CN101988058A - Gene expression composition, PRRS (Porcine Reproductive and Respiratory Syndrome) oral vaccine and preparation methods thereof - Google Patents

Abstract

The invention discloses a gene expression composition capable of expressing an allergen protein ORF5 (Open Reading Frame) of a PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), a vaccine expression vector thereof and an oral vaccine containing the same. The gene expression composition comprises a promoter, polynucleotide for coding the ORF5 peptide of the PRRSV, a terminator NOS and polynucleotide of a banana heavy ubiquitin gene 3' MAR. The invention also provides the preparation methods of the gene expression composition, the vaccine expression vector thereof and the oral vaccine containing the same. In addition, the invention also provides a method for preventing the PRRS. The vaccine expression vector is transferred into a plant through a transgenic technology, transgenic plants is fed to pigs generate an antibody resisting the PRRSV so as to prevent the PRRS.

Description

A kind of genetic expression composition, pig reproduction and respiratory complication oral vaccine and preparation method thereof

Technical field

The present invention relates to a kind ofly express genetic expression composition, its vaccine expression vector of the antigen protein of pig reproduction and breathing syndrome virus and contain oral vaccine of its genetic expression composition and preparation method thereof.

Background technology

(Porcine reproductive and respiratory syndrome is very important in recent years pig virus disease PRRS), causes the American-European serious economy loss all over the world that reaches for pig reproduction and respiratory syndrome.PRRS is an a kind of pig transmissible disease based on sow breeding difficulty and the respiratory tract infection of all ages pig, and easy concurrent secondary sexuality is dyed and death.And its complication of piglet shoat is serious more more, causes mortality ratio to increase, thereby causes very big economically loss.

This disease at first in 1987 in the U.S.'s most of pig farms outburst, because of this disease causes the hemostasis of a pig limb end and ear or blue sometimes, so be called blue otopathy (blue ear disease) again.The lungs of the main infected pigs of PRRS and cause interstitial pneumonia, cause pig only except having difficulty in breathing, because of the scavenger cell of lung and general and with the infected destruction of monocyte, thereby cause the acquired immune deficiency syndrome of pig, therefore easy infection secondary venereal disease is former in salmonella, p pestic or other viral cause of diseases (Chiou et al., 2000).

And find that at the viral separation rate of the serum antibody positive or lungs and hilar lymph node PRRSV generally infects domestic pig farm.The Causative virus that this is sick, pig reproduction and breathing syndrome virus (Porcine reproductiveand respiratory syndrome virus, PRRSV), for forward, sub-thread, have the RNA viruses of lipid big envelope, can influence the activity of a pig lungs scavenger cell, make its pseudopodium shorten, reduce or be lobate cell rupture death, and engulfing with sterilizing power of lungs scavenger cell also obviously is suppressed (Qiu etc., 1998).

The virus of the RNA of report demonstration in recent years malicious vaccine alive is returned change, caused the pig farm outburst epidemic situation of many use vaccines, show that traditional RNA malicious vaccine alive is perhaps unreliable and its danger is arranged, with the plant is this viral multivalence of foundational development time unit vaccine, for having approach economic and oral throwing easily and mode immunity concurrently, if can develop smoothly and finish, can reduce the harm of pig reproduction and respiratory complication virus, and releasing traditional vaccine all scrupulous on packing, conveying, storage, purifying.

Summary of the invention

Purpose of the present invention promptly is to provide a kind of pig reproduction and respiratory complication (PPRS) oral vaccine with plant production.

Of the present invention time a purpose is to provide various reproduction of plant production pig and the antigenic expression vectors of breathing syndrome virus (PRRSV) of being applicable to.

Another object of the present invention is to provide these can express the purposes of pig reproduction and the antigenic expression vector of breathing syndrome virus (PRRSV) in plant materials, this purposes comprises with plant materials produces virus antigen, transgenic plant, oral vaccine etc.

For achieving the above object, the present invention has taked following technical scheme:

A kind of genetic expression composition (expression cassette) comprises:

Promotor;

Coding pig reproduction and breathing syndrome virus (PRRSV) ORF5 win the polynucleotide of peptide;

NOS terminates sub; And

The polynucleotide of the heavy ubiquitin gene 3 ' MAR of banana, it has the nucleotide sequence as SEQ ID No:2; Wherein 5 ' end of the polynucleotide of this coding PRRSV ORF5 victory peptide is connected in 3 ' end of this promotor, and this coding PRRSV ORF5 wins the 5 ' end that 3 ' end of the polynucleotide of peptide is connected in this NOS termination, and 3 ' end of this NOS termination is connected with 5 ' end of the polynucleotide of the heavy ubiquitin gene 3 ' MAR of banana again; This promotor can drive the Transcription (transcription) that the downstream coding wins the polynucleotide of peptide in the organism that contains this genetic expression composition.

The polynucleotide that wherein should coding PRRSV ORF5 wins peptide, its 3 ' end can be further keep the 5 ' end that message (HDEL) wins the polynucleotide of peptide with the coding endoplasmic reticulum and are connected; 3 ' the end that this coding HDEL wins the polynucleotide of peptide is connected with 5 ' end of aforesaid NOS termination, can make vaccine expression vector, as: vaccine expression vector pGKU-35PRRSV provided by the present invention, pGKU-79PRRSV.

Wherein this vaccine expression vector pGKU-35PRRSV, pGKU-79PRRSV can further comprise polynucleotide, the 2nd NOS that second promotor (win the promotor that 5 ' end of the polynucleotide of peptide is connected with coding PRRSV ORF5, claim first promotor), one yard PPRSV ORF6 win peptide respectively and terminate sub; Wherein 3 ' of this second promotor end is connected with the 5 ' end that this coding PPRSV ORF6 wins the polynucleotide of peptide; 3 ' the end that this coding PPRSV ORF6 wins the polynucleotide of peptide is connected with 5 ' end of the 2nd NOS termination; Can prepare the vaccine expression vector (as: vaccine expression vector pGKU-5-6 provided by the present invention) that contains two or more promotors, wherein these promotors can be identical promotor or different promotors.

5 ' the end that the polynucleotide that wherein should coding PPRSV ORF5 wins peptide, its 3 ' end can be further with coding PPRSVORF6 wins the polynucleotide of peptide is connected; This coding PPRSV ORF6 wins the polynucleotide of peptide, and its 3 ' end can be further keeps the 5 ' end that message (HDEL) wins the polynucleotide of peptide with the coding endoplasmic reticulum and is connected; 3 ' the end that this coding HDEL wins the polynucleotide of peptide is connected with 5 ' end of aforesaid NOS termination, can make vaccine expression vector, as: vaccine expression vector pGKU-56Fusion provided by the present invention.

Wherein this promotor 3 ' end can further (heat-labile toxinB subunit LTB) 5 ' of the polynucleotide of victory peptide holds connection with coding heat-labile toxin B sub-cell; Its 3 ' end is connected with the 5 ' end that coding L wins the polynucleotide of peptide; 3 ' the end that this coding L wins the polynucleotide of peptide is connected with the 5 ' end that aforesaid coding PPRSV ORF5 wins the polynucleotide of peptide; 3 ' the end that this coding PPRSV ORF5 wins the polynucleotide of peptide is connected with the 5 ' end that coding endoplasmic reticulum reservation message (HDEL) wins the polynucleotide of peptide; 3 ' the end that this coding HDEL wins the polynucleotide of peptide is connected with 5 ' end of aforesaid NOS termination, can make vaccine expression vector, as: vaccine expression vector pLTB-L2-ORF5 provided by the present invention, pLTB-L4-ORF5 and pLTB-L6-ORF5.

The PRRSV ORF5 that wherein encodes wins the polynucleotide of peptide, and it has the nucleotide sequence as SEQ ID No:1; This promotor (first promotor or second promotor) is including but not limited to: CaMV35S, the heavy ubiquitin gene promotor of banana, and it has can be in the suitable promotor of this biology expression in vivo target gene as the nucleotide sequence of SEQ ID No:3 or other; This coding HDEL wins the polynucleotide of peptide, has the nucleotide sequence as SEQ IDNo:4 or SEQ ID No:5; The polynucleotide of this coding LTB victory peptide has the nucleotide sequence as SEQ IDNo:6; This coding L wins the polynucleotide of peptide, and its polynucleotide sequence is selected among SEQ IDNo:7, SEQ ID No:8, the SEQ ID No:9 at least a.

The present invention also provides by the expressed recombination fusion protein that goes out of these vaccine expression vector, it comprises: (heat-labile toxin B subunit LTB) wins peptide, L victory peptide, pig reproduction and breathing syndrome virus (PRRSV) ORF5 victory peptide, pig reproduction and breathing syndrome virus (PRRSV) ORF6 victory peptide and endoplasmic reticulum reservation message (HDEL) and wins peptide heat-labile toxin B sub-cell.

Wherein this LTB wins the N end that peptide is positioned at recombination fusion protein; The N end that this L wins peptide is connected with the C end that LTB wins peptide; The C end that this L wins peptide is connected with the N end that this PRRSV ORF5 wins peptide; The C end that this PRRSVORF5 wins peptide is connected with the N end that this HDEL wins peptide; This HDEL wins the C end that peptide is positioned at recombination fusion protein.

Wherein this LTB victory peptide has the aminoacid sequence as SEQ ID No:10; This L wins peptide, and its aminoacid sequence is selected among SEQ ID No:11, SEQ ID No:12, the SEQ ID No:13 at least a; This HDEL victory peptide has the aminoacid sequence as SEQ ID No:14.

