CN1624141A - Process for raising expressing content of colyliform virus VPT or VP4 in plant and its product - Google Patents

Abstract

A method for increasing the expression content of the antigen protein of the rotavirus VP7 or VP4 causing the diarrhea of baby in transgenic plant is disclosed. Said transgenic plant or the antigen protein extracted from the transgenic plant can be used to cause the immune response reaction of people or mammal.

Description

Improve rotavirus vp 7Or VP 4The method of expression of plants content and product

The present invention relates to improve the infantile diarrhea rotavirus vp ₇Or VP ₄The method of antigen protein expression contents in transgenic plant, thus and and then utilize these transgenic plant that contain human rotavirus's antigen protein and derivative thereof to cause that Mammals is to the proteic immune response of this virus antigen by direct mode edible or enterally administering.

(Human rotavirus HRV) is the main pathogen that causes infant's infectivity severe diarrhoea in the world wide to rotavirus, and is particularly outstanding in developing country, and the Asia, Africa and Latin America area hundred million person-times of 30-50 takes place approximately at every year, causes more than 300 ten thousand death of child.Also widely popular every year in Chinese rotavirus diarrhea, account for children's diarrhae inpatient's 60-64%, about 100,000 the death of having an appointment every year.Though the people at any age can be subjected to the infection of rotavirus, have only the infant of 6 monthly age-2 year old manifest symptom to occur, the less generation of the infected more than 2 years old is seriously suffered from diarrhoea.Virus is mainly through fecal-oral transmission, and epidemic season mainly occurs in autumn and winter.

The human rotavirus belongs to the Reoviridae rotavirus, and complete virion is spherical in shape, and for having 20 body structures of three layers of protein shell, diameter is about 75nm, and the rough type particle of no outer capsid is 60nm, and interior nuclear diameter is 40nm.Because virion inner casing protein protomer under Electronic Speculum is more than 20 spoke-like arrangement, seem the wheel of neat in edge, so gain the name.The viral genome that is wrapped in the protein shell is made up of 11 double-stranded RNAs (dsRNA) sections, and total molecular weight is about 12 * 10 ⁶Each sections is a gene, and the polypeptide of encoding comprises 4 in core and inner capsid albumen, 5 of 2 in outer capsid albumen and Nonstructural Proteins.The proteic VP of outer capsid ₇And VP ₄, be called main in and antigen and less important in and antigen, the antigen site that is used for the rotavirus somatotype is positioned at this two albumen ^[1]

VP ₇Being a kind of glycoprotein (Glycoprotein), accounting for 30% of viral protein amount, can be encoded by gene segment 9,8 or 7, is the major protein of outer capsid, and molecular weight 38KD is according to VP ₇Antigenic difference, rotavirus are divided into 14 serotypes, but 10 infected person (G wherein ₁-G ₆, G ₈-G ₁₀And G ₁₂).That the epidemiology significance is arranged is G ₁, G ₂, G ₃And G ₄Four types.

VP ₄By the 4th genes encoding, be the less important albumen of outer capsid, only account for 1.5% of viral protein amount, it is distributed on the virus coat with the spinous process form, the about 88KD of molecular weight.VP ₄To the proteolytic enzyme sensitivity, under the pancreatin effect, dissociate into VP ₅(60KD) and VP ₈(28KD).According to VP ₄The difference of gene nucleotide series adopts RT-PCR to distinguish rotavirus, has determined 20 P genotype at present, but 9 infected person wherein.Have the epidemiology significance be P[8] and P[4].

The relation of rotavirus G type and P type shows according to China and somatotype data all over the world, the modal P[8 that is combined as of G type and P type] G ₁, P[8] G ₃, P[8] G ₄And P [4] G ₂ ^[2]

The virus serotype investigation is crucial to development and application Rotavirus Vaccine.The known person rotavirus has 14 G serotypes, but 10 infected person wherein.In the world, there are documents and materials to show that 95% rotavirus strain isolated is G ₁-G ₄Type accounts for 54%, 18%, 12% and 11% respectively.2000, Fang Zhaoyin researcher has carried out somatotype research to the virgin rotavirus of 1293 strain shares that China 10 areas (Beijing, Changchun, Qinhuangdao, Zhengzhou, Hangzhou, Foochow, Guangzhou, Lanzhou, Chengdu and Kunming) is separated to, found that above-mentioned 10 regional 1998-2000 colyliform viruses are with G ₁Type is main epidemic strain, accounts for 72.7%, is G secondly ₃(14.2%), G ₂(12.1%) and G ₄(2.5%), 3.1% sample being arranged is polyinfection.In conjunction with the somatotype result in many years of 1982-1997, the Chinese Infant and Children rotavirus is also with G ₁, G ₂, G ₃And G ₄Serotype is the most common, but because of the time and from different places, and these four serotypes take place and shared ratio difference to some extent again.Above-mentioned result of study provides more clear and epidemiology background information system for the development and application of Chinese Rotavirus Vaccine ^[2]

The infantile diarrhea that causes for rotavirus does not at present have specifics, can only take the oral rehydration method, to reduce because of the death toll due to the diarrheal dehydration, nutritional status is little to human rotavirus's initiation potential influence, improve sanitary condition is very ineffective to the prevention rotavirus, the infant below 3 years old that developed country and developing country all have an appointment more than 90% all infected HRV, and therefore, development vaccine control HRV diarrhoea is subjected to the attention of countries in the world unanimity.

The natural infection of rotavirus can not be protected human rotavirus's infection again, but can prevent serious diarrhoea.Therefore the target that develops Rotavirus Vaccine is that serious diarrhoea takes place the prevention infant.Enteron aisle sIgA anti-rotavirus infects more even more important than serum antibody IgG, and the sIgA on small intestine epithelium surface is significant on anti-infectious mechanism, so people are striving to find a kind of oral vaccine that can directly stimulate intestinal mucosa always.Yet, although people are doing many work aspect the exploration production infantile diarrhea Rotavirus Vaccine, and develop oral attenuated live vaccines of the first-generation and s-generation multivalence reprovision body vaccine in succession, but in clinical use, all find to exist poor stability, effect instability and the more high problems of production cost, therefore fail to promote the use of in the world.

Several stages has been passed through in the research of Rotavirus Vaccine, the earliest be oral attenuated live vaccines, tested several vaccines: the Wa (G that can adapt to tissue culture growth ₁) strain, from the isolating low pathogenicity M37 of newborn infant (G ₁), 1076 (G2), MCN13 (G ₃), ST3 (G ₄) and acclimatization to cold strain W179 (G ₁) and SC2 (G ₂) etc.It is pathogenic that these strains have a potential to the infant, and the acclimatization to cold strain then causes immunogenicity relatively poor because of passage number too much.The rotavirus of also having tested animal-origin in addition prepares vaccine, as bovine rota RIT4237 (G ₆) strain, UK (G ₆) strain and WC3 (G ₆) strain and monkey rotavirus MMC18006 (G ₃) strain.Though the Rotavirus Vaccine strain toxicity of animal-origin is lower; but the vaccine strain protectiveness instability of these candidates; easy excitated people's body does not produce the neutralizing antibody at the human rotavirus; its reason may be the serotype difference of the epidemic strain of different areas, and the animal strain can not excite special-shaped protection sometimes.Therefore, though above-mentioned vaccine research early, also fails to obtain approval up to now ^[3]China's Lanzhou institute of Biological Products separates the sheep rotavirus LLR-85 (G that goes down to posterity and cultivate ₁₀Serotype) strain, after the children to 6 monthly age-6 year old once inoculated, the long positive rate of serum neutralizing antibody 4 multiplications reached about 60%.All objects of observation are not found symptoms such as heating, diarrhoea and vomiting ^[4]Its immune effect is better, has obtained first class national new drug certificate and trial production code now.The shortcoming of this vaccine be price still than higher, average family still is difficult to bear.

S-generation Rotavirus Vaccine is a multivalence reassortant vaccine, and wherein comparatively successful is monkey rotavirus (RRV) tetravalence reassortant vaccine.When monkey rotavirus and human rotavirus's while cells infected, reprovision can take place in two cover gene segments each other, screens single VP ₇Gene is wherein had only VP by the monkey rotavirus reassortant that the human rotavirus replaces ₇Gene comes from the human rotavirus, and other 10 gene segments all come from the monkey rotavirus, this strain is attenuations to the mankind, and the characteristics that the human rotavirus is difficult for incubation growth that overcome are arranged simultaneously.By a difference expressing human rotavirus G ₁, G ₂And G ₄Serotype VP ₇Reassortant and parent monkey rotavirus G ₃The VP of serotype ₇Reassortant mix, constitute tetravalence reassortant vaccine.Finding to the cacatory protection ratio of infant to be 80% in the clinical study of the U.S., Finland and Venezuela, is 48-68% to the protection ratio of general diarrhoea ^[3]This vaccine is developed by NIH (NIH), in August, 1998, U.S. Wyeth-Ayerst company obtained U.S. food and drug administration (FDA) production permit, find that after nearly 1 year 1500000 children taking children the danger of intussusception take place increase 10-14 doubly, therefore U.S.'s disease prevention and control center (CDC) advise that this vaccine stops to produce, and the vaccine of developing other form then is subjected to bigger attention.

Third generation Rotavirus Vaccine is to utilize genetic engineering technique to express corresponding viral sub-units composition, as the vaccine immunity body.As in recombinant baculovirus, expressing VP respectively ₂, VP ₆, VP ₇And VP ₄In albumen and be assembled into the virion of free nucleic acid, or express their fusion rotein respectively, the immunization experiment animal can be brought out the generation of specific antibody.In recent years, dna vaccination ^[5]With the microencapsulation vaccine ^[6]Research also obtained certain progress, but in the further work that awaits of the research aspect human rotavirus's vaccine.

A large amount of experimental studies shows that people or other Mammals can produce immune response to resist the invasion of this cause of disease after being subjected to cause of disease and infecting.This process relates to one of following three kinds of immunologic mechanisms at least: mucous membrane, body fluid or cell.Mucosal immune response produces secretor type antibody I gA, can protect respiratory tract, digestive tube, reproductive system and gland surface not to be infected.Thereby mucoantibody can stop cause of disease to be settled down at mucomembranous surface constitutes the first road defensive barrier.The generation of mucoantibody can obtain by the local immunity of secretion sexual gland body and tissue, also can produce by stimulating enteron aisle and lymphatic tissue of respiratory tract.Humoral immunization relates generally to IgG and IgM, and they participate in invading engulfing of cause of disease in blood, the viral cytotoxicity that neutralizes or complement-mediated is arranged.

The researchist has been noted that oral vaccination can induce the generation of blood and mucoantibody, and common situation is that oral vaccination needs many antigens, and this is because antigen is adsorbed by reality and the quantity utilized is lower.Therefore, the needed antigen quantity of oral vaccination is considerably beyond injection inoculation ⁽⁷⁾Yet, exception is also arranged, have found that, some albumen only need orally just can produce body fluid and mucosal immune response on a small quantity, and these albumen usually can combine with the glycolipid or the glycoprotein specificity on mucomembranous cell film surface, therefore, these albumen are also referred to as mucous membrane antigen, for example viral hemagglutinin.Oral and intramuscular injection dosage comparative experiments shows that mucous membrane class antigen also can cause humoral immunization very effectively, compares with intramuscular injection, has only slight difference between the two.Rotavirus vp ₇And VP ₄Albumen may also belong to mucous membrane antigen.

The latest developments of genetically engineered research field make the expression of plants foreign gene become possibility.Contain that the vegetable cell of foreign gene is renewable to go out whole plant, and give the offspring gene genetic.Some unifacial leaf and dicotyledons have all carried out successful conversion so far.For example tobacco, potato, tomato, soybean and corn etc.

The dicotyledons of Agrobacterium (Agrobacterium tumefaciens) mediation transforms existing numerous reports.Agriculture bacillus mediated leaf disc transformation method can carry out transgenosis, selection and regeneration effectively.

Monocotyledons is relatively more difficult through agrobacterium mediation converted, but also can utilize other method for transformation such as PEG and electro fusion method.Someone successfully imports foreign gene in the protoplastis of corn, paddy rice and wheat.Go out complete plant from the protoplast regeneration that transforms then.Method for transformation that some are new such as microtubule injection and particle bombardment may be more effective in monocotyledons transforms and regenerates.

Plant is a kind of variation, low-cost and reproducible material resources, developing rapidly of biotechnology then makes us further widen the use range of plant, in plant, adopt the method for this " molecule agricultural " abroad, successfully produced increasing useful matter, comprising some high value pharmaceutical protein polypeptide, cell growth factor as the people, erythropoietin, Interferon, rabbit, tethelin, monoclonal antibody and can be used as antigen protein that vaccine uses etc., some research institutions and company have begun to obtain huge economic benefit from the production of these pharmaceutical proteins.

It is worthy of note especially, in April, 1998, (the NationalInstitute of Allergy and Infectious Diseases of American National allergy and transmissible disease institute, NIAID) reported first can produce antibody behind the edible transgenic Rhizoma Solani tuber osi of people in the body at intestinal bacteria heat-labile toxin B subunit, have in 11 volunteers that antibody has increased by 4 times in 10 people (91%) serum, have 6 people (55%) mucoantibody also to increase by 4 times.This result of study clearly illustrates that:

(1) protein gene that efficiently expresses some cause of diseases in plant is feasible.

(2) cause of disease albumen can be finished translation post-treatment process in plant, space as glycosylation and higher structure is folding, thereby make this recombinant protein polypeptide have identical immunogenicity with native protein, this is that the prokaryotic expression system is incomparable, and the latter can't carry out glycosylation to being derived from Eukaryotic albumen.

(3) some antigen proteins that produce in the plant can not degraded by stomach en-by the digestive tube of animal safely, and reason is that they can and stimulate animal body to produce immunne response with the lip-deep glycoprotein molecule receptors bind of intestinal mucosa cells film.This proteinoid is also by appellation mucous membrane antigen (mucosalimmunogen), but not all albumen all has this ability, now only finds that minority virus and bacterium contain this antigen protein.

(4) research has confirmed that also mucous membrane antigen only needs oral a small amount of albumen, just can produce good immune response, and is big unlike the intramuscularly requirement.

These result of study indication plants will become another new protein expression system after microbial fermentation system and animal cell culture system.And utilize plant expression system production pathogenetic bacteria or virus antigen albumen to have the following advantages:

(1) safer: except that only having increased the recombinant antigen protein, do not change other genetic composition of plant in the transgenic plant, therefore, it will not contain harmful composition, has also got rid of fully and has polluted other viral possibility.

(2) price is cheaper: because it does not need purifying and refrigeration, the production of antigen protein may be to plant farm crop simply, therefore can reduce production costs greatly.

(3) use is more convenient: vaccination ways just allows simply and eats some product that contains the proteic plant edible food part of this pathogen antigen or processed by this plant, saved the injection process of trouble, this vaccination ways is highly suitable for China or other developing country.

(4) immunizing power is more lasting: people or other Mammals eat this transgenic plant at regular intervals or by some product that it processes, can make internal antibody maintain a higher level all the time, have strengthened the resistivity to communicable disease.

Existing people attempts expressing hepatitis B virus coating small protein (S) in plant, the hepatitis B surface antigen that discovery produces in plant (HBsAg) antigen has similar antigenic characteristic and physical property to HBsAg in being derived from human blood or gene engineering yeast, and and then the notion of " food vaccine " has been proposed, think can produce immune response behind the directly edible transgenic plant that contain this proteantigen of people or Mammals.For this reason, also applied for relevant patent ⁽⁸⁾Yet before the present invention, also nobody proposes to suffer from diarrhoea four common serotype G of rotavirus ₁, G ₂, G ₃And G ₄VP ₇Gene and P[4] and P[8] genotypic VP ₄Antigenic protein gene imports plant respectively or simultaneously, to produce the rotavirus oral vaccine of plant origin.Express proteic transgenic plant of a certain serotype antigen such as G ₂VP ₇, when independent oral vaccination, this serotype virus stain can be prevented, and after will expressing the proteic transgenic plant composition mixing of several serotype antigens, diarrhea virus multivalence oral vaccine might be developed, this is that other production system can't be accomplished at present.

Utilizing transgenic plant to produce one of the biggest problem that oral vaccine may run into is that external source recombinant antigen protein expression contents in transgenic plant is lower, and body can not produce and could produce immune effect after immune response maybe need eat very many transgenic plant behind this transgenic plant of infant or animal edible.Before the present invention, also nobody proposes rotavirus vp ₇Or VP ₄The codon that gene is had a preference for according to plant is optimized (nucleotide sequence changes), and the VP after optimizing ₇Or VP ₄The coded antigen protein aminoacid sequence of antigenic protein gene is without any variation, but protein expression content improves greatly, can significantly improve the immune effect behind the transgenic plant or derivatives thereof oral vaccination thus.

Although before the present invention, the part non-coding sequence (Ω sequence) that existing people utilizes tobacco mosaic virus (TMV) 5 ' end as regulating and controlling sequence to strengthen the expression level of foreign gene in plant.But nobody insert simultaneously at the goal gene two ends 5 of plant virus (being meant marmor upsilon here) ' end and 3 ' end non-coding region nucleotide sequence as regulating and controlling sequence with the enhancing rotavirus vp ₇Or VP ₄The expression efficiency of antigenic protein gene in transgenic plant combining with codon optimized measure, can further improve VP thus ₇Or VP ₄Expression contents in transgenic plant.

Before the present invention, although existing people proposes the notion of " food vaccine ", but in concrete operations, there is very big difficulty, because the recombinant antigen protein content of the expression in the plant is uncertain, and there is very big-difference between the Different Organs of plant Different Individual or same plant individual, the directly edible this transgenic plant of infant or animal will produce certain risk, the long-time edible vegetable material that contains a small amount of recombinant antigen protein can cause immunotolerance, and edible too much antigen of short period of time can cause allergy.The present invention proposes to contain the edible part of transgenic plant of diarrhoea rotavirus recombinant antigen protein or the method that its extract is processed into electuary, oral liquid or other preparation, it is quantitative to carry out strictness to wherein contained recombinant antigen protein thus, thereby, overcome the above-mentioned shortcoming that directly edible transgenic plant exist thus to people or mammiferous edible pitch time and amount clear and definite regulation having been arranged.

Also there are problems in existing human rotavirus's living vaccine at aspects such as security, protection domain, vaccine price and inoculation comfort levels; therefore; exploitation and develop new can be safely, cheapness and effectively prevent infantile diarrhea rotavirus polyvalent vaccine just to seem very necessary and urgent; utilizing transgenic plant to produce the rotavirus oral vaccine is a kind of selection preferably; it may be resisted various communicable diseases for human and Mammals a new way is provided, and its key is at first to improve recombinant antigen protein VP ₇Or VP ₄Expression contents in transgenic plant.

The invention provides and improve human rotavirus VP ₇Or VP ₄The method of antigen protein expression contents in transgenic plant and how to use the method that contains such recombinant antigen protein transgenic plant.This invention is specially adapted to China or other developing country.

The present invention utilizes edible transgenic plant to produce human rotavirus's antigen protein, and these recombinant antigen proteins are similar with native protein aspect immunogenicity.According to a preferential embodiment, the invention provides the method that improves human rotavirus's antigen protein expression contents in transgenic plant exactly and how specifically to use the method that these contain the recombinant antigen protein transgenic plant.Here human rotavirus's antigen protein of indication is a rotavirus vp ₇Or VP ₄Structural protein, plant refer to the daily edible plant of people.

The invention provides and improve human rotavirus VP ₇Or VP ₄The method of antigen protein expression contents in transgenic plant at first is coding human rotavirus VP ₇Or VP ₄The gene of antigen protein is separated, gene is after the codon of having a preference for according to plant is modified and transformed, its 5 ' end and 3 ' end add the regulating and controlling sequence that can strengthen in the plant-derived virus of expression of plants efficient respectively, to modify then and improved wheel virus antigen protein gene is inserted on the plasmid that contains selective marker, promotor is positioned at the upstream of this gene, and it handles the expression of this gene in plant.After foreign gene is expressed in each organ of plant or edible part, this part plant tissue of infant's edible.

The human rotavirus VP of indication of the present invention ₇Or VP ₄Antigen protein is meant to be produced in transgenic plant; the shortcoming of some virus antigen protein production methods such as microbial fermentation and animal cell culture before it has overcome; it utilizes transgenic plant to produce human rotavirus's antigen protein; if directly eat or be processed into electuary, oral liquid or other preparation; then have cheapness, characteristics safe and easy to use, more people is protected.According to a preferential embodiment, the infant only need eat the other parts of fruit, juice, root, stem, leaf or the plant of these transgenic plant, or utilizes electuary, oral liquid or other preparation of the preparation of these transgenic plant or transgenic plant crude extract just can obtain immunological competence to this transmissible disease in the mode of enterally administering.The plant here is meant that people are daily and eats, owing to avoided purification process, reduced production cost and eliminated some other unfavorable factor that exists simultaneously.

According to an embodiment, the present invention relates to utilize transgenic plant to produce the method that contains antigen protein electuary, oral liquid or other preparation.The transgenic plant material of producing this electuary, oral liquid or other preparation can have multiple mode, it can be complete plant, or the part of plant such as fruit, leaf, stem and stem tuber, or a part of crude extract of plant or through purifying and partial purification are derived from the virus antigen albumen of transgenic plant fully.In a word, adopt which kind of mode to depend primarily on to produce mucosal immune response to react required and thisly be derived from the antigenic dosage of transgenic plant and what of material of contained interference immune response in this composition, as carbohydrate, pyrogen and toxin etc., and reduce the generation of immunotolerance as far as possible.

