CN1295129A - Method of producing human insulin by utilizing transgenosis tomato - Google Patents
Method of producing human insulin by utilizing transgenosis tomato Download PDFInfo
- Publication number
- CN1295129A CN1295129A CN00122802A CN00122802A CN1295129A CN 1295129 A CN1295129 A CN 1295129A CN 00122802 A CN00122802 A CN 00122802A CN 00122802 A CN00122802 A CN 00122802A CN 1295129 A CN1295129 A CN 1295129A
- Authority
- CN
- China
- Prior art keywords
- gene
- insulin
- expression
- plant
- tomato
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention uses transgenic tomato as biological reactor of producing human insulin. Based on plant favoritism principle, insulin gene codon is designed, the gene is artificially synthesized and placed downstream specific expression fruit promoter before transferred into tomato. Large amount of human insulin produced by tomato fruit may be used as oral product for preventing and curing some types of diabetes and used to extract insulin injecta. By means of the positioning recombination system applied in gene controlling technology and the control of plant hormone in seed growth expression, the tomato seed is made to abort or insulin gene erased to avoid the repeated utilization of progeny seed.
Description
The present invention utilizes tamato fruit to come manufacturer's Regular Insulin as a kind of plant bioreactor.This insulin human can enter blood circulation as a kind of oral preparations by mucocutaneous membrane on the digestive tube by eating something rare tomato.Also can extract the insulin human of high expression level amount simultaneously, and obtain the injection insulin human from this tamato fruit.
The point of main innovation of the present invention is that (1) select the codon of plant-preference for use, the synthetic human insulin gene, and (2) adopt tamato fruit specificity promotor, make the expressive site of Regular Insulin concentrate on fruit, and content are very high; (3) the location recombination system is regulated in the seed development process the regulation and control and the plant hormone of foreign gene, make the tamato fruit of producing Regular Insulin not contain the seed that can continue to plant, or the human insulin gene among the seed embryo is deleted.This technology has prevented that not only human insulin gene from entering common tomato in some way, and has guaranteed insulin human tomato production person's lasting economic interests.
Bio-reactor is the field that recently not many biotechnologys research and development department extremely pays close attention to.Produce people's somatomedin such as milk cow, milk goat galactophore biological reactor.The research that utilizes potato tuber to produce Hepatitis B virus vaccine in plant bioreactor research is subjected to paying attention to widely.Tomato produces oral polypeptide drug with it and has very big application potential as eating vegetables or fruit.The insulin human is crucial for the urine patient's of insulin-dependent treatment.Some diabetic subject's metabolism adjusting is relevant with the autoimmunity function.
Oral insulin be proved to be the urine of some type sick treatment be effectively (Reddy S etat, Panereas 2000,20 (1): 55-60; Ploix C et at, Diabetes1999,48 (1) 2150-6; Ramiya VK, Maclaren NK, Horm Res 1997,48 suppl 4:67-70)
One of technical essential of this invention is the synthetic of insulin gene.Why not adopting natural human insulin's dna sequence dna, is because we adopt the codon of plant-preference can improve the expression amount of this gene in plant.The encoded amino-acid sequence of natural insulin A chain and B chain of our used human insulin gene, but the DNA sequence of DNA sequence and natural human insulin's gene and inequality.
Two of the main points of this invention are clone and the application that started about the tamato fruit specificity.The data of existing tamato fruit specificity promotor in the document (Mark JJ et at, 1993 Plant Mol Biol21:625-640) has wherein comprised the nucleotide sequence of 4Kb.We synthesize and have designed the sequence of a pair of primer amplification 851bp.Experimental results show that this promoter region not only has very special fruit specificity, and expression amount is apparently higher than general composition type expression promoter CaMV35S.
