CN1772902A - One coding gene of rotavirus VP6 protein and its application - Google Patents

Abstract

The present invention discloses one coding gene of rotavirus VP6 protein and its application, and aims at providing one coding gene of rotavirus VP6 protein with high expression amount in dicotyledonous plant, especially clover, method of producing oral rotavirus vaccine with the coding gene and the obtained oral vaccine. The rotavirus VP6 protein coding gene has one of the following nucleotide sequences: 1. DNA sequence of SEQ ID No. 1 in the sequence list; 2. DNA sequence of SEQ ID No. 2 in the sequence list; 3. the nucleotide sequence capable of hybridizing in high strict condition with the DNA sequence limited by SEQ ID No. 1 or SEQ ID No. 2; and 4. DNA sequence possessing over 80 % homology with the SEQ ID No. 1 or SEQ ID No. 2 limited DNA sequence and high expression amount in dicotyledonous plant. The oral rotavirus vaccine is used in preventing various rotavirus infection caused acute diarrhea of infant and young animal.

Description

Rotavirus vp 6 proteic a kind of encoding gene and application thereof

Technical field

The present invention relates to rotavirus vp 6 proteic a kind of encoding gene and application thereof, particularly utilize this encoding gene to produce the method for rotavirus oral vaccine and the rotavirus oral vaccine that obtains.

Background technology

The infection of rotavirus is ubiquity in the mankind, Mammals, birds, is the main diseases original of viral diarrhea.Rotavirus is divided into 7 groups (A-G), and wherein the A group rotavirus is the major cause that causes infant and young animal diarrhoea, worldwide is widely current.No matter developed country or developing country, the infant more than 90% had been subjected to the infection of rotavirus before 3 years old.In developing country, annual nearly 18,000,000 children are in hospital because of infecting rotavirus, and wherein about megadeath causes enormous economic loss (Dodet etal.1997), occupies second in China infant cause of the death.In addition, rotavirus also is one of lethal major reason of children livestock in age (ox, sheep, pig).Calf infects pilosity and was born in 3 days-15 ages in week, and case fatality rate can reach 50%, the concurrent pneumonia that has, and lethality rate raises.The piglet that lacks the maternal antibody protection, sickness rate is higher in one week of birth, and case fatality rate is up to 100%.This shows, induce passive immunization to seem and be even more important for the development Rotavirus Vaccine.

Rotavirus belongs to Reoviridae, and its genome is a double-stranded RNA, comprises 11 gene fragments.Each fragment contains an open reading frame, encode respectively 6 structural protein (VP1, VP2, VP3, VP4, VP6, VP7) and 5 Nonstructural Proteins (NSP1-NSP5).The shell of rotavirus is divided into three layers, and outermost layer is made of VP4 and VP7, and the middle layer is made of VP6, internal layer by VP2 constitute (Raming R F, Annu.Rev.Microbiol., 1997,51:225-255).VP4 and VP7 can induce body to produce neutrality antibody, and the serotype of decision rotavirus is main species specificity antigen, and the vaccine that utilizes VP4 and VP7 to make can not produce cross protection.VP6 is group or subgroup specific antigens, accounts for 51% of virion, and the antibody that the protein induced body of VP6 produces has very strong cross reactivity, expresses the proteic vaccine of VP6 the allos provide protection can be provided.Though VP6 albumen can not stimulate body to produce neutrality antibody, it can strengthen homology and allogenic VP4 and the proteic immunogenicity of VP7, and raising neutralizing antibody level (Esquivel FR, et.al.Arch.Virol., 2000,145:813-825).People such as Feng N (J.Clin.Invest., 2002, result of study 109:1203-1213) shows: VP6 antibody I gA is in vivo by the provide protection of the early transcription realization in the inhibition virus replication to body.Therefore, when the development Rotavirus Vaccine, VP6 albumen can be produced the shared vaccine of people and animals efficiently as candidate albumen.

U.S. FAD approval end user one monkey rotavirus tetravalence reprovision vaccine (RRV-TV) in 1998; this vaccine is better to severe diarrhoea protectiveness; to the barrier effect of common diarrhoea about 70%; but 1999 because discovery causes infant's intussusception and be stopped use (Centers for Disease Control.MMWR; 1999,48:577-581).The mandate although subunit vaccine of now developing and dna vaccination have patented abroad because there are problems such as preparation technology, security, production cost, is not all gone public at present.The nonreplicative recombinant adenovirus of expressing human rotavirus VP 7 was applied for a patent (application number 02104125.3) at home in 2002.Because VP7 is a species specificity antigen, the vaccine made from a kind of VP7 of serotype often can not make body produce the ability of the rotavirus of other serotype of opposing, because there is different serotype in rotavirus in different areas, so this vaccine is unfavorable for the big area popularization.

Utilize plant to produce the edible vaccine and opened up a new way at biological technical field as bio-reactor.1992, and Mason etc. (Proc Natl Acad Sci USA, 1992,89:11745-11749) proposed to produce the new approaches of vaccine with transgenic plant, up to now, there have been multiple virus and bacterial antigens in plant, to express in order to the production oral vaccine.Rotavirus infects and breeds through intestinal mucosa and is confined to enteron aisle, and therefore inducing the localised protection mucosal immunity is the key point of development Rotavirus Vaccine.Applying transgene plant production edible vaccine has great potential, because it not only can induce the mucous membrane IgA of body generation secretor type, or a low cost system of producing antigen protein.There are some researches show, oral route is easier to induce mucosa-immune than other parenteral routes, the antigen protein that utilizes transgenic plant to produce is caught cell-M cell (microfold cell) identification and transhipment by antigen special in the enteron aisle lymphocytic epithelium after arriving enteron aisle, further under the assistance of antigen presenting cell and T cell, activate the B cell and discharge secretory IgA (sIgA).After soluble antigen in sIgA and the mucous membrane, bacterium, virus etc. combine, may seal the site that they combine with acceptor on the M cell, thereby stop it to enter body; In addition, sIgA also can enter intestinal epithelial cell through blood circulation, to just at the virus performance interception function of infects epithelial.So, mucous membrane IgA not only can resist those and grow microorganism at mucous membrane surface surely, can also resist those and enter body through mucous membrane surfaces but the pathogenic micro-organism (Lamm M E, the Annu Rev Microbiol that cause systemic disease, 1997,51:311-340).