The invention provides the method for in vegetable cell, producing human rotavirus's recombinant protein, can cause that under native state the people produces immune response and these albumen are known.In other words, the rotavirus vp of indication of the present invention ₇Or VP ₄Recombinant protein can be used as antigen, has and be derived from the immunogenicity of this antigen protein as yeast or bacterium of people or other conventional expression system generation.Perhaps more definite theory, antigen protein of the present invention is the same with this antigen protein that is derived from people and other conventional expression system generation, all can cause immune response by oral and mode enterally administering.According to a preferential embodiment, this human rotavirus's recombinant protein is a kind of antigen protein, and it can express generation in vegetable cell, and has and be derived from people or the similar immunogenicity of this antigen protein that other conventional expression system produced.

Antigen of the present invention is derived from the human rotavirus, and it can express generation in plant.According to a preferential embodiment, antigen is derived from human rotavirus's structural protein VP ₇Or VP ₄

Human rotavirus VP of the present invention ₇Or VP ₄Antigen can be expressed generation in plant, and express to produce this antigenic plant have at least a part be can directly eat or pass through after the roughing edible.The edible part of this kind of plant or plant should not have toxicity, and therefore, many common food plants all are included within the defined plant scope of the present invention.In addition, in some cases, as potato, also be considered to edible plants here, some part that let it be to the greatest extent is considered to deleterious (the potato ball stem portion is nontoxic, therefore belong within the claim scope of the present invention, but its fruit is deleterious), in this case, this kind of plant also should be included within the claim scope of the present invention.

Recombinant antigen protein of the present invention can be human rotavirus's natural antigen albumen VP ₇Or VP ₄Whole.According to a preferential embodiment, the VP of different serotypes ₇Antigen protein (G for example ₁, G ₂, G ₃And G ₄) or genotypic VP ₄Antigen protein (for example P[4] and P[8]) can in plant, express respectively earlier, and then partly or entirely mix according to demand to satisfy different therapeutic purpose.According to the more preferential embodiment of another one, these antigens also can 2 kinds or are two or morely expressed simultaneously in same plant, have so just saved later mixing process.In some specific embodiments, antigen also may be the part of this native protein molecule.Sometimes, antigen can be combined together to form fusion rotein with another one polypeptide or protein molecular such as bacteria pathogeny protein polypeptide.Proteic fusion can be passed through protein translation post-treatment, covalently bound mode, perhaps by the DNA recombinant technology gene is just linked together before translation.These two kinds of technology are technology that the expert of the art knows already.

In other embodiments of the present invention, the transgenic plant of one or more expressing human rotavirus recombinant antigen proteins are fabricated.For the present invention, a kind of transgenic plant should have been expressed certain virus antigen at least in the part cell of this plant.According to some preferential embodiment of the present invention, it is edible that transgenic plant of the present invention have a part at least, and its expressed antigen is a certain serotype VP of rotavirus ₇Or genotypic VP ₄, or the VP of multiple serotype ₇With genotypic VP ₄, it or they can cause immune response, particularly mucosal immune response reaction is just as the antigen protein of native protein or the generation of other expression system.

The present invention relates to a kind of composition problem of food, contained some edible part of transgenic plant in this food at least, the transgenic plant here should be able to expressing human wheel virus antigen albumen.For the present invention, certain part of plant or plant is considered to have some nutritive value, and it can provide metabolism required energy for people or other Mammals, VITAMIN and other cofactors.Although fruit does not provide energy, protein, carbohydrate and fat in a large number, with regard to specially edible by people because it contains human rotavirus's recombinant antigen protein, fruit also should be included in the claim scope of the present invention.

The present invention relates to the composition problem of a kind of electuary, oral liquid or other preparation, at least contain some edible part of transgenic plant in electuary, oral liquid or other preparation, the transgenic plant here should be able to be expressed a kind of human rotavirus's recombinant antigen protein.For the present invention, the antigen protein that contains in electuary, oral liquid or other preparation should be able to accurate quantification, to avoid producing immunotolerance behind the infants.According to a preferential embodiment of the present invention, the antigen that electuary, oral liquid or other preparation of the present invention contained is the mixture of a kind of antigen protein of human rotavirus or several antigen proteins, it can cause immune response, particularly mucosal immune response reaction is just as the antigen protein of native protein or the generation of other expression system.

As mentioned above, according to certain embodiments of the present invention, the food here will comprise the food that contains the mucous membrane antigen protein, and this mucous membrane antigen protein can combine with mucomembranous cell surface glycosylation molecular receptor, and it is a kind of human rotavirus's of being derived from a antigen protein.Therefore, according to a specific embodiment more, the food of indication of the present invention will comprise the part of transgenic plant at least, it has some nutritive value, and contain a certain serotype of human rotavirus or genotypic antigen protein, and this antigen protein can combine with the glycosylation molecular receptor on mucomembranous cell surface.Under any circumstance, food of the present invention all comprises root, stem, leaf, fruit or the said plant seed of plant.

To the relevant item with other of the method used in the present invention structure of some plasmid vectors very importantly, they play an important role in the processed and applied of transgenic plant that obtain indication of the present invention and transgenic plant.The plasmid vector that is used for transforming plant by coding a certain serotype of human rotavirus or genotypic dna sequence dna, handle the plant promoter that this dna sequence dna expresses said plant and can strengthen expression regulation sequence and form.In some specific embodiments, plasmid vector comprises that also one is selected or marker gene, is mainly used in the detection of transgenic plant or cell.In some specific embodiments, plant promoter of the present invention is the cauliflower mosaic virus 35S promoter.As mentioned above, according to some specific embodiment, plasmid vector of the present invention is used to transform edible plants, the antigen of its coding is a kind of mucous membrane antigen, furtherly, the coded antigen of plasmid vector can induce immune response reaction, particularly mucous membrane immune response, as native protein be derived from this antigen protein that other expression system produces.In a preferential embodiment, the plasmid vector that the present invention is used to transform plant contains a certain serotype VP of coding human rotavirus ₇Or genotype VP ₄The enhancing expression regulation sequence of the gene of antigen protein, plant-derived virus and a plant promoter, the proteantigen of this genes encoding can cause that immune response, particularly mucosal immune response reaction are just as native protein or be derived from this antigen protein of other expression system, it is to strengthen this genetic expression that plant-derived viral nucleotide sequences mainly acts on, and plant promoter mainly is to handle the expression of this gene in plant.In a similar embodiment, dna fragmentation provided by the invention can be used for particle bombardment and transforms plant.

The present invention also provides the method that obtains transgenic plant cells, and it may further comprise the steps: 1. make up a plasmid vector or section of DNA sequence, wherein all contain a certain serotype VP of coding human rotavirus ₇Or genotype VP ₄The gene of antigen protein adds the enhancing expression regulation sequence at these gene two ends, then above-mentioned dna sequence dna is placed under the manipulation of a plant promoter; 2. with plasmid vector or above-mentioned dna fragmentation transformed plant cells.According to a preferential embodiment, also comprise the method that goes out complete transfer-gen plant from plant transformed cell regeneration.

The invention provides the method for transformation of various vegetable cells or plant.Although only utilized specific method for transformation in the preferential embodiment of some that is described below, but it is evident that, other methods for plant transformation that the expert of the art knew already also should be included within the claim scope of the present invention, and these methods comprise that microtubule injection, PEG merge, electricity merges.According to some preferential embodiment, use be agriculture bacillus mediated method for transformation, refer in particular to the Agrobacterium transformation system that Ti-plasmids participates in.In some other preferential embodiment, can also use particle bombardment transformed plant cells or plant.

The present invention has only mentioned potato and tomato when introducing method for transformation in detail, yet as the methods for plant transformation that the expert of the art knew already, many kind of plant can utilize method provided by the invention to transform.Therefore, all these plants all should be included within the claim scope of the present invention, and these plants can be monocotyledons or dicotyledons.

Will describe in detail in following example, used plasmid vector is a binary vector system in the methods for plant transformation of the present invention.According to an embodiment, the plasmid that the present invention uses is a conformability carrier.According to a more preferential embodiment, plasmid used in the present invention is pBI121.

The invention provides and contain a kind of serotype VP of human rotavirus ₇Or genotype VP ₄The production method of antigen protein transgene food.It may further comprise the steps: 1. make up a plasmid vector or section of DNA sequence, wherein all contain an a kind of serotype VP of coding human rotavirus ₇Or genotype VP ₄The gene of antigen protein, and these gene two ends are placed under the plant promoter manipulation after having inserted the enhancing expression regulation sequence then; 2. with plasmid vector or above-mentioned dna fragmentation transformed plant cells; 3. from transgenic plant cells, bear complete transgenic plant again; 4. gather in the crops the edible part of transgenic plant; 5. edible a certain amount of this transgenic plant avoid producing immunotolerance as far as possible, to reach the effect of prevention this serotype of human rotavirus or diarrhoea that genotype is caused.

The invention provides the production method that contains multiple serotype of human rotavirus or genotype antigen protein transgene food.It may further comprise the steps: make up a plasmid vector or section of DNA sequence 1., wherein all contain the coding serotype of human rotavirus more than 2 or 2 or the gene of genotype antigen protein, the two ends of gene all are inserted with plant virus and strengthen expression regulation sequence, and these genes are respectively placed under different plant promoters or the manipulation of same plant promoter; 2. with plasmid vector or above-mentioned dna fragmentation transformed plant cells; 3. from transgenic plant cells, bear complete transgenic plant again; 4. gather in the crops the edible part of transgenic plant; 5. will contain human rotavirus's different serotypes or genotype antigen protein plant edible food part equivalent or inequality mixes; 6. edible a certain amount of this transgenic plant mixture is to reach the effect of prevention human rotavirus diarrhoea that multiple serotype is caused.

The present invention also provides the human rotavirus a kind of serotype VP ₇Or genotype VP ₄The production method of electuary, oral liquid or other preparation that antigen protein content is determined, it may further comprise the steps: make up a plasmid vector or section of DNA sequence 1., the gene that wherein all contains coding a kind of serotype of human rotavirus or genotype antigen protein, the two ends of gene are placed under the plant promoter manipulation after inserting the regulating and controlling sequence that strengthens expression; 2. with plasmid vector or above-mentioned dna fragmentation transformed plant cells; 3. from transgenic plant cells, bear complete transgenic plant again; 4. in transgenic plant cell or complete plant, extract antigen protein and be processed into electuary, oral liquid or other preparation.According to a preferential embodiment, complete transgenic plant or its some edible part of containing antigen protein can directly be carried out freezing, dry and pulverizing, are processed into electuary, oral liquid or other preparation then, and determine the wherein content of antigen protein; 5. edible a certain amount of this electuary, oral liquid or other preparation avoid producing immunotolerance as far as possible, to reach the effect of prevention human rotavirus diarrhoea that this serotype is caused.

The present invention also provides the production method of electuary, oral liquid or other preparation that the multiple serotype of human rotavirus or genotype antigen protein content determines, it may further comprise the steps: make up a plasmid vector or section of DNA sequence 1., the gene that wherein all contains coding a kind of serotype of human rotavirus or genotype antigen protein, the two ends of gene are placed under the plant promoter manipulation after inserting the regulating and controlling sequence that strengthens expression; 2. with plasmid vector or above-mentioned dna fragmentation transformed plant cells; 3. from transgenic plant cells, bear complete transgenic plant again; 4. in transgenic plant cell or complete plant, extract antigen protein; 5. will contain human rotavirus's different serotypes or genotype antigen protein extract equivalent or inequality and mix, and then be processed into electuary, oral liquid or other preparation.According to a preferential embodiment, complete transgenic plant or its some edible part of containing antigen protein can directly be carried out freezing, dry and pulverizing, are processed into electuary, oral liquid or other preparation then, and determine the wherein content quantity of antigen protein; 6. edible a certain amount of this electuary, oral liquid or other preparation avoid producing immunotolerance as far as possible, to reach the effect of prevention human rotavirus diarrhoea that multiple serotype is caused.

In order to describe some preferential embodiment of the present invention in detail, and for referencial use with corresponding drawing:

Fig. 1 rotavirus vp ₇And VP ₄The clone of gene.

Fig. 2 A rotavirus G ₂VP ₇Nucleotide sequence sequence before and after genetic modification and the modification relatively.The natural rotavirus serotype G that the N representative obtains by the RT-PCR amplification ₂VP ₇Gene; M represents the rotavirus MG of complete synthetic ₂VP ₇Genes encoding part and plant virus regulating and controlling sequence, sequence total length 1209bp, coding region totally 978 Nucleotide wherein, 326 amino acid of encoding, modify the back and changed 221 Nucleotide altogether, see to mark part in the sequence, horizontal line represents not change sequence, and sequence is artificial synthetic marmor upsilon 5 ' end and 3 ' end non-coding region (enhancing expression regulation sequence) in the framework; The underscore sequence is represented BamHI or SacI point of contact; Natural G ₂VP ₇Gene G+C% content is 31.3% (306/978), modifies back MG ₂VP ₇Gene G+C% content is 53.9% (527/978); The aminoacid sequence of codon optimized front and back does not change.

Fig. 2 B rotavirus G ₁VP ₄Nucleotide sequence sequence before and after genetic modification and the modification relatively.The natural rotavirus serotype G that the N representative obtains by the RT-PCR amplification ₁VP ₄Gene; M represents the rotavirus MG of complete synthetic ₁VP ₄Genes encoding part and plant virus regulating and controlling sequence, the sequence total length is 2559bp, coding region totally 2325 Nucleotide wherein, 776 amino acid of encoding, modify the back and changed 510 Nucleotide altogether, see to mark part in the sequence, horizontal line represents not change sequence, and sequence is artificial synthetic marmor upsilon 5 ' end and 3 ' end non-coding region (enhancing expression regulation sequence) in the framework; The underscore sequence is represented BamHI or SacI point of contact; Natural G ₁VP ₄Gene G+C% content is 32% (306/978), modifies back MG ₁VP ₄Gene G+C% content is 53.9% (527/978); The aminoacid sequence of codon optimized front and back does not change.

Modified and the improved rotavirus vp of Fig. 3 ₇And VP ₄The clone of gene.L represents marmor upsilon 5 ' end parts non-coding area sequence; Tail represents marmor upsilon 3 ' end parts non-coding area sequence; MG ₂VP ₇Represent the G after codon optimized ₂VP ₇Gene; MG ₁P[8] VP ₄Represent the G after codon optimized ₁P[8] VP ₄Gene.

Fig. 4 pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇And pBMG ₁VP ₄The plant expression vector construction synoptic diagram.GUS represents β-Glucuronidase (glycuronidase) gene

Fig. 5 pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇And pBMG ₁VP ₄Expression of plants carries structural representation.N-Pro represents rouge alkali synthetase promoter; NptII represents neomycin phosphotransferase gene; N-Ter represents the rouge alkali synthetase terminator; 35S-Pro represents the cauliflower mosaic virus 35S promoter; L represents marmor upsilon 5 ' end non-coding region partial sequence; T represents marmor upsilon 3 ' end non-coding region partial sequence.G ₂VP ₇And G ₁VP ₄Represent the G of unmodified and transformation ₂VP ₇And G ₁VP ₄Gene; MG ₂VP ₇And MG ₁VP ₄The G of representative after codon optimized ₂VP ₇And G ₁VP ₄Gene.

G in Fig. 6 potato plant blade ₂VP ₇, G ₁VP ₄, MG ₂VP ₇And MG ₁VP ₄The comparison of the content of mRNA.Fig. 6 A1 and 6B1 are negative control potato RNA; Fig. 6 A2 is pBG ₂VP ₇Transform the transgenic Rhizoma Solani tuber osi RNA that the back obtains; Fig. 6 A3 is pBMG ₂VP ₇Transform the transgenic Rhizoma Solani tuber osi RNA that the back obtains; Fig. 6 B2 is pBG ₂VP ₇Transform the transgenic Rhizoma Solani tuber osi RNA that the back obtains; Fig. 6 B3 is pBMG ₂VP ₇Transform the transgenic Rhizoma Solani tuber osi RNA that the back obtains;

G in the different transgenic potato plant blades of Fig. 7 ₂VP ₇And G ₁VP ₄Recombinant antigen protein content.1 and 2 negative control plants; 3 is pBMG ₂VP ₇Transform the potato transfer-gen plant that the back obtains; 4 is pBG ₂VP ₂Transform the potato transfer-gen plant that the back obtains; 5 is pBMG ₁VP ₄Transform the potato transfer-gen plant that the back obtains; 6 is pBG ₁VP ₄Transform the potato transfer-gen plant that the back obtains.

Fig. 8 PCR detects G ₂VP ₇, G ₁VP ₄, MG ₂VP ₇And MG ₁VP ₄The integration situation of gene in different tomatoes.Fig. 8 AM is the standard DNA molecular weight; Fig. 8 A1 is pBG ₂VP ₂Transform the amplified production of the transgenic Fructus Lycopersici esculenti of back acquisition; Fig. 8 A2 is pBMG ₂VP ₂Transform the amplified production of the transgenic Fructus Lycopersici esculenti of back acquisition; The amplified production of the negative control plant of Fig. 8 A3; Fig. 8 A4 is with pG ₂VP ₂Plasmid is made the amplified production (positive control) of template.Fig. 8 BM is the standard DNA molecular weight; Fig. 8 B1 is pBG ₁VP ₄Transform the amplified production of the transgenic Fructus Lycopersici esculenti of back acquisition; Fig. 8 B2 is pBMG ₁VP ₄Transform the amplified production of the transgenic Fructus Lycopersici esculenti of back acquisition; The amplified production of the negative control plant of Fig. 8 B3; Fig. 8 B4 is with pG ₁VP ₄Plasmid is made the amplified production (positive control) of template.

G in Fig. 9 tomato plant blade ₂VP ₇, G ₁VP ₄, MG ₂VP ₇And MG ₁VP ₄The comparison of the content of mRNA.Fig. 9 A1 and 9B1 are the negative control tomato RNAs; Fig. 9 A2 is pBG ₂VP ₇Transform the transgenic Fructus Lycopersici esculenti RNA that the back obtains; Fig. 9 A3 is pBMG ₂VP ₇Transform the transgenic Fructus Lycopersici esculenti RNA that the back obtains; Fig. 9 B2 is pBG ₂VP ₇Transform the transgenic Fructus Lycopersici esculenti RNA that the back obtains; Fig. 9 B3 is pBMG ₂VP ₇Transform the transgenic Fructus Lycopersici esculenti RNA that the back obtains.

G in the different transgenic Fructus Lycopersici esculenti plant leafs of Figure 10 ₂VP ₇And G ₁VP ₄Recombinant antigen protein content.1 and 2 negative control plants; 3 is pBMG ₂VP ₇Transform the tomato transfer-gen plant that the back obtains; 4 is pBG ₂VP ₂Transform the tomato transfer-gen plant that the back obtains; 5 is pBMG ₁VP ₄Transform the tomato transfer-gen plant that the back obtains; 6 is pBG ₁VP ₄Transform the tomato transfer-gen plant that the back obtains.

Figure 11 rotavirus G ₂VP ₇The stability of recombinant antigen protein in water is measured.Contain G ₂VP ₇After the potato tuber of recombinant antigen protein (0.1g) ground in the 400ul phosphoric acid buffer, (15-20 ℃) placed after 1-7 days under the room temperature, measured OD after getting 100ul supernatant liquor enzyme linked immunoassay ₄₅₀The nm absorbance value.

Figure 12 rotavirus G ₂VP ₇Recombinant antigen protein is restrained oneself degree to high temperature in water.Contain G ₂VP ₇After the potato tuber of recombinant antigen protein (0.1g) grinds in the 400ul phosphoric acid buffer, under differing temps, place after 3 hours, measure OD after getting 100ul supernatant liquor enzyme linked immunoassay ₄₅₀The nm absorbance value.

The G that Figure 13 goes out through affinitive layer purification ₂VP ₇And G ₁VP ₄The PAGE of recombinant antigen protein analyzes.1 is the protein standard molecular weight; 2 is from transgenic Fructus Lycopersici esculenti (pBM G through affinity chromatography ₁VP ₄Conversion) G that is purified in the blade ₁VP ₄Recombinant antigen protein; 3 is transgenic Fructus Lycopersici esculenti (pBM G ₁VP ₄Transform) the blade total protein; 4 negative contrast tomato leaf total proteins; 5 is from transgenic Rhizoma Solani tuber osi (pBM G through affinity chromatography ₂VP ₇Conversion) G that is purified in the stem tuber ₂VP ₇Recombinant antigen protein; 6 is transgenic Rhizoma Solani tuber osi (pBM G ₂VP ₇Transform) the stem tuber total protein; 7 negative contrast potato tuber total proteins; 8 is the contained albumen of rotavirus plastochondria (positive control).

The present invention includes following integral part: 1. utilize the DNA recombinant technology to make up efficient plasmid expression vector, it contains wheel virus antigen protein gene VP ₇Or VP ₄The plant virus regulating and controlling sequence of or derivatives thereof and reinforcing gene expression, the infant might produce the suffer from diarrhoea specificity neutralizing antibody of rotavirus at the people behind the antigen protein of edible these genes encodings; 2. select suitable recipient plant to transform, be integrated in the karyomit(e) of recipient plant with making the proteic stable gene of coding for antigens and also can entail the offspring; 3. from the recipient plant cell that transforms, bear complete transgenic plant again, and wheel virus antigen albumen can be stablized, be produced efficiently to this plant; The directly edible this transgenic plant of infant or be main the active ingredient electuary, oral liquid or other preparation that process so that the mode of enterally administering is oral with these transgenic plant after, might prevent a kind of serotype or multiple serotype people the infecting of rotavirus of suffering from diarrhoea.