In addition, the present invention also provides the expression vector that comprises aforementioned genetic expression composition (vector), and utilizes these genetic expression compositions or its carrier to change the application of growing the aspect in gene.This application comprises: a kind of pig reproduction and respiratory complication (PPRS) oral vaccine with plant production, and the method for a kind of prevention pig infected pigs's reproduction and respiratory complication (PPRS).

The preparation method of this oral vaccine wherein comprises:

Step 1: constructing the genetic expression that contains aforementioned genetic expression composition changes and to grow carrier (transfer vector) and change and grow plastid to obtain reorganization;

Step 2: the reorganization of step 1 changeed grow plastid and change and grow, change to obtain containing this reorganization that the plant that grows plastid changes cell colonization or tissue is grown in the plant commentaries on classics into vegetable cell or tissue;

Step 3: the plant of culturing step 2 gained changes cell colonization or tissue is grown in the plant commentaries on classics, part organ, tissue or the cell that plant is grown in plant or commentaries on classics grown in the commentaries on classics that contains aforementioned genetic expression composition with generation, and wherein this commentaries on classics is grown part organ, tissue or the cell that plant or commentaries on classics grow plant and is direct for the only oral vaccine of pig.

Wherein should prevent the method for pig infected pigs's reproduction and respiratory complication (PPRS), comprise:

Grow these expression vectors in plant by commentaries on classics, to express PRRSV ORF5 or other following desire expressed proteins in plant materials, only again these commentaries on classics are grown gene plant feeding pig, so that it is to pig reproduction and breathing syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) produce antibody, with reproduction of preventing infection pig and respiratory complication (PPRS).

Further, the present invention also provides a kind of list from the polynucleotide sequence that weighs the 3 ' lateral areas (3 ' Flanking region, 3 ' MAR) of ubiquitin gene (Mh-UBQ1) from banana, has the sequence as SEQ ID No:2; The GenBank accession number of the heavy ubiquitin gene of this banana is AF502575 (SEQ ID No:15).The matrix association regions sequence (matrix attachment region, MAR) be one section with nuclear matrix have specificity bonded dna sequence dna, its sequence characteristic is the chromatin fringe region that is rich in the AT base and is positioned at the gene two ends mostly.It is relevant whether MAR and chromatinic winding arrangement are untied, and infers that therefore MAR participates in the expression (Gasser et al., 1989) of regulatory gene.Generally speaking, MAR has carrying out that is beneficial to Transcription, represents that promptly this gene has higher expression.

Above-mentioned nucleotide sequence, aminoacid sequence, including but not limited to: with sequence provided by the present invention, through sudden change (mutation), deletion (deletion), insertion (insertion) or the existing modes such as (replacement) of replacement, with 1 to a plurality of Nucleotide or amino acid change, but still be suitable for the object of the invention.The preferably, it should have 80% sequence complementarity (complementary) at least, or at least 90% sequence identity (Identitty).

Wherein above-mentioned gene changes grows expression vector, includes but not limited to: pBI101, pBI121, pBIN19 (ClonTech), pCAMBIA1301, pCAMBIA1305, pGREEN (GenBank Accession No:AJ007829), pGREEN II (GenBank Accession No:EF590266) (www.pGreen.ac.uk), pGreen0029 (John Innes Centre).

The mode that gene is grown in above-mentioned commentaries on classics includes but not limited to: the Agrobacterium mediator method, gene recombined virus infects method, the sub-carrier that jumps changes the method for growing, particle gun changes the method for growing, electroporation, microinjection, the pollen tube method, the liposome media changes the method for growing, the ultrasound media changes the method for growing, the silicon carbide fiber media changes the method (silicon carbidefiber-mediated transformation) of growing, electrophoretic method (electrophoresis), laser microbeam (lasermicrobeam), macrogol (polyethylene glycol, PEG), calcium phosphate changes the method for growing, DEAE-dextran changes the method etc. of growing.

Pig reproduction and respiratory complication oral vaccine and uses thereof with plant production provided by the present invention with other prior aries mutually relatively the time, has more following advantage:

1. vaccine expression vector provided by the present invention except that can be in plant interior expression antigen, and be grown gene engineering by commentaries on classics and can be produced the gene that contains it and change and grow plant, can be as oral vaccine.

2. oral vaccine provided by the present invention compared to traditional preparation process, removes the problem of malicious vaccine safety of can avoiding living, also can remove on the traditional preparation process vaccine all scrupulous on packing, conveying, storage, purifying.

Description of drawings

Figure 1A is the policy map of constructing of intermediate carrier PRRSV-35PGHT, and wherein p35PGHT carries out partially digested with Sac I after Nco I enzyme is cut again; Figure 1B is the policy map of constructing of vaccine expression vector pGKU-35PRRSV;

Fig. 2 A is the policy map of constructing of intermediate carrier PRRSV-79PSGHT, and wherein p79PSGHT carries out partially digested with Sac I after the NcoI enzyme is cut again; Fig. 2 B is the policy map of constructing of vaccine expression vector pGKU-79PRRSV;

Fig. 3 A is the policy map of constructing of intermediate carrier ORF6-79PSGHT, and wherein p79PSGHT carries out partially digested with Sac I after Nco I enzyme is cut again; Fig. 3 B is the policy map of constructing of intermediate carrier intermediate carrier P-ORF6-T; Fig. 3 C is the policy map of constructing of vaccine expression vector pGKU-5-6; Fig. 3 D is the policy map of constructing of intermediate carrier ORF56F-79PSGHT; Fig. 3 E is the policy map of constructing of vaccine expression vector pGKU-56Fusion;

Fig. 4 A is the policy map of constructing of intermediate carrier 79LTB-L2-ORF5-PSGHT, and wherein p79PSGHT cuts with Nco I enzyme after Sac I is partially digested again; Fig. 4 B is the policy map of constructing of vaccine expression vector pLTB-L2-ORF5; Fig. 4 C is the policy map of constructing of intermediate carrier 79LTB-L4-ORF5-PSGHT, and wherein p79PSGHT cuts with Nco I enzyme after Sac I is partially digested again; Fig. 4 D is the policy map of constructing of vaccine expression vector pLTB-L4-ORF5; Fig. 4 E is the policy map of constructing of intermediate carrier 79LTB-L6-ORF5-PSGHT, and wherein p79PSGHT cuts with Nco I enzyme after Sac I is partially digested again; Fig. 4 F is the policy map of constructing of vaccine expression vector pLTB-L6-ORF5;

Fig. 5 A intends changeing the pcr analysis result who grows plant (1,2,3) for changeing the tobacco that grows vaccine expression vector pGKU-35PRRSV, and wherein P is the pcr analysis result of vaccine expression vector pGKU-35PRRSV; Fig. 5 B intends changeing the pcr analysis result who grows plant (1,2) for changeing the tobacco that grows vaccine expression vector pGKU-79PRRSV, and wherein P is the pcr analysis result of vaccine expression vector pGKU-79PRRSV; Fig. 5 C intends changeing the Nan Fangshi hybridization analysis result who grows plant (1,2,3,4,5,6) for changeing the tobacco grow vaccine expression vector pGKU-35PRRSV, and wherein P is that Nan Fangshi hybridization analysis result, the WT of vaccine expression vector pGKU-35PRRSV represents not change and grow plant; Fig. 5 D intends changeing the Nan Fangshi hybridization analysis result who grows plant (1,2) for changeing the tobacco grow vaccine expression vector pGKU-79PRRSV, and wherein P is that Nan Fangshi hybridization analysis result, the WT of vaccine expression vector pGKU-79PRRSV represents not change and grow plant;

Fig. 6 A is that PRRSV ORF5 changes the RT-PCR analytical results of growing plant (pGKU-35PRRSV (1,2,3,4,5), pGKU-79PRRSV (6)) in tobacco; Fig. 6 B is that PRRSV ORF5 grows the RT-PCR analytical results of plant (pGKU-35PRRSV (1,2,3,4,5,6)) in the tobacco commentaries on classics.

Fig. 7 A is that PRRSV ORF5 grows immunity commentaries on classics stain analysis (Immunoblot analysis) result that plant (1,2,3,4,5) or pGKU-79PRRSV (6) are grown in plant pGKU-35PRRSV commentaries on classics in the tobacco commentaries on classics; Wherein WT represents not change and grows plant; The recombinant protein that E.coli expresses is as positive controls, and its size is about 42kD; Fig. 7 B is that PRRSV ORF5 grows immunity commentaries on classics stain analysis (Immunoblot analysis) result that plant (1,2,3,4,5,6) is grown in plant pGKU-35PRRSV commentaries on classics in the tobacco commentaries on classics;

Fig. 8 A changes the ELISA result who grows plant (1,2,3,4,5,6) for the pGKU-35PRRSV tobacco, and wherein WT grows plant for not changeing; Fig. 8 B is the result of Fig. 8 A after quantitatively, and wherein * represents that the tool statistics is showing meaning (P＜0.05), and WT grows plant for commentaries on classics not;

Fig. 9 A for change with pGKU-35PRRSV grow tobacco plant (GP5-T group) feeding pig only after, the result who carries out lymph corpuscle proliferative response (DCPM) analysis; Fig. 9 B for change with pGKU-35PRRSV grow tobacco plant (GP5-T group) feeding pig only after, detect the result of IgG antibody amount in each time point; Fig. 9 C for change with pGKU-35PRRSV grow tobacco plant (GP5-T group) feeding pig only after, detect the result of IgA antibody amount in each time point;