2000, and O ' Brien G J etc. (Virology, 2000,270:444-453) utilize virus vector in tobacco, to express rotavirus vp 6 albumen and in plant materials, successfully be assembled into virion.2002, Matsumura, T. etc. (Arch Virol, 2002,147:1263-1270) use agriculture bacillus mediated conversion system and in transgenic Rhizoma Solani tuber osi, expressed bovine rota VP6 gene.Yu J in 2003 etc. in transgenic Rhizoma Solani tuber osi, expressed mouse rotavirus vp 6 genes (Transgenic Research, 2003,12:163-169).But in above-mentioned research, the proteic expression amount of VP6 is all very low, and high-content only reaches 0.1% of potato tuber total soluble protein.Because the antigen of low expression level is the oral immunological tolerance that may cause body directly, so increase the key factor that the expression amount of antigen gene in the transfer-gen plant has become development edible vaccine.

Up to now, existing multiple dicotyledons and monocotyledons are used to produce vaccine, the proteic vegetable material of having reported at present of antigen expressed mainly concentrates on tobacco and potato, this mainly is because the genetic transformation easy handling of this two kind of plant, but contain harmful compositions such as Nicotine in the tobacco, and potato should not be eaten raw, can cause antigen protein content to reduce by 50% after boiling, and produces oral vaccine so this two kind of plant is unsuitable for commercialization.

The diarrhoea of the humans and animals that causes for rotavirus infection does not still have effectively treatment and preventive means at present, and this area presses for develops a kind of vaccine safely and efficiently.

Summary of the invention

The purpose of this invention is to provide in dicotyledons, particularly in clover, have rotavirus vp 6 proteic a kind of encoding genes of higher expression amount.

Rotavirus vp 6 protein coding genes provided by the present invention can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the sequence table: 2 dna sequence dna;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of 1 or SEQ ID №: the 2 dna sequence dnas hybridization that limit;

4) with sequence table in SEQ ID №: 1 or SEQ ID №: 2 dna sequence dnas that limit have 80% above homology, and have the dna sequence dna of higher expression amount in dicotyledons.

The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

Sequence 1 in the sequence table is by 1194 based compositions, its open reading frame (ORF) be from 5 ' the 1st at end is to 1194 bit bases, the VP6 of 397 amino-acid residues of coding, name is called the sVP6 gene, the molecular weight of its single stranded DNA is 367.86, and the molecular weight of double-stranded DNA is 736.0.

Sequence 2 in the sequence table is Kozak sequence (AACC by 1216 based compositions from 5 of sequence 2 ' end 1-8 bit base ATGG), from 5 of sequence 2 ' end 5-1195 bit base is the sVP6 encoding sequence, from 5 of sequence 2 ' end 1196-1213 bit base is an endoplasmic reticulum maintenance signal peptide SEKDEL sequence (TCT GAG AAA GAT GAG CTA), from 5 of sequence 2 ' end 1213-1216 bit base is terminator codon (TAA), with the nucleotide sequence called after sVP6-SEKDEL gene of sequence in the sequence table 2.

Another object of the present invention provides a kind of method of utilizing above-mentioned rotavirus vp 6 protein coding genes to produce the rotavirus oral vaccine.

The method of production rotavirus oral vaccine provided by the present invention, be that above-mentioned any rotavirus vp 6 protein coding genes are imported the dicotyledons cell, obtain expressing rotavirus vp 6 proteic dicotyledonous transgenic plant, extract the total soluble protein of described dicotyledonous transgenic plant, obtain the rotavirus oral vaccine.

Described dicotyledons comprises clover, soybean, tomato, apple, banana and Radix Dauci Sativae etc.

Described dicotyledons is preferably clover.

Described rotavirus vp 6 protein coding genes can utilize any carrier that can guide foreign gene to express in plant, it is imported vegetable cell.Described rotavirus vp 6 protein coding genes can add any general promotor, strengthen promotor or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.For the ease of transgenic plant or transgenic plant cells being identified and being screened, can process employed carrier, as the antibiotic marker gene (gentamicin, kantlex etc.) that adds alternative mark (gus gene, GFP and luciferase gene etc.) or have resistance.For the security that transgenic plant discharge, when making up plant expression vector, also can not carry any marker gene, carry out specific PCR molecular marker screening in seedling stage.

Contain the expression vector of described rotavirus vp 6 protein coding genes can be by using conventional biological method transformed plant cells or tissues such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, agriculture bacillus mediated or particle gun, and the plant transformed tissue cultivating become plant.

In order to improve rotavirus vp 6 proteic expression amounts, 5 of described rotavirus vp 6 protein coding genes ' end is connected with following element:

1) 5 of described rotavirus vp 6 protein coding genes ' end closely is connected with Kozak sequence (AACCATGGCT);

2) 5 of described Kozak sequence ' end closely is connected with tobacco etch virus 5 ' non-translational region;

Described tobacco etch virus 5 ' non-translational region can have the nucleotide sequence of sequence 3 in the sequence table; This tobacco etch virus 5 ' non-translational region can be cut carrier pRTL2 with restriction enzyme XhoI or EcoR I and Nco I enzyme and obtain;

3) 5 of described tobacco etch virus 5 ' non-translational region ' end is connected with the cauliflower mosaic virus 35S promoter of the two enhansers of band;

The cauliflower mosaic virus 35S promoter of the two enhansers of described band is one of following nucleotide sequence;

1) SEQ ID № in the sequence table: 4 dna sequence dna;

2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 4 dna sequence dnas hybridization that limit;

3) cut the nucleotide fragments of the cauliflower mosaic virus 35S promoter of the two enhansers of band that carrier pRTL2 obtains with restriction enzyme HindIII or SphI or PstI or HincII and XhoI or EcoRI enzyme;

In the sequence 3, being the recognition site of XhoI from the 1st to the 6th deoxynucleotide of 5 ' end, is the recognition site of EcoRI from the 6th to the 11st deoxynucleotide of 5 ' end, is the recognition site of NcoI from the 139th to the 144th deoxynucleotide of 5 ' end.In the sequence 4, from the 1st to the 6th deoxynucleotide of 5 ' end is the recognition site of HindIII, from the 7th to the 12nd deoxynucleotide of 5 ' end is the recognition site of SphI, from the 13rd to the 18th deoxynucleotide of 5 ' end is the recognition site of PstI, from the 19th to the 24th deoxynucleotide of 5 ' end is the recognition site of HincII, from the 770th to the 775th deoxynucleotide of 5 ' end is the recognition site of XhoI, is the recognition site of EcoRI from the 775th to the 780th deoxynucleotide of 5 ' end.

Described rotavirus vp 6 protein coding genes import vegetable cell by expression vector pBsVP6.

Utilize the rotavirus oral vaccine of aforesaid method production also to belong to protection scope of the present invention.