The invention provides and efficiently express wheel virus antigen albumen VP ₇Or VP ₄The construction process of its derivative transgenic plant, this transgenic plant might be caused infant's immunne response as food or after being processed into electuary, oral liquid or other preparation by infants.The result of this immunne response makes the infant have resistivity to the suffer from diarrhoea infection of rotavirus of people.This immunne response is the result who produces behind the wheel virus antigen albumen owing to efficiently expressing in transgenic plant, and rotavirus vp ₇Or VP ₄The generation of antigen protein then is to strengthen the result that can stablize, express efficiently under the common regulation and control of expressed sequence because these antigenic protein genes are integrated in the genome of plant and in promotor and plant virus.

1. utilize transgenic plant to produce rotavirus vp ₇Or VP ₄Antigen protein

In fact, before modern medicine produced, people were just conscious or utilize some natural components in plant or the plant to cure human diseases unconsciously.

In recent ten years, the progress along with biotechnology, special plant genetic engineering field makes the mankind obtain to handle the ability that foreign gene is expressed in plant.1992, American C.J.Arntzen and H.S.Mason took the lead in having proposed to produce with transgenic plant the new approaches of vaccine ^[9], this thinking has been facilitated the rise that utilizes transgenic plant to produce vaccine research.After this, hepatitis B surface antigen has been expressed in succession in domestic and international a plurality of laboratory in tobacco, potato, tomato, clover and lettuce ^[9,10], the coli heat-sensitive toxin B subunit ^[11,12], b subunit of cholera toxin ^[13,14,15], the norwalk virus coat protein ^[16]With antigens such as rabies virus G proteins ^[17], and utilize the antigen of in plant, expressing to carry out animal and human's immunization experiment, and obtained a large amount of valuable datas, established good basis (seeing Table 1) for utilizing transgenic plant to produce vaccine from now on.

The humans and animals vaccine antigen protein that table 1 utilizes transgenic plant to produce ^[18]

Transmissible disease cause of disease target gene transgenic plant immunization ways conceptual phase

Cub diarrhoea enteritis virus spinous process protein maize oral vaccination is grinding aftosa Foot-and-mouth disease VP1 arabidopsis, the outer membrane glycoprotein G tomato of the hemorrhage virus structural protein VP60 of clover oral vaccination preclinical phase rabbit hemorrhage rabbit potato injection inoculation preclinical phase rabies lyssaviruse--preclinical phase diarrhoea norwalk virus coat protein potato oral vaccination preclinical phase carious tooth Streptococcus mutans monoclonal antibody tobacco local application of property bleb human nature herpesviral monoclonal antibody soybean local application preclinical phase hepatitis B HBsAg potato, albumen potato in the tobacco oral vaccination I phase clinical lettuce hepatitis B hepatitis B film, tomato oral vaccination preclinical phase cholera comma bacillus b subunit of cholera toxin potato oral vaccination preclinical phase diarrhoea toxin producing coli heat-sensitive toxin B subunit potato oral vaccination I phase clinical diarrhoea toxin producing coli heat-sensitive toxin B subunit corn oral vaccination preclinical phase pulmonary tuberculosis tubercle bacillus secretory protein MPT64 carrot--

The invention provides and significantly improve the infantile diarrhea rotavirus vp ₇And VP ₄The method of antigen protein expression contents in transgenic plant, in addition, the coding rotavirus vp ₇Or VP ₄The gene of antigen protein also can be used in the present invention after through disappearance or variation.For example, expression vector contains through the wheel virus antigen protein gene of modifying, and the result causes the one or more amino-acid residues of antigen protein to change, thereby has also changed the immunological competence of this antigen protein.Expression vector also can contain successive 2 or 2 above different serotypes or genotypic wheel virus antigen albumen coded sequence simultaneously, these genetic expressions form a big fusion rotein, not only change the immunogenicity of antigen protein, also saved later hybrid process process.Expression vector also can only contain rotavirus vp ₇Or VP ₄The part encoding sequence of antigen protein, the result only produces a bit of polypeptide or as the sub-fraction of fusion rotein, in this case, they also belong within the claim scope of this patent.

The present invention not only allows to produce a kind of rotavirus vp in a kind of plant ₇Or VP ₄Antigen protein, and can in a kind of plant, produce multiple rotavirus vp simultaneously ₇Or VP ₄Antigen protein.Gene constructed on same expression vector with multiple antigen protein utilizes it to transform plant then, thereby can express multiple wheel virus antigen albumen simultaneously in a transgenic plant.In addition, transgenic plant also can obtain by the method that repeatedly transforms or transform simultaneously with a plurality of expression vectors that contain different antigenic protein genes simultaneously.Rotavirus has 4 different serotypes and 2 different genotype at least, and each serotype or genotypic antigen protein are different.According to a preferential embodiment, in a kind of plant, express a kind of serotype or genotypic antigen protein, the transgenic plant equivalent or the inequality that will contain different serotypes or genotype antigen protein then mix, after being reprocessed into electuary, oral liquid and other preparation, can resist the infection of different virus hypotype after eating simultaneously.According to not preferential embodiment, be in a kind of transgenic plant, to express these several different serotypes or genotypic antigen protein simultaneously, edible this transgenic plant or be the major ingredient electuary, oral liquid and other preparation that process with these transgenic plant after, also can resist different virus serotype or genotypic infection simultaneously.

2. improve the method for foreign gene at expression of plants content

Significantly improve foreign gene and comprise modification and the transformation of selecting plant strongly expressed promotor, using the enhancing expression regulation sequence and carry out goal gene according to the codon that plant is had a preference in the method for transgenic plant expression contents plant.

The codon that the present invention at first has a preference for according to plant, foreign protein genes is optimized, under the precursor that does not change the foreign protein aminoacid sequence, improved the content of G+C% in the gene, make it more near the ratio of plant autogene G+C%, can be more stable when transcribing formation mRNA.

5 of plant virus justice RNA ' end non-coding region and 3 ' end non-coding region to its superpowerly in vegetable cell transcribe, duplicate, stability and translation play important effect.Therefore, carry out codon optimized to goal gene removing, the non-coding sequence that the present invention also proposes to add plant virus 5 ' end and 3 ' end at the goal gene two ends can further improve the expression contents of foreign gene in plant as strengthening the regulating and controlling sequence of goal gene in expression of plants efficient.According to preferential scheme of implementing of the present invention, marmor upsilon 5 ' end and 3 ' end parts non-coding region are selected as strengthening the regulating and controlling sequence of expressing.

3. the selection of recipient plant

Utilize different foreign DNA transfer techniques, now transformed various plants, comprise many dicotyledonss and some monocotyledonss.Consider the complexity of genetic transformation operation, the present invention is more prone to use dicotyledons as recipient plant, although some monocotyledons such as wheat, corn and paddy rice may be more suitable for as people's food or be processed into electuary, oral liquid or other preparation.

Select recipient plant to carry out genetic transformation and will consider whether antigen protein can express in its edible part.Because, if after antigen protein is expressed in certain part reality, leaf, stem, seed or the root of plant, the plant tissue of direct-edible this part of people or after processing, make electuary, oral liquid or other preparation is oral for people.According to not preferential embodiment, antigen protein also can be expressed in the plant that anorexia is used, and therefrom is purified into required antigen protein then fully and is processed into electuary, oral liquid and other preparation.

When selecting recipient plant, also to consider some other factor.The edible part of recipient plant preferably need not heat just edible, because heating can cause the antigen protein sex change, thereby reduces its immunogenicity.In addition, because wheel virus antigen albumen VP ₇And VP ₄The generation of the easiest induce immune response reaction when the people just is born, therefore, the mode that the plant part that contains these antigen proteins preferably can be processed into liquid is drunk.

The recipient plant that is suitable for the present invention's conversion comprises dicotyledons and the monocotyledons that all can be used as human or animal's food, part or all edible of plant, these plants comprise potato, tomato, Radix Dauci Sativae, lettuce, cucumber, wheat, soybean, paddy rice, corn, banana and papaya etc., but are not limited only to this.

4. methods for plant transformation

There are many kinds of methods foreign gene can be imported monocotyledons and dicotyledons.The main method of the foreign gene stable integration being gone into the plant chromosome genomic dna comprises: 1) agrobacterium-mediated transformation; 2) dna direct is taken in method, comprises protoplasma fusion method, electrization, microtubule injection, the pollen tube passage method of PEG mediation and uses particle gun that dna direct is sent into vegetable cell and tissue etc.

Agrobacterium-mediated transformation is to utilize plasmid vector that specific dna fragmentation is integrated into the plant chromosome genomic dna.The method for transformation of plant tissue is different because of plant and Agrobacterium system.The most frequently used method is Ye Panfa, and it is applicable to any plant explants that is easy to the regeneration whole plant.Conversion method for agrobacterium is specially adapted to the conversion of dicotyledons.

Take in method for transformation as the top various dna directs of having mentioned, electrization is protoplastis to be placed to be placed on a highfield earlier, under galvanic action foreign DNA is sent in the protoplastis.The microtubule injection is with microtubule dna direct to be injected in the cell.Particle bombardment is that DNA is adsorbed on earlier on sal epsom or the tungsten particle, and these particles are accelerated to be injected in vegetable cell or the tissue.

According to a preferential embodiment of the present invention, Agrobacterium Ti-plasmids system is selected.Contain one section in the Ti-plasmids of Agrobacterium and be called transfer DNA (T-DNA), it can enter in the vegetable cell and be integrated in the karyomit(e) of plant.The structure of transfer vector was divided into for two steps, at first was to make up the plasmid that can duplicate in intestinal bacteria, and this plasmid contains rotavirus vp ₄Or VP ₇Antigenic protein gene (VP ₇Be meant G here ₁, G ₂, G ₃Or G ₄The VP of serotype ₇Albumen, VP ₄Be P[4] or P[8] genotypic VP ₄Albumen); The T-DNA border sequence is contained at the two ends of antigen gene, and it is responsible for the insertion site of goal gene in Plant Genome.Usually another one selected marker gene (as kalamycin resistance gene) also is comprised in the left and right sides border sequence of T-DNA, can provide a selective marker to confirm whether plant or vegetable cell contain the T-DNA sequence of integration after this gene is expressed in vegetable cell.Second step of vector construction is that plasmid is transferred to the Agrobacterium from intestinal bacteria.This can finish by the mode or the dna direct lead-in mode of triparental mating.For the transfer of T-DNA, also to contain a cover induced gene in the used agrobacterium strains, they are transferred in the vegetable cell at T-DNA and play an important role.

The people who is familiar with the plant genetic conversion will be appreciated that the agrobacterium strains that transforms usefulness can have multiple choices, and plasmid construction also has multiple mode, and its purpose all is in order more to help the genetic transformation of plant.Equally, people also will be appreciated that except agrobacterium tumefaciens in some cases, to also have other Agrobacterium such as root of hair type Agrobacterium to be more suitable for the conversion of some plants.

The method for transformation of plant tissue is different because of plant and agriculture bacillus mediated system.The most frequently used is the c4 plant leaf discs conversion method, and it is suitable for the multiple regenerated plant explants that is easy to.In some cases, also need to add some helpers.Other method for transformation comprises protoplast transformation, goes out vegetable cell and whole plant by protoplast regeneration then.

The present invention is not limited only to use Agrobacterium Ti-plasmids conversion system, should comprise that also the means of other physical property directly import foreign gene method and other method with exogenous gene transfered plant cell of vegetable cell or protoplastis.

5. genetic expression promotor

After recipient plant and method for transformation are determined, need to select a composing type, grow expression adjustment type or the organizing specific type and start and give just foreign gene and can in plant, express.

All are known can handle the promotor that foreign gene transcribes and can use in the present invention in vegetable cell.These promotors can obtain from plant or virus, as cauliflower mosaic virus 35S promoter (comprise through splicing, double and the improved 35S promoter that suddenlys change); Photoinduction gene promoter such as ribose bisphosphate carboxylase small subunit (rbcS); Adversity inducible gene such as ethanol dehydrogenase (Adh1) or maize actin gene promoter etc., but be not limited only to this.The present invention also can utilize known promotor such as the potato plastogene promotor that efficiently expresses at the plant edible portion.

The plasmid that is used for Plant Transformation contains a selected marker gene usually.Many have the gene of this function to be developed.

Below with transgenic Rhizoma Solani tuber osi and tomato production wheel virus antigen albumen VP ₇Or VP ₄Be example, describe some preferential embodiments, but application of the present invention be not limited only to this.

Selecting potato and tomato is to illustrate for example in the different piece of plant to express wheel virus antigen albumen VP as recipient plant ₇Or VP ₄

Embodiment 1

1. rotavirus vp ₇And VP ₄The structure of antigenic protein gene high-efficiency plant expression vector

As shown in Figure 1, rotavirus serotype G ₁And G ₂(or G ₃And G ₄Serotype) nucleic acid is separated as masterplate from infantile rotavirus enteritis patient's ight soil or artificial culture thing (the CDC virus disease provides), according to each serotype DNA nucleotide sequence of known human rotavirus ⁽¹⁹⁾Synthetic two pairs of specific primers.Amplification G ₂VP ₇Gene, 5 ' end primer sequence of use is 5 '-A GGATCCATGTACGGTTATGAATATACCACAATTCTAAC-3 ', 3 ' end primer sequence is 5 '-A GAGCTCCTAAATTCTATAAAACGCAGCTGTGTC-3 '.Amplification G ₁P[8] VP ₄Gene, 5 ' end primer of use is 5 '-A GGATCCATGGCTTCACTCATTTATAGACAGCTTCTCAC-3 ', 3 ' end primer is 5 '-A GAGCTCTCACAATTTACATTGTAGAATTAACTGCTCAATTC-3 '.Obtained rotavirus serotype G respectively by trans polymerase chain reaction (RT-PCR) amplification ₂VP ₇Gene and genotype G ₁P[8] VP ₄Gene (is annotated: if with rotavirus serotype G ₂Nucleic acid be template, utilize above-mentioned two pairs of specific primers, can obtain G respectively by RT-PCR ₂The VP of serotype ₇Gene and genotype P[4] VP ₄Gene; G ₃Can obtain G ₃Serotype VP ₇Gene and genotype P[8] VP ₄Gene; G ₄Can obtain G ₄The VP of serotype ₇Gene and genotype P[8] VP ₄Gene).Amplified fragments directly is inserted in pGEM-T (Promega company) plasmid, obtains PG ₂VP ₇And PG ₁VP ₄Two plasmid vectors then, by nucleotide sequence analysis, are determined rotavirus G ₂VP ₇And G ₁VP ₄Antigenic protein gene whether correct and complete (Fig. 2 A, 2B).

In order to improve rotavirus G ₂VP ₇And G ₁VP ₄The expression contents of gene in transgenic plant taked following three kinds of measures: 1) rotavirus G ₂VP ₇And G ₁VP ₄Gene is modified and is transformed by the codon of having a preference for according to plant respectively, improved MG ₂VP ₇Gene order G+C% content brings up to 53.9%, MG by 31.3% ₁VP ₄Bring up to 53.9% by 32%, gene order after the change and codon are seen Fig. 2 A and 2B; 2) at rotavirus MG ₂VP ₇And MG ₁VP ₄Increase by one section nucleotide sequence (seeing Fig. 2 A and the 2B) conduct that is derived from marmor upsilon (PVY) 5 ' end non-coding region before the gene start codon ATG respectively and strengthened expression regulation sequence; 3) at rotavirus MG ₂VP ₇And MG ₁VP ₄Increase by one section nucleotide sequence (seeing Fig. 2 A and the 2B) conduct that is derived from marmor upsilon 3 ' end non-coding region behind gene terminator codon TAG or the TGA and strengthened expression regulation sequence.Rotavirus MG ₂VP ₇And MG ₁VP ₄The optimization of gene codon and finish by Takara Biotech company with 5 ' end and 3 ' end and the fusion work of PVY nucleotide sequence.Improved MG ₂VP ₇And MG ₁VP ₄Gene is respectively inserted in pGEM-T (Promega company) plasmid, obtains pMG ₂VP ₇And pMG ₁VP ₄Two plasmid vector (see figure 3)s.

Because in primer 5 ' end, introduced Bam HI site, in primer 3 ' end, introduced the SacI site, so can be with rotavirus vp with Bam HI and Sac I double digestion ₇Or VP ₄Antigenic protein gene is respectively from PG ₂VP ₇And pG ₁VP ₄Scale off on two plasmid vectors, separate to obtain little dna fragmentation, be inserted into subsequently that (Bam HI and Sac I double digestion) just is built into two binary expression vector pBG respectively on the former gus gene of the plasmid pBI121 site by agarose gel electrophoresis ₂VP ₇And pBG ₁VP ₄(Fig. 4), their expression contents in plant in fact will be as object of reference to determine goal gene VP ₇Or VP ₄Modified having or not with transformation back expression contents in transgenic plant significantly improves.。

At artificial reconstructed rotavirus MG ₂VP ₇And MG ₁VP ₄Gene and when add strengthening expression regulation sequence (plant-derived virus) is held at the 5 ' end and 3 ' of this nucleotide sequence and also have been added BamHI and SacI site (see figure 3) respectively, so can be with improved rotavirus MG with Bam HI and Sac I double digestion ₂VP ₇Or MG ₁VP ₄Antigenic protein gene is respectively from pMG ₂VP ₇And pMG ₁VP ₄Scale off on above-mentioned two plasmid vectors, separate to obtain little dna fragmentation, be inserted into subsequently that (Bam HI and Sac I double digestion) just is built into two binary expression vector pBMG respectively on the former gus gene of the plasmid pBI121 site by agarose gel electrophoresis ₂VP ₇And pBMG ₁VP ₄(Fig. 4).

Plasmid pBI121 is available from U.S. Clonetech company, and its Bam HI and Sac I restriction enzyme site lay respectively between cauliflower mosaic virus 35S promoter and the gus gene initiation codon and between gus gene terminator codon and the NOS terminator.Select for use plasmid pBI121 to be because gus gene can be deleted, and can insert the another one protein gene subsequently with Bam HI and Sac I double digestion.New gene can be expressed in vegetable cell.Plasmid pBI121 also contains neomycin phosphotransferase II gene (NPT II), and the enzyme of its coding can be vegetable cell provides kalamycin resistance, thereby cell and the tissue that contains T-DNA can be screened.NPT II gene has promotor and the poly gland acid signal sequence of oneself, and they are derived from nopaline (Nopaline) synthetic enzyme (NOS) gene.

Plasmid pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇And pBMG ₁VP ₄Contain respectively: 1) Xin Meisu synthase gene II (NPT II), it offers the vegetable cell kalamycin resistance; 2) wheel virus antigen protein gene and handle the cauliflower mosaic virus 35S promoter of this gene; 3) T-DNA left and right sides border sequence, it can be with NPTII gene and the transgenosis of wheel virus antigen protein gene in plant materials and be incorporated in the plant chromosome; 4) at pBMG ₂VP ₇And pBMG ₁VP ₄In, at MG ₂VP ₇Or MG ₁VP ₄Plant virus (PVY) regulating and controlling sequence of energy reinforcing gene expression is also contained at the gene two ends.PBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇And pBMG ₁VP ₄Structure as shown in Figure 5.

2. plant expression vector is imported Agrobacterium (A.tumefaciens)

Contain rotavirus G ₂VP ₇, G ₁VP ₄, MG ₂VP ₇Or MG ₁VP ₄The plasmid pBG of antigenic protein gene ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄Transferred in the Agrobacterium LBA4404 bacterial strain by the mode of triparental mating respectively, this bacterial strain is available from U.S. Clonetech company.It is that it contains complete Vir gene because it is an improved Agrobacterium that bacterial strain LBA4404 now has been widely used, but T-DNA is deleted.The Vir gene can trans adjusting T-DNA from plasmid pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄Transfer in vegetable cell.

Agrobacterium contain 25mg/L Streptomycin sulphate 50ml YEP (peptone 10 gram, yeast extract 10 grams, NaCl 5 grams, adding distil water to 1 liter, pH7.0) shaking culture in the substratum is at OD _600nmWhen value reached 0.4-0.7, the centrifugal collection bacterial cell of 4000g was suspended in 1ml again with agrobatcerium cell and does not contain in any antibiotic YEP substratum stand-by.Contain expression vector pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄DH5 α (available from U.S. GIBCO company) and the HB101 (available from U.S. Clonetech company) that contains helper plasmid pRK2013 (available from U.S. Clonetech company) be seeded in respectively and contain 50mg/L kantlex 50mlLB (peptone 5 grams, yeast extract 10 grams, NaCl 10 grams, adding distil water to 1 liter, pH7.0) shaking culture in the substratum is at OD _600nmWhen value reached 0.4-0.7, the centrifugal collection bacterial cell of 4000g was suspended in cell respectively 1ml then and does not contain in any antibiotic LB substratum stand-by.Get above-mentioned three kinds of each 200ul of cell suspending liquid, place same 1.5ml centrifuge tube, 28 ℃ leave standstill overnight incubation, get this culture 5-10ul, evenly spread upon on the YEP solid medium flat board, contain 25mg/L Streptomycin sulphate and 50mg/L kantlex in this substratum simultaneously, cultivate after 2 days, contain pBG for 28 ℃ ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄The Agrobacterium bacterium colony of plasmid begins to produce.

From Agrobacterium, extract plasmid DNA with alkaline lysis, and can detect pBG with digestion with restriction enzyme ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄Whether plasmid DNA exists.

3. agriculture bacillus mediated plant genetic transforms

The genetic transformation of plant at first is that plant tissue organ or cell and Agrobacterium are cultivated altogether, after cultivating about 2 days, the plant explants dislocation is selected in the substratum accordingly.Plant explants can be protoplastis, callus or other organ-tissue, because of plant different.Selecting the organ of band leaf for use is the most frequently used method.