Figure 10 A to Figure 10 D is respectively banana changes the GUS active mass chemical staining analytical results of growing the plant different sites;

Figure 11 A intends changeing the pcr analysis result who grows plant (1,2,3,4,5) for changeing the banana of growing vaccine expression vector pGKU-35PRRSV, and wherein P is the pcr analysis result of vaccine expression vector pGKU-35PRRSV; Figure 11 B intends changeing the pcr analysis result who grows plant (1,2,3,4) for changeing the banana of growing vaccine expression vector pGKU-79PRRSV, and wherein P is the pcr analysis result of vaccine expression vector pGKU-79PRRSV;

Figure 12 intends changeing the Nan Fangshi hybridization analysis result who grows plant (1,2,3,4,5) for changeing the banana grow vaccine expression vector pGKU-35PRRSV, and wherein P is that Nan Fangshi hybridization analysis result, the WT of vaccine expression vector pGKU-35PRRSV represents not change and grow plant;

Figure 13 grows the RT-PCR analytical results of plant (pGKU-35PRRSV (1,2,3,4,5)) for PRRSV ORF5 changes in banana;

Figure 14 is that PRRSV ORF5 grows immunity commentaries on classics stain analysis (Immunoblot analysis) result that plant (1,2,3,4,5) is grown in plant pGKU-35PRRSV commentaries on classics in the banana commentaries on classics; Wherein WT represents not change and grows plant; The recombinant protein that E.coli expresses is as positive controls;

The ELISA result that Figure 15 A grows PRRSV ORF5 (GP5) antigen presentation of plant (1,2,3,4,5) for the commentaries on classics of pGKU-35PRRSV banana, wherein WT grows plant for not changeing; Figure 15 B is the result of Figure 15 A after quantitatively, and wherein * represents that the tool statistics is showing meaning (P＜0.05), and WT grows plant for commentaries on classics not;

Figure 16 for Fig. 9 B for change with pGKU-35PRRSV grow banana plant (GP5-B group) feeding pig only after, detect the result of IgG antibody amount in each time point;

Figure 17 for change with pGKU-35PRRSV grow banana plant (GP5-B group) feeding pig only after, the result who carries out lymph corpuscle proliferative response (DCPM) analysis;

Figure 18 A is for being that the vaccine expression vector body pGKU-56Fusion tobacco of probe intend to change the Nan Fangshi hybridization analysis result who grows plant (1 to 7) with the ORF5 gene; Figure 18 B is for being that the vaccine expression vector body pGKU-56Fusion tobacco of probe intend to change the Nan Fangshi hybridization analysis result who grows plant (1 to 7) with the ORF6 gene, and wherein P is that the Nan Fangshi hybridization analysis result, WT of pGKU-56Fusion grows plant for commentaries on classics not;

Figure 19 A is for being that the vaccine expression vector body pGKU-5-6 tobacco of probe intend to change the Nan Fangshi hybridization analysis result who grows plant (1 to 5) with the ORF5 gene; Figure 19 B is for being that the vaccine expression vector pGKU-5-6 tobacco of probe intend to change the Nan Fangshi hybridization analysis result who grows plant (1 to 5) with the ORF6 gene, and wherein P is that the Nan Fangshi hybridization analysis result, WT of pGKU-5-6 grows plant for commentaries on classics not;

Figure 20 A is that PRRSV ORF5-ORF6 fusion gene is grown the RT-PCR analytical results of plant (pGKU-56Fusion (1 to 7)) in the tobacco commentaries on classics; Figure 20 B is that tobacco changes and to grow the RT-PCR analytical results of plant (pGKU-5-6 (1 to 5)), wherein is that the specificity primer with the ORF5 gene carries out RT-PCR; Figure 20 C is that tobacco changes and to grow the RT-PCR analytical results of plant (pGKU-5-6 (1 to 5)), wherein is that the specificity primer with the ORF6 gene carries out RT-PCR;

Figure 21 A intends changeing the pcr analysis result who grows plant (1 to 4) for changeing the tobacco grow vaccine expression vector pLTB-L2-ORF5, and wherein P is that the pcr analysis result, WT of vaccine expression vector pLTB-L2-ORF5 grows plant for commentaries on classics not; Figure 21 B intends changeing the pcr analysis result who grows plant (1 to 4) for changeing the tobacco grow vaccine expression vector pLTB-L4-ORF5, and wherein P is that the pcr analysis result, WT of vaccine expression vector pLTB-L4-ORF5 grows plant for commentaries on classics not; Figure 21 C intends changeing the pcr analysis result who grows plant (1 to 8) for changeing the tobacco grow vaccine expression vector pLTB-L6-ORF5, and wherein P is that the pcr analysis result, WT of vaccine expression vector pLTB-L6-ORF5 grows plant for commentaries on classics not;

Figure 22 A intends changeing for changeing the tobacco grow vaccine expression vector pLTB-L6-ORF5 that to grow plant (1 to 8) be the Nan Fangshi hybridization analysis result of probe (0.6kb) with the ORF gene, and wherein P is that Nan Fangshi hybridization analysis result, the WT of vaccine expression vector pLTB-L6-ORF5 represents not change and grow plant; Figure 22 B intends changeing for changeing the tobacco grow vaccine expression vector pLTB-L6-ORF5 that to grow plant (1 to 8) be the Nan Fangshi hybridization analysis result of probe (0.4kb) with the LTB gene, and wherein P is that Nan Fangshi hybridization analysis result, the WT of vaccine expression vector pLTB-L6-ORF5 represents not change and grow plant;

Figure 23 A is that pLTB-L2-ORF5 grows the RT-PCR analytical results of plant (1 to 4) in the tobacco commentaries on classics; Figure 23 B is that pLTB-L4-ORF5 grows the RT-PCR analytical results of plant (1 to 4) in the tobacco commentaries on classics; Figure 23 C is that pLTB-L6-ORF5 grows the RT-PCR analytical results of plant (1 to 4) in the tobacco commentaries on classics;

Figure 24 A is the ELISA result of PRRSV ORF5 (GP5) antigen presentation of tobacco transformed strain (pGKU-35PRRSV (ORF5), pLTB-L2-ORF5 (LTB-ORF5)), and wherein WT grows plant for not changeing; Figure 24 B is the result of Figure 24 A after quantitatively;

Figure 25 A for respectively with pGKU-PRRSV-ORF5-LTB, pGKU-PRRSV-ORF5 change grow tobacco plant feeding pig only after, carry out the result that lymph corpuscle proliferative response (CPM) is analyzed; Figure 25 B for respectively with pGKU-PRRSV-ORF5-LTB, pGKU-PRRSV-ORF5 change grow tobacco plant feeding pig only after, in the result of each time point detecting IgA antibody amount; Figure 25 C for respectively with pGKU-PRRSV-ORF5-LTB, pGKU-PRRSV-ORF5 change grow tobacco plant feeding pig only after, in the result of each time point detecting IgG antibody amount; Wherein W-T grows plant for not changeing.

Embodiment

Technical and the scientific terminology of described in this specification sheets all is unless definition to some extent in addition is all this affiliated field and has the meaning that common skill person can understand jointly.

The present invention system is demonstrated with the following examples to illustrate, but the present invention is not limited by following embodiment.Used medicine, the biomaterial of the present invention is all commercially available to be easy to obtain.

Constructing of embodiment 1 vaccine expression vector

(Shu) material and method

A. plastid material

Plastid p35PGHT: total length 8.1kb contains CaMV 35S promoter, GUS reporter gene, NOS termination and the heavy ubiquitin gene 3 ' MAR of banana of 0.4kb.

Plastid p79PSGHT: total length 9.1kb contains heavy ubiquitin gene (Mh-UBQ1) promotor of banana, GUS reporter gene, NOS termination and the heavy ubiquitin gene 3 ' MAR of banana.

Plastid pGKU: total length 7.4kb contains left border and right border, drives screening-gene nptII and reporter gene GUS respectively with the CaMV 35S promoter.

Plastid pBluescript II SK (-): total length 2.9kb has multiple cloning site (MCS).

Wherein plastid pGKU and plastid pBluescript II SK (-) are all the commercially available plastid that is easy to obtain; Plastid p35PGHT and plastid p79PSGHT then by following construct the strategy with:

See also Taiwan patent application case (TW patent appl.No.097131520).At first, reach suitable primer to BMARR and BMARF, grow the polynucleotide fragment that banana weighs the 3 ' lateral areas (3 ' Flanking region, 3 ' MAR) of ubiquitin gene (Mh-UBQ1), have sequence as SEQ ID No:2 with choosing by PCR; The GenBank accession number of the heavy ubiquitin gene of this banana (Mh-UBQ1) is AF502575 (SEQ ID No:15); This 3 ' MAR is positioned at 3 ' terminal sequence stop code downstream 2968bp fragment of the heavy ubiquitin gene of banana (Mh-UBQ1).Choosing being grown 3 ' the MAR fragment that carries out after enzyme cuts with EcoRI and SalI, construct in pMhUBQ1p-GUS (the middle plastid of TW patent appl.No.097131520), to obtain having p35PGHT plastid and the p79PSGHT plastid shown in Fig. 2 A shown in Figure 1A.Wherein the promotor of this p35PGHT is the CaMY 35S promoter; The promotor of this p79PSGHT is the promotor of Mh-UBQ1, has the polynucleotide sequence (TW patent appl.No.097131520) as SEQ ID No:3.