The present invention adopts the codon of dicotyledons preference to transform the partial nucleotide sequence of people A group rotavirus VP6 gene, increased substantially the expression amount (content up to 0.28% of total soluble protein) of VP6 albumen in the transgenic plant blade, antigen gene commercially producing of rotavirus plant vaccine that rise to of expression amount in transgenic plant provides important assurance; With the female mouse of rotavirus oral vaccine oral immunity BALB/c of the present invention, produced serum IgG and the mucous membrane IgA antibody of high-caliber anti-VP6 in female mouse body; With ape rotavirus SA-11 the mouse that immune stepmother mouse is given birth to is attacked poison, the result confirms that the VP6 antibody that female mouse produces can pass to mouse, and can provide the allos cross protection for mouse.Rotavirus oral vaccine of the present invention can induce humans and animals to produce mucosa-immune by oral mode, can be used in infant that the multiple rotavirus infection of prevention causes and the acute diarrhea of children livestock in age.

The method of production rotavirus oral vaccine of the present invention has the following advantages:

1. cost is low, and easily promote: plant is the proteinic low cost system of scale operation, in case obtain to efficiently express the transgenic plant of antigen protein, just can form industrialized scale by vegetative propagation rapidly at short notice.Plant vaccine is easy to preparation, need not refrigeration, can reduce production costs greatly.

2. safer: as only to have increased rotavirus vp 6 antigen proteins in the rotavirus oral vaccine of the present invention, do not changed other genetic composition of plant, do not contained harmful composition.Oral vaccine does not need to use injector to inject in addition, and the unclean pollution that brings of syringe has not only reduced cost but also avoided.

3. more efficient: antigen protein can effectively be translated post-treatment in transgenic plant, space as glycosylation, phosphorylation, amidation and higher structure is folding, thereby make this recombinant protein have the immunogenicity identical with native protein, this is that the prokaryotic expression system is incomparable, and the latter can't translate post-treatment to being derived from Eukaryotic albumen.

4, the first-selected clover of the present invention is as recipient plant, mainly be biological yield height, protein content height (protein content in every 100g tender leaf is 5.9g), the good palatability of considering clover, the clover tender leaf not only can also can be made the oral liquid human consumption through the simple course of processing as animal-feed.

Description of drawings

Fig. 1 is that the nucleotide sequence of VP6 gene and improved sVP6 gene compares

Fig. 2 A is the structure synoptic diagram of plant expression vector pBsVP6 plasmid

Fig. 2 B is the structural representation in the T-DNA district of pBsVP6 plasmid

Fig. 3 A is that the PCR of transgenic alfalfa detects

Fig. 3 B is that the Southern hybridization of transgenic alfalfa detects

Fig. 3 C is that the RT-PCR of the total RNA of transgenic alfalfa detects

Fig. 4 is the structure synoptic diagram of prokaryotic expression carrier pGVP6 and pGsVP6

Fig. 5 A is the GST-VP6 that expresses and the SDS-PAGE electrophorogram of GST-sP6 fusion rotein in E.coli BL21

Fig. 5 B is the VP6 and the proteic SDS-PAGE electrophorogram of sVP6 of purifying

Fig. 6 is the VP6 in the Western hybridization detection transgenic alfalfa blade

Fig. 7 is a VP6 protein content in the ELISA quantitative assay transgenic alfalfa blade

Fig. 8 detects the transgenic alfalfa of asexual propagation for pcr amplification

Female mouse just immune back 36th day the anti-VP6 serum IgG antibody of Fig. 9 A for detecting oral immunity

Fig. 9 B just exempts from the 60th day the anti-VP6 small intestinal mucosa IgA antibody in back for the female mouse that detects oral immunity

Fig. 9 C just exempts from the continuous anti-VP6 saliva IgA antibody all around in back for the female mouse that detects oral immunity

Fig. 9 D just exempts from the continuous anti-VP6 ight soil IgA antibody all around in back for the female mouse that detects oral immunity

Figure 10 is the provide protection of transgenic plant vaccine to the mouse of passive immunization

Embodiment

Method in the following experiment is ordinary method if no special instructions.

Embodiment 1, utilize dicotyledons preference codon synthetic people A group rotavirus VP6 gene-sVP6 gene and sVP6-SEKDEL gene

With DNAMAN software the coding region sequence of people A group rotavirus VP6 gene (GenBank AF260931) is analyzed, analyzed its A+T percentage composition, restriction endonuclease sites, the aminoacid sequence of translating, codon uses preferences.The difference that compares the codon-bias of VP6 genes encoding region sequence and dicotyledons, under the prerequisite that does not change an aminoacid sequence, the VP6 coding region sequence is transformed, made its codon-bias, avoid using rare codon NCG and NTA as far as possible near dicotyledons.

In addition, in order to guarantee to produce the sophisticated mRNA that translates, transform following sequence

1) transform six polyadenylic acids (Poly (A)) signal sequence in the VP6 genes encoding region sequence, so as not at poly (A) thus the 20bp-30bp mRNA of place in downstream is cut the mRNA that can not produce total length.These sequences comprise: the GATAAA of the 69-74 position of AF260931; The AATATT of 280-285 position; The GATAAA of 401-406 position; The AACCAA of 532-537 position; The AATAAT of 792-797 position; The AATGAA of 1038-1043 position.

2) the possible mRNA degraded sequence (ATTTA of the 122-126 position of AF260931 of four in the transformation VP6 genes encoding region sequence; The ATTTA of 181-185 position; The ATTTA of 392-396 position; The ATTTA of 716-720 position.

3) the rna plymerase ii terminator sequence (AGTAGAA of the 803-809 position of AF260931) in the transformation VP6 genes encoding region sequence, sequence A GTNNAA (N is any base) may cause mRNA to synthesize premature termination.

With improved sequence input computer, analyze its G+C content, aminoacid sequence, restriction enzyme site, and carry out the comparison of nucleotide homology rate and amino acid homology rate with former sequence.The result shows, the codon transformation makes the G+C content of gene be increased to 44% from 33%, approaches the G+C content (45%) of dicotyledons.Improved unnamed gene is " a sVP6 gene ", and the nucleotide sequence of sVP6 gene is shown in the sequence in the sequence table 1, and the nucleotide sequence homology of this gene and VP6 gene is 87.94%, and the amino acid sequence homologous rate is 100% (Fig. 1).In order further to optimize the expression of foreign gene in plant, hold at 5 ' of sVP6 gene and synthesized Kozak sequence (AACC ATGG); At the preceding endoplasmic reticulum maintenance signal sequence SEKDEL (TCT GAG AAA GAT GAG CTA) that inserted of the terminator codon (TAA) of sVP6 gene, obtained sVP6-SEKDEL gene (sequence 2).The nucleotide sequence of sVP6 gene (sequence 1) and sVP6-SEKDEL gene (sequence 2) is given birth to worker bio-engineering corporation by Shanghai and is synthesized.SVP6-SEKDEL gene (sequence 2) is connected to the BamHI/SacI site of pUC19 plasmid, obtains to contain the recombinant plasmid pUsVP6 of sVP6-SEKDEL gene (sequence 2).