Blade transforms main method according to people such as Horsch ⁽²⁰⁾[kind is No. 3, middle potato to the potato aseptic seedling, Vegetable ﹠ Flower Inst., Chinese Academy of Agriculture Science provides] be kept in 22 ℃ of illumination boxs, be aided with under the medium intensity of illumination, every 3 week clip top young shoot, again cuttage is not in containing any antibiotic MS solid medium, and the blade in 2 weeks of growing is suitable for transforming most.Tomato (kind is middle vegetables No. 5, and Vegetable ﹠ Flower Inst., Chinese Academy of Agriculture Science provides) then transforms with the cotyledon or the hypocotyl aseptic explant of 7-10 age in days.

No matter potato still is a tomato all is converted into best with blade.Contain expression vector pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄Agrobacterium LBA4404 be seeded in the 50ml YEP substratum (containing 50mg/ml kantlex and 25mg/ml Streptomycin sulphate) 28 ℃ and cultivated 2 days, 4000g collected thalline in centrifugal 5 minutes, with 25ml liquid MS nutrient solution (not containing microbiotic) washing once, be suspended into OD again with MS again _600nm=0.2-0.4.To there be in the Agrobacterium nutrient solution after the blade of wound immerses dilution several minutes, blot bacterium liquid unnecessary on the blade, and then the blade dislocation not contained on any antibiotic regeneration culture medium 23-25 ℃ and cultivated altogether 2 days with aseptic filter paper.Cultivate on the selection substratum that the blade dislocation is fresh, contain 100-200ug/ml kantlex and 500ug/ml Pyocianil in this substratum, later per two weeks are changed a subculture.When blade wound callus has young shoot to form and grows to when enough big, young shoot cutting-out (usually in 6-10 week after the conversion) is transferred on the root media and grown.After root occurs, will grow under the regeneration plant dislocation aseptic condition or transfer in the soil, the growth down of the temperature that suits, illumination and nutritional condition.

4. express the screening of wheel virus antigen protein gene engineering plant

After transforming about four months (tomato produces fruit and needs 10 months).Can detect transfer-gen plant and whether contain wheel virus antigen albumen VP ₇And VP ₄

1) biological chemistry and immunology detection

Blade, stem, stem tuber or fruit collected specimens from transgenic plant.And sample is cushioned liquid (1.59g Na at bag ₂CO ₃, 2.93g NaHCO ₃, adding distil water to 1 liter) in grind with the ratio of 1: 2 (W/V) after, homogenate centrifugal 10 minutes at 10000g is got supernatant liquor, except that staying sub-fraction to be used for the Lowry standard measure soluble proteins, all the other are used for rotavirus vp ₇Or VP ₄The antigen protein Determination on content.By the VP in the affine euzymelinked immunosorbent assay (ELISA) mensuration of the immunity transgenic plant ₇Or VP ₄Protein content.At first transfer-gen plant supernatant liquor (comprising the negative control plant) is joined respectively in each aperture of enzyme-linked reaction plate, 100ul in each aperture establishes two repetitions.Next day is with PBST damping fluid (8.0g NaCl, 0.2g KH ₂PO ₄, 2.9g Na ₂HPO ₄12H ₂O, 0.2g kCl, 0.5ml Tween20, adding distil water to 1 liter pH7.4) is washed four times, each 2 minutes, after the drying, adds the anti-G through 1/200 times of dilution of PBST (containing 0.2%BSA) ₂VP ₇Mouse monoclonal antibody (Chemicon company product) (is annotated: VP ₄What protein determination was used is the human rotavirus sheep polyclonal antibody of Chemicon company, can with G ₂VP ₄And G ₁VP ₄Special serological reaction takes place), 37 ℃ were reacted 2 hours.Wash 4 times with PBST, dry the back and add the anti-mouse enzyme labelled antibody of rabbit through the alkali phosphatase enzyme mark of 1/5000 times of dilution of PBST (containing 0.2%BSA) (to VP ₇) or the anti-sheep enzyme labelled antibody of rabbit (to VP ₄) (being Sigma company product) 100ul, 37 ℃ were reacted 2 hours, wash 5 times with PBST, dry the back and add 100ul substrate buffer solution [9.7ml diethanolamine, 60mg p-nitrophenyl (Sigma company), adding distil water to 100 milliliter, pH9.8], reaction is 30 minutes under the room temperature, adds 50ul 3M NaOH termination reaction then, reads each aperture 450nm absorbance value.

2) rotavirus vp ₇Or VP ₄The detection of protein gene

VP in the transgenic plant ₇Or VP ₄The integration situation of protein gene can detect by pcr amplification purpose fragment gene or the reaction of Southern hybridization trace, in transgenic plant and control plant, extract its genomic dna respectively, according to this as masterplate, increase with albumen primer in the specific coating, or with behind BamH I and the Sac I double digestion, the electrophoretic separation dna fragmentation is after transferring on the nylon membrane, with the VP of digoxin (Digoxigenin) mark ₇Or VP ₄The protein nucleic acid encoding sequence is as probe.In addition, can collect the transfer-gen plant selfed seed, the foreign gene of sprouting experiment or its offspring of pcr analysis by the kalamycin resistance situation of isozygotying.

5. expression rotavirus vp ₇Or VP ₄The breeding of protein transgene plant and cultivation

Filtering out efficient and stably express rotavirus vp ₇Or VP ₄Behind proteic transgenic Rhizoma Solani tuber osi or the tomato plant, must a large amount of these transfer-gen plants of breeding for producing this antigen protein.Therefore potato, can just can obtain a large amount of seedling and micro potato at short notice by the method for cuttage, and needn't worry to cause owing to syngenesis offspring's genetic recombination problem by nourishing and generating.Tomato or other rely on the transgenic plant of seminal propagation will obtain the offspring of stably express, then need the long period, because seminal propagation has shortcoming, its offspring lacks homogeneity, and foreign gene is passed to the offspring according to mendelian inheritance.Basically, be different in each seed heredity, have its hereditary property.Therefore, transgenic plant preferably will be through the several generations screening, and after obtaining pure lines, foreign gene could stably be passed to the offspring.

6. contain rotavirus vp ₇Or VP ₄The preparation of proteantigen genetically modified food

Rotavirus vp ₇Or VP ₄Albumen can be produced by a large amount of breeding transgene plants, and plant is direct-edible or be processed into capsule, electuary and other preparation after results.Though potato tuber can not be eaten something rare, after lyophilize, wear into fine powder and be processed into capsule, electuary or other preparation.Tamato fruit then can be processed into bottled tomato juice conveniently to be drunk.Within a certain period of time, people are by oral a certain amount of capsule, electuary or tomato juice or directly edible tomato (one or many within a certain period of time), the rotavirus vp that wherein contains ₇Or VP ₄Albumen might produce immune response by exciting human, and people have also just possessed the immunological competence to rotavirus.

Embodiment 2

1. the conversion of potato

Utilize leaf dish method to transform potato down agriculture bacillus mediated, by selecting cultured continuously and screening on the substratum, complete kalamycin resistance potato plant is reproduced out then.

According to people's such as Horsch method, utilize leaf dish method to transform potato.The potato aseptic seedling is cut blade from the petiole position that links to each other with stem with scissors after cuttage grew for 2 weeks in the MS solid medium again.Contain expression vector pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄Agrobacterium strains LBA4404 be seeded among the 50ml YEP, cultivated 2 days for 28 ℃, centrifugal 5 minutes of 4000g collects thalline, is suspended in again then in the MS substratum of liquid to OD _600nm=0.4, with in the Agrobacterium of cutting after blade immerses dilution 5 minutes, take out then, to blot with filter paper, the blade face places on the MS solid medium up, cultivates altogether 2 days for 28 ℃.On then that the blade dislocation is the fresh selection substratum, this substratum contains 200ug/ml kantlex and 500ug/ml carboxylic Bian penicillin, can suppress the growth of agrobatcerium cell and the plant transformed cell is selectively grown.Grow callus and differentiate seedling up to the petiole wound every changing a selective medium 2 week later on.When seedling occurs and long to enough greatly the time, seedling is scaled off (cultivating 4-8 week of back altogether usually), move on on the root media and (contain the 100ug/ml kantlex), after root grew, seedling was moved in the aseptic nutrition soil growth and is aided with suitable temperature and illumination condition.

2. the RNA of transgenic Rhizoma Solani tuber osi analyzes

Utilize the G of digoxigenin labeled ₂VP ₇, G ₁VP ₄, MG ₂VP ₇Or MG ₁VP ₄The protein gene probe is to pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄Kalamycin resistance potato plant RNA after expression vector transforms has carried out hybridization analysis.

At pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄Expression vector transforms in the transfer-gen plant that is obtained, and chooses VP ₇Or VP ₄The total RNA of the blade of the transgenic potato plant that expression contents is the highest is extracted, the method document that sees reference ⁽²¹⁾Approximately the 1.0g blade is clayed into power with mortar in liquid nitrogen, adds 10ml RNA and extracts damping fluid (0.2M Tris-HCl, pH6.8; 0.2M NaCl; 20mM EDTA; 2%SDS), add 10ml saturated phenol damping fluid (10mM Tris-HCl, pH8.0 immediately; 1mM EDTA is saturated) carry out extracting, use the chloroform extracting of 10ml then.3000g is centrifugal, collect the upper strata water, the dehydrated alcohol that adds 0.3M Potassium ethanoate (pH5.2) damping fluid and 2.5 times of volumes, the 10000g centrifugal collecting precipitation, after air-dry, precipitation is suspended in the water of 2ml again, adds ammonium acetate buffer and the 10ml dehydrated alcohol of 2ml 6M subsequently, the 10000g centrifugal collecting precipitation.After being deposited in drying, be suspended in again in the 0.4ml water, RAN concentration can estimate that the RNA light absorption value of supposing 1mg/ml is 25 units by the light absorption value of measuring 260nm.

Get 10ug RNA sample, about 5ul, after 2 times of volume RNA sample buffers (10ml deionized formamide, 3.5ml 37% formaldehyde, 2ml 5X MOPS) mixing, 65 ℃ were heated 5 minutes, after the cooling, add 3ulRNA sample loading buffer (50% glycerine, 1mM EDTA, 0.4% tetrabromophenol sulfonphthalein), then through 1% RNA agarose gel electrophoresis.Nucleic acid is transferred on the nylon membrane (Hybond N+) by the hair pipette method, and used damping fluid is 20XSSC, pH7.2 (3M sodium-chlor, 0.3M Trisodium Citrate), and be 16 hours transfer time.Nucleic acid by uviolizing after by commissure on nylon membrane, film is placed prehybridization solution [5XSSC, 0.1% (w/v) N-Lauroylsarcosine, 0.02%SDS, 100ug/m[salmon sperm dna], 68 ℃ of prehybridizations 3 hours, hybridization solution [the 5XSSC that more renews then, 0.1% (w/v) N-Lauroylsarcosine, 0.02%SDS, 1% encapsulant (providing in the Roche company test kit), the DNA pin 60ng that adds the digoxigenin labeled of sex change in the 5ml hybridization solution is hybridized, and probe length is respectively 978bp (G ₂VP ₇), 2328bp (G ₁VP ₄), they are respectively from plasmid pG ₂VP ₇, and pG ₁VP ₄, pMG ₂VP ₇Or pMG ₁VP ₄Pcr amplification product (amplimer is to seeing 1 among the embodiment 1), comprised rotavirus vp ₇Or VP ₄Whole encoding sequences of protein gene (comprising the sequence after gene codon is optimized).68 ℃ of hybridization were taken out Hybond membrane after 24 hours in hybridization solution, in 200ml 2XSSC, washed film under the room temperature 2 times in the 0.1%SDS damping fluid, then in 100ml 0.1XSSC, washed film 2 times for 68 ℃ in the 0.1%SDS damping fluid.Color reaction is substantially according to the method that provides in the test kit (Roche company), and nylon membrane is prior to elution buffer [0.1M Maleic acid, pH7.5; 0.15M NaCl; 3%Tween 20 (w/v)] in balance 5 minutes, with 50ml 1X sealing damping fluid sealing 30 minutes, DigiTAb to the concentration that adds alkali phosphatase enzyme mark was 75mU/ml then, reaction is 30 minutes under the room temperature, with elution buffer 2 times, each 15 minutes.With film dislocation 20ml colour developing damping fluid (0.1M Tris-HCl, pH9.5; 0.1M NaCl; 50mMMgCl ₂) in balance 5 minutes, the substrate buffer solution that more renews (200ul NBT/BCIP substrate adds 10ml colour developing damping fluid), colour developing is 16 hours under the shading condition, treat that desired band occurs after, the water termination reaction.

Through plasmid pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄Transfer-gen plant that transforms and the RNA results of hybridization of negative control plant as shown 6.PBMG ₂VP ₇Or pMBG ₁VP ₄The RNA hybridization signal of transfer-gen plant is pBG respectively ₂VP ₇And pBG ₁VP ₄Strong a lot, this may be because: 1) VP in these transfer-gen plants ₇And VP ₄Gene is optimized by the codon of having a preference for according to plant respectively; 2) gene 5 ' end and 3 ' end have increased the Nucleotide regulating and controlling sequence that is derived from PVY respectively.Compare with the standard rna molecular weight, the RNA that transcribes has 1Kb (G approximately ₂VP ₇), 1.2Kb (MG ₂VP ₇), 2.3Kb (G ₁VP ₄) and 2.6Kb (MG ₁VP ₄) long, with pre-estimate consistent.The RNA of negative control plant does not observe hybridization signal.At pBMG ₂VP ₇Or pBMG ₁VP ₄In the transfer-gen plant, stable, the great expression of mRNA and can with the coding rotavirus vp ₇Or VP ₄The result of pyrenoids nucleotide sequence probe specificity hybridization shows that the content of purpose mRNA is significantly improved in these transfer-gen plants.

3. transgenic potato plant VP ₇Or VP ₄The mensuration of protein content

Get different transgenic potato plant blade 0.1g, each adds the 1.5ml deionized water, grinds with glass homogenizer under 4 ℃.Centrifugal 5 minutes of 4 ℃ of homogenate 10000g utilize Lowry method (protein determination kit is available from Sigma company, and article No. is P5656), get the part supernatant liquor and measure soluble protein content.Utilize enzyme-linked immunosorbent assay (seeing 4 among the embodiment 1) to measure VP in the transgenic plant supernatant liquor ₇Or VP ₄Protein content.Human rotavirus G with standard content ₂The positive contrast of serotype (Argene company product) is because can't obtain the G of standard content ₂VP ₇Or G ₁VP ₄Albumen, below the G that measured ₂VP ₇Or G ₁VP ₄Expressing quantity is an approximation.Positive control becomes different protein content levels (1280.0-10.0ng) with doubling dilution, behind color reaction, reads the 450nm absorbance value, prepares a standard absorption curves.As shown in Figure 7, by finding that relatively (post 1,2) do not find to contain VP in the negative control plant ₇Or VP ₄Albumen then can detect the G of different content in transgenic potato plant ₂VP ₇Or G ₁VP ₄Albumen, wherein pBMG ₂VP ₇Transfer-gen plant VP ₇Recombinant protein content is about 720ng, accounts for 0.15% of plant soluble proteins.Compare pBG ₂VP ₇0.01% improve about 15 times (post 3 and strain 4).PBMG ₁VP ₄VP in the transfer-gen plant ₄Recombinant protein content is about 480ng, accounts for 0.1% of plant soluble proteins, compares pBG ₁VP ₄0.005% improve about 20 times (post 5 and post 6).

4. transgenic Rhizoma Solani tuber osi different tissues organ rotavirus vp ₇Or VP ₄The mensuration of protein content

Get transgenic Rhizoma Solani tuber osi blade, stem and stem tuber and organize each 0.1g, processing and measuring method are the same.Listed transgenic Rhizoma Solani tuber osi (pBMG in the table 2 ₂VP ₇And pBMG ₁VP ₄) VP in the different tissues organ ₇Or VP ₄Proteic content.

VP in table 2 transgenic Rhizoma Solani tuber osi leaf, stem and the stem tuber ₇Or VP ₄Protein content

As table 2, the G in the potato leaf ₂VP ₇The highest 0.15% of the soluble proteins that is about of recombinant antigen protein expression amount reaches 720ng/g fresh weight blade.G in the stem ₂VP ₇Although the albumen expression amount accounts for 0.3% of plant soluble proteins, because of the soluble proteins amount that contains in the stem is lower, so every g fresh weight only contains 340ng G ₂VP ₇Albumen is about half of blade.G in the potato tuber ₂VP ₇Protein content is the highest, is about the 2450ng/g fresh weight, and this is just for processing and utilizing potato tuber that great convenience is provided.

5. expression rotavirus vp ₇And VP ₄The breeding of recombinant antigen protein transgenic Rhizoma Solani tuber osi

The breeding of transgenic Rhizoma Solani tuber osi is described in embodiment 1.

Embodiment 3

1. the conversion of tomato

In tomato (Lycopersicom esculentum) kind method for transformation of vegetables No. 5 (available from Vegetable ﹠ Flower Inst., Chinese Academy of Agriculture Science) as embodiment 21 described in, also be under Agrobacterium LBA4404 mediation, to utilize Ye Panfa to carry out genetic transformation.Cotyledon or hypocotyl aseptic explant after Agrobacterium is infected and grew 7-10 days, this explant does not need pre-cultivation, but after cutting-out, immersed immediately in the Agrobacterium bacterium liquid 5-10 minute, take out the back and blot unnecessary bacterium liquid with filter paper, back dislocation Z1 substratum (adding the 1mg/L zeatin in the MS minimum medium) was cultivated 2 days under 25 ℃ of shading conditions altogether.Then explant being transferred to SM substratum (adding 1mg/L zeatin, 100mg/L kantlex and 500mg/L Pyocianil in the MS minimum medium) evoked callus forms and the seedling differentiation.The seedling of downcutting is in RM substratum (adding 0.2mg/L NAA, 100mg/L kantlex and 500mg/L Pyocianil in the 1/2MS minimum medium) root induction, and certain altitude to be grown to is transferred in the sterile soil then, the condition incubation growth that suits.

2.PCR detect VP ₇And VP ₄The integration situation of antigenic protein gene in tomato

The extracting method of tomato dna group DNA is substantially according to the method for Chee ⁽²²⁾Get 3g tomato fresh leaves, put into the mortar liquid nitrogen grinding, in the powder dislocation 20ml centrifuge tube after grinding, (100mM Tris-HCl, pH7.5 contain 5M NaCl, 10mM EDTA to add 9ml CTAB extracting solution then, the 10ml/L beta-mercaptoethanol, 10g/L CTAB), thermal agitation 1 minute makes it abundant mixing.Temperature was bathed 90 minutes in 60 ℃ of water-baths, during vibration in per 30 minutes once.From water-bath, take out centrifuge tube, placed 4-5 minute under the room temperature, make it cooling, add the 4.5ml chloroform then, shake gently, make it abundant mixing.5000g is centrifugal 10 minutes under the room temperature.Collect supernatant liquor in another 20ml centrifuge tube, add the 4.5ml chloroform, repeat above-mentioned steps.Collect supernatant liquor, add the ice-cold Virahol of 10ml, jog 10-15 minute, centrifugal 5 minutes of 5000g, collecting precipitation contains 0.2M NaOAc and 2ml 76% ethanol contains 10mM NH with 3ml 76% ethanol ₄OAc respectively washes precipitation once, adds TE (pH8.0) damping fluid dissolving DNA at last, and DNA concentration is transferred to 1ug/ul.

Get the about 1ul of above-mentioned tomato dna group DNA 1ug as masterplate, add rotavirus G respectively ₂VP ₇Or G ₁VP ₄Protein gene nucleotide coding sequence Auele Specific Primer is to (seeing 1 among the embodiment 1) each 1ul, 10XPCR damping fluid 3ul, and dNTP (each 2.5mM) 3ul, the about 1.5U of Taq enzyme 0.5ul adds water to cumulative volume 30ul.94 ℃ 30 seconds, 50 ℃ 45 seconds, 72 ℃ 1.5 minutes, carry out 35 circulations altogether.After reaction finishes, get 5ul amplification sample and carry out the detection of 1% agarose gel electrophoresis.Shown in Fig. 8 A and 8B, (swimming lane 3) do not find specific band in the negative control plant, and at G ₂VP ₇Then can amplify the specific band (swimming lane 1 and 2) of 1 about 1.0kb in the transgenic Fructus Lycopersici esculenti plant, at G ₁VP ₄Then can amplify the specific band (swimming lane 1 and 2) of 1 about 2.3kb in the transgenic Fructus Lycopersici esculenti plant, this result shows rotavirus vp ₇Or VP ₄Antigenic protein gene has been integrated among the tomato dna group DNA.

3. the RNA in the transgenic Fructus Lycopersici esculenti immature fruit analyzes

The extracting method of RNA is as 2 among the embodiment 2 in the transgenic Fructus Lycopersici esculenti immature fruit.The electrophoresis of RNA sample, commentaries on classics film, hybridization and color reaction are as 2 among the embodiment 2.

Through plasmid pBG ₂VP ₇, pBG ₁VP ₄, pBMG ₂VP ₇Or pBMG ₁VP ₄Transfer-gen plant that transforms and the RNA results of hybridization of negative control plant as shown 9.PBMG ₂VP ₇Or pBMG ₁VP ₄The RNA hybridization signal of transfer-gen plant is pBG respectively ₂VP ₇Or pBG ₁VP ₄Strong a lot, and similar with the analytical results of transgenic Rhizoma Solani tuber osi blade RNA, show that the content of purpose mRNA is significantly improved in these transfer-gen plants.

The content of RNA is very low in mature fruit, can not carry out rna blot analysis.