Three of the main points of this invention are to utilize location recombination system (Cre-Lox, FLP-FRT, and R-RsGin-gix) to control the behavior of foreign gene (human insulin gene under the fruit specific promoter driving) in filial generation.The application principle of this location recombination system is at Odell JT and BussellSH, Rusell:Use of Site-Specific Recombination Systems in Plants, (" application of location recombination system in external gene activity regulation and control " sees the loyal equality of woods and compiles J Passkowski (ed) Homologous Recombination and Gene Silencing in Plants219-270 and this invention applicant's book: " moving towards the molecular biology of plants of 21 century ", narration was once arranged the P66-71 of Science Press), but the experimental design in this application is novel.We make fruit specific promoter and insulin human's fusion gene be in two in the same way can be by between the site (as lox P) of recombinase (as Cre) identification.Simultaneously designed the recombinase gene (as Cre) that is blocked in the another one parent, the upstream extremity of the blocking-up sequence of this gene (blocking fragment) has fetal development specificity promotor.When the blocking-up sequence is deleted under the effect of (inducing as cupric ion) gene expression regulation system of (the handling as heat shock) of physics or chemistry, recombinase gene is just started working, under the identification and catalysis of recombinase, people's pancreas islet gene that fruit specific promoter drives is just deleted.So F
1Contain human insulin gene and expression product thereof in the fruit for tomato plant, and at its seed (F
2) in the Regular Insulin fusion gene deleted.
Four of the main points of foregoing invention are that the fusion gene that tamato fruit specificity promotor human insulin gene is constituted is placed one of parent.In second parent, be provided with simultaneously phytohormone Regulation gene or lethal gene under the ovule development-specific promoters driven.After two parent's mating, F
1Regular Insulin can be produced for fruit, but the seed that to educate can not be formed.In order to make the actually operating in the production process become more capable.We make and also carry a recombinase gene (as Cre) among first parent; Phytohormone Regulation gene or lethal gene are under the state that is blocked, are provided with at blocking-up sequence two ends that two parents all can normally raise up seed the recognition site (as lox P).And their filial generation is just because of the activation of phytohormone Regulation gene or lethal gene, though can the result can not form the seed that can educate.
Here the phytohormone Regulation gene can be isopentenyl transferase genes or the indolylacetic acid synthesis related gene iaa M from agrobatcerium T-DNA, and lethal gene can be the barnase gene from genus bacillus.
All the other technical key points comprise agriculture bacillus mediated to genetic transformation technology of tomato or other genetic transformation technology, and to the separation and the purification technique of Regular Insulin, the mensuration inspection technology of Regular Insulin etc.
In one embodiment of the invention, about synthesizing of human insulin gene, select the codon of plant-preference for use.In 63 bases of insulin human A chain, 13 bases have been changed for this reason.In 90 bases of B chain, 18 bases have been changed.We have designed 6 amino acid whose junction fragments between A chain and B chain.The principle of design of the amino acid kind of this junction fragment and order is to keep the natural Three Dimensions Structure of insulin human.We clone EcoRI and BamHI site in the pGEM-11Z carrier with comprise the insulin human A chain and B chain and the middle segment order of connection of synthetic, and are determined this plasmid called after pllZ-ins through the analysis and the sequential analysis of plasmid enzyme restriction collection of illustrative plates.In this embodiment, the human insulin gene fragment can obtain by pcr amplification, and during amplification, the primer of use is seen the sequence 1 and 2 in the sequence table.
In one embodiment of the invention, we place human insulin gene tamato fruit specificity expression's promotor (P
2a12) regulation and control under, finish after the structure of an intermediate carrier, place a kind of double base table carrier (see figure 1) again.The increase primer of this promotor of PCR program is seen sequence 3 and 4 in the sequence table.
In one embodiment of the invention, human insulin gene places under the regulation and control of composition type expression promoter CaMV35S promotor.Change in the intermediate carrier again.Further change binary expression vector again over to, finish the structure (see figure 2) of a plant expression vector.
In one embodiment of the invention, adopt location recombination system Cre-lox and FLP-FRT to control insulin gene at F
2The deletion in generation, (see figure 3).
By the overexpression of prenyltransferase (ipt) gene in seed development is crossed kind, cause seeds abortion in one embodiment of the invention.This technology once had report in document (Nadia Ficcadenti etad 1999 Molecular Breeding 5 (5) 463-470).But utilize the expression of recombination system controlling plant hormone, cause seeds abortion, make F
2Can not utilize for seed is a place of the present invention innovation again.This The Application of Technology is controlled effectively the production process of the tomato of insulin-containing.Human insulin gene is remained in certain kind more easily and certain parent in.This embodiment is seen shown in 4 figure.