B. introduction

Used primer among the present invention, as shown in table 1:

The tabulation of table 1 primer

The primer title	Forwards/reverse	Primer sequence	SEQ?ID?No：?
				PRF?	Forward	5’-CAg ATgCATTggggAACTgCTTg-3’ NSi?I?	16?
PRR?	Oppositely	5’-TgC gAgCTCTCA AAgCTC ATCgTggggACgACCCCATCg-3’ Sac?I?*?L?E?D?H?	17?
				5ORF6?	Forward	5’-TAAg CCATggggTCgTCCCTA-3’ Nco?I?	18?
3ORF6?	Oppositely	5’-ggC gAgCTCTTA CAATTC ATCATgTTTggCATATTTgAC-3’ Sac?I?*?L?E?D?H?	19?
				5ORF5Sc2?	Forward	5’-AAT CCgCggCAACCCCTgTA-3’ Sac?II?	20?
3ORF5N?	Oppositely	5’-TAA CCATgggACgACCC-3’ Nco?I?	21?
				5ORF52?	Forward	5’-TTA gggCCCATgTTggggAACT-3’ Apa?I?	22?
5ORF54?	Forward	5’-TTA gggCCCggACCTATgTTggggAACT-3’ Apa?I?	23?
				5ORF56?	Forward	5’-TTA gggCCCggACCTggTCCAATgTTggggAACT-3’	24?

?

?

Apa?I?

?

BMARR?	Oppositely	5’-actgaattcgtccgctgctttgctgt-3	28?
				BMARF?	Forward	5’-gcgcgtcgaccaaatttttgttagattgcatg-3’	29?

C. vegetable material

The present invention is with tobacco (Nicotiana tabacum L.cv Wisc.38) and northern any of several broadleaf plants (Musa spp.cvPei-Chiao, AAA group) change the test materials of growing as the Agrobacterium gene, but any kind of the present invention and commentaries on classics culturing method of being applicable to all is contained in the applicable scope of the present invention.

D. gene changes culturing method

The commentaries on classics of tobacco leaf disk is grown and is screened

Present method is revised from (1985) such as Horsch, and test materials is tobacco (Nicotiana tabacum L.cvWisc.38).Get the blade of aseptic seeding tobacco plant, directly cutting leaf disk under agrobacterium liquid changes and grows.Leaf disk places N01B1 substratum (MS Macro (1X), MS Micro (1X), MS vitamins (1X), 0.1mg1 ^-1NAA, 1mgl ^-1BA, 30gl ^-1Sucrose, 7gl ^-1Agar, pH5.7) on, cultivated altogether three days under the photoenvironment in 25 ℃, 16 hours.After leaf disk cleaned with the N01B1 that contains 250mg/L cefotaxime, dislocation contains on the N01B1 solid medium of 250mg/L cefotaxime (cefotaxime) and 100mg/L kalamycin (kanamycin), screens about three weeks in 25 ℃, 16 hours under the photoenvironment.After treating that leaf disk grows indefinite bud, move on the N01B1 solid medium that contains 250mg/L cefotaxime and 200mg/L kanamycin, carried out time screening and culturing in 25 ℃, 16 hours under the photoenvironment.Cut the back through the indefinite bud of screening and move into and contain in the N01B1 solid medium of 250mg/L cefotaxime and 200mg/L kanamycin, treat behind its root of hair extensible bottle, carry out subsequent analysis.

The commentaries on classics of banana suspension cell is grown and is screened

Present method is according to woods (1997), the banana suspended culture cell is after natural subsidence, after being prepared into 50% (v/v) enchylema and agrobacterium liquid uniform mixing, after cultivating three days altogether, cell moved to SHGC solid medium (the SH Macro (1X) that contains 200mg/L cefotaxime and 50mg/L G418, SH Micro (1X), MS vitamins (1X), 100mgl ^-1Glutamine, 1mgl ^-1Biotin, 230mgl ^-1Proline, 10gl ^-1Lactose, 45gl ^-1Sucrose, 0.1gl ^-1Maltextract, 0.2mgl ^-1NAA, 0.2mgl ^-12ip, 0.1mgl ^-1Kinetin, 0.05mgl ^-1Zeatin, 200mgl ^-1Cefotaxime, 50mgl ^-1G418,3gl ^-1Gelrite, pH5.3) enterprising row filter, and treat that it is grown is the body embryo.Again the body embryo moved to the cultivation of germinateing afterwards and continuing screening and culturing down, after treating its germination and becoming plant, carrying out subsequent analysis based on 25 ℃, 16 hours photoenvironments.

(2) the ORF5 gene constructs

Desire of the present invention is expressed the coded main overcoat glycoprotein 5 (ORF5-encoded majorenvelop glycoprotein 5) of PRRSV ORF in plant materials, and then with these commentaries on classics grow gene plant as oral vaccine with feeding animals, the antibody of PRRSV so that it can create antagonism, and then antagonism PPRS.Therefore, carry out following constructing:

See also Figure 1A, Figure 1B, Fig. 2 A, shown in Fig. 2 B, with pig reproduction and respiratory complication virus (poreine reproductive and respiratory syndrome virus, PRRSV) ORF5 gene fragment is as template, utilize primer PRF and PRR to carry out the synthetic end of PCR and have the segmental ORF5 gene fragment of endoplasmic reticulum reservation message HDEL, cutting the back with the NsiI enzyme handles 3 ' end-grain cutting flat with Klenow, cut the fragment that 0.6kb is reclaimed in the back with the SacI enzyme again, carrier p35PGHT and p79PSGHT that access is cut through NcoI and SacI enzyme can get intermediate carrier PRRSV-35PGHT and PRRSV-79PSGHT.Intermediate carrier PRRSV-35PGHT and PRRSV-79PSGHT reclaim 4.2kb and 5.2kb fragment respectively after the PstI enzyme is cut, the pGKU that access is cut with the PstI enzyme, obtain weighing by CaMV 35S promoter and banana respectively the vaccine expression vector pGKU-35PRRSV and the pGKU-79PRRSV of ubiquitin gene (Mh-UBQ1) promoters driven ORF5 gene, and all have the contiguous sequence of banana ubiquitin gene 3 ' end that contains matrix association regions (Mh-UBQ 1flanking region, MAR), reporter gene GUS and NOS termination.

(3) express constructing of ORF5 and ORF6 gene simultaneously

See also shown in Fig. 3 A to Fig. 3 B, with PRRSV ORF6 gene as template, utilize primer 5ORF6 and 3ORF6 to carry out the synthetic end of PCR and have the segmental ORF6 gene fragment of endoplasmic reticulum reservation message HDEL, cut the fragment that 0.5kb is reclaimed in the back with NcoI and SacI enzyme, the carrier p79PSGHT that access is cut through NcoI and SacI enzyme can get intermediate carrier ORF6-79PSGHT.Intermediate carrier ORF6-79PSGHT cuts the back with HindIII and EcoRI enzyme and inserts among the carrier pSK (-), behind NOS termination (T), add that PstI cuts the position, and remove the contiguous sequence of banana ubiquitin gene 3 ' end contain matrix association regions (Mh-UBQ13 ' franking region), can obtain intermediate carrier P-ORF6-T; Cut the back with the PstI enzyme again and reclaim 2.3kb, the 5.2kb fragment that after the PstI enzyme is cut, reclaims with intermediate carrier PRRSV-79PSGHT, insert the pGKU that cuts with the PstI enzyme simultaneously, can obtain weighing by banana respectively the vaccine expression vector pGKU-5-6 (Fig. 3 C) of ubiquitin gene (Mh-UBQ1) promoters driven ORF5 and ORF6 gene.

See also shown in Fig. 3 D, utilize primer 5ORF5Sc2 and 3ORF5N to carry out PCR, synthesize and have the ORF5 gene latter end fragment that NcoI cuts the position, after cutting, NcoI and SacII enzyme reclaim the 50bp fragment, with the intermediate carrier ORF6-79PSGHT that cuts through HindIII and NcoI enzyme, and cut the 0.7kb fragment that is reclaimed behind the pGKU-79PRRSV with HindIII and SacII enzyme and carry out three sections joint reactions, obtain intermediate carrier ORF56F-79PSGHT.See also shown in Fig. 3 E, intermediate carrier ORF56F-79PSGHT reclaims the 5.7kb fragment after the PstI enzyme is cut, the pGKU that access is cut with the PstI enzyme, obtain vaccine expression vector pGKU-56Fusion, and have the contiguous sequence of banana ubiquitin gene 3 ' end, reporter gene GUS and NOS termination that contains matrix association regions by heavy ubiquitin gene (Mh-UBQ1) promoters driven ORF5 of banana and ORF6 fusion gene.