4. VP in leaf and the fruit ₇Or VP ₄The mensuration of middle protein content

With mortar plant tissue is ground, in attrition process, add liquid nitrogen, add 10 times of volume sterilization deionized waters in the plant tissue powder and dilute.Centrifugal 5 minutes of 4 ℃ of plant homogenate 10000g, (Sigam, the method that p5656) provides are got the part supernatant liquor and are measured soluble protein content according to test kit.Protein determination and quantivative approach are with 3 among the embodiment 2.As shown in Figure 10, by finding that relatively (post 1,2) do not find to contain G in the negative control plant ₂VP ₇Or G ₁VP ₄Albumen then can detect the G of different content in the transgenic Fructus Lycopersici esculenti plant ₂VP ₇Or G ₁VP ₄Albumen, wherein pBMG ₂VP ₇VP in the transgenic Fructus Lycopersici esculenti plant leaf ₇Recombinant protein content is about 3200ng, accounts for 0.13% of plant soluble proteins.Compare pBG ₂VP ₇0.02% improve about 6.5 times (post 3 and strain 4).PBMG ₁VP ₄VP in the transgenic Fructus Lycopersici esculenti plant leaf ₄Recombinant protein content is about 1560ng, accounts for 0.067% of plant soluble proteins, compares pBG ₁VP ₄0.005% improve about 13.4 times (post 5 and post 6).The above results shows, uses plant-derived virus (PVY) nucleotide sequence as the enhancing expression regulation sequence, and the measures such as optimization of binding purposes gene codon, can improve VP equally in transgenic Fructus Lycopersici esculenti ₇Or VP ₄The expression contents of gene in plant.

Listed transgenic Fructus Lycopersici esculenti (pBMG in the table 3 ₂VP ₇And pBMG ₁VP ₄) G in leaf and the fruit ₂VP ₇Or G ₁VP ₄The content of recombinant antigen protein.The greenery and the erythrocarpus that are taken at the transgenic Fructus Lycopersici esculenti of growing in the greenhouse carry out plant soluble proteins and G ₂VP ₇Or G ₁VP ₄The recombinant protein Determination on content is as above-mentioned described.Detect less than G in non-transgenic plant leaf and the fruit ₂VP ₇Or G ₁VP ₄Albumen.

Rotavirus vp in table 3 transgenic Fructus Lycopersici esculenti leaf and the fruit ₇Or VP ₄Recombinant protein content

Protein content is far above potato leaf in the coating in the tomato leaf, and expression amount is 0.13% of a soluble proteins.G in mature fruit ₂VP ₇The content of recombinant protein accounts for 0.0091% of soluble proteins, or is 182ng/g fruit fresh weight.Because the density of fruit is than higher, the VP in the tamato fruit ₇Or VP ₄Expressing quantity has been concentrated tomato plant VP ₇Or VP ₄Major part,, one heavy 100 the gram tamato fruit contain about 18.2ug G ₂VP ₇Or 7.4ug G ₁VP ₄Recombinant antigen protein.

Embodiment 4

1. rotavirus vp ₇Or VP ₄The stability of recombinant antigen protein in plant tissue is measured

Rotavirus G ₂VP ₇Or G ₁VP ₄Recombinant antigen protein can be preserved in complete vegetable cell for a long time.As with 4 ℃ of following cryopreservation of potato tuber mode, preservation period reaches 1 year, and antigen protein does not have any loss yet.But after plant cell structures was destroyed, the various proteolytic enzyme in the organoid will be released, contained recombinant antigen protein in their energy degrading plant juice.Therefore, detect the stability of recombinant antigen protein in water, for guaranteeing as far as possible in the course of processing that from now on antigen protein do not suffer a loss most important.As can be seen from Figure 11, rotavirus vp ₇Or VP ₄The time that recombinant antigen protein is placed in water is long more, and proteic loss will be big more.At room temperature place after 1 day and G in the new juice that grinds ₂VP ₇Antigen protein content differs 20%, and from the 2nd day, the amount of antigen protein can sharply reduce, and after the 7th day, has detected less than antigen protein basically.This result shows, in the transgenic Rhizoma Solani tuber osi stem tuber course of processing, should it be dehydrated, and can prevent effectively that just the proteasome degradation that recombinant antigen protein can not discharged in the cell from destroying.

, high temperature dehydrates rotavirus G although can quickening the transgenic Rhizoma Solani tuber osi stem tuber ₂VP ₇Or G ₁VP ₄Recombinant antigen protein is limited to the pyritous suffertibility.As can be seen from Figure 12, when temperature surpassed 50 ℃, the treatment time reached 3 hours, can detected G in the water ₂VP ₇The quantity of antigen protein sharply descends, and this shows that the endurable limit high temperature of this antigen protein is 50 ℃.Surpass this temperature, albumen can sex change, thereby loses the response capacity with specific antibody.

Comprehensively above-mentioned, can draw as drawing a conclusion, dry potato tuber can effectively protect recombinant antigen protein not suffer a loss with low temperature, the most suitable fast with this understanding.

2. contain rotavirus vp ₇Or VP ₄The preparation of recombinant antigen protein electuary

Potato tuber is not easy to eat, and can consider to be processed into electuary.1000g fresh weight potato tuber can obtain the about 150g of dry-matter after lyophilize, this dry-matter and then worn into 150-200 purpose dry powder at low temperatures after measured, contains 1.5ug VP in the dry powder that this transgenic Rhizoma Solani tuber osi stem tuber of every 100mg is made approximately ₇Or 0.54ug VP ₄Recombinant antigen protein.For ease of infant taking, can adopt the electuary packaged form, the dry powder that the 1-5g that packs in every bag is made by the transgenic Rhizoma Solani tuber osi stem tuber.Be packaged as example with 5g, contain VP ₇Or VP ₄About respectively 75ug of recombinant antigen protein and 27ug.Children only need take 1 bag below 3 years old, just may obtain enough immunizing doses.When taking, cause protein denaturation for avoiding high temperature, can accompany by warm boiling water dilution back clothes down.

3. contain rotavirus vp ₇Or VP ₄Other preparation of recombinant antigen protein

Contain VP ₇Or VP ₄There are some problems in the transgenic Fructus Lycopersici esculenti fruit of recombinant antigen protein when directly eating, at first be that infant below 3 years old can't directly eat something rare tomato, secondly because can not measure the antigen protein content in each tomato, thereby it is comparatively suitable just can't to determine that also each infant eats several tamato fruits at every turn.In this case, can consider that tamato fruit is processed into fruit tea or other drinks form, only need carry out sampling Detection, just can be used as the guidance foundation of infant taking dosage every batch of product.Certainly, the mode that potato tuber or other transgenic plant also can be processed into beverage is convenient to take, and its prerequisite is to guarantee VP under the liquid situation ₇Or VP ₄Recombinant antigen protein can not degraded by proteolytic enzyme, at ambient temperature can standing storage.

The VP of plant origin ₇Or VP ₄Recombinant antigen protein can also have other application mode, and for example, antigen protein can be used as foodstuff additive through behind complete purifying or the partial purification, mixes in food such as milk powder, bean powder, fruit juice and sour milk and the beverage and is prepared into various specific functionality food.

Embodiment 5

1. utilize the affinity chromatography rotavirus vp of from transgenic Rhizoma Solani tuber osi or tomato, purifying ₇Or VP ₄Recombinant protein

Rotavirus sheep polyclonal antibody is available from U.S. Chemicon company, this antibody can with rotavirus G ₁, G ₂, G ₃And G ₄VP Deng four serotypes ₇And genotype P[4] and P[8] VP ₄Albumen produces immunological response.At first confirm to contain the material that can produce specific reaction in transgenic Rhizoma Solani tuber osi stem tuber or the tomato leaf extract, pass through the size of recombinant protein in the SDS-PAGE electrophoresis detection plant then with this antibody by enzyme linked immunosorbent assay.

According to the method that provides in the product description, rotavirus sheep polyclonal antibody is fixed on CNBr activatory Sepharose 4B (Pharmacia Biotech company), obtains activatory antibody-gel composite.Get 1000 gram pBMG respectively ₂VP ₇Transgenic Rhizoma Solani tuber osi stem tuber or pBMG ₁VP ₄Tomato leaf adds equivalent PBS damping fluid and (contains 0.1%Triton X-100; 0.5mM PMSF), 4 ℃ of following homogenate.Homogenate is through 40, and 000g is centrifugal, and supernatant liquor is collected in the back.Supernatant liquor is dissolved in 20ml TSA damping fluid (20mM Tris-HCl, pH8.0,140mM NaCl, 0.025%NaN again after lyophilize ₃) in, then with above-mentioned activatory antibody-gel composite 4 ℃ of following thorough mixing 16 hours.Behind the reactant upper prop, Tris damping fluid (50mM Tris-HCl with 5 times of column volume pH8.0 and pH9.0,0.1%Triton X-100,500mM NaCl) washes post respectively, then with 10ml trolamine damping fluid (50mM trolamine, pH11.5,0.1%TritonX-100,150mM NaCl) carry out wash-out, elutriant is collected in respectively in 10 1.5ml centrifuge tubes.Detect having or not of antigen protein in each centrifuge tube by the absorption reaction of enzyme connection.To have immunoreactive elutriant to pool together, and carry out 4 ℃ of dialysed overnight in the dialysis tubing of packing into, dialysis buffer liquid is 2 liters of PBS.The dialysis finish after from dialysis tubing the sucking-off dialysate, after lyophilize, be dissolved in again in the 1ml PBS damping fluid, therefrom take out 20ul then and carry out the SDS-PAGE electrophoresis.The SDS-PAGE electrophoresis detection is found the G that recombinates in the transgenic plant ₂VP ₇Proteic molecular weight is about 38KD, G ₁VP ₄Be 88KD, respectively with G as the rotavirus (Argene company product) of positive control ₁VP ₇Albumen (molecular weight 38KD) and G ₁VP ₄Albumen (88KD) similar (seeing Figure 13).

2. the injecting immune inoculation test of mouse

For examination small white mouse (BALB/c) available from former Beijing Medical University (existing Department Of Medicine, Peking University), two kinds of sexes, but that the mouse of every batch of each treatment group of experiment is is male or female, body weight is about the 15-20 gram.Get the rotavirus G of 100ng behind affinity chromatography ₂VP ₇(being derived from potato tuber) or G ₁VP ₄Antigen protein (being derived from tomato leaf) adds the PBS damping fluid respectively to final volume 200ul, does not add any adjuvant, with every mouse of abdominal injection mode immunity, 5 mouse of every winding kind.Every 10 days injection inoculations 1 time, inoculate back 10 days and get the blood mode in the 5th and collect mouse resisting anteserum by eye.

Sero-fast tiring measured by the enzyme linked immunological absorption reaction, rotavirus culture G ₁Or G ₂Serotype (Argene company product) is cushioned liquid dilution (1.59g Na through bag ₂CO ₃, 2.93g NaHCO ₃, adding distil water to 1 liter) after, add 100ul (containing antigen 50ng) in each aperture, 4 ℃ down bag spent the night.Next day is with PBST damping fluid (8.0g NaCl, 0.2g KH ₂PO ₄, 2.9g Na ₂HPO ₄12H ₂O, 0.2g kCl, 0.5mlTween20, adding distil water to 1 liter pH7.4) is washed four times, each 2 minutes, after the drying, adds the mouse resisting anteserum 100ul that is diluted to (containing 0.2%BSA) different concns through PBST, 37 ℃ of reactions 2 hours.Wash 4 times with PBST, dry rabbit anti-sheep enzyme labelled antibody (the Sigma company product) 100ul of back adding through the alkali phosphatase enzyme mark of 1/5000 times of dilution of PBST (containing 0.2%BSA), 37 ℃ were reacted 2 hours, wash 5 times with PBST, dry the back and add 100ul substrate buffer solution [9.7ml diethanolamine, 60mg p-nitrophenyl (Sigma company), adding distil water to 100 milliliter, pH9.8], reaction is 30 minutes under the room temperature, add 50ul 3M NaOH termination reaction then, read each aperture 450nm absorbance value.

The comparison that the different immunogen mouse resisting anteserums of table 4 are tired

(1) numeral is the mean value (OD450nm light absorption value) of 3 experiments in the table

As shown in table 4, under the proteic situation of the same doses of antigen of injection inoculation, the VP of plant origin ₇And VP ₄Recombinant antigen protein all can produce specific rotavirus antibody.When detecting the wheel virus antigen of same quantity with it, under identical dilution situation, VP ₄The light absorption value of its OD450nm of antiserum(antisera) compares VP ₇Want high.This shows VP ₄Immunogenicity as if than VP ₇Stronger, this may be more relevant with this molecular weight of albumen.VP ₇And VP ₄Antiserum(antisera) does not all produce immunological response with negative control albumen under various extent of dilution.

3. the oral immunization of mouse experiment

Mouse for examination is identical with top injection inoculation experiment.Can't standard determine the immunizing dose (amount that each mouse eats is inconsistent) of each mouse because of getting food naturally, so the method that oral immunization takes disposable oral cavity to inject.Weigh 1g G respectively ₂VP ₇The transgenic Rhizoma Solani tuber osi stem tuber (contains 2.4ug VP approximately ₇Recombinant antigen protein) or 1gG ₁VP ₄The transgenic Rhizoma Solani tuber osi stem tuber (contains 0.96ug VP approximately ₄Recombinant antigen protein), after the 1mlPBS damping fluid mixes, utilizes glass homogenizer to grind to form fine particulate, add the PBS damping fluid to final volume 2ml.Weigh 1g G ₂VP ₇Transgenic Rhizoma Solani tuber osi stem tuber and 1g G ₁VP ₄The transgenic Rhizoma Solani tuber osi stem tuber utilizes glass homogenizer to grind to form fine particulate after mixing, and adds the PBS damping fluid to final volume 2ml.Utilize needle point (to avoid the mouse digestive tube is caused any harm) then in operating process for the syringe (available from chemical reagent shop, Beijing) of spheroidal (can avoid the mouse esophagus is damaged) carefully injects thick homogenate in the mouse esophagus by the mouse oral cavity.Mouse before oral vaccination hungry 24 hours earlier inoculates within back 2 hours and will not intake.

All treatment group all every 10 days oral vaccinations 1 time, are inoculated 8 times altogether.3 of blood are got from mouse tail vein in behind each oral vaccination the 7th day, and the blood of every group of (5 mouse) mouse is pooled together, with after 100ul PBS damping fluid mixes, placed 1 hour under the room temperature then, and centrifugal collection top serum ,-20 ℃ preservation is to be measured down.The production of mouse antibodies detects by enzyme-linked immunosorbent assay, and concrete operation method is as 2 among the embodiment 5.

Detected result is as shown in table 5, with containing G respectively ₂VP ₇And G ₁VP ₄The transgenic Rhizoma Solani tuber osi stem tuber oral vaccination mouse of plant recombinant antigen protein can inducing specific antibody produces, but antibody titers than injection inoculation a little less than, and the time that the first immunisation responsing reaction occurs is also than later.To contain G ₂VP ₇And G ₁VP ₄Oral vaccination mouse again after the transgenic Rhizoma Solani tuber osi stem tuber of plant recombinant antigen protein mixes and grinds, specific antibody-antigen-reactive not only can take place with rotavirus serotype G1 in the antiserum(antisera) that obtains, can also specific serum take place with serotype G2 learn reaction simultaneously, this shows in transgenosis expresses two kinds of different serotypes or genotypic recombinant antigen protein at the same time during oral vaccination, can induce respectively at immunogenic specific antibody separately, this shows simultaneously that also it is feasible utilizing this mode to produce infant's rotavirus multivalence oral vaccine.Similar immunological response does not appear in the mouse of edible negative control plant.

Table 5 transgenic plant are with the comparison of oral way inoculation back mouse internal antibody production

(1) numeral is the mean value (OD450nm light absorption value) of 3 experiments in the table

4. the safety evaluation behind the edible transgenic Rhizoma Solani tuber osi of mouse

At every turn feed transgenic Rhizoma Solani tuber osi stem tuber (0.5g) juice of 1ml of mouse (BALB/c) was fed 1 time every 10 days, and feed half a year continuously, and after this observing half a year, no matter be G ₂VP ₇Or G ₁VP ₄Treatment group does not all find have distortion, death or other abnormal conditions to take place for the examination mouse.Similarly, fed in per 3 days 2ml transgenic Rhizoma Solani tuber osi stem tuber (1g) juice 1 time of mouse was fed for 2 weeks continuously, and no matter observation after this is G ₂VP ₇Or G ₁VP ₄Treatment group does not find that mouse has distortion, death and other abnormal conditions to take place, and sees Table 6 yet.These results show the edible G that contains ₂VP ₇Or G ₁VP ₄The transgenic plant of recombinant antigen protein are safe.

Safety evaluation behind the edible transgenic Rhizoma Solani tuber osi of table 6 mouse

(1) every group of mouse for examination has 5.(2) "-" representative does not have

Reference

1. the Hou Yun moral is outstanding, Molecular Virology, Xueyuan Press, 1990

2. the side starts the third of the twelve Earthly Branches Qi Jin, Yang Hui etc., viral journal, 2001,17 (1): 17-23

3. river will Kui and Li Zhongduo, the Chinese biological goods are learned magazine, 2000,13 (1): 61-62

4. old winter plum, Bai Zhisheng, Liu Su etc., the Chinese biological goods are learned magazine, 1994,7 (2): 53-56

5. grandson is luxuriant, Giambiagi S. and Safrikanova I. etc., and the Chinese biological goods are learned magazine, 1998,11 (3): 129-132

6.Chen?S.C.，Jones?D.H.，Fynan?E.F.，et?al.，Journal?of?Virology，1998，72(7)：5757-5766

7.de?Aizpurua?and?Russell-Jones，J.Exp.Med.，1988，167：440-451

8.Man-kit，L.D.，Arntzen，C.J.and?Mason，H.S.，WO?94/20135，1994

9.Mason?H.S.，Lam?D.M.-K.and?Arntzen?C.J.，Proc.Natl.Acad.Sci.USA，1992，89(12)：11745-11749

10. Liu Yu is happy, Wang Jinfang and Qiu Bingsheng etc., Chinese science (B collects),, 23 (3): 57-60 in 1993

11.Tacket?C.，Mason?H.，Losonsky?G.，Clement?J.，Levine?M.and?ArntzenC.，Nature?Medicine，1998，4：607-609

12.Mason?H.，Haq?T.，Clements?J.and?Arntzen?C.，Vaccine，1998，16：1336-1343

13.Arakawa?T.，Chong?D.，Merritt?J.and?Langridge?W，Trans.Res.，1997，6：403-423

14.Arakawa?T.，Chong?D.，and?Langridge?W，Nat.Biotechnol.，1998，16：292-297

15. Wang Xin state, Cheong Kuoc Va, Fang Rongxiang etc., biotechnology communication, 2000,11 (2): 98-102

16.Mason?H.S.，Ball?J.M.，Shi?J.J.，et?al.，Proc.Natl.Acad.Sci.USA，1996，93：5335-5340

17.McGarvey?P.B.，Hammond?J.，Dienelt?M.M.，et?al.，Bio/Technology.，1995，1484-1487

18. the Liu De tiger, biotechnology circular, 1999,4:1-5

19.Chen?S.C.，Jones?D.H.，Fynan?E.F.，et?al.，Journal?of?Virology，1998，72(7)：5757-5766

20.Horsch，R.B.，et?al.，Science，1985，227：1229-1231

21.Mason，H.，et?al.，Plant?Molecular?Biology，1988，11：845-856

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Just some the special preferential embodiments that further describe of the present invention are according to the patent application requirement with in order to explain and illustrate the content of this patent.Obviously, do not deviating within the spirit and scope of the present invention, can do further improvement and variation on this basis.

Sequence table

＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences

＜120〉improve rotavirus vp ₇Or VP ₄The method of expression of plants content and product

<140>200310117119.9

<141>2003-12-03

<160>8

<210>1

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<221>5’UTP

<222>(1)...(28)

＜223〉design according to marmor upsilon mRNA 5 ' end non-coding region, to improve rotavirus vp ₇Or VP ₄

The expression contents of antigenic protein gene in transgenic plant.