Embodiment 2, preparation rotavirus oral vaccine

1, expresses the plant conversion carrier structure of sVP6 gene

Utilize restriction enzyme NcoI and SacI digestion pUsVP6 plasmid, reclaiming size is the sVP6-SEKDEL gene fragment of 1.2kb, and be inserted into carrier pRTL2 (J.Virol., 1990, NcoI/SacI site 64:1590-1597) obtains containing the recombinant plasmid pRsVP6 of sVP6-SEKDEL gene.Use HindIII and Sac I double digestion pRsVP6 again, reclaim size and be the containing of 2.4kb " cauliflower mosaic virus of two enhansers (CaMV) 35S promoter+tobacco etch virus (TEV) 5 ' non-translational region (5 '-UTR)+Kozak sequence+sVP6-SEKDEL " fragment, be connected with pBI121 plasmid through same double digestion, obtain binary expression vector pBsVP6, vector construction figure is shown in Fig. 2 A.

The T-DNA district of pBsVP6 plasmid comprises two expression casettes: NptII expression casette and sVP6 expression casette.The NptII expression of gene is given plant kalamycin resistance (Kan R), as the selection markers of transfer-gen plant.The sVP6 expression of gene is driven by the CaMV 35S promoter with two enhansers; The gene 5 ' end is connected with TEV 5 '-UTR sequence and Kozak sequence, in order to optimized gene translate initial; The endoplasmic reticulum maintenance signal sequence SEKDEL of gene 3 ' end participates in the interior location of cell of target protein, thereby strengthens proteic stability and expression amount.The structural representation in the T-DNA district of pBsVP6 plasmid is shown in Fig. 2 B.

2, expression and the detection of sVP6 gene in clover

Utilize freeze-thaw method that the pBsVP6 plasmid is introduced Agrobacterium tumefaciens (Agrobacterium tumefaciens) LBA4404.LBA4404 is a kind of improved Agrobacterium, and the plasmid pAL4404 that it contains has complete vir gene, but the T-DNA district is deleted.PAL4404 and pBsVP6 plasmid constitute the binary vector system, and the T-DNA district stable integration among the mediation pBsVP6 is to plant chromosome.

After 10 times of the fresh Agrobacterium LBA4404 bacterium liquid dilutions that contains pBsVP6, add pre-cotyledon and the hypocotyl of cultivating Baoding alfalfa of 1-2 days, soak 3-5min, blot residual bacterium liquid with filter paper, cotyledon and hypocotyl be placed on do not contain antibiotic SH callus and keep substratum (SH substratum+0.025mg/L KT+2mg/L2 was cultivated 3-4 days on 4-D) altogether; Blade is changed over to again and contain in the antibiotic SH selection substratum (SH substratum+0.05mg/L KT+2mg/L 2,4-D+25mg/L Kan+500mg/L Cb), per two weeks are changed subcultures.When callus has embryoid to form, embryoid is transferred to division culture medium (SH substratum+0.4mg/L KT+50mg/L Kan+500mg/L Cb) go up growth, when breaking up the young shoot that produces more than the 1cm, young shoot is transferred on the root media (1/2MS minimum medium+50mg/L Kan+500mg/L Cb), cultivated about 20 days, every strain seedling can grow the root that the 3-5 bar is about 10cm, approximately 10-12 week can obtain the kalamycin resistance plant, to take root and well-grown resistant plant is transplanted in the vermiculite soil, plant the greenhouse after 4 weeks.

In order to detect in the genome whether the sVP6 gene be incorporated into transgenic alfalfa, with CTAB method (Wang Guanlin etc., plant genetic engineering philosophy and technique, first version, 1998, Beijing, Science Press) from the tender transgenic alfalfa blade of children, extract plant genome DNA.With the plant genome DNA is template, utilizes Auele Specific Primer P 1 (5 '-ATGGAGGTTCTGTACTCATTGTC-3 ') and Auele Specific Primer P2 (5 '-AGACTTGATCAACATGCTTCT-3 ') pcr amplification sVP6 gene.The result shows have 58 strains to amplify the 1.2kb band (Fig. 3 A) of an expection size in the 127 strain kalamycin resistance plant.Among Fig. 3 A, the clover that 1-10 transforms for the pBsVP6 plasmid, 11 is unconverted clover (negative control), and 12 is pBsVP6 plasmid (positive control), and M is 1kb DNA ladder.

With the genomic dna of Nco I and Sac I difference double digestion pBsVP6 plasmid conversion alfalfa plants and unconverted plant, behind agarose gel electrophoresis, transfer to Hybond N ⁺On the nylon membrane, adopt Promaga company's random primer labelling test kit mark sVP6 gene (sequence 1), with α- ³²The sVP6 gene of P mark carries out Southern hybridization as probe.The result shows to obtain a specific hybridization band in the sativa genomic dna after the conversion shown in Fig. 3 B, and amixia band in the unconverted sativa genomic dna.Among Fig. 3 B, 1 is the PCR product (positive control) of pBsVP6 plasmid, and 2 is unconverted clover (negative control), the clover that 3-6 transforms for the pBsVP6 plasmid.

With SDS method (Wang Guanlin etc., the plant genetic engineering philosophy and technique, first version, 1998, Beijing, Science Press) extracts Southern hybridization and present the transfer-gen plant of positive signal and total RNA of unconverted plant, be template with the total RNA of plant respectively, adopt RT-PCR test kit and primer P1, the P2 of TaKaRa company to carry out RT-PCR, the result is shown in Fig. 3 C, show that transfer-gen plant amplification that Southern hybridization presents positive signal has obtained the band of a 1.2kb, and unconverted plant does not obtain band.Among Fig. 3 C, the clover that 1-6 transforms for the pBsVP6 plasmid, 7 is unconverted clover (negative control), 8 is the PCR product (positive control) of pBsVP6 plasmid, M is 1kb DNA ladder, and these results show: the sVP6 gene has been incorporated in the karyomit(e) of transgenic alfalfa and has expressed complete mRNA.

3, VP6 and sVP6 albumen expression and the purifying in intestinal bacteria

Sequence and pGEX-4T-1 plasmid vector (Invitrigen according to VP6 gene, sVP6 gene, USA) restriction enzyme site, design upstream primer and downstream primer, for the ease of being connected with the pGEX-4T-1 carrier, primer 5 ' end is introduced BamH I or Xho I restriction enzyme site respectively.Genomic dna with the A group rotavirus is a template, utilizes pr imer3 (5 '-TACGGATCCAGAACCATGGAGGTTCT GTAC-3 ') and pr imer4 (5 '-ACGCTCGAGTCACTTAATCAACATGCTTCTAATG-3 ') pcr amplification to obtain the VP6 gene of 1.2kb; With the pUsVP6 plasmid is template, utilizes the sVP6 gene of primer3 (5 '-TACGGATCCA GAACCATGGAGGTTCTGTAC-3 ') and primer5 (5 '-ACGCTCGAGTCACTTAATCAACATGCTTCTAATG-3 ') pcr amplification 1.2kb.