(4) express constructing of ORF5 and LTB fusion gene

See also shown in Fig. 4 A to 4B, LTB gene and ORF5 gene joined in the PCR mode, and make and have glycine between two gene (glycine, G) and proline(Pro) (proline, P).Assert that sequence is gggCCC because ApaI cuts the position, the amino acid of translating is GP, connects LTB and ORF5 gene so use ApaI to cut the position.Utilize that primer 5LTB and 3LTB2 are synthetic to have BspHI and ApaI cuts the LTB gene fragment of position, and utilize primer 5ORF52 and the synthetic front end of PRR to have the ORF5 gene fragment that GP (SEQ ID No:11), end have HDEL.The LTB gene fragment is cut the fragment that 0.4kb is reclaimed in the back with BspHI and ApaI enzyme, and cuts the ORF5 gene fragment that reclaim the back through ApaI and SacI enzyme, and the carrier p79PSGHT that common access is cut through NcoI and SacI enzyme can get intermediate carrier 79LTB-L2-ORF5-PSGHT.Intermediate carrier 79LTB-L2-ORF5-PSGHT reclaims the 5.6kb fragment after the PstI enzyme is cut afterwards, the pGKU that access is cut with the PstI enzyme, obtain vaccine expression vector pLTB-L2-ORF5, and have the contiguous sequence of banana ubiquitin gene 3 ' end, reporter gene GUS and NOS termination that contains matrix association regions by heavy ubiquitin gene (Mh-UBQ1) promoters driven LTB of banana and ORF5 fusion gene; Wherein the contained LTB-L2-ORF5 nucleotide fragments of vaccine expression vector pLTB-L2-ORF5 has the sequence as SEQ ID No:25.See also shown in Fig. 4 C to 4F, utilize the synthetic front end of primer 5ORF54 and PRR, 5ORF56 and PRR to have two respectively and repeat GP (SEQ ID No:12), three repetition GP (SEQ ID No:13), and the terminal ORF5 gene fragment that has HDEL.With the common pGKU that cuts with the PstI enzyme that inserts of LTB gene, obtain vaccine expression vector pLTB-L4-ORF5 and pLTB-L6-ORF5 in the above described manner by heavy ubiquitin gene (Mh-UBQ1) promoters driven LTB of banana and ORF5 fusion gene; Wherein the contained LTB-L4-ORF5 nucleotide fragments of vaccine expression vector pLTB-L4-ORF5 has the sequence as SEQ ID No:26; Wherein the contained LTB-L6-ORF5 nucleotide fragments of vaccine expression vector pLTB-L6-ORF5 has the sequence as SEQ ID No:27.

Aforementioned each vaccine expression vector that makes is carried out the gene commentaries on classics respectively at tobacco, banana grow, grow plant to obtain the gene commentaries on classics that contains those vaccine expression vector.This gene commentaries on classics culturing method can be had by this field knows the knowledgeable usually, carries out the gene commentaries on classics with existing skill and grows, and also can consult the Taiwan patent application case (applying date: on December 26th, 2008, application number: 097150905) disclosed method.

Embodiment 2 expresses the ORF5 genetic tobacco changes the analysis of growing plant

Tobacco changes the molecule checking of growing plant

Grow plant through the tobacco plan commentaries on classics that antibiotic-screening obtains, its GUS active mass chemical staining analytical results presents blue positive reaction.Extract and intend changeing the genomic dna of growing plant leaf, carry out PCR with the primer that is suitable for.PCR result shows, can intend changeing in pGKU-35PRRSV and grow plant (Fig. 5 A) and pGKU-79PRRSV and intend changeing and grow the synthetic fragment that detects expection in the plant (Fig. 5 B); And Nan Fangshi hybridization analysis result also has the signal fragment of expection, and wherein Fig. 5 C is that the plant analytical results is grown in the commentaries on classics of pGKU-35PRRSV plan, Fig. 5 D is that the plant analytical results is grown in the commentaries on classics of pGKU-79PRRSV plan.

Tobacco changes the gene expression analysis of growing plant

Extract the tobacco transformed strain blade total amount RNA that expresses PRRSV ORF5 gene, and in PRRSVORF5 gene fragment 5 ' end and 3 ' end design specificity sequence as primer, carry out reverse transcription polymerase chain reaction (RT-PCR), see also shown in Fig. 6 A, the 1st road (lane) to the 5th road is that plant is grown in the commentaries on classics of pGKU-35PRRSV plan, the 6th road is that plant is grown in the commentaries on classics of pGKU-79PRRSV plan, all has the 0.6kb fragment of expection length to synthesize; See also shown in Fig. 6 B, the 1st road (lane) is that pGKU-35PRRSV intend to change and to grow plant and all have the 0.6kb fragment of expection length synthetic to the 6th road.

Tobacco changes GP5 (ORF5-encoded major envelop glycoprotein5) protein expression and the quantitative analysis of the PRRSV that grows plant

Extract the tobacco transformed strain leaf protein of expressing PRRSV ORF5 gene, (the sodium dodecyl s μ lfate polyacrylamide gelelectrophoresis of 12 sodium alkyl sulfate polyacrylamide gel electrophoresises through 10%, SDS-PAGE) the back commentaries on classics is steeped on film, carries out immunity with the specificity antibody (anti-PRRSV rabbit serum) that corresponds to PRRSV and changes stain analysis; The result shows no matter grow plant (1,2,3,4,5) or pGKU-79PRRSV (6) (shown in Fig. 7 A) or pGKU-35PRRSV commentaries on classics in the pGKU-35PRRSV commentaries on classics and grow the expression that plant (1,2,3,6) (shown in Fig. 7 B) all can detect GP5.(enzyme-linked immunosorbent assay ELISA) changes the antigen expressed quantitative analysis of growing tobacco and with enzyme linked immunosorbent assay analysis method; With the GP5 recombinant protein of an amount of escherichia coli expression as quantitative criterion, each pGKU-35PRRSV (1,2,3,4,5,6) change the ELISA measuring result grow tobacco, shown in Fig. 8 A, will it quantitative after, the pGKU-35PRRSV commentaries on classics is grown the expressed GP5 protein content of tobacco and is about 112ng/mgTSP (total soluble protein), accounts for 0.01% (shown in Fig. 8 B) of total water-soluble protein.Change and grow tobacco in pig oral immunity test result only

Clip pGKU-35PRRSV commentaries on classics is grown tobacco plant (GP5-T group) blade or is changeed and grow tobacco plant (WT) blade 50g feeding piglet respectively, feeding four times (the 0th day, the 14th day, the 28th day, and the 42nd day) altogether, each two weeks at interval, and respectively at collected in the 1st, 6,13,20,27,34,41,48 day serum, saliva sample, and peripheral blood monocyte cell (peripheral blood mononuclear cells is PBMCs) to carry out the analysis of immunological competence; Wherein measure the total IgG of anti-PRRSV in the sample, total IgA amount respectively, reach specificity lymph corpuscle proliferative response (PRRSV-specific lymphocyte blastogenicresponse) PRRSV.This lymph corpuscle proliferative response changes (difference in counts per minute, DCPM) representative with the metering of per minute.

The result shows, behind feeding GP5-T (test group) or WT blade (control group), there is no that breathing appears in piglet or other clinical symptom, and this promptly confirms to take to change grows tobacco plant or change and grow the safety of anxiety tobacco plant does not have to(for) piglet.With the collected PBMC of aforementioned each time point, it is stimulated with 104TCID50PRRSV virus strain MD-001.After stimulating in 72 hours, will [ ³H]-thymidine adds wherein, waits to cultivate can measure the per minute variable after 18 hours (counts per minute CPM), and then calculates DCPM.See also shown in Fig. 9 A, single-mindedly can promptly can be observed in the 13rd day (behind the oral vaccination of the 1st day GP5-T blade) the earliest the lymph corpuscle proliferative response that PRRSV produced, and along with the 2nd time to the 4th GP5-T blade oral vaccination, this lymph corpuscle proliferative response (DCPM) also obviously increases (P＜0.05) thereupon.

In addition, see also shown in Fig. 9 B, be the amount of the IgG antibody of antagonism PRRSV GP5 in the collected serum of aforementioned each time point.In the 1st day and the 13rd day (days post-initial oral vaccination, DPIOV) during, the amount of the IgG antibody of this antagonism PRRSV GP5 does not have considerable change.Yet, the pig that can be observed feeding GP5-T blade in (behind the 2nd oral vaccination 6 days) after the 20th day only, IgG antibody of its antagonism PRRSV GP5 obviously increases.Along with the 2nd time to the 4th GP5-T blade oral vaccination, total IgG antibody amount of this antagonism PRRSVGP5 also increases thereupon gradually.

See also shown in Fig. 9 C, the earliest can be after the 20th day (DPIOV) can be observed feeding GP5-T blade pig only, IgA antibody of its antagonism PRRSV GP5 obviously increases.Along with the 2nd time to the 4th GP5-T blade oral vaccination, total IgA antibody amount of this antagonism PRRSV GP5 also increases thereupon gradually.

The above results shows, oral feeding (inoculation) can be expressed the proteic commentaries on classics of PRRSV GP5 and be grown plant and give pig, can make its generation can resist the immune response of PRRSV, and then the PRRS that avoids infection.

Embodiment 3 expresses ORF5 gene banana changes the analysis of growing plant

Banana changes the staining analysis of growing plant

Prepared pGKU-35PRRSV, the commentaries on classics of pGKU-79PRRSV plastid among the embodiment 1 are grown into banana, as northern any of several broadleaf plants (Pei Chiao), and with GUS active mass chemical staining detect this banana change grow each position of plant with analyze this whether banana change and grow plant and express PRRSV GP5 albumen.See also Figure 10 A to Figure 10 D, the result shows that Figure 10 A is the root of control group; Figure 10 B is that pGKU-79PRRSV changes the root of growing the banana plant seedling; On behalf of pGKU-35PRRSV, Figure 10 C and Figure 10 D change root and the blade of growing the banana plant seedling respectively; Can present blue positive reaction in root and blade respectively.