<400>1

atcacttcca?accaatttca?gatcaaca????28

<210>2

<211>981

<212>DNA

＜213〉human rotavirus (huamn rotavirus)

<220>

<221>CDS

<222>(1)...(981)

<400>2

atg?tac?ggt?tat?gaa?tat?acc?aca?att?cta?acc?att?ctg?ata?ttt?atc?48

Met?Tyr?Gly?Tyr?Glu?Tyr?Thr?Thr?Ile?Leu?Thr?Ile?Leu?Ile?Phe?Ile

1???????????????5????????????????????10??????????????????15

att?cta?ctc?aac?tat?ata?tta?aaa?act?ata?acc?aat?acg?atg?gac?tac?96

Ile?Leu?Leu?Asn?Tyr?Ile?Leu?Lys?Thr?Ile?Thr?Asn?Thr?Met?Asp?Tyr

20??????????????????25??????????????????30

att?ata?ttt?aga?ttt?ttg?tta?ctc?atc?gct?ttg?atg?tca?cca?ttt?gtg?144

Ile?Ile?Phe?Arg?Phe?Leu?Leu?Leu?Ile?Ala?Leu?Met?Ser?Pro?Phe?Val

35??????????????????40??????????????????45

agg?aca?cag?aac?tat?gga?atg?tat?tta?cca?ata?aca?gga?tca?cta?gat?192

Arg?Thr?Gln?Asn?Tyr?Gly?Met?Tyr?Leu?Pro?Ile?Thr?Gly?Ser?Leu?Asp

50??????????????????55??????????????????60

gct?gta?tac?aca?aac?tct?act?agt?gga?gaa?tca?ttt?cta?aca?tcc?aca?240

Ala?Val?Tyr?Thr?Asn?Ser?Thr?Ser?Gly?Glu?Ser?Phe?Leu?Thr?Ser?Thr

65??????????????????70??????????????????75??????????????????80

tta?tgt?ttg?tat?tat?cca?act?gaa?gca?aaa?aat?ggg?atc?agt?gat?aat?288

Leu?Cys?Leu?Tyr?Tyr?Pro?Thr?Glu?Ala?Lys?Asn?Gly?Ile?Ser?Asp?Asn

85??????????????????90??????????????????95

gaa?tgg?gaa?aat?act?tta?tcg?caa?tta?ttt?tta?aca?aaa?ggt?tgg?cca?336

Glu?Trp?Glu?Asn?Thr?Leu?Ser?Gln?Leu?Phe?Leu?Thr?Lys?Gly?Trp?Pro

100?????????????????105?????????????????110

aca?gga?tca?gtc?tat?ttt?aaa?gac?tac?aat?gat?att?act?act?ttt?tcc?384

Thr?Gly?Ser?Val?Tyr?Phe?lys?Asp?Tyr?Asn?Asp?Ile?Thr?Thr?Phe?Ser

115?????????????????120?????????????????125

atg?aat?cca?caa?tta?tat?tgt?gat?tat?aac?ata?gta?cta?atg?aga?tat?432

Met?Asn?Pro?Gln?Leu?Tyr?Cys?Asp?Tyr?Asn?Ile?Val?Leu?Met?Arg?Tyr

130?????????????????135?????????????????140

gat?aat?aca?tct?gaa?tta?gat?gga?tca?gaa?tta?gct?gat?ttg?ata?ttg?480

Asp?Asn?Thr?Ser?Glu?Leu?Asp?Gly?Ser?Glu?Leu?Ala?Asp?Leu?Ile?Leu

145?????????????????150?????????????????155?????????????????160

aat?gaa?tgg?tta?tgt?aat?cca?atg?gat?ata?tca?tta?tat?cat?tac?caa?528

Asn?Glu?Trp?Leu?Cys?Asn?Pro?Met?Asp?Ile?Ser?Leu?Tyr?His?Tyr?Gln

165?????????????????170?????????????????175

caa?aat?agt?gaa?tca?aat?aag?tgg?ata?tca?atg?gga?aca?gac?tgt?act?576

Gln?Asn?Ser?Glu?Ser?Asn?Lys?Trp?Ile?Ser?Met?Gly?Thr?Asp?Cys?Thr

180?????????????????185?????????????????190

gtg?aaa?gtg?tgt?cca?ctg?aat?aca?caa?agc?ttg?gga?ata?ggt?tgt?aaa?624

Val?Lys?Val?Cys?Pro?Leu?Asn?Thr?Gln?Ser?Leu?Gly?Ile?Gly?Cys?Lys

195?????????????????200?????????????????205

aca?acg?gac?gta?gac?aca?ttt?gaa?att?gtc?gct?tcg?tct?gaa?aaa?tta?672

Thr?Thr?Asp?Val?Asp?Thr?Phe?Glu?Ile?Val?Ala?Ser?Ser?Glu?Lys?Leu

210?????????????????215?????????????????220

gta?ata?act?gat?gtc?gtt?aat?ggg?gtt?aat?cat?aaa?ata?aat?att?tca?720

Val?Ile?Thr?Asp?Val?Val?Asn?Gly?Val?Asn?His?Lys?Ile?Asn?Ile?Ser

225?????????????????230?????????????????235?????????????????240

ata?agt?aca?tgt?act?att?cga?aat?tgt?aat?aag?tta?ggt?cca?aga?gag?768

Ile?Ser?Thr?Cys?Thr?Ile?Arg?Asn?Cys?Asn?Lys?Leu?Gly?Pro?Arg?Glu

245?????????????????250?????????????????255

aat?gta?gct?ata?ata?caa?gtt?ggt?ggc?ccg?aat?gca?tta?gac?ata?aca?816

Asn?Val?Ala?Ile?Ile?Gln?Val?Gly?Gly?Pro?Asn?Ala?Leu?Asp?Ile?Thr

260?????????????????265?????????????????270

gct?gat?cca?acg?act?gtt?cca?caa?gtt?caa?aga?atc?atg?aga?gtg?aat?864

Ala?Asp?Pro?Thr?Thr?Val?Pro?Gln?Val?Gln?Arg?Ile?Met?Arg?Val?Asn

275?????????????????280?????????????????285

tgg?aaa?aaa?tgg?tgg?caa?gta?ttt?tat?act?gta?gta?gat?tat?att?aat?912

Trp?Lys?Lys?Trp?Trp?Gln?Val?Phe?Tyr?Thr?Val?Val?Asp?Tyr?Ile?Asn

290?????????????????295?????????????????300

cag?att?ata?cag?gtt?atg?tcc?aaa?aga?tca?aga?tca?tta?gac?aca?gct?960

Gln?Ile?Ile?Gln?Val?Met?Ser?Lys?Arg?Ser?Arg?Ser?Leu?Asp?Thr?Ala

305?????????????????310?????????????????315?????????????????320

gcg?ttt?tat?tat?aga?att?tag?????????????????????????????????????981

Ala?Phe?Tyr?Tyr?Arg?Ile??*

325

<210>3

<211>981

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(981)

＜223〉according to human rotavirus's serotype G ₂VP ₇Gene and designing is not changing coded aminoacid sequence

Prerequisite under, the codon that all codons are all had a preference for according to plant has carried out revising and manually closing

Become.

<400>3

atg?tac?ggt?tac?gac?tac?acc?acc?atc?ctc?acc?atc?ctc?atc?ttc?atc?48

Met?Tyr?Gly?Tyr?Glu?Tyr?Thr?Thr?Ile?Leu?Thr?Ile?Leu?Ile?Phe?Ile

1???????????????5???????????????????10??????????????????15

atc?ctc?ctc?aac?tac?atc?ctc?aaa?acc?atc?acc?aat?acc?atg?gac?tac?96

Ile?Leu?Leu?Asn?Tyr?Ile?Leu?Lys?Thr?Ile?Thr?Asn?Thr?Met?Asp?Tyr

20??????????????????25??????????????????30

att?atc?ttc?agg?ttc?ctc?ctc?ctc?atc?gcc?ctc?atg?tcg?cca?ttc?gtg?144

Ile?Ile?Phe?Arg?Phe?Leu?Leu?Leu?Ile?Ala?Leu?Met?Ser?Pro?Phe?Val

35??????????????????40??????????????????45

agg?acc?cag?aac?tac?gga?atg?tac?ctc?cca?atc?acc?gga?tcg?ctc?gat?192

Arg?Thr?Gln?Asn?Tyr?Gly?Met?Tyr?Leu?Pro?Ile?Thr?Gly?Ser?Leu?Asp

50??????????????????55??????????????????60

gcc?gtg?tac?acc?aac?tcg?acc?agg?gga?gac?tcg?ttc?ctc?acc?tcg?acc?240

Ala?Val?Tyr?Thr?Asn?Ser?Thr?Ser?Gly?Glu?Ser?Phe?Leu?Thr?Ser?Thr

65??????????????????70??????????????????75??????????????????80

ctc?tgc?ctc?tac?tac?cca?acc?gac?gca?aag?aat?ggg?atc?agg?gac?aat?288

Leu?Cys?Leu?Tyr?Tyr?Pro?Thr?Glu?Ala?Lys?Asn?Gly?Ile?Ser?Asp?Asn

85??????????????????90??????????????????95

gac?tgg?gac?aat?acg?ctc?tcg?cag?ctc?ttt?ctc?aca?aag?ggt?tgg?cca?336

Glu?Trp?Glu?Asn?Thr?Leu?Ser?Gln?Leu?Phe?Leu?Thr?Lys?Gly?Trp?Pro

100?????????????????105?????????????????110

acc?gga?tcg?gtg?tat?ttt?aag?gac?tac?aat?gac?atc?acc?acc?ttc?tcg?384

Thr?Gly?Ser?Val?Tyr?Phe?lys?Asp?Tyr?Asn?Asp?Ile?Thr?Thr?Phe?Ser

115?????????????????120?????????????????125

atg?aac?cca?cag?ctc?tac?tgc?gac?tac?aac?atc?gtg?ctc?atg?agg?tac?432

Met?Asn?Pro?Gln?Leu?Tyr?Cys?Asp?Tyr?Asn?Ile?Val?Leu?Met?Arg?Tyr

130?????????????????135?????????????????140

gac?aat?acc?tcg?gac?ctc?gac?gga?tcg?gag?ctc?gct?gac?ctc?atc?ctc?480

Asp?Asn?Thr?Ser?Glu?Leu?Asp?Gly?Ser?Glu?Leu?Ala?Asp?Leu?Ile?Leu

145?????????????????150?????????????????155?????????????????160

aat?gag?tgg?ctc?tgc?aat?cca?atg?gac?atc?tcc?ctc?tat?cag?tac?cag?528

Asn?Glu?Trp?Leu?Cys?Asn?Pro?Met?Asp?Ile?Ser?Leu?Tyr?His?Tyr?Gln

165?????????????????170?????????????????175

cag?aat?agc?gag?tcg?aat?aag?tgg?atc?tcg?atg?gga?acc?gac?tgc?acc?576

Gln?Asn?Ser?Glu?Ser?Asn?Lys?Trp?Ile?Ser?Met?Gly?Thr?Asp?Cys?Thr

180?????????????????185?????????????????190

gtg?aag?gtg?tgt?cca?ctg?aat?acc?cag?agc?ttg?gga?ata?ggt?tgc?aag?624

Val?Lys?Val?Cys?Pro?Leu?Asn?Thr?Gln?Ser?Leu?Gly?Ile?Gly?Cys?Lys

195?????????????????200?????????????????205

acc?acc?gac?gtg?gac?acc?ttc?gac?atc?gtg?gct?tcg?tcg?gac?aag?ctc?672

Thr?Thr?Asp?Val?Asp?Thr?Phe?Glu?Ile?Val?Ala?Ser?Ser?Glu?Lys?Leu

210?????????????????215?????????????????220

gtc?atc?act?gac?gtg?gtg?aat?ggg?gtg?aat?cag?aag?atc?aat?ctc?tcc?720

Val?Ile?Thr?Asp?Val?Val?Asn?Gly?Val?Asn?His?Lys?Ile?Asn?Ile?Ser

225?????????????????230?????????????????235?????????????????240

atc?agc?acc?tgc?act?atc?cga?aat?tgc?aat?aag?ctc?ggt?cca?aga?gag?768

Ile?Ser?Thr?Cys?Thr?Ile?Arg?Asn?Cys?Asn?Lys?Leu?Gly?Pro?Arg?Glu

245?????????????????250?????????????????255

aat?gtg?gct?atg?atc?cag?gtg?ggt?ggc?ccg?aat?gca?ctc?gac?atc?aca?816

Asn?Val?Ala?Ile?Ile?Gln?Val?Gly?Gly?Pro?Asn?Ala?Leu?Asp?Ile?Thr

260?????????????????265?????????????????270

gct?gac?cca?acc?acc?gtg?cca?cag?gtg?cag?agg?atc?atg?agg?gtg?aat?864

Ala?Asp?Pro?Thr?Thr?Val?Pro?Gln?Val?G?ln?Arg?Ile?Met?Arg?Val?Asn

275?????????????????280??????????????????285

tgg?aag?aag?tgg?tgg?cag?gtg?ttc?tac?act?gtg?gtg?gac?tac?atc?aat?912

Trp?Lys?Lys?Trp?Trp?Gln?Val?Phe?Tyr?Thr?Val?Val?Asp?Tyr?Ile?Asn

290?????????????????295?????????????????300

cag?atc?atc?cag?gtg?atg?tcg?aag?agg?tcg?agg?tcg?ctc?gac?acc?gct?960

Gln?Ile?Ile?Gln?Val?Met?Ser?Lys?Arg?Ser?Arg?Ser?Leu?Asp?Thr?Ala

305?????????????????310?????????????????315?????????????????320

gcg?ttt?tac?tac?agg?atc?tag?????????????????????????????????????981

Ala?Phe?Tyr?Tyr?Arg?Ile??*

325

<210>4

<211>326

<212>PRT

＜213〉human rotavirus (human rotavirus)

<400>4

Met?Tyr?Gly?Tyr?Glu?Tyr?Thr?Thr?Ile?Leu?Thr?Ile?Leu?Ile?Phe?Ile

1???????????????5???????????????????10??????????????????15

Ile?Leu?Leu?Asn?Tyr?Ile?Leu?Lys?Thr?Ile?Thr?Asn?Thr?Met?Asp?Tyr

20??????????????????25??????????????????30

Ile?Ile?Phe?Arg?Phe?Leu?Leu?Leu?Ile?Ala?Leu?Met?Ser?Pro?Phe?Val

35??????????????????40??????????????????45

Arg?Thr?Gln?Asn?Tyr?Gly?Met?Tyr?Leu?Pro?Ile?Thr?Gly?Ser?Leu?Asp

50??????????????????55??????????????????60

Ala?Val?Tyr?Thr?Asn?Ser?Thr?Ser?Gly?Glu?Ser?Phe?Leu?Thr?Ser?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Cys?Leu?Tyr?Tyr?Pro?Thr?Glu?Ala?Lys?Asn?Gly?Ile?Ser?Asp?Asn

85??????????????????90??????????????????95

Glu?Trp?Glu?Asn?Thr?Leu?Ser?Gln?Leu?Phe?Leu?Thr?Lys?Gly?Trp?Pro

100?????????????????105?????????????????110

Thr?Gly?Ser?Val?Tyr?Phe?lys?Asp?Tyr?Asn?Asp?Ile?Thr?Thr?Phe?Ser

115?????????????????120?????????????????125

Met?Asn?Pro?Gln?Leu?Tyr?Cys?Asp?Tyr?Asn?Ile?Val?Leu?Met?Arg?Tyr

130?????????????????135?????????????????140

Asp?Asn?Thr?Ser?Glu?Leu?Asp?Gly?Ser?Glu?Leu?Ala?Asp?Leu?Ile?Leu

145?????????????????150?????????????????155?????????????????160

Asn?Glu?Trp?Leu?Cys?Asn?Pro?Met?Asp?Ile?Ser?Leu?Tyr?His?Tyr?Gln

165?????????????????170?????????????????175

Gln?Asn?Ser?Glu?Ser?Asn?Lys?Trp?Ile?Ser?Met?Gly?Thr?Asp?Cys?Thr

180?????????????????185?????????????????190

Val?Lys?Val?Cys?Pro?Leu?Asn?Thr?Gln?Ser?Leu?Gly?Ile?Gly?Cys?Lys

195?????????????????200?????????????????205

Thr?Thr?Asp?Val?Asp?Thr?Phe?Glu?Ile?Val?Ala?Ser?Ser?Glu?Lys?Leu

210?????????????????215?????????????????220

Val?Ile?Thr?Asp?Val?Val?Asn?Gly?Val?Asn?His?Lys?Ile?Asn?Ile?Ser

225?????????????????230?????????????????235?????????????????240

Ile?Ser?Thr?Cys?Thr?Ile?Arg?Asn?Cys?Asn?Lys?Leu?Gly?Pro?Arg?Glu

245?????????????????250?????????????????255

Asn?Val?Ala?Ile?Ile?Gln?Val?Gly?Gly?Pro?Asn?Ala?Leu?Asp?Ile?Thr

260?????????????????265?????????????????270

Ala?Asp?Pro?Thr?Thr?Val?Pro?Gln?Val?Gln?Arg?Ile?Met?Arg?Val?Asn

275?????????????????280?????????????????285

Trp?Lys?Lys?Trp?Trp?Gln?Val?Phe?Tyr?Thr?Val?Val?Asp?Tyr?Ile?Asn

290?????????????????295?????????????????300

Gln?Ile?Ile?Gln?Val?Met?Ser?Lys?Arg?Ser?Arg?Ser?Leu?Asp?Thr?Ala

305?????????????????310?????????????????315?????????????????320

Ala?Phe?Tyr?Tyr?Arg?Ile

325

<210>5

<211>200

<212>DNA

＜213〉artificial sequence

<220>

<221>3’UTP

<222>(1)...(200)

＜223〉design according to marmor upsilon mRNA 3 ' end non-coding region, to improve rotavirus vp ₇Or VP ₄

The expression contents of antigenic protein gene in transgenic plant.

<400>5

ggcctgactc?tatctggtta?attccgcatt?attaagtttt?gaataaaaag?tgccgggttg?60

tcgttgttgt?ggatgattca?tcgattaggt?gatgttgcga?ttctgtcgta?gcagtaacta?120

tgtctggatc?tatctgcttg?ggtggtgttg?tgatttcgtc?ataacagtga?ctgtaaactt?180

caatcaggag?acaaaaaaaa?????????????????????????????????????????????200

<210>6

<211>2328

<212>DNA

＜213〉human rotavirus (human rotavirus)

<220>

<221>CDS

<222>(1)...(2328)