Claims (9)
1, the present invention produces insulin human's method for tamato fruit.This method utilizes the human insulin gene of the external source of synthetic to exist
Under the driving of fruit specific promoter or other high efficient expression starters, express in the tomato, at this moment comprised the insulin human
Gene imports tomato by genetically modified approach, and in fruit and other edible position great expression.This external source imports
Gene be present among the suitable parent, be delivered to filial generation (F again
1Generation), meanwhile utilize the location recombination system to make this
The genetic transcription unit that produces the insulin human is at F
2Generation is deleted to be fallen or and utilizes the hormone regulating and controlling gene (as iaa M gene, ipt
Gene) or certain pernicious gene (as certain RNase gene) stop F
2Normal development for seed.This people's pancreas
The plant that the island plain gene is expressed should be not limited only to tomato, should expand to other greengrocery and melon or fruit type crops such as cucumber,
Watermelon, muskmelon, eggplant, cucumber, pumpkin etc.Also should expand to other fruit plant, as banana, apple, pears,
Pineapple etc.Also should expand to piece root, tuberous plant, as Hu Luobu, radish, sweet potato, cassava, potato etc.
2, in 1 of the claim the about the human insulin gene of synthetic.The synthetic of this gene will be not limited only to plant
The use of the codon of preference (comprises the use of monocotyledons preference codon and making of dicotyledons preference codon
With), should expand to the transformation that helps expression amount raising and more effective human insulin gene structure.
3, the transformation about gene structure should expand to about the annexation between insulin human A chain and the B chain in the claim 2.
Comprise the design of fault structure autonomous between A chain and the B chain, and A chain and B chain encoding order are placed on not respectively
Among the transcription unit together.
4, the application of the related promotor of claim 1 is not limited only to tamato fruit specificity promotor, also comprises in other organ high
The promotor of horizontal expression, as stem tuber root or other storage organ (as seed) but the promotor of great expression, though not
It is specificity expression's promotor.
5, the application about the location recombination system comprises Cre-lox, FLP-FRT, R-RS and Gin-gix in the narration of claim 1
Use the fit applications that a kind of system also comprises location not of the same race recombination system separately in four kinds of location recombination systems.
6, not only relate to tomato one kind of plant in the claim 1.When being applied to tomato, will in the embodiment that we have adopted
Foreign gene places different parents, by first-filial generation (F
1) obtain the Regular Insulin of commercial applications.Plant in difference
When using in the thing, will be not limited to foreign gene is placed among two parents, can be simultaneously or be placed on earlier foreign gene
In the same plant, logical again other gene regulating approach reaches regulatable efficiently manufacturer's Regular Insulin.
7, relate to the control human insulin gene in claim 1 and 5 the narration at F
2For the method that can not be reused in the seed.
Seed is not unique organ of multiplication.This by locating the method for recombination system deletion specific objective DNA, should may extend into other tissue and organ.
8, claim 1 and 4.When adopting the hormone regulating and controlling gene or cause the gene (as the RNase gene) of plant tissue decline, should not be limited to expression at the seed position yet, should comprise the expression of some other organ (as bud or regeneration bud).