The VP6 gene that pcr amplification obtains is connected to the BamH I/Xho I site of prokaryotic expression carrier pGEX-4T-1 carrier behind BamH I and Xho I double digestion, make up gained prokaryotic expression plasmid called after pGVP6; The sVP6 gene that pcr amplification obtains is connected to the BamH I/Xho I site of prokaryotic expression carrier pGEX-4T-1 carrier, gained prokaryotic expression plasmid called after pGsVP6, vector construction figure such as Fig. 4 behind BamH I and Xho I double digestion.

Use recombinant plasmid pGVP6 and pGsVP6 Transformed E .coli BL21 competent cell (day for epoch, China) respectively, induce target protein to express through IPTG for 37 ℃, analyze the target protein expression with SDS-polyacrylamide gel electrophoresis (SDS-PAGE).In Fig. 5 A, M is a protein molecular weight standard, 1-3 be respectively E.coli that the pGEX-4T-1 plasmid transforms do not induce, IPTG induces 3hr, expression product during 5hr, 4-6 be respectively E.coli that pGVP6 transforms do not induce, IPTG induces 3hr, expression product during 5hr, 7-9 be respectively E.coli that pGsVP6 transforms do not induce, IPTG induces 3hr, expression product during 5hr.The result shows: pGVP6 and pGsVP6 plasmid are all efficiently expressed in the BL21 cell, and the GST-VP6 that pGVP6 expresses is consistent with the GST-sVP6 fusion rotein size that pGsVP6 expresses, and is 65kDa.

With Triptide-agarose resin chromatography column absorption GST-VP6 and GST-sVP6 fusion rotein, fusion rotein is cut the wash-out with damping fluid L through 20U/ml zymoplasm enzyme, eluted product is through the FPLC 900 (Amershan of fast protein liquid chromatography system, USA) further separation and purification, the SDS-PAGE electrophoresis result shows VP6 albumen and the sVP6 albumen (Fig. 5 B) that has obtained 42kDa.In Fig. 5 B, 1 is the VP6 albumen of purifying from the E.coli BL21 that pGVP6 transforms, and 2 is the sVP6 albumen of purifying from the E.coli BL21 that pGsVP6 transforms, and M is a protein molecular weight standard.

4, the proteic qualitative and quantitative analysis of VP6 of transgenic plant expression

Employing Western is hybridized and the quantitative ELISA technology is qualitative and the expression amount of detection by quantitative VP6 albumen in transgenic alfalfa.

Get pBsVP6 transgenic plant transformed blade 1g and place mortar, add 1ml and extract damping fluid [10mM sodium phosphate buffer (pH7.2), 105mM NaCl, 0.05%Tween, 40mM sodium ascorbate, 20mM disodium ethylene diamine tetraacetate, 1mM PMSF], liquid nitrogen grinding.The centrifugal 10min of 12000r/min gets supernatant, and supernatant liquor is through 30% and 60% saturated (NH ₄) ₂SO ₄Fractionation precipitation, the plant total soluble protein (TSP) of acquisition return and are dissolved in 0.1M phosphate buffered saline buffer (PBS).TSP is used for immunology detection and mouse oral immunity behind dialysis 24hr.The plant total soluble protein separates through the SDS-PAGE gel electrophoresis, and application electrotransfer device is transferred to albumen and carries out Western Blot immunoblotting reaction on the nylon membrane.The first antibody of Western hybridization is rotavirus vp 6 rabbit anti-serums of 1000 times of dilutions, this antiserum(antisera) is to be the antigen immune rabbit with rotavirus vp 6 albumen, according to a conventional method preparation; Second antibody be the alkali phosphatase enzyme mark of 5000 times of dilutions goat anti-rabbit igg (Santa Cruz, USA), after the Hybond membrane washing 3 times, with NBT/BCIP colour developing, observations.The Western results of hybridization shows to have detected complete VP6 albumen in transgenic alfalfa that its molecular weight is consistent with the VP6 albumen of purifying, and is 42kDa as shown in Figure 6 from E.coli.Among Fig. 6,1 is the VP6 albumen (positive control) of purifying from the E.coli of reorganization, and 2 is unconverted clover blade (negative control), and 3-8 is the clover blade that pBsVP6 transforms, and M is a protein molecular weight standard.

The quantitative ELISA analysis is the relative content that calculates VP6 albumen/plant total soluble protein (TSP) in the transfer-gen plant.Get conversion and unconverted plant leaf 0.2g, put in the liquid nitrogen and grind, grinding product changes Eppendorf tube rapidly over to, adds the 1.0ml bag and is cushioned liquid (1.5g/L Na ₂CO ₃, 2.93g/L NaHCO ₃), mixing, the centrifugal 15min of 12000rpm gets supernatant (plant crude protein extracting solution), for examination.At first, add Coomassie brilliant blue solution with plant crude protein extracting solution dilution, mixing, room temperature is placed 2min, measures the light absorption value of 595nm, finds the content of the total soluble protein (TSP) the sample from the BSA typical curve of serial dilution.Then, get the plant total soluble protein sample slightly carried and be cushioned the VP6 standard model 100 μ l of liquid serial dilution, join the enzyme plate aperture, 37 ℃ of insulation 3hr with bag.Detersive enzyme yoke plate three times adds rotavirus vp 6 rabbit anti-serums (this antiserum(antisera) is to be the antigen immune rabbit with rotavirus vp 6 albumen, preparation) according to a conventional method of 1000 times of dilutions, 37 ℃ of reaction 1hr.Wash the goat anti-rabbit igg that adds the horseradish peroxidase-labeled of 5000 times of dilutions behind the plate (Santa Cruz, USA), 37 ℃ of reaction 1hr.O-Phenylene Diamine-H ₂O ₂(pH5.0) colour developing, H ₂SO ₄Termination reaction is measured the light absorption value of every hole sample at 492nm with microplate reader.On same elisa plate, measure the proteic light absorption value of the VP6 that from E.coli, purifies of serial dilution, set up typical curve,, determine the content of VP6 albumen in transfer-gen plant light absorption value and its comparison of plant sample.Calculate the relative content (%) of VP6 albumen/TSP in the transfer-gen plant at last.Quantitative ELISA is the result show, the proteic content of VP6 accounts for the 0.06%-0.28% of plant total soluble protein in the transgenic alfalfa plants, high expression level amount be 0.28% (no.14) (Fig. 7).Among Fig. 7,1 is unconverted plant (negative control), and 2-19 is the pBsVP6 transformed plant.