<400>6

atg?gct?tca?ctc?att?tat?aga?cag?ctt?ctc?act?aat?tca?tat?tcg?gta?48

Met?Ala?Ser?Leu?Ile?Tyr?Arg?Gln?Leu?Leu?Thr?Asn?Ser?Tyr?Ser?Val

1???????????????5???????????????????10??????????????????15

gac?ttg?cat?gac?gaa?ata?gaa?cag?att?gga?tcg?gag?aaa?act?caa?aat?96

Asp?Leu?His?Asp?Glu?Ile?Glu?Gln?Ile?Gly?Ser?Glu?Lys?Thr?Gln?Asn

20??????????????????25??????????????????30

gtg?acg?gta?aat?cca?ggc?cca?ttt?gca?cag?act?aga?tat?gct?cca?gtt?144

Val?Thr?Val?Asn?Pro?Gly?Pro?Phe?Ala?Gln?Thr?Arg?Tyr?Ala?Pro?Val

35??????????????????40??????????????????45

aat?tgg?gga?cac?gga?gag?att?aat?gat?tca?act?aca?gtg?gaa?cca?gtt?192

Asn?Trp?Gly?His?Gly?Glu?Ile?Asn?Asp?Ser?Thr?Thr?Val?Glu?Pro?Val

50??????????????????55??????????????????60

tta?gat?ggt?cct?tat?caa?ccg?act?aca?ttc?aaa?cca?ccc?aat?gat?tat?240

Leu?Asp?Gly?Pro?Tyr?Gln?Pro?Thr?Thr?Phe?Lys?Pro?Pro?Asn?Asp?Tyr

65??????????????????70??????????????????75??????????????????80

tgg?ctg?ctt?att?agt?tca?aat?aca?gat?gga?gta?gtt?tat?gag?agt?aca?288

Trp?Leu?Leu?Ile?Ser?Ser?Asn?Thr?Asp?Gly?Val?Val?Tyr?Glu?Ser?Thr

85??????????????????90??????????????????95

aat?aat?agt?gac?ttt?tgg?aca?gca?gtt?atc?gct?gtc?gaa?cca?cat?gtc?336

Asn?Asn?Ser?Asp?Phe?Trp?Thr?Ala?Val?Ile?Ala?Val?Glu?Pro?His?Val

100?????????????????105?????????????????110

agt?caa?aca?aat?agg?caa?tat?gtt?tta?ttt?ggt?gag?aat?aag?cag?ttt?384

Ser?Gln?Thr?Asn?Arg?Gln?Tyr?Val?Leu?Phe?Gly?Glu?Asn?Lys?Gln?Phe

115?????????????????120?????????????????125

aac?gta?gaa?aat?agt?tca?gat?aaa?tgg?aaa?ttt?ttc?gaa?atg?ttt?aaa?432

Asn?Val?Glu?Asn?Ser?Ser?Asp?Lys?Trp?Lys?Phe?Phe?Glu?Met?Phe?Lys

130?????????????????135?????????????????140

ggt?aat?agt?cag?agt?gat?ttt?tct?aat?aga?cgg?act?tta?acc?tct?aat?480

Gly?Asn?Ser?Gln?Ser?Asp?phe?Ser?Asn?Arg?Arg?Thr?Leu?Thr?Ser?Asn

145?????????????????150?????????????????155?????????????????160

aat?aga?ctt?gta?gga?atg?cta?aaa?tat?ggt?gga?aga?gta?tgg?acg?ttt?528

Asn?Arg?Leu?Val?Gly?Met?Leu?Lys?Tyr?Gly?Gly?Arg?Val?Trp?Thr?Phe

165?????????????????170?????????????????175

cat?ggt?gaa?aca?cca?aga?gct?act?act?gat?agt?tcg?aat?act?gcg?gat?576

His?Gly?Glu?Thr?Pro?Arg?Ala?Thr?Thr?Asp?Ser?Ser?Asn?Thr?Ala?Asp

180?????????????????185?????????????????190

tta?aat?aat?ata?tca?att?ata?att?cat?tca?gag?ttt?tat?att?att?cca?624

Leu?Asn?Asn?Ile?Ser?Ile?Ile?Ile?His?Ser?Glu?Phe?Tyr?Ile?Ile?Pro

195?????????????????200?????????????????205

aga?tcc?caa?gaa?tct?aaa?tgt?aat?gaa?tat?att?aat?aat?ggt?tta?cca?672

Arg?Ser?Gln?Glu?Ser?Lys?Cys?Asn?Glu?Tyr?Ile?Asn?Asn?Gly?Leu?Pro

210?????????????????215?????????????????220

cca?att?caa?aat?act?aga?aat?gta?gtt?ccg?tta?tct?cta?tca?tcc?aga?720

Pro?Ile?Gln?Asn?Thr?Arg?Asn?Val?Val?Pro?Leu?Ser?Leu?Ser?Ser?Arg

225?????????????????230?????????????????235?????????????????240

tct?att?caa?tat?aga?aga?gca?caa?gtt?aat?gaa?gat?att?aca?att?tca?768

Ser?Ile?Gln?Tyr?Arg?Arg?Ala?Gln?Val?Asn?Glu?Asp?Ile?Thr?Ile?Ser

245?????????????????250?????????????????255

aaa?act?tca?tta?tgg?aag?gaa?atg?caa?tat?aat?aga?gat?att?ata?ata?816

Lys?Thr?Ser?Leu?Trp?Lys?Glu?Met?Gln?Tyr?Asn?Arg?Asp?Ile?Ile?Ile

260?????????????????265?????????????????270

aga?ttt?aaa?ttt?ggt?aat?agt?gtc?ata?aaa?tta?gga?gga?ttg?gga?tat?864

Arg?Phe?Lys?Phe?Gly?Asn?Ser?Val?Ile?Lys?Leu?Gly?Gly?Leu?Gly?Tyr

275?????????????????280?????????????????285

aaa?tgg?tct?gaa?ata?tca?tac?aaa?gca?gcg?aat?tat?caa?tat?agc?tat?912

Lys?Trp?Ser?Glu?Ile?Ser?Tyr?Lys?Ala?Ala?Asn?Tyr?Gln?Tyr?Ser?Tyr

290?????????????????295?????????????????300

tcg?cgt?gat?ggt?gaa?caa?gtt?act?gca?cat?acc?act?tgt?tca?gta?aat?960

Ser?Arg?Asp?Gly?Glu?Gln?Val?Thr?Ala?His?Thr?Thr?Cys?Ser?Val?Asn

305?????????????????310?????????????????315?????????????????320

gga?gta?aat?aat?ttt?agc?tat?aat?gga?ggt?tca?cta?cct?act?gat?ttc?1008

Gly?Val?Asn?Asn?Phe?Ser?Tyr?Asn?Gly?Gly?Ser?Leu?Pro?Thr?Asp?Phe

325?????????????????330?????????????????335

agt?att?tcg?aga?tat?gag?gtt?att?aaa?gaa?aat?tct?tat?gta?tat?ata?1056

Ser?Ile?Ser?Arg?Tyr?Glu?Val?Ile?Lys?Glu?Asn?Ser?Tyr?Val?Tyr?Ile

340?????????????????345?????????????????350

gat?tac?tgg?gat?gat?tca?aaa?gca?ttt?aga?aat?atg?gta?tat?gtt?aga?1104

Asp?Tyr?Trp?Asp?Asp?Ser?Lys?Ala?Phe?Arg?Asn?Met?Val?Tyr?Val?Arg

355?????????????????360?????????????????365

tcg?tta?gca?gca?aat?ttg?aat?tca?gtg?aaa?tgt?gta?ggt?ggg?agt?tat?1152

Ser?Leu?Ala?Ala?Asn?Leu?Asn?Ser?Val?Lys?Cys?Val?Gly?Gly?Ser?Tyr

370?????????????????375????????????????380

gat?ttt?aga?tta?cct?gta?ggt?gaa?tgg?cct?att?atg?aat?ggc?ggt?gct?1200

Asp?Phe?Arg?Leu?Pro?Val?Gly?Glu?Trp?Pro?Ile?Met?Asn?Gly?Gly?Ala

385?????????????????390?????????????????395?????????????????400

gta?tca?tta?cat?ttt?gct?gga?gtt?aca?tta?tct?aca?cag?ttc?act?gat?1248

Val?Ser?Leu?His?Phe?Ala?Gly?Val?Thr?Leu?Ser?Thr?Gln?Phe?Thr?Asp

405?????????????????410?????????????????415

ttt?gta?tca?ttg?aat?tcg?cta?cga?ttt?aga?ttc?agt?tta?aca?gta?gat?1296

Phe?Val?Ser?Leu?Asn?Ser?Leu?Arg?Phe?Arg?Phe?Ser?Leu?Thr?Val?Asp

420?????????????????425?????????????????430

gaa?cca?tct?ttc?tca?ata?ata?cga?aca?cgt?aca?atg?aac?tta?tac?gga?1344

Glu?Pro?Ser?Phe?Ser?Ile?Ile?Arg?Thr?Arg?Thr?Met?Asn?Leu?Tyr?Gly

435?????????????????440?????????????????445

tta?cca?gca?gct?aat?cca?aac?aat?gga?aat?gaa?tac?tat?gaa?gtg?tca?1392

Leu?Pro?Ala?Ala?Asn?Pro?Asn?Asn?Gly?Asn?Glu?Tyr?Tyr?Glu?Val?Ser

450?????????????????455?????????????????460

gga?agg?ttc?tca?ctt?att?tct?tta?gtt?cca?acc?aat?gat?gat?tat?caa?1440

Gly?Arg?Phe?Ser?Leu?Ile?Ser?Leu?Val?Pro?Thr?Asn?Asp?Asp?Tyr?Gln

465?????????????????470?????????????????475?????????????????480

act?cca?att?atg?aat?tca?gta?aca?gta?agg?caa?gat?tta?gaa?cgc?cag?1488

Thr?Pro?Ile?Met?Asn?Ser?Val?Thr?Val?Arg?Gln?Asp?Leu?Glu?Arg?Gln

485?????????????????490?????????????????495

ctt?aat?gat?tta?cga?gaa?gag?ttt?aat?tca?ttg?tca?caa?gaa?ata?gct?1536

Leu?Asn?Asp?Leu?Arg?Glu?Glu?Phe?Asn?Ser?Leu?Ser?Gln?Glu?Ile?Ala

500?????????????????505?????????????????510

atg?tca?caa?ttg?att?gat?tta?gca?tta?tta?cct?tta?gat?atg?ttt?cct?1584

Met?Ser?Gln?Leu?Ile?Asp?Leu?Ala?Leu?Leu?Pro?Leu?Asp?Met?Phe?Pro

515?????????????????520?????????????????525

atg?ttt?tcg?ggg?ata?aaa?agt?act?att?gat?ctg?acc?aag?tca?atg?gca?1632

Met?Phe?Ser?Gly?Ile?Lys?Ser?Thr?Ile?Asp?Leu?Thr?Lys?Ser?Met?Ala

530?????????????????535?????????????????540

act?agt?gta?ata?aaa?aaa?ttt?aga?aaa?tca?aaa?tta?gcc?aca?tca?att?1680

Thr?Ser?Val?Ile?Lys?Lys?Phe?Arg?Lys?Ser?Lys?Leu?Ala?Thr?Ser?Ile

545?????????????????550?????????????????555?????????????????560

tca?gaa?atg?act?aat?tca?ttg?tca?gat?gcg?gct?tcg?tcg?gca?tca?aga?1728

Ser?Glu?Met?Thr?Asn?Ser?Leu?Ser?Asp?Ala?Ala?Ser?Ser?Ala?Ser?Arg

565?????????????????570?????????????????575

agt?gct?tct?att?aga?tca?aat?tta?tcg?acg?act?tca?aat?tgg?tct?aat?1776

Ser?Ala?Ser?Ile?Arg?Ser?Asn?Leu?Ser?Thr?Thr?Ser?Asn?Trp?Ser?Asn

580?????????????????585?????????????????590

gct?tca?aaa?agt?gta?tta?aat?gta?act?gac?tca?gta?aat?gat?gtt?tca?1824

Ala?Ser?Lys?Ser?Val?Leu?Asn?Val?Thr?Asp?Ser?Val?Asn?Asp?Val?Ser

595?????????????????600?????????????????605

aca?caa?aca?tct?acc?att?agt?aag?aaa?ctt?aga?tta?aaa?gag?atg?att?1872

Thr?Gln?Thr?Ser?Thr?Ile?Ser?Lys?Lys?Leu?Arg?Leu?Lys?Glu?Met?Ile

610?????????????????615?????????????????620

act?caa?act?gaa?gga?att?agt?ttt?gac?gat?att?tca?gca?gcc?gta?ttg?1920

Thr?Gln?Thr?Glu?Gly?Ile?Ser?Phe?Asp?Asp?Ile?Ser?Ala?Ala?Val?Leu

625?????????????????630?????????????????635?????????????????640

aga?acg?aaa?ata?gat?atg?tcc?aca?caa?att?gga?aaa?aat?acc?tta?cct?1968

Arg?Thr?Lys?Ile?Asp?Met?Ser?Thr?Gln?Ile?Gly?Lys?Asn?Thr?Leu?Pro

645?????????????????650?????????????????655

gat?ata?gtt?act?gaa?gca?act?gaa?aag?ttt?att?cca?aaa?cga?tca?tat?2016

Asp?Ile?Val?Thr?Glu?Ala?Thr?Glu?Lys?Phe?Ile?Pro?Lys?Arg?Ser?Tyr

660?????????????????665?????????????????670

cga?gtg?tta?aaa?gat?gat?gaa?gtg?atg?gaa?gtt?aac?act?gaa?gga?aag?2064

Arg?Val?Leu?Lys?Asp?Asp?Glu?Val?Met?Glu?Val?Asn?Thr?Glu?Gly?Lys

675?????????????????680?????????????????685

ttt?ttt?gct?tat?aaa?gtg?gat?aca?ctt?aat?gag?atc?cca?ttt?gat?ata?2113

Phe?Phe?Ala?Tyr?Lys?Val?Asp?Thr?Leu?Asn?Glu?Ile?Pro?Phe?Asp?Ile

690?????????????????695?????????????????700

aat?aaa?ttc?gct?gaa?ctt?gtg?acg?gat?tct?cca?gtt?ata?tca?gcg?ata?2160

Asn?Lys?Phe?Ala?Glu?Leu?Val?Thr?Asp?Ser?Pro?Val?Ile?Ser?Ala?Ile

705?????????????????710?????????????????715?????????????????720

ata?gat?ttt?aaa?acg?cta?aaa?aat?tta?aac?gat?aat?tat?gga?att?acc?2208

Ile?Asp?Phe?Lys?Thr?Leu?Lys?Asn?Leu?Asn?Asp?Asn?Tyr?Gly?Ile?Thr

725?????????????????730?????????????????735

cgc?ata?gaa?gca?ctt?aat?tta?att?aaa?tcg?aat?ccg?aat?gta?cta?cgt?2256

Arg?Ile?Glu?Ala?Leu?Asn?Leu?Ile?Lys?Ser?Asn?Pro?Asn?Val?Leu?Arg

740?????????????????745?????????????????750

aat?ttt?att?aat?caa?aat?aat?cca?att?ata?aga?aat?aga?att?gag?cag?2304

Asn?Phe?Ile?Asn?Gln?Asn?Asn?Pro?Ile?Ile?Arg?Asn?Arg?Ile?Glu?Gln

755?????????????????760?????????????????765

tta?att?cta?caa?tgt?aaa?ttg?tga?????????????????????????????????2328

Leu?Ile?Leu?Gln?Cys?Lys?Leu??*

770?????????????????775

<210>7

<21l>2328

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(2328)

＜223〉according to human rotavirus's serotype G ₁VP ₄Gene and designing is not changing coded aminoacid sequence

Prerequisite under, the codon that all codons are all had a preference for according to plant has carried out revising and manually closing

Become.