Banana changes the molecule checking of growing plant

In addition, extract the banana transformed strain genomic dna of expressing PRRSV ORF5 gene, and hold design specificity sequence as primer, carry out polymerase chain reaction in PRRSVORF5 gene fragment 5 ' end and 3 '.Seeing also Figure 11 A changes the polymerase chain reaction analytical results of growing plant for the pGKU-35PRRSV banana, and the result shows that plant (1,2,3,4,5,6) is grown in the commentaries on classics of banana plan all has the 0.6kb fragment of expection length to be synthesized; See also Figure 11 B and change the polymerase chain reaction analytical results of growing plant for the pGKU-79PRRSV banana, the result shows, plant (2,3,4) is grown in the commentaries on classics of banana plan all has the 0.6kb fragment of expection length to be synthesized, and the confirmation commentaries on classics is grown gene and inserted really in the plant genome.And Nan Fangshi hybridization analysis result also has the signal fragment (Figure 12) of expection.

Banana changes the gene expression analysis of growing plant

Extract the banana commentaries on classics and grow plant pGKU-35PRRSV (1,2,3,4,5) blade total amount RNA, and in PRRSV ORF5 gene fragment 5 ' end and 3 ' end design specificity sequence as primer, carry out the reverse transcription polymerase chain reaction, all show the 0.6kb fragment synthetic (Figure 13) of expection length.

Banana changes GP5 (ORF5-encoded major envelop glycoprotein5) protein expression and the quantitative analysis of the PRRSV that grows plant

Extract the banana commentaries on classics and grow the leaf protein of plant pGKU-35PRRSV (1,2,3,4,5), behind 10% SDS-PAGE, change and steep on film, carry out immunity with the specificity antibody that corresponds to PRRSV and change stain analysis, also can detect the expression (Figure 14) of GP5.And change the antigen expressed quantitative analysis of growing banana with ELISA, with the GP5 recombinant protein of an amount of escherichia coli expression as quantitative criterion, each pGKU-35PRRSV (1,2,3,4,5,6) change the ELISA measuring result of growing banana, shown in Figure 15 A, with it quantitatively after, pGKU-35PRRSV changes and grows the expressed GP5 protein content of banana and be about 285ng/mg TSP, accounts for 0.03% (Figure 15 B) of total water-soluble protein.

Change and grow banana in pig oral immunity test result only

Detect PRRSV specificity lymphproliferation response with MTS (Promega) method.In the experiment during respectively at the 0th, 14,28 day with oral way, pGKU-35PRRSV changeed grow banana plant (GP5-B) or change and grow banana plant (WT) blade, only (no pig reproduction and breathing syndrome virus infect to give 5-6 pig in age in week, PRRSV-free), simultaneously by beginning in the 7th day, gather blood, saliva sample every 14 days, detect the ability to express of peripheral blood monocyte (PBMC) internal specific T cell afterwards again in the MTS mode.Detection mode is as follows, adds 100 μ l PBMCs (2 * 10 in three multiple modes in the 96wells flat chassis ⁵Cells/well), and stimulate, in 37 ℃, 5%CO with 100 μ l, 104TCID50PRRSV ₂Under cultivate three days after, add 20 μ l detection reagent and its numerical value of interpretation under OD490nm.StimulationIndex (SI)=the average light absorption value of gained/no PRRSV stimulates the average light absorption value of gained down to have PRRSV to stimulate down.

See also shown in Figure 16, the results are shown in promptly can be observed feeding GP5-B blade on the 21st day pig only, IgG antibody of its antagonism PRRSV GP5 obviously increases.Along with the 2nd time to the 3rd time GP5-B blade oral vaccination, the total IgG antibody amount of this antagonism PRRSV GP5 also increases thereupon gradually.With the collected PBMC of aforementioned each time point, it is stimulated with 104TCID50PRRSV virus strain MD-001.After stimulating in 72 hours, MTS is added wherein, and measure its light absorption value (OD value).See also shown in Figure 17, single-mindedly can promptly can be observed in the 21st day (behind the oral vaccination of the 1st GP5-B blade) the earliest the lymph corpuscle proliferative response that PRRSV produced, and along with the 2nd time to the 3rd time GP5-B blade oral vaccination, this lymph corpuscle proliferative response also obviously increases (P＜0.05) thereupon.

In sum, the commentaries on classics of expressing the ORF5 gene grow tobacco, banana through the feeding pig only after, can in a pig serum and saliva, detect corresponding IgG and IgA and produce.Tire and virus neutralizing capacity for further improving the antibody generation, the present invention also carries out constructing of new a collection of vaccine expression vector.

The characteristic of novel vaccine expression vector

Enterotoxigenic Escherichia coli (enterotoxigenic Escherichia coli, ETEC) heat-labile toxin B sub-cell (heat-labile toxin B subunit, LTB) be the albumen of five rings shape structure, because of it can combine with the small intestinal mucosa cell, event can help antigen to enter and further bring out the intestine immunity reaction, uses so often be used as adjuvant (adjuvant).In addition, research points out that PRRSV ORF6 can utilize halfcystine (cysteine) and ORF5 to form special-shaped double base body (heterodimer), if express ORF5 and ORF6 simultaneously, then the corresponding antibody of Chan Shenging has preferable virus neutralizing capacity (Jiang et al., 2006).

Therefore, the present invention is by the characteristic of LTB and ORF6, choosing grows LTB gene and ORF6 gene, resulting choosing is grown through sequence alignment, (Chueh and Lee conforms to the LTB gene (Accession No.M17873) of NCBI database and document, 2001), it is constructed in new a collection of vaccine expression vector (see also embodiment 1 (four) and express constructing of ORF5 and LTB fusion gene), and carry out analyses such as genetic expression, molecule checking, immunological competence respectively in embodiment 4 and embodiment 5.

The tobacco that embodiment 4 expresses ORF5 and ORF6 gene simultaneously changes the analysis of growing plant

Tobacco changes the molecule checking of growing plant

The tobacco that antibiotic-screening obtains intends changeing and grows plant, and its blade of clip carries out GUS active mass chemical staining and is blue positive reaction.Further extract the genomic dna of following plant: 1. tobacco (pGKU-56Fusion) is grown in the commentaries on classics of expressing ORF5 and ORF6 fusion gene, 2. expresses the commentaries on classics of ORF5 and ORF6 fusion gene simultaneously and grows tobacco (pGKU-5-6)); Respectively with ORF5 and ORF6 gene fragment as probe, its Nan Fangshi hybridization analysis result all has the signal fragment (respectively shown in Figure 18 A, 18B, 19A, 19B) of expection.Wherein Figure 18 A is that the tobacco of probe (0.6kb) changes and to grow plastid pGKU-56Fusion and change the Nan Fangshi hybridization analysis result who grows plant with the ORF5 gene; Figure 18 B is that the tobacco of probe (0.5kb) changes and to grow plastid pGKU-56Fusion and change the Nan Fangshi hybridization analysis result who grows plant with the ORF6 gene; Figure 19 A is that the tobacco of probe (0.6kb) changes and to grow plastid pGKU-5-6 and change the Nan Fangshi hybridization analysis result who grows plant with the ORF5 gene; Figure 19 B is that the tobacco of probe (0.5kb) changes and to grow plastid pGKU-5-6 and change the Nan Fangshi hybridization analysis result who grows plant with the ORF6 gene.

Tobacco changes the gene expression analysis of growing plant

Extract tobacco pGKU-56Fusion commentaries on classics and grow plant leaf total amount RNA, and carry out the reverse transcription polymerase chain reaction to confirm the target gene normal expression, can synthesize the 1.1kb fragment the (the 1st of expection with ORF5 and ORF6 gene specificity primer, 2,3,4,7 roads) (Figure 20 A); Extracting tobacco pGKU-5-6 changes and grows plant leaf total amount RNA, carries out the reverse transcription polymerase chain reaction with the specificity primer of ORF5 and ORF6 gene respectively and all can synthesize and expect 0.6 and the 0.5kb fragment (Figure 20 B, Figure 20 C) of length.

The tobacco that embodiment 5 expresses ORF5 and LTB fusion gene changes the analysis of growing plant

Tobacco changes the molecule checking of growing plant

See also shown in Figure 21 A to 21C, grow the genomic dna of tobacco, carry out polymerase chain reaction with the specificity primer of LTB and ORF5 gene with expressing LTB and the plan commentaries on classics of ORF5 fusion gene.PCR result shows, can change in the plan of expressing LTB and ORF5 fusion gene and grow the synthetic fragment (1kb) that detects expection in the plant; Wherein Figure 21 A is that pLTB-L2-ORF5 intends changeing the pcr analysis result who grows plant; Wherein Figure 21 B is that pLTB-L4-ORF5 intends changeing the pcr analysis result who grows plant; Wherein Figure 21 C is that pLTB-L6-ORF5 intends changeing the pcr analysis result who grows plant.

In addition, see also shown in Figure 22 A to 22B, respectively with LTB and ORF5 gene fragment as probe, its Nan Fangshi hybridization analysis result also has the signal fragment of expection; Wherein Figure 22 A is that pLTB-L6-ORF5 intend to change that to grow plant be the Nan Fangshi hybridization analysis result of probe (0.6kb) with the ORF gene; Wherein Figure 22 B is that pLTB-L6-ORF5 intend to change that to grow plant be the Nan Fangshi hybridization analysis result of probe (0.4kb) with the LTB gene.