The transgenic alfalfa plants of 5 quick asexual propagations

In order to obtain a large amount of transgenic alfalfas fast, the transgenic alfalfa plants that the VP6 expressing quantity is higher ( lane 4,5,6,8,13,14,15,16,19) carries out vegetative propagation.Get the blade of transgenic alfalfa, 75% alcohol disinfecting 10s, 0.1% mercury chloride sterilization 4min uses aseptic water washing 5～6 times again; Be cut into small pieces to be placed on the SH callus substratum and cultivate, change the SH division culture medium all around over to, cultivated for three weeks, cut resistant buds and change root induction on the 1/2MS substratum over to, three weeks just can be transplanted in the flowerpot that vermiculite soil is housed, and flowerpot is grown and two weeks can be planted in the land for growing field crops.Amount to three months and just obtained a large amount of high expression level plant, be enough to satisfy the demand of animal immune.

Transgenic alfalfa (no.14) obtains 79 strain alfalfa plants altogether through asexual propagation, is template with this 79 strain plant genome DNA, utilizes P1 and P2PCR amplification sVP6 gene.Agarose gel electrophoresis is the result show, all plant have all obtained the target fragment (Fig. 8) of 1.2kb through pcr amplification, illustrates that the numerous plant of a large amount of expansions of the blade that utilizes transgenic alfalfa can't cause losing of target gene.Among Fig. 8,9 is unconverted plant (negative control), and 1-8 is the pBsVP6 transformed plant, and 10 is pBsVP6, as positive control.Alfalfa is the perennial plant of cross-pollination, karyomit(e) is allotrtraploid, obtain homozygote and need the long cycle, a large amount of transgenic alfalfas have just been obtained in a short time by the blade asexual propagation, having saved loaded down with trivial details selfing breeding process, is a kind of suitable plant vaccine expression system from a side proof alfalfa.

Embodiment 3, the female mouse of expression rotavirus vp 6 proteic transgenic alfalfa oral immunities can make generation mice obtain protection

As immunological adjuvant, its sequence is TCCATGACGTT CCTGACGTT (CpG ODN 1826) to this research employing trunk through the CpG of thiophosphoric acid modification ODN, gives birth to worker bio-engineering corporation by Shanghai and synthesizes, and using dosage is 10 μ g/ time.The proteic transgenic alfalfa of high expression level sVP6 can obtain the immunity that wide variety of materials is used for mouse in three months through after the vegetative propagation.(NH through 30% and 60% ₄) ₂SO ₄The transgenic alfalfa base of leaf total soluble protein (TSP) that fractionation precipitation obtains returns and is dissolved in 0.1M PBS, and TSP is used for the mouse oral immunity behind normal saline dialysis 24hr.

The female mouse of BALB/c in 30 6-8 age in week is divided into 3 groups, 10 every group, weekly the intragastrically immunity once, 4 weeks of continuous immunity (the 0th, 7,14,21d).Mix 10 μ gCpG ODN adjuvants with 10mg non-transgenic clover TSP and irritate first group of female mouse (group1) of stomach immunity, as negative control; Mix 10 μ g CpG ODN adjuvants with 10mg transgenic alfalfa TSP (containing VP6 protein 24 μ g) and irritate second group of female mouse (group2) of stomach immunity; The VP6 albumen of purifying with 24 μ g mixes the 3rd group of female mouse (group3) of 10 μ g CpG ODN adjuvants filling stomach immunity, as positive control.

It is by elisa plate with VP6 protein sample (the 20 μ g/ml) bag of purifying that ELISA detects, anti-measure anti-VP6 IgG antibody titer with the sheep anti-mouse igg of the horseradish peroxidase-labeled of 5000 times of dilutions as two, and with the sheep anti mouse IgA of the horseradish peroxidase-labeled of 2000 times of dilutions as the two anti-anti-VP6IgA antibody titers of measuring the mouse generation.With the light absorption value of microplate reader working sample at 492nm; The light absorption value of sample is negative control more than 2 times of light absorption value of (non-transgenic clover TSP mixes 10 μ g CpG ODN and irritates the stomach immune mouse), then is judged to be positive findings, and antibody titers is represented with the inverse that sample obtains the highly diluted multiple of positive findings.Inverse with the mean value of the antibody titers of every group of mouse is done column diagram, and indicates the analytical results of standard deviation in the drawings.

Just exempt from back 36d,, collect serum the female rathole socket of the eye blood sampling of immunity.Second group of female mouse all detected the serum IgG antibody of the specific anti-VP6 of high titre in female mouse body after 10mg transgenic alfalfa TSP immunity 4 times, antibody titers is 1/7800-1/9600; The 3rd group of female mouse (antibody titers is 1/4800-1/7200) (Fig. 9 A) apparently higher than the VP6 protein immunization of purifying with 24 μ g.

(the 0th, 7,14,21, the 28d) saliva of collecting every female mouse is measured mucous membrane IgA antibody titers weekly, and the result shows that after each immunity, antibody titers all strengthens to some extent; Just exempt from back 28d, the saliva IgA antibody titers of second group of female mouse reaches as high as 1/480, a little more than the 3rd group of female mouse (the highest antibody titers 1/400) (Fig. 9 C).

(the 0th, 7,14,21,28d) ight soil of collecting every female mouse is measured mucous membrane IgA antibody titers weekly, and the result shows that antibody titers all strengthens to some extent after each immunity; Just exempt from back 28d, the ight soil IgA antibody titers of second group of female mouse reaches as high as 1/240, with the 3rd group of female mouse (the highest antibody titers 1/240) fair (Fig. 9 D); The immunological tolerance phenomenon does not appear in the female mouse that continues to increase the explanation immunity of saliva and fecal antibody titre.

Just exempt from back 60d, put to death 4 female mouse for every group, collect small intestine and homogenate, ELISA measures intestinal mucosa IgA antibody titers; The result confirms, all produced the mucous membrane IgA antibody of specific anti-VP6 in second group of female mouse enteron aisle, and titre is 1/300-1/640, apparently higher than the 3rd group of female mouse (antibody titers is 1/,160 one 1/480) (Fig. 9 B).All in all, mucous membrane of small intestine IgA antibody titers is higher than saliva and ight soil IgA antibody titers.

All do not detect IgG and the IgA antibody of specific anti-rotavirus VP6 in first group 10 female mouse bodies with unconverted clover TSP immunity.

Above result of study shows that the rotavirus edible vaccine of the sVP6 gene of express transforming can induce body to produce humoral immunization and mucosal immunity; As oral adjuvant, can effectively strengthen humoral immunization and the mucosal immunity of mouse with CpG ODN.