<400>7

atg?gct?tcg?ctc?atc?tac?agg?cag?ctc?ctc?acc?aat?tcg?tac?tcg?gtg?48

Met?Ala?Ser?Leu?Ile?Tyr?Arg?Gln?Leu?Leu?Thr?Asn?Ser?Tyr?Ser?Val

I???????????????5???????????????????10??????????????????15

gac?ttg?cat?gac?gag?atc?gag?cag?atc?gga?tcg?gag?aag?acc?cag?aat?96

Asp?Leu?His?Asp?Glu?Ile?Glu?Gln?Ile?Gly?Ser?Glu?Lys?Thr?Gln?Asn

20??????????????????25??????????????????30

gtg?acg?gta?aat?ccc?ggc?cca?ttc?gca?cac?act?aga?tag?gct?cca?gtt?144

Val?Thr?Val?Asn?Pro?Gly?Pro?Phe?Ala?Gln?Thr?Arg?Tyr?Ala?Pro?Val

35??????????????????40??????????????????45

aat?tgg?ggg?cac?gga?gag?atc?aat?gac?tcg?acc?acc?gtg?gag?cca?gtg?192

Asn?Trp?Gly?His?Gly?Glu?Ile?Asn?Asp?Ser?Thr?Thr?Val?Glu?Pro?Val

50??????????????????55??????????????????60

tta?gac?ggt?cct?tac?cag?ccg?acc?acc?ttc?aag?cca?ccc?aat?gac?tac?240

Leu?Asp?Gly?Pro?Tyr?Gln?Pro?Thr?Thr?Phe?Lys?Pro?Pro?Asn?Asp?Tyr

65??????????????????70??????????????????75??????????????????80

tgg?ctg?ctc?atc?agc?tcg?aat?acc?gac?gga?gtg?gtg?tac?gag?agc?acc?288

Trp?Leu?Leu?Ile?Ser?Ser?Asn?Thr?Asp?Gly?Val?Val?Tyr?Glu?Ser?Thr

85??????????????????90??????????????????95

aat?aat?agc?gac?ttc?tgg?acc?gca?gtg?atc?gct?gtg?gag?cca?cac?gtg?336

Asn?Asn?Ser?Asp?Phe?Trp?Thr?Ala?Val?Ile?Ala?Val?Glu?Pro?His?Val

100?????????????????105?????????????????110

agc?cag?acc?aat?agg?cag?tac?gtg?ctc?ttc?ggt?gag?aat?aag?cag?ttc?384

Ser?Gln?Thr?Asn?Arg?Gln?Tyr?Val?Leu?Phe?Gly?Glu?Asn?Lys?Gln?Phe

115?????????????????120?????????????????125

aac?gtg?gag?aat?agc?tcg?gac?aag?tgg?aag?ttt?ttc?gaa?atg?ttt?aag?432

Asn?Val?Glu?Asn?Ser?Ser?Asp?Lys?Trp?Lys?Phe?Phe?Glu?Met?Phe?Lys

130?????????????????135?????????????????140

ggt?aat?agt?cag?agc?gac?ttc?tcg?aat?agg?cgg?acc?tta?acc?tcg?aat?480

Gly?Asn?Ser?Gln?Ser?Asp?phe?Ser?Asn?Arg?Arg?Thr?Leu?Thr?Ser?Asn

145?????????????????150?????????????????155?????????????????160

aat?agg?ctc?gtg?gga?atg?ctc?aag?tac?ggt?gga?agg?gtg?tgg?acg?ttt?528

Asn?Arg?Leu?Val?Gly?Met?Leu?Lys?Tyr?Gly?Gly?Arg?Val?Trp?Thr?Phe

165?????????????????170?????????????????175

cac?ggt?gag?acc?cca?agg?gct?acc?acc?gac?agc?tcg?aat?acc?gcg?gac?576

His?Gly?Glu?Thr?Pro?Arg?Ala?Thr?Thr?Asp?Ser?Ser?Asn?Thr?Ala?Asp

180?????????????????185?????????????????190

ctc?aat?aat?atc?tcg?atc?atc?atc?cac?tcg?gag?ttc?tac?atc?atc?cca?624

Leu?Asn?Asn?Ile?Ser?Ile?Ile?Ile?His?Ser?Glu?Phe?Tyr?Ile?Ile?Pro

195?????????????????200?????????????????205

aga?tcc?caa?gaa?tct?aaa?tgt?aat?gaa?tat?att?aat?aat?ggt?tta?cca?672

Arg?Ser?Gln?Glu?Ser?Lys?Cys?Asn?Glu?Tyr?Ile?Asn?Asn?Gly?Leu?Pro

210?????????????????215?????????????????220

cca?atc?cag?aat?acc?agg?aat?gtg?gtg?ccg?ctc?tcg?ctc?tcg?tcg?agg?720

Pro?Ile?Gln?Asn?Thr?Arg?Asn?Val?Val?Pro?Leu?Ser?Leu?Ser?Ser?Arg

225?????????????????230?????????????????235?????????????????240

tcg?atc?cag?tac?agg?agg?gca?cag?gtg?aat?gag?gac?atc?acc?atc?tcg?768

Ser?Ile?Gln?Tyr?Arg?Arg?Ala?Gln?Val?Asn?Glu?Asp?Ile?Thr?Ile?Ser

245?????????????????250?????????????????255

aag?acc?tcg?ctc?tgg?aag?gag?atg?cag?tac?aat?agg?gac?atc?atc?atc?816

Lys?Thr?Ser?Leu?Trp?Lys?Glu?Met?Gln?Tyr?Asn?Arg?Asp?Ile?Ile?Ile

260?????????????????265?????????????????270

aga?ttc?aag?ttc?ggt?aat?agc?gtg?atc?aag?ctc?gga?gga?ctc?gga?tac?864

Arg?Phe?Lys?Phe?Gly?Asn?Ser?Val?Ile?Lys?Leu?Gly?Gly?Leu?Gly?Tyr

275?????????????????280?????????????????285

aag?tgg?tcg?gag?atc?tcg?tac?aag?gca?gcg?aat?tac?caa?tac?agc?tac?912

Lys?Trp?Ser?Glu?Ile?Ser?Tyr?Lys?Ala?Ala?Asn?Tyr?Gln?Tyr?Ser?Tyr

290?????????????????295?????????????????300

tcg?cgt?gac?ggt?gag?cag?gtg?acc?gca?cac?acc?acc?tgc?tcg?gtg?aat?960

Ser?Arg?Asp?Gly?Glu?Gln?Val?Thr?Ala?His?Thr?Thr?Cys?Ser?Val?Asn

305?????????????????310?????????????????315?????????????????320

gga?gtg?aat?aat?ttc?agc?tac?aat?gga?ggt?tcg?ctc?cct?acc?gac?ttc?1008

Gly?Val?Asn?Asn?Phe?Ser?Tyr?Asn?Gly?Gly?Ser?Leu?Pro?Thr?Asp?Phe

325?????????????????330?????????????????335

agc?atc?tcg?agg?tac?gag?gtg?atc?aag?gag?aat?tcg?tac?gtg?tac?atc?1056

Ser?Ile?Ser?Arg?Tyr?Glu?Val?Ile?Lys?Glu?Asn?Ser?Tyr?Val?Tyr?Ile

340?????????????????345?????????????????350

gac?tac?tgg?gac?gac?tcg?aag?gca?ttc?agg?aat?atg?gtg?tac?gtt?agg?1104

Asp?Tyr?Trp?Asp?Asp?Ser?Lys?Ala?Phe?Arg?Asn?Met?Val?Tyr?Val?Arg

355?????????????????360?????????????????365

tcg?ctc?gca?gca?aat?ctc?aat?tcg?gtg?aag?tgt?gtg?ggt?ggg?agc?tac?1152

Ser?Leu?Ala?Ala?Asn?Leu?Asn?Ser?Val?Lys?Cys?Val?Gly?Gly?Ser?Tyr

370?????????????????375?????????????????380

gac?ttc?agg?ctc?cct?gtg?ggt?gag?tgg?cct?atc?atg?aat?ggc?ggt?gct?1200

Asp?Phe?Arg?Leu?Pro?Val?Gly?Glu?Trp?Pro?Ile?Met?Asn?Gly?Gly?Ala

385?????????????????390?????????????????395?????????????????400

gtg?tcg?ctc?cac?ttc?gct?gga?gtg?acc?ctc?tcg?acc?cag?ttc?acc?gac?1248

Val?Ser?Leu?His?Phe?Ala?Gly?Val?Thr?Leu?Ser?Thr?Gln?Phe?Thr?Asp

405?????????????????410?????????????????415

ttc?gtg?tcg?ctc?aat?tcg?ctc?cga?ttc?agg?ttc?agc?ctc?acc?gtg?gac?1296

Phe?Val?Ser?Leu?Asn?Ser?Leu?Arg?Phe?Arg?Phe?Ser?Leu?Thr?Val?Asp

420?????????????????425?????????????????430

gag?cca?tct?ttc?tcg?atc?atc?cga?acc?cgt?acc?atg?aac?ctc?tac?gga?1344

Glu?Pro?Ser?Phe?Ser?Ile?Ile?Arg?Thr?Arg?Thr?Met?Asn?Leu?Tyr?Gly

435?????????????????440?????????????????445

ctc?cca?gca?gct?aat?cca?aac?aat?gga?aat?gag?tac?tac?gag?gtg?tcg?1392

Leu?Pro?Ala?Ala?Asn?Pro?Asn?Asn?Gly?Asn?Glu?Tyr?Tyr?Glu?Val?Ser

450?????????????????455?????????????????460

gga?agg?ttc?tcg?ctc?atc?tcg?ctc?gtg?cca?acc?aat?gac?gac?tac?cag?1440

Gly?Arg?Phe?Ser?Leu?Ile?Ser?Leu?Val?Pro?Thr?Asn?Asp?Asp?Tyr?Gln

465?????????????????470?????????????????475?????????????????480

acc?cca?atc?atg?aat?tcg?gtg?acc?gtg?agg?cag?gat?ctc?gag?cgc?cag?1488

Thr?Pro?Ile?Met?Asn?Ser?Val?Thr?Val?Arg?Gln?Asp?Leu?Glu?Arg?Gln

485?????????????????490?????????????????495

ctc?aat?gac?ctc?cga?gag?gag?ttc?aat?tcg?ctc?tcg?cag?gag?atc?gct?1536

Leu?Asn?Asp?Leu?Arg?Glu?Glu?Phe?Asn?Ser?Leu?Ser?Gln?Glu?Ile?Ala

500?????????????????505?????????????????510

atg?tcg?cag?ctc?atc?gac?ctc?gca?ctc?ctc?cct?ctc?gac?atg?ttc?cct?1584

Met?Ser?Gln?Leu?Ile?Asp?Leu?Ala?Leu?Leu?Pro?Leu?Asp?Met?Phe?Pro

515?????????????????520?????????????????525

atg?ttc?tcg?ggg?atc?aag?agc?acc?atc?gac?ctc?acc?aag?tcg?atg?gca?1632

Met?Phe?Ser?Gly?Ile?Lys?Ser?Thr?Ile?Asp?Leu?Thr?Lys?Ser?Met?Ala

530?????????????????535?????????????????540

acc?agc?gtg?atc?aag?aag?ttc?agg?aag?tcg?aag?ctc?gcc?acc?tcg?atc?1680

Thr?Ser?Val?Ile?Lys?Lys?Phe?Arg?Lys?Ser?Lys?Leu?Ala?Thr?Ser?Ile

545?????????????????550?????????????????555?????????????????560

tcg?gag?atg?acc?aat?tcg?ctc?tcg?gac?gcg?gct?tcg?tcg?gca?tcg?agg?1728

Ser?Glu?Met?Thr?Asn?Ser?Leu?Ser?Asp?Ala?Ala?Ser?Ser?Ala?Ser?Arg

565?????????????????570?????????????????575

agc?gct?tcg?att?agg?tcg?aat?ctc?tcg?acc?acc?tcg?aat?tgg?tcg?aat?1776

Ser?Ala?Ser?Ile?Arg?Ser?Asn?Leu?Ser?Thr?Thr?Ser?Asn?Trp?Ser?Asn

580?????????????????585?????????????????590

gct?tcg?aag?agc?gtg?ctc?aat?gtg?acc?gac?tcg?gtg?aat?gac?gtg?tcg?1824

Ala?Ser?Lys?Ser?Val?Leu?Asn?Val?Thr?Asp?Ser?Val?Asn?Asp?Val?Ser

595?????????????????600?????????????????605

acc?cag?acc?tcg?acc?atc?agc?aag?aag?ctc?agg?ctc?aag?gag?atg?atc?1872

Thr?Gln?Thr?Ser?Thr?Ile?Ser?Lys?Lys?Leu?Arg?Leu?Lys?Glu?Met?Ile

610?????????????????615?????????????????620

acc?cag?acc?gag?gga?atc?agc?ttc?gac?gac?atc?tcg?gca?gcc?gtg?ttg?1920

Thr?Gln?Thr?Glu?Gly?Ile?Ser?Phe?Asp?Asp?Ile?Ser?Ala?Ala?Val?Leu

625?????????????????630?????????????????635?????????????????640

agg?acc?aag?atc?gac?atg?tcg?acc?cag?atc?gga?aag?aat?acc?ctc?cct?1968

Arg?Thr?Lys?Ile?Asp?Met?Ser?Thr?Gln?Ile?Gly?Lys?Asn?Thr?Leu?Pro

645?????????????????650?????????????????655

gac?atc?gtg?acc?gag?gca?acc?gag?aag?ttc?atc?cca?aag?cga?tcg?tac?2016

Asp?Ile?Val?Thr?Glu?Ala?Thr?Glu?Lys?Phe?Ile?Pro?Lys?Arg?Ser?Tyr

660?????????????????665?????????????????670

cga?gtg?ctc?aag?gac?gat?gag?gtg?atg?gac?gtg?aac?acc?gag?gga?aag?2064

Arg?Val?Leu?Lys?Asp?Asp?Glu?Val?Met?Glu?Val?Asn?Thr?Glu?Gly?Lys

675?????????????????680?????????????????685

ttc?ttc?gct?tac?aag?gtg?gac?acc?ctc?aat?gag?atc?cca?ttc?gac?atc?2112

Phe?Phe?Ala?Tyr?Lys?Val?Asp?Thr?Leu?Asn?Glu?Ile?Pro?Phe?Asp?Ile

690?????????????????695?????????????????700

aat?aag?ttc?gct?gag?ctc?gtg?acc?gac?tcg?cca?gtg?atc?tcg?gcg?atc?2160

Asn?Lys?Phe?Ala?Glu?Leu?Val?Thr?Asp?Ser?Pro?Val?Ile?Ser?Ala?Ile

705?????????????????710?????????????????715?????????????????720

atc?gac?ttc?aag?acc?ctc?aag?aat?ctc?aac?gac?aat?tac?gga?atc?acc?2208

Ile?Asp?Phe?Lys?Thr?Leu?Lys?Asn?Leu?Asn?Asp?Asn?Tyr?Gly?Ile?Thr

725?????????????????730?????????????????735

cgc?atc?gag?gca?ctc?aat?ctc?atc?aag?tcg?aat?ccg?aat?gtg?ctc?cgg?2256

Arg?Ile?Glu?Ala?Leu?Asn?Leu?Ile?Lys?Ser?Asn?Pro?Asn?Val?Leu?Arg

740?????????????????745?????????????????750

aat?ttc?atc?aat?cag?aat?aat?cca?atc?atc?agg?aat?agg?atc?gag?cag?2304

Asn?Phe?Ile?Asn?Gln?Asn?Asn?Pro?Ile?Ile?Arg?Asn?Arg?Ile?Glu?Gln

755?????????????????760?????????????????765

ctc?atc?ctc?cag?tgc?aag?ctc?tga?????????????????????????????????2328

Leu?Ile?Leu?Gln?Cys?Lys?Leu??*

770?????????????775

<210>8

<211>775

<212>PRT

＜213〉human rotavirus (human rotavirus)

<400>8

Met?Ala?Ser?Leu?Ile?Tyr?Arg?Gln?Leu?Leu?Thr?Asn?Ser?Tyr?Ser?Val

1???????????????5???????????????????10??????????????????15

Asp?Leu?His?Asp?Glu?Ile?Glu?Gln?Ile?Gly?Ser?Glu?Lys?Thr?Gln?Asn

20??????????????????25??????????????????30

Val?Thr?Val?Asn?Pro?Gly?Pro?Phe?Ala?Gln?Thr?Arg?Tyr?Ala?Pro?Val

35??????????????????40??????????????????45

Asn?Trp?Gly?His?Gly?Glu?Ile?Asn?Asp?Ser?Thr?Thr?Val?Glu?Pro?Val

50??????????????????55??????????????????60

Leu?Asp?Gly?Pro?Tyr?Gln?Pro?Thr?Thr?Phe?Lys?Pro?Pro?Asn?Asp?Tyr

65??????????????????70??????????????????75??????????????????80

Trp?Leu?Leu?Ile?Ser?Ser?Asn?Thr?Asp?Gly?Val?Val?Tyr?Glu?Ser?Thr

85??????????????????90??????????????????95

Asn?Asn?Ser?Asp?Phe?Trp?Thr?Ala?Val?Ile?Ala?Val?Glu?Pro?His?Val

100?????????????????105?????????????????110

Ser?Gln?Thr?Asn?Arg?Gln?Tyr?Val?Leu?Phe?Gly?Glu?Asn?Lys?Gln?Phe

115?????????????????120?????????????????125

Asn?Val?Glu?Asn?Ser?Ser?Asp?Lys?Trp?Lys?Phe?Phe?Glu?Met?Phe?Lys

130?????????????????135?????????????????140

Gly?Asn?Ser?Gln?Ser?Asp?phe?Ser?Asn?Arg?Arg?Thr?Leu?Thr?Ser?Asn

145?????????????????150?????????????????155?????????????????160

Asn?Arg?Leu?Val?Gly?Met?Leu?Lys?Tyr?Gly?Gly?Arg?Val?Trp?Thr?Phe

165?????????????????170?????????????????175

His?Gly?Glu?Thr?Pro?Arg?Ala?Thr?Thr?Asp?Ser?Ser?Asn?Thr?Ala?Asp

180?????????????????185?????????????????190

Leu?Asn?Asn?Ile?Ser?Ile?Ile?Ile?His?Ser?Glu?Phe?Tyr?Ile?Ile?Pro

195?????????????????200?????????????????205

Arg?Ser?Gln?Glu?Ser?Lys?Cys?Asn?Glu?Tyr?Ile?Asn?Asn?Gly?Leu?Pro

210?????????????????215?????????????????220

Pro?Ile?Gln?Asn?Thr?Arg?Asn?Val?Val?Pro?Leu?Ser?Leu?Ser?Ser?Arg

225?????????????????230?????????????????235?????????????????240

Ser?Ile?Gln?Tyr?Arg?Arg?Ala?Gln?Val?Asn?Glu?Asp?Ile?Thr?Ile?Ser

245?????????????????250?????????????????255

Lys?Thr?Ser?Leu?Trp?Lys?Glu?Met?Gln?Tyr?Asn?Arg?Asp?Ile?Ile?Ile

260?????????????????265?????????????????270

Arg?Phe?Lys?Phe?Gly?Asn?Ser?Val?Ile?Lys?Leu?Gly?Gly?Leu?Gly?Tyr

275?????????????????280?????????????????285

Lys?Trp?Ser?Glu?Ile?Ser?Tyr?Lys?Ala?Ala?Asn?Tyr?Gln?Tyr?Ser?Tyr

290?????????????????295?????????????????300

Ser?Arg?Asp?Gly?Glu?Gln?Val?Thr?Ala?His?Thr?Thr?Cys?Ser?Val?Asn

305?????????????????310?????????????????315?????????????????320

Gly?Val?Asn?Asn?Phe?Ser?Tyr?Asn?Gly?Gly?Ser?Leu?Pro?Thr?Asp?Phe

325?????????????????330??????????????????335

Ser?Ile?Ser?Arg?Tyr?Glu?Val?Ile?Lys?Glu?Asn?Ser?Tyr?Val?Tyr?Ile

340?????????????????345?????????????????350

Asp?Tyr?Trp?Asp?Asp?Ser?Lys?Ala?Phe?Arg?Asn?Met?Val?Tyr?Val?Arg

355?????????????????360?????????????????365

Ser?Leu?Ala?Ala?Asn?Leu?Asn?Ser?Val?Lys?Cys?Val?Gly?Gly?Ser?Tyr

370?????????????????375?????????????????380

Asp?Phe?Arg?Leu?Pro?Val?Gly?Glu?Trp?Pro?Ile?Met?Asn?Gly?Gly?Ala

385?????????????????390?????????????????395?????????????????400

Val?Ser?Leu?His?Phe?Ala?Gly?Val?Thr?Leu?Ser?Thr?Gln?Phe?Thr?Asp

405?????????????????410?????????????????415

Phe?Val?Ser?Leu?Asn?Ser?Leu?Arg?Phe?Arg?Phe?Ser?Leu?Thr?Val?Asp

420?????????????????425?????????????????430

Glu?Pro?Ser?Phe?Ser?Ile?Ile?Arg?Thr?Arg?Thr?Met?Asn?Leu?Tyr?Gly

435?????????????????440?????????????????445

Leu?Pro?Ala?Ala?Asn?Pro?Asn?Asn?Gly?Asn?Glu?Tyr?Tyr?Glu?Val?Ser

450?????????????????455?????????????????460

Gly?Arg?Phe?Ser?Leu?Ile?Ser?Leu?Val?Pro?Thr?Asn?Asp?Asp?Tyr?Gln

465?????????????????470?????????????????475?????????????????480

Thr?Pro?Ile?Met?Asn?Ser?Val?Thr?Val?Arg?Gln?Asp?Leu?Glu?Arg?Gln

485?????????????????490?????????????????495

Leu?Asn?Asp?Leu?Arg?Glu?Glu?Phe?Asn?Ser?Leu?Ser?Gln?Glu?Ile?Ala

500?????????????????505?????????????????510

Met?Ser?Gln?Leu?Ile?Asp?Leu?Ala?Leu?Leu?Pro?Leu?Asp?Met?Phe?Pro

515?????????????????520?????????????????525

Met?Phe?Ser?Gly?Ile?Lys?Ser?Thr?Ile?Asp?Leu?Thr?Lys?Ser?Met?Ala

530?????????????????535?????????????????540

Thr?Ser?Val?Ile?Lys?Lys?Phe?Arg?Lys?Ser?Lys?Leu?Ala?Thr?Ser?Ile

545?????????????????550?????????????????555?????????????????560

Ser?Glu?Met?Thr?Asn?Ser?Leu?Ser?Asp?Ala?Ala?Ser?Ser?Ala?Ser?Arg

565?????????????????570?????????????????575

Ser?Ala?Ser?Ile?Arg?Ser?Asn?Leu?Ser?Thr?Thr?Ser?Asn?Trp?Ser?Asn

580?????????????????585?????????????????590

Ala?Ser?Lys?Ser?Val?Leu?Asn?Val?Thr?Asp?Ser?Val?Asn?Asp?Val?Ser

595?????????????????600?????????????????605

Thr?Gln?Thr?Ser?Thr?Ile?Ser?Lys?Lys?Leu?Arg?Leu?Lys?Glu?Met?Ile

610?????????????????615?????????????????620

Thr?Gln?Thr?Glu?Gly?Ile?Ser?Phe?Asp?Asp?Ile?Ser?Ala?Ala?Val?Leu

625?????????????????630?????????????????635?????????????????640

Arg?Thr?Lys?Ile?Asp?Met?Ser?Thr?Gln?Ile?Gly?Lys?Asn?Thr?Leu?Pro

645?????????????????650?????????????????655

Asp?Ile?Val?Thr?Glu?Ala?Thr?Glu?Lys?Phe?Ile?Pro?Lys?Arg?Ser?Tyr

660?????????????????665?????????????????670

Arg?Val?Leu?Lys?Asp?Asp?Glu?Val?Met?Glu?Val?Asn?Thr?Glu?Gly?Lys

675?????????????????680?????????????????685

Phe?Phe?Ala?Tyr?Lys?Val?Asp?Thr?Leu?Asn?Glu?Ile?Pro?Phe?Asp?Ile

690?????????????????695?????????????????700

Asn?Lys?Phe?Ala?Glu?Leu?Val?Thr?Asp?Ser?Pro?Val?Ile?Ser?Ala?Ile

705?????????????????710?????????????????715?????????????????720

Ile?Asp?Phe?Lys?Thr?Leu?Lys?Asn?Leu?Asn?Asp?Asn?Tyr?Gly?Ile?Thr

725?????????????????730?????????????????735

Arg?Ile?Glu?Ala?Leu?Asn?Leu?Ile?Lys?Ser?Asn?Pro?Asn?Val?Leu?Arg

740?????????????????745?????????????????750

Asn?Phe?Ile?Asn?Gln?Asn?Asn?Pro?Ile?Ile?Arg?Asn?Arg?Ile?Glu?Gln

755?????????????????760?????????????????765

Leu?Ile?Leu?Gln?Cys?Lys?Leu

770?????????????????775

Claims (40)

1. method that improves wheel virus antigen albumen expression contents in transgenic plant, this method comprise the optimization of goal gene codon and use the part non-coding sequence of plant virus 5 ' end and 3 ' end to strengthen the regulating and controlling sequence of expression as goal gene in plant.

2. as the method in the claim 1, it is edible that wherein said plant has a part at least.

3. as the method in the claim 1, wherein said virus antigen albumen is human rotavirus VP ₇, VP ₄The antigen protein or derivatives thereof.

4. as the method in the claim 1, wherein said plant virus is a marmor upsilon, and said enhancing expression regulation sequence is the 5 ' end of marmor upsilon RNA and the part non-coding sequence of 3 ' end.

5. one kind causes that people or Mammals produce the method for immunne response to wheel virus antigen albumen, and this method comprises that using effective and definite immunizing dose, as to derive from the transgenic plant that can produce this antigen protein or derivatives thereof vegetable material or its to process extract carries out oral or enterally administering to this Mammals.

6. as the method in the claim 5, it is edible that wherein said plant has a part at least.

7. as the method in the claim 5, wherein said wheel virus antigen albumen is VP ₇, VP ₄The antigen protein or derivatives thereof.

8. antigen is from a kind of virus, and wherein said antigen can be expressed production in plant, and this antigen is protein, has immunogenicity under state of nature.

9. as the antigen in the claim 8, it is edible that wherein said plant has a part at least.

10. as the antigen in the claim 8, wherein said virus is the human rotavirus who causes diarrhoea.

11. transgenic plant are the plants that can express one or more viral proteins, these albumen have immunogenicity under state of nature.

12. as the transgenic plant in the claim 11, it is edible that wherein said plant has a part at least.

13. as the transgenic plant in the claim 11, wherein said virus is people's rotavirus of suffering from diarrhoea.

Wheel virus antigen is expressed in plant 14. a people suffers from diarrhoea, and wherein said antigen is protein, and it has immunogenicity under native state, and can be used as a kind of effective integral part of medicine.

15. a food is made up of certain part of transgenic plant at least, it is eaten is that wherein said plant is to show one or more human rotavirus VP because it has certain nutrient value ₇Or VP ₄The plant of antigen protein, these antigen proteins have immunogenicity under state of nature.

16. as any food in the claim 15, wherein said plant edible food partly comprises fruit, leaf, stem, root or said plant seed.

17. a plasmid vector that is used to transform plant comprises at least:

One section coding people antigenic dna sequence dna of rotavirus protein of suffering from diarrhoea, this sequence be the codon had a preference for according to plant through what optimize, this albumen has immunogenicity under native state.

The coding people suffer from diarrhoea the antigenic dna sequence dna of rotavirus protein two ends respectively be derived from marmor upsilon 5 ' end and 3 ' the part non-coding sequence of holding links to each other.

The plant promoter that the dna sequence dna of modifying with above-mentioned process is connected, this promotor is handled said gene and is expressed in said plant.

18. as the plasmid vector in the claim 17, it contains a selection or marker gene.

19. as the plasmid vector in the claim 17, wherein said plant promoter is the cauliflower mosaic virus 35S promoter.

20. as the plasmid vector in the claim 17, it is edibility that wherein said plant has a part at least.

21. as the plasmid vector in the claim 17, wherein said viral protein antigen is people's rotavirus vp of suffering from diarrhoea ₇, VP ₄The antigen protein or derivatives thereof.

22. one section dna sequence dna that is used for particle bombardment conversion plant comprises at least:

One section coding people antigenic dna sequence dna of rotavirus protein of suffering from diarrhoea, this sequence be the codon had a preference for according to plant through what optimize, this albumen has immunogenicity under native state.

The coding people suffer from diarrhoea the antigenic dna sequence dna of rotavirus protein two ends respectively be derived from marmor upsilon 5 ' end and 3 ' the part non-coding sequence of holding links to each other.

The plant promoter that the dna sequence dna of modifying with above-mentioned process is connected, this promotor is handled said gene and is expressed in said plant.

23. as the dna sequence dna in the claim 22, it contains a selection or marker gene.

24. as the dna sequence dna in the claim 22, wherein said plant promoter is the cauliflower mosaic virus 35S promoter.

25. as the dna sequence dna in the claim 22, it is edibility that wherein said plant has a part at least.

26. as the dna sequence dna in the claim 22, wherein said viral protein antigen is the people wheel virus antigen albumen VP that suffers from diarrhoea ₄Or VP ₇

27. a method that makes up transgenic plant cells comprises:

Make up a plasmid vector or section of DNA sequence, wherein all contain one or more codons of coding through the people who the optimizes wheel virus antigen albumen VP that suffers from diarrhoea ₇, VP ₄The gene or derivatives thereof, be derived from marmor upsilon 5 ' end and the 3 ' non-coding area sequence of holding and can handle the plant promoter that these genes are expressed in said plant, these albumen have immunogenicity under state of nature.With this plasmid vector or this dna sequence dna transformed plant cells.

28. as the method in the claim 27, it comprises bears complete transfer-gen plant again from said transgenic plant cells.

29. the method for a kind of electuary, oral liquid or other preparation of producing comprises:

Make up a plasmid vector or section of DNA sequence, wherein all contain one or more codons of coding through the people who the optimizes wheel virus antigen albumen VP that suffers from diarrhoea ₇Or VP ₄The gene or derivatives thereof, be derived from marmor upsilon 5 ' end and the 3 ' non-coding area sequence of holding and can handle the plant promoter that these genes are expressed in said plant, these albumen have immunogenicity under native state.

With this plasmid vector or this dna sequence dna transformed plant cells.

The edible part of transgenic plant is freezing, dry and grind, directly or after mixing be made into electuary, oral liquid or other preparation then.

30. as the method for claim 29, wherein also be included in and utilize transgenic plant as food and be processed into before electuary, oral liquid or other preparation, should from said transgenic plant cells, bear complete transfer-gen plant again.

31., wherein also comprise from said vegetable cell partial purification or be purified into said antigen protein fully then directly or mix the effective integral part of back as electuary, oral liquid or other preparation as the method in the claim 29.

32. as the method in the claim 30, comprising certain part of gathering in the crops said transgenic plant at least.

33. as the method in the claim 29, wherein said vegetable cell transforms by the Agrobacterium system.

34. as the method in the claim 33, wherein said Agrobacterium system is an Agrobacterium Ti-plasmids system.

35. as the method in the claim 29, wherein said plant is tomato, potato, lettuce, Radix Dauci Sativae, banana, apple, pawpaw, soybean, clover or other edible plant.

36. as the method in the claim 29, wherein said plasmid vector is a binary vector system.

37. as the method in the claim 29, wherein said plasmid vector is a conformability carrier.

38. as the method in the claim 29, wherein said plasmid vector is pBI121.

39. as the method in the claim 29, wherein said vegetable cell is to transform as methods such as particle bombardment, microtubule injection, PEG absorption process and electro fusion methods by other method for transformation except that Agrobacterium transformation system.

40. as the method in the claim 29, wherein said vegetable cell is the cell of a dicotyledons, or a monocotyledonous cell.

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