Tobacco changes the gene expression analysis of growing plant

See also shown in Figure 23 A to 23C, (the tool coding repeats the polynucleotide sequence of glycine and proline(Pro) to extract tobacco pLTB-L2-ORF5 respectively, be called for short the polynucleotide sequence that coding L wins peptide), pLTB-L4-ORF5 (two polynucleotide sequences that repeat glycine and proline(Pro) of tool coding), pLTB-L6-ORF5 (three polynucleotide sequences that repeat glycine and proline(Pro) of tool coding) changes and grows plant leaf total amount RNA, and carry out the reverse transcription polymerase chain reaction to confirm the target gene normal expression, all can synthesize to expect the 1kb fragment (Figure 23 A to 23C) of length with LTB and ORF5 gene specificity primer respectively.Tobacco changes GP5 (ORF5-encoded major envelop glycoprotein5) protein expression and the quantitative analysis of the PRRSV that grows plant

See also shown in Figure 24 A to 24B, extract the tobacco transformed strain (pGKU-35PRRSV (ORF5) that expresses LTB and ORF5 fusion gene, pLTB-L2-ORF5 (LTB-ORF5)) leaf protein, change the antigen expressed quantitative analysis of growing tobacco with ELISA, antibody is: anti-GP5 monoclonal antibody (Figure 24 A), use the expressed GP5 recombinant protein of intestinal bacteria as quantitative criterion, after quantitatively, it is 155ng/mg TSP (total solubleprotein) that the expressed GP5 protein content of tobacco pLTB-L2-ORF5 transformed strain is grown in commentaries on classics, accounts for 0.015% (Figure 24 B) of total water-soluble protein.

Change and grow tobacco in pig oral immunity test result only

Clip changes grows tobacco plant blade 50g feeding piglet, and feeding is three times altogether, each two weeks at interval.See also shown in Figure 25 A, in the experiment during respectively at the 0th, 14,28 day with oral way, 50g PRRSV-ORF5 gene changeed grow tobacco (pGKU-PRRSV-ORF5-LTB, pGKU-PRRSV-ORF5) and give not have for eight ages in week the pig (n=4) that pig reproduction and breathing syndrome virus infect respectively, simultaneously by beginning in the 7th day, gathered blood every 14 days with the EDTA anticoagulant tube, detect the multiplication capacity (CPM) (detecting the collection of sample, as described in embodiment 2) that peripheral blood monocyte (PBMC) internal specific lymph is asked in the blastogenesis mode more afterwards.The result shows, compared to W-T (control group), no matter be pGKU-PRRSV-ORF5-LTB or pGKU-PRRSV-ORF5 change grow tobacco all can be behind first time oral vaccination, the specificity lymph corpuscle proliferative response (CPM) that can be observed in about the 3rd week PRRSV obviously increases, and along with the 2nd time to the 3rd time oral vaccination, this lymph corpuscle proliferative response also obviously increases thereupon.

In addition, see also shown in Figure 25 B and Figure 25 C, during respectively at the 0th, 14,28 day with oral way, the 50g gene changeed grow tobacco (pGKU-PRRSV-ORF5-LTB, pGKU-PRRSV-ORF5) and give not have for eight ages in week the pig (n=4) that pig reproduction and breathing syndrome virus infect respectively, simultaneously by beginning in the 7th day, gather blood once every 14 days, detect the power valency of PRRSV specific antibody (IgA) and specific antibody (IgG) in the saliva afterwards again in the ELISA mode.The result shows, compared to W-T (control group), no matter be pGKU-PRRSV-ORF5-LTB or pGKU-PRRSV-ORF5 change grow tobacco all can be behind first time oral vaccination, specific antibody (IgA) amount, specific antibody (IgG) amount that can be observed in about the 3rd week PRRSV obviously increase, and along with the 2nd time to the 3rd time oral vaccination, this specific antibody (IgA) amount, specific antibody (IgG) amount also obviously increase thereupon.

Above-listed detailed description is at the specifying of possible embodiments of the present invention, and this embodiment is not in order to limiting claim of the present invention, does not allly break away from equivalence of the present invention and implements or change, all should be contained in the claim of the present invention.

Sequence table

＜110〉Univ Nat Taiwan

 

＜120〉a kind of genetic expression composition, pig reproduction and respiratory complication oral vaccine and preparation method thereof

 

<160>29

 

<210>1

<211>615

<212>DNA

＜213〉pig reproduction and breathing syndrome virus

 

<400>1

atgttgggga?actgcttgac?cgtgggctgt?tgctcgcggt?cgcttttttt?gtggtgtatt 60

gtgccgttct?gtttagctgc?gctcgtcagc?gccaacggaa?acagcagctc?ttattcccag 120

ttgatttata?acctgacgct?atgtgagctg?aatggcacag?attggctggc?agacagattt 180

gattgggcag?tggaaacttt?tgtcatcttt?cccgtgatta?ctcacatcgt?ttcctacggt 240

gcccttacta?ccagccacct?ccttgataca?gtcggtctgg?tcactgtatc?caccgccggg 300

ttctatcacg?ggcggtatgt?cttgagcagc?atctacgcgg?tctgtgccct?ggctgcgtta 360

atttgcttcg?tcattagact?ggcgaagaat?tgcatgtcct?ggcgctactc?atgtaccagg 420

tacaccaact?ttcttctaga?cactaagggc?agaatttatc?gttggcgatc?gcccgtcatc 480

atagagaaag?ggggtaaagt?tgaggtcgaa?ggccatttga?tcgacctcaa?gagagttgtg 540

cttgatggtt?ccgcggcaac?ccctgtaacc?agaattccag?cggaacgatg?gggtcgtccc 600

cacgatgagc?tttga 615

 

<210>2

<211>2968

<212>DNA

＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))

 