9, the expression product of the human insulin gene of the synthetic mentioned of claim 1.This gene expression product should expand to human insulin analogue or other polypeptide product that the insulin human receptor site is worked.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00122802A CN1295129A (en) | 2000-08-25 | 2000-08-25 | Method of producing human insulin by utilizing transgenosis tomato |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00122802A CN1295129A (en) | 2000-08-25 | 2000-08-25 | Method of producing human insulin by utilizing transgenosis tomato |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1295129A true CN1295129A (en) | 2001-05-16 |
Family
ID=4589323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00122802A Pending CN1295129A (en) | 2000-08-25 | 2000-08-25 | Method of producing human insulin by utilizing transgenosis tomato |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1295129A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004111244A3 (en) * | 2003-06-17 | 2005-02-03 | Sembiosys Genetics Inc | Methods for the production of insulin in plants |
| US7393998B2 (en) | 2002-03-08 | 2008-07-01 | Prodigene, Inc. | Commercial production of insulin and insulin-like proteins in plants |
| CN101684468B (en) * | 2003-04-14 | 2011-11-16 | 林忠平 | Synthetic human insulin gene and use thereof in cultivation of transgenic tomatoes |
-
2000
- 2000-08-25 CN CN00122802A patent/CN1295129A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7393998B2 (en) | 2002-03-08 | 2008-07-01 | Prodigene, Inc. | Commercial production of insulin and insulin-like proteins in plants |
| US7554006B2 (en) | 2002-03-08 | 2009-06-30 | Prodigeue, Inc. | Commercial production of insulin and insulin-like protein in plants |
| CN101684468B (en) * | 2003-04-14 | 2011-11-16 | 林忠平 | Synthetic human insulin gene and use thereof in cultivation of transgenic tomatoes |
| WO2004111244A3 (en) * | 2003-06-17 | 2005-02-03 | Sembiosys Genetics Inc | Methods for the production of insulin in plants |
| JP2007523611A (en) * | 2003-06-17 | 2007-08-23 | セムバイオシス ジェネティクス インコーポレイテッド | Method for producing insulin in plants |
| EP1959017A1 (en) * | 2003-06-17 | 2008-08-20 | SemBioSys Genetics Inc. | Methods for the production of insulin in plants |
| CN1836047B (en) * | 2003-06-17 | 2010-12-22 | 赛姆生物系统遗传公司 | Methods for the production of insulin in plants |
| EA014887B1 (en) * | 2003-06-17 | 2011-02-28 | Сембайосиз Джинетикс Инк. | Method for the expression of insulin in plant seeds, a method for obtaining plant seeds comprising insulin and a plant capable of setting seeds comprising insulin |
| JP4864701B2 (en) * | 2003-06-17 | 2012-02-01 | セムバイオシス ジェネティクス インコーポレイテッド | Method for producing insulin in plants |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Moon et al. | Development of systems for the production of plant-derived biopharmaceuticals | |
| Daniell et al. | Chloroplast-derived vaccine antigens and other therapeutic proteins | |
| Boehm | Bioproduction of therapeutic proteins in the 21st century and the role of plants and plant cells as production platforms | |
| Park et al. | Potential of plants to produce recombinant protein products | |
| Streatfield et al. | Plant production systems for vaccines | |
| RU2004109918A (en) | MODIFIED CRY3A TOXINS AND THE NUCLEOTIDE SEQUENCE ENCODING THEM | |
| CN101815784A (en) | Production of foreign nucleic acids and polypeptides in plant systems | |
| Jani et al. | Studies on the immunogenic potential of plant-expressed cholera toxin B subunit | |
| Daniell et al. | Chloroplast genetic engineering | |
| JP2003515350A5 (en) | ||
| CN1295129A (en) | Method of producing human insulin by utilizing transgenosis tomato | |
| Mohebodini et al. | Agrobacterium-mediated transformation of lettuce (Lactuca sativa L.) to express IgG-binding protein A and human pro-insulin as a fusion protein | |
| US7554006B2 (en) | Commercial production of insulin and insulin-like protein in plants | |
| CN106146628B (en) | A kind of artificial synthesized insect resistance protein and its relevant biological material and application | |
| CN100540666C (en) | Utilize transgenic lettuce, tomato and tobacco to produce insulin human's method | |
| CN102146377A (en) | Method for preparing protein by embryogenic cell tissue | |
| CN104630228A (en) | Promoter of cotton heat shock transcription factor GhHsf 39 genes and application of promoter | |
| JP6493393B2 (en) | Method for producing useful protein by plant | |
| CA1337280C (en) | Production of proteins in plants | |
| SP et al. | Molecular Pharming: A New Approach for a Healthy Future by a Vast Development in the Pharmaceutical Industry | |
| KR102046117B1 (en) | Production method of recombinant human insulin-like growth factor-I protein using plant callus | |
| CN107760709B (en) | Gene for regulating and controlling heat resistance of plant and application of gene in plant improvement | |
| Omo-Ikerodah et al. | An overview of the potentials of natural rubber (Hevea brasiliensis) engineering for the production of valuable proteins | |
| Schillberg et al. | Contained molecular farming using plant cell and tissue cultures | |
| CN101684468B (en) | Synthetic human insulin gene and use thereof in cultivation of transgenic tomatoes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |