as a raw material by using transformed rice grains containing OsrbFGF;
(2) filtering the OsrbFGF extract using a positive pressure filtration apparatus to obtain an OsrbFGF filtrate;
(3) separating and extracting the OsrbFGF filtrate on a heparin 6 Fast Flow column, removing impurity proteins from the OsrbFGF filtrate bound to the heparin 6 Fast Flow column through two-step washing;
(4) A method comprising eluting OsrbFGF bound to a heparin 6 Fast Flow column with an OsrbFGF elution buffer to obtain OsrbFGF purified at high purity. 11. The method of claim 10,
The method comprising:
(1) OsrbFGF extract was prepared with extraction buffer containing 50 mM PB (pH 7.5), 1 mM EDTA, 1 mM L-reduced glutathione, and 250 mM NaCl using transformed rice seeds containing OsrbFGF ;
(2) in order to obtain the OsrbFGF extract, the extract OsrbFGF and filtered through a pressure filtration apparatus and 3㎛ 0.22㎛ amount sequentially;
(3) isolating and purifying the OsrbFGF extract on a heparin 6 Fast Flow column and equilibrating the column with 50 mM PB (pH 7.5) containing 250 mM NaCl and 1 mM L-reduced glutathione; Removing the impurity protein from the OsrbFGF extract through a two step rinse;
(4) an order to obtain the OsrbFGF purified in high purity, eluting the OsrbFGF coupled to the heparin 6 Fast Flow column with elution buffer OsrbFGF,
Wash buffer 1 contained 50 mM PB (pH 7.5), 600 mM NaCl, 1 mM L-reduced glutathione, Wash buffer 2 contained 50 mM PB (pH 7.5), 900 mM NaCl, 1 mM L-reduced glutathione;
Wherein said OsrbFGF elution buffer contains 10 mM Tris-HCl (pH 7.5), 1.7 mM NaCl, 1 mM L-reduced glutathione. A transformed rice comprising a rice genetic codon-optimized gene expressing the human basic fibroblast growth factor of claim 1.

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