The public mouse mating of every group of female mouse of remaining 6 BALB/c and non-immune BALB/c, through about 19-20 days period of pregnancy, every female mouse can give birth to 4-9 mouse.30 suckling mouses have been educated in first group of female mouse symbiosis with the immunity of non-transgenic clover, have educated 32 suckling mouses with second group of female mouse symbiosis of transgenic alfalfa immunity, have educated 24 suckling mouses with the 3rd group of female mouse symbiosis of the VP6 protein immunization of purifying.In whole experiment, suckling mouse is always by female mouse lactation.

Because the diarrhoea sensitivity that suckling mouse only caused rotavirus within postnatal 15 days, so suckling mouse postnatal the 5th day, SA-11 attacks poison to it with the ape rotavirus, and dosage is 100 tissue cultureinfective dose (TCID) ₅₀, the diarrhoea situation of observation in continuous 10 days and record mouse.

After ape rotavirus SA-11 attacks poison, observe the anus of 3 suckling mouses every day,, do not suffer from diarrhoea just be judged to be, otherwise just be judged to be diarrhoea if 3 times all do not have watery stools; The suckling mouse anus of diarrhoea becomes redness, and is stained with a large amount of ight soil, and the symptom difference that the diarrhoea and the suckling mouse of not suffering from diarrhoea show is tangible; Qualitatively judge the diarrhoea intensity of suckling mouse according to anus degree that reddens and the ight soil amount that is stained with.

The symptom of diarrhea of the suckling mouse that female mouse gave birth to of transgenic alfalfa immunity obviously alleviates, and degree that anus reddens and the ight soil amount that is stained with are starkly lower than control group (the not suckling mouse that female mouse gave birth to of transgenic alfalfa immunity).Compare with control group, the diarrhoea time length of the suckling mouse that female mouse gave birth to of transgenic alfalfa immunity obviously shortens, diarrhea rate (the diarrhoea suckling mouse accounts for the percentage ratio of all attacking malicious suckling mouse) obviously reduces, attacking the poison back the 5th day, all suckling mouses are no longer suffered from diarrhoea, and diarrhoea decrement (the not diarrhea rate of the suckling mouse that female mouse gave birth to of the diarrhea rate of the suckling mouse that female mouse gave birth to of transgenic alfalfa immunity-transgenic alfalfa immunity) reaches 60%; And the diarrhoea time length of the suckling mouse that female mouse gave birth to of the VP6 protein immunization of purifying slightly shortens, diarrhea rate slightly reduces, attacking the poison back the 7th day, all suckling mouses are no longer suffered from diarrhoea, and diarrhoea decrement (the not diarrhea rate of the suckling mouse that female mouse gave birth to of the VP6 protein immunization of diarrhea rate-purifying of the suckling mouse that female mouse gave birth to of transgenic alfalfa immunity) is 25% (Figure 10).Among Figure 10, the fractional denominator is a used test suckling mouse number, and molecule is diarrhoea suckling mouse number.

This result of study shows that the anti-people A group rotavirus VP6 antibody that produces in female mouse body of transgenic plant vaccine oral immunity can pass to mouse, and the allos cross protection of other A group rotavirus of opposing can be provided for mouse.Express safety that codon optimized rotavirus vp 6 proteic transgenic plant vaccines promise to be infant that prevention causes by the A group rotavirus and cub diarrhoea and vaccine efficiently.

The processing of embodiment 4, rotavirus oral vaccine

With the clover is the preparation that example is introduced the rotavirus oral vaccine, and different transgenic plant need different complete processings to produce oral vaccine.

Clover is a kind of perennial leguminous plants, and the good reputation of " king of herbage " is arranged, and the biomass height can adopt the method that cradles, and obtains a large amount of transgenic alfalfa materials in short duration.The clover of cultivating in the greenhouse can cradle nine times in 1 year, and the clover tender leaf of results can be processed into the edible vaccine.

The complete processing of alfalfa leaf protein is as follows: at first adopt the multi-functional double spiral squeezer that integrates pulverizing, pulls an oar and squeeze to handle the clover blade, adopt the heating coacervation to extract leaf protein then, be heated to 80 ℃-90 ℃ earlier, be cooled fast to 40 ℃, the condensation product that leaches is the cytoplasm protein of good mouthfeel, can be directly edible for the human or animal.As long as the VP6 antigen protein content to the transgenic alfalfa of each batch extraction is sampled, just can obtain the plant vaccine of definite dosage, be used for commercialization production.

Sequence table

<160>4

<210>1

<211>1194

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

atggaggttc?tgtactcatt?gtctaagact?cttaaagatg?ctagagacaa?gattgttgaa 60

ggcacactct?attccaatgt?tagcgatctc?attcagcaat?tcaatcaaat?gatcatgact 120

atgaatggaa?atgacttcca?aactggaggc?attggtaacc?ttccagttag?gaattggatc 180

tttgattttg?gtcttttggg?tacaaccctt?cttaacttgg?atgctaacta?tgttgaaaat 240

gccagaacta?ccattgagta?cttcattgac?tttattgaca?atgtctgcat?ggatgaaatg 300

gcaagagagt?ctcaaagaaa?tggagtggct?cctcaatctg?aggccttgag?gaaactctca 360

gggatcaaat?tcaagaggat?caactttgat?aactcctcag?aatacataga?aaactggaat 420

ctccaaaaca?gacgccagcg?tactggattt?gtcttccata?agcctaacat?atttccctac 480

tcagccagct?tcaccttgaa?tagatctcag?cccatgcatg?ataacttgat?gggaactatg 540

tggcttaatg?ctggaagtga?gatccaagtg?gctggttttg?actattcctg?tgctataaac 600

gcaccagcca?acatacagca?gtttgagcac?attgtccagc?ttagacgtgc?actcaccaca 660

gctactatca?ccttgcttcc?tgatgcagaa?agattcagtt?tcccaagggt?tatcaacagt 720

gctgacgggg?caactacctg?gttctttaat?cctgtcattc?ttaggccaaa?caatgtggag 780

gtggagttct?tgttgaatgg?acagattatc?aacacatacc?aggctcgctt?tggtaccatt 840

atcgcaggaa?actttgatac?aattcgattg?agcttccagt?tgatgcgtcc?acccaacatg 900

acaccagctg?ttaacgcact?cttcccacaa?gcccaacctt?tccaacacca?tgccacagtt 960

ggactcacct?tgcgcattga?atctgctgtc?tgcgaatcag?tgcttgccga?tgccaatgag 1020

actctgcttg?ccaatgtgac?cgcagtgcgt?caagaatatg?ctataccagt?tggtcctgtt 1080

ttcccacctg?gcatgaactg?gactgagctt?atcactaact?actctccatc?cagggaagac 1140

aacctgcaac?gtgtctttac?agtggcttcc?attagaagca?tgttgatcaa?gtaa 1194

<210>2

<211>1216

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

aaccatggag?gttctgtact?cattgtctaa?gactcttaaa?gatgctagag?acaagattgt 60

tgaaggcaca?ctctattcca?atgttagcga?tctcattcag?caattcaatc?aaatgatcat 120

gactatgaat?ggaaatgact?tccaaactgg?aggcattggt?aaccttccag?ttaggaattg 180

gatctttgat?tttggtcttt?tgggtacaac?ccttcttaac?ttggatgcta?actatgttga 240

aaatgccaga?actaccattg?agtacttcat?tgactttatt?gacaatgtct?gcatggatga 300

aatggcaaga?gagtctcaaa?gaaatggagt?ggctcctcaa?tctgaggcct?tgaggaaact 360

ctcagggatc?aaattcaaga?ggatcaactt?tgataactcc?tcagaataca?tagaaaactg 420

gaatctccaa?aacagacgcc?agcgtactgg?atttgtcttc?cataagccta?acatatttcc 480

ctactcagcc?agcttcacct?tgaatagatc?tcagcccatg?catgataact?tgatgggaac 540

tatgtggctt?aatgctggaa?gtgagatcca?agtggctggt?tttgactatt?cctgtgctat 600

aaacgcacca?gccaacatac?agcagtttga?gcacattgtc?cagcttagac?gtgcactcac 660

cacagctact?atcaccttgc?ttcctgatgc?agaaagattc?agtttcccaa?gggttatcaa 720

cagtgctgac?ggggcaacta?cctggttctt?taatcctgtc?attcttaggc?caaacaatgt 780

ggaggtggag?ttcttgttga?atggacagat?tatcaacaca?taccaggctc?gctttggtac 840

cattatcgca?ggaaactttg?atacaattcg?attgagcttc?cagttgatgc?gtccacccaa 900

catgacacca?gctgttaacg?cactcttccc?acaagcccaa?cctttccaac?accatgccac 960

agttggactc?accttgcgca?ttgaatctgc?tgtctgcgaa?tcagtgcttg?ccgatgccaa 1020

tgagactctg?cttgccaatg?tgaccgcagt?gcgtcaagaa?tatgctatac?cagttggtcc 1080

tgttttccca?cctggcatga?actggactga?gcttatcact?aactactctc?catccaggga 1140

agacaacctg?caacgtgtct?ttacagtggc?ttccattaga?agcatgttga?tcaagtctga 1200

gaaagatgag?ctataa 1216

<210>3

<211>144

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

ctcgagaatt?caacacaaca?tatacaaaac?aaacgaatct?caagcaatca?agcattctac 60

ttctattgca?gcaatttaaa?tcatttcttt?taaagcaaaa?gcaattttct?gaaaattttc 120

accatttacg?aacgatagcc?atgg 144

<210>4

<211>780

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

aagcttgcat?gcctgcaggt?caacatggtg?gagcacgaca?ctctcgtcta?ctccaagaat 60

atcaaagata?cagtctcaga?agaccagagg?gctattgaga?cttttcaaca?aagggtaata 120

tcgggaaacc?tcctcggatt?ccattgccca?gctatctgtc?acttcatcga?aaggacagta 180

gaaaaggaag?atggcttcta?caaatgccat?cattgcgata?aaggaaaggc?tatcgttcaa 240

gaatgcctct?accgacagtg?gtcccaaaga?tggaccccca?cccacgagga?acatcgtgga 300

aaaagaagac?gttccaacca?cgtcttcaaa?gcaagtggat?tgatgtgata?acatggtgga 360

gcacgacact?ctcgtctact?ccaagaatat?caaagataca?gtctcagaag?accagagggc 420

tattgagact?tttcaacaaa?gggtaatatc?gggaaacctc?ctcggattcc?attgcccagc 480

tatctgtcac?ttcatcgaaa?ggacagtaga?aaaggaagat?ggcttctaca?aatgccatca 540

ttgcgataaa?ggaaaggcta?tcgttcaaga?atgcctctac?cgacagtggt?cccaaagatg 600

gacccccacc?cacgaggaac?atcgtggaaa?aagaagacgt?tccaaccacg?tcttcaaagc 660

aagtggattg?atgtgatatc?tccactgacg?taagggatga?cgcacaatcc?cactatcctt 720

cgcaagaccc?ttcctctata?taaggaagtt?catttcattt?ggagaggacc?tcgagaattc 780

Claims (10)

1, rotavirus vp 6 protein coding genes have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the sequence table: 2 dna sequence dna;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of 1 or SEQ ID №: the 2 dna sequence dnas hybridization that limit;

4) with sequence table in SEQ ID №: 1 or SEQ ID №: 2 dna sequence dnas that limit have 80% above homology, and have the dna sequence dna of higher expression amount in dicotyledons.

2, a kind of method of producing the rotavirus oral vaccine, be that described any rotavirus vp 6 protein coding genes of claim 1 are imported the dicotyledons cell, obtain expressing rotavirus vp 6 proteic dicotyledonous transgenic plant, extract the total soluble protein of described dicotyledonous transgenic plant, obtain the rotavirus oral vaccine.

3, method according to claim 2 is characterized in that: described dicotyledons comprises clover, soybean, tomato, apple, banana and Radix Dauci Sativae; Described dicotyledons is preferably clover.

4, according to claim 2 or 3 described methods, it is characterized in that: 5 of described rotavirus vp 6 protein coding genes ' end closely is connected with the Kozak sequence.

5, method according to claim 4 is characterized in that: 5 of described Kozak sequence ' end closely is connected with tobacco etch virus 5 ' non-translational region.

6, method according to claim 5 is characterized in that: described tobacco etch virus 5 ' non-translational region is sequence 3 in sequence table.

7, method according to claim 6 is characterized in that: 5 of described tobacco etch virus 5 ' non-translational region ' end is connected with the cauliflower mosaic virus 35S promoter of the two enhansers of band.

8, method according to claim 7 is characterized in that: the cauliflower mosaic virus 35S promoter of the two enhansers of described band is one of following nucleotide sequence;

1) SEQ ID № in the sequence table: 4 dna sequence dna;

2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 4 dna sequence dnas hybridization that limit;

3) cut the nucleotide fragments of the cauliflower mosaic virus 35S promoter of the two enhansers of band that carrier pRTL2 obtains with restriction enzyme HindIII or SphI or PstI or HincII and XhoI or EcoRI enzyme.

9, method according to claim 8 is characterized in that: described rotavirus vp 6 protein coding genes import vegetable cell by expression vector pBsVP6.

10, the rotavirus oral vaccine that the described method of arbitrary claim is produced among the claim 2-9.

Images