<400>2

aattcgtccg?ctgctttgct?gtcttagtgt?tctctctgtt?ggtgtgcgtg?gaagtcctat 60

cggaggtctg?aaggaataag?cattggtatc?gttggcatgt?attgttgaca?tctttgtttg 120

gtgaatgtct?atggttgtct?tgtggagtct?ctggtgttct?ataacttgtg?tcagtgccat 180

tatgttttta?tcgactgctg?ctgcccttct?cttgctaatg?tacgatgctg?atgctgatgc 240

tgatgttgct?actgctgttt?tatatattta?atgtatccaa?ttaattatgt?gtgttcagtg 300

gtgttcgatc?aacacaggtg?aaaacttttc?aagggagttt?ttttggtatt?gaaatatttt 360

gtttagatca?tgtgtacgaa?gatactcatt?ctatgctgtt?caaaagcata?acgtaaagtg 420

tataggaggt?ggttgttaaa?aaagatttag?tctacttctt?ctcgcattta?tagagaacat 480

tataaattta?tcatattata?ttgggtatta?gatcttggta?ttagattttt?atctaactac 540

tcatcacact?tagattaatg?aatttttgac?tcgtattgga?tttcatctat?agaatttaac 600

aagtttactg?aatatccaat?aagataaggg?tttcgacgga?tatctaatat?cctctaggtc 660

aaatattgag?tttgtcgtga?gtcatacata?tcagaactct?ttctagatca?gtgaaaacgt 720

actaggtcac?tctccaaatg?atatatgaaa?tgtaatctat?acatatcaga?actctttcta 780

gattagtgaa?aacgtactag?gtcactctcc?aaatgatata?tgaaatgtaa?tctattggac 840

atatctgtcc?ttaattatat?gtatttatag?tttcttatcc?atctaatatc?ttaaagatta 900

cattccgggt?atagtgttat?taaacccata?tgatttctac?tcgagcctca?ctctaatcgg 960

attcttctgg?agaaggattt?gtttgagcaa?aaacatataa?gatattattc?tcataacact 1020

gagagtgaat?aatcttctat?caacactcaa?tagctctcat?aaaattgatt?gtcacttttg 1080

acggttagtt?gtgctagatc?tggaacttcc?agagttacaa?gttcagcatc?aaagagtgga 1140

gtactcatat?atgacatctt?tggtatttaa?tgatcggatt?caccactagg?acgacagaat 1200

cactgtctga?caatgatgta?tcatcatgtt?atgactgttt?cgacactaag?gggtgaatta 1260

gtgttttcgt?aaaaacatcg?gtttaaaaat?tttgaactat?aaaatcataa?cgaaaatacg 1320

tttaaactta?aagtgcaagt?aagagtagta?gacaattaaa?tcaataatca?attacaatga 1380

aaagcacaat?ataaatgtag?atcgagttta?tagtgattcg?gtcatcgtaa?cctacgtcca 1440

ctctcgattc?ctctttcgag?gccatcggcg?ttcactaacg?atcttctttc?aatgggtaaa 1500

gcctaactac?tctcttacaa?ctctttctcc?tttttacaaa?tttaagagaa?aacatttacg 1560

agtctcatac?ctccgttaga?tagaatttta?aacttttgaa?aaaaaatgag?aggaactctc 1620

aacacttttc?aagattacaa?ctttgattca?ctagcatttt?agttcactct?ttattgtgtc 1680

ttctcagcaa?ggataagtga?ggtatttata?gaccccaaat?gatttcaaaa?ataaagtcaa 1740

aaatttaaat?ttctgagtat?ttcgggtatg?agcggtacca?ccactgtgct?gggcggtact 1800

accgcttggt?agagcatcga?agacggggct?ctagcggtac?taccgccagc?ttgggcgata 1860

ccatcacctg?ccagcattaa?tgctgagggt?accatcatct?agtctggatg?gtactatcat 1920

cgaacagcct?ggaaaagtgt?gttcttgact?tttccaactc?ataggcggtt?ccaccgcttg 1980

attcaatctt?tgggtcacta?aataggcctc?caaatgagct?caaatcagtt?ccaatggacc 2040

cttaattgag?ttaacattgt?tacacttgaa?tctaactcaa?ttaagatcta?aaactacgac 2100

gatcatgact?taaatgatta?caaaacatat?aatattatgt?tgtccgacat?atcattggtt 2160

cattcaaact?ctcgatgcat?catgctcttt?ttcggtgtat?tgctcgatct?atcgacatgt 2220

tgactctcac?aacgtccaat?cttgatgcat?gtccaattct?tccggcccga?tgcctgatct 2280

ctaacttcag?ctcaatattt?gattcttcta?cttcaattgt?tttgcctttt?catgatcaaa 2340

gttagtcctt?tgcatcactt?ttcttaaaat?ccagattata?ttgtaactca?tcaattaata 2400

tcatcatcaa?aatccgggat?tcacacatca?ccatctagca?ttccattagt?ggatcaatca 2460

gttaactcat?tctccaataa?tcacttgcac?gatatcccta?gtgtctccat?acaagtagtt 2520

atgagaccaa?ccgcctccat?catatagacg?ggtatacaac?gcaccaattt?atccagttat 2580

ctcaatgacc?tctcaagcaa?cctatgacta?agatcattta?ggatttatat?ttaaaggtaa 2640

atcaatctta?ttattatgat?cttatcataa?tctgattctc?agtatataga?tccatgaaca 2700

tgtgtaacgg?acaacataaa?atgagaaaaa?aattataata?aataattata?aaaaaaataa 2760

tatgtaacgt?gtcagatgtc?tcatcactca?tcttgtaact?cacgtaattg?atctcgtcga 2820

aagtgagtgt?tttggtaaga?gtggctccac?taggtaccac?ttaaaggaaa?tcctaagtga 2880

attgctttac?ttcacgctgc?tgaattgttc?tgcagtttgt?atcacccttt?gccatgttaa 2940

tgcatgcatg?caatctaaca?aaaatttg 2968

 

<210>3

<211>3093

<212>DNA

＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))

 

<400>3

ggatccacat?gttctgcaga?tagatagata?ccatcttctg?aggctttttg?tagttccaag 60

ttgtaggcta?attctcggtt?cttccaagaa?aatatgatga?acactcacta?attctttcta 120

ccctgtccac?catgcaggtt?tagcttctca?acaatgtttt?tcgcacatag?taagtgagat 180

tcacgatacg?tctgatacaa?tttaaacaac?catttcctaa?ttattggtac?aaattagctc 240

caacatacat?ttacgagttt?ttgtgtgtga?aaattgtgca?ggggagaaca?cagtcagtac 300

gctcgggcga?ttcgtgttga?tagtatggat?gttcgtagtc?ctaattatca?actcgagcta 360

cacagccagc?ttgacgtcaa?tcctcacagt?tcaacagctc?tcatcaggga?ttactgggct 420

tgacagcttg?ctctcgtcct?ctgaacctat?tggctaccag?aaggggaagt?tttcgaggaa 480

ttacatgata?gaagagctca?acattcctgc?gtccagactc?gtacctctga?actcccctgc 540

agagtatgct?agggctctcc?ggctggggcc?aaagggtggt?ggtgtggcag?ccatcgtcga 600

tgagattcca?tacgtcgaga?tcttcttgtc?tgcctactgc?cagttcaaga?tcgtgggtca 660

agagttcacc?aaaaatggat?ggggatttgt?aagtatctcg?atccaagaat?gcaagcatcg 720

ttaccctctg?ttaacatcaa?cacatcttta?tggtaacagc?agctcaggat?tcattggcac 780

tcgaagcatt?actcttttct?tttaagagca?attccagtat?catttgaact?gttttcttca 840

tttcagattc?tcttagagat?tcagaattga?tagacaacaa?aaagtcttag?caagtcttca 900

ttgttatatg?gctacgaaga?cgaacaaaca?aacatgacaa?gtgcaaataa?tgaactccca 960

agcaaatctc?tatcatcttc?ttgtgtgaaa?agtgaagctt?tacattacga?caatagtcga 1020

gatgtgaacc?tctactcgat?atttctgaag?gctactttca?tgaaataagt?tcaaccaaga 1080

atcgattagt?acgatgtagt?cagtctgtca?tgcaatatcc?tttgtgcgga?tacagttttg 1140

tcgaatctat?agaattttaa?tgtggacttc?aattttcatt?ctccgacagg?cattccagag 1200

ggactctcct?cttgcggttg?acctgtccac?tgcaatcctg?gcactatccg?agaatggcga 1260

tctccagacg?atccacgaga?aatggttgtc?gcgtgctgga?tgtccttccc?aaggtgttga 1320

agaagaagcg?aaccggctaa?gtctcagtag?cttctggggt?ctcttcctcc?tcagtggcat 1380

cgtgtgtttt?cttgctctca?tccttttctg?cataaaggtg?tgctatcagt?atgccatgta 1440

cagcagcgca?gaggtcgaca?agcccagaga?aaacgacttg?atcgacggaa?gccaacatgc 1500

cctatgcaag?ttaaagagca?tcaaggcttt?gattcgcttc?ttcgacatga?aggaagaaga 1560

aatcaacaaa?gtcatcacga?aaaaaccgag?tggtacacaa?aatggtcctc?caacttcgga 1620

tgatgggcat?tcgctgccat?cttcatagaa?cgtggtaaga?agatgcagaa?atctttctta 1680

ccaaaagaac?atcagctatt?ttgcattagg?aaatgcgatt?acttggtaga?cttgatgaag 1740

actgcgactg?cgactgtttc?acaaagtgcg?agttcttaat?ttgtcttgaa?tctcttataa 1800

agcaatccag?aacatacata?ttttctagtg?ctggatgtaa?ctcgatatgt?agctgcaaca 1860

aaatcaaagg?aaatttaagc?tgcacaaaag?atgatcgtct?tcttttgatg?aaaagaatga 1920

gcatatcgtg?gaatcgctac?gtcgtctctt?cttaccgctg?ccatcaccaa?ggatcgcagg 1980

taaatgagac?acttctctta?catcccacgc?ggatacccaa?ctagtccagg?agggatagga 2040

ggaggactga?acttgctggg?tcccacatga?ggttttgtcc?gatcgccaaa?tcaactgggc 2100

ccacagctgt?ggcatccgag?atggaagcct?gacgcctgtc?aagggcacgt?gcatgtgatt 2160

tcttctgcgt?gcggtcacct?ctggcgtact?cgggaaaaat?ctggcaacgg?acgaattcct 2220

tctcgaatgc?aacgccgtta?tctttgaacg?gcacccgtgc?cacgagagag?taatgctcgc 2280

ctcacctacc?agcttgctgg?ttggcgaagc?aacatggaac?ccacgcgtga?gtcatgaact 2340

gtaatgtggg?aataggacgg?atacacgtta?ctccgacagc?gacgtcctac?cttgcggtag 2400

caggatacgt?ctcttatatc?gtccgtccac?ttgtgcggcg?atcactggct?gaactctttt 2460

taggtaacat?caccggtagg?taatctcacg?ccagaagatg?gctataaata?aggctcctca 2520

ggcccgatct?caatgcgctc?tattcaatat?tgtcgaaagg?cttgcaagtt?ttcttccttt 2580

gcttcaaggt?atgttttcgc?ctatacctga?gtatttccct?attttaggcc?ttttttgcct 2640

tttttatttt?tattttctgt?agcttcgatg?ttaatggatg?aaggggtaga?tcaatcgttt 2700

tctttctatt?ttattgatgt?tgttgttaac?aattgtctga?ttctttgccg?actcctttga 2760

gagatttggt?aatcgtttgc?gattttcttt?tttgtgatat?gttagttggt?ctataaaagt 2820

cgattcttat?attgttgtga?cacgatctac?cacgtcttgc?atatgttttc?ttgtaggaag 2880

cttcgtctca?gatttgaagc?atattgatgt?gtcgcttttg?atcatttagt?cgaagtggtt 2940

gattttaatt?ttaggtcatt?catttttttt?cttgtacact?ttgcatgatc?cgtctcagat 3000

ctgatcaatt?gttgttagtc?cagtagtcct?tttttgtgta?ttagatctga?tggttgtttt 3060

ctgactcctt?tcttctttta?tctctttgat?cag 3093

 

<210>4

<211>12

<212>DNA

＜213〉artificial sequence

<220>

＜223〉HDEL of synthetic wins the nucleotide sequence of peptide

 

<400>4

gtgctactcg?aa 12

 

<210>5

<211>12

<212>DNA

＜213〉artificial sequence

<220>

＜223〉HDEL of synthetic wins the nucleotide sequence of peptide

 

<400>5

gtactactta?ac 12

 

<210>6

<211>375

<212>DNA

＜213〉enterotoxigenic Escherichia coli (enterotoxigenic Escherichia coli)

<220>

＜223〉nucleotide sequence of heat-labile toxin B sub-cell (heat-labile toxin B subunit)

 

<400>6

atgaataaag?taaaatgtta?tgttttattt?acggcgttac?tatcctctct?atatgcacac 60

ggagctcccc?agactattac?agaactatgt?tcggaatatc?gcaacacaca?aatatatacg 120

ataaatgaca?agatactatc?atatacggaa?tcgatggcag?gcaaaagaga?aatggttatc 180

attacattta?agagcggcga?aacatttcag?gtcgaagtcc?cgggcagtca?acatatagac 240

tcccagaaaa?aagccattga?aaggatgaag?gacacattaa?gaatcacata?tctgaccgag 300

accaaaattg?ataaattatg?tgtatggaat?aataaaaccc?ccaattcaat?tgcggcaatc 360

agtatgaaaa?actag 375

 

<210>7

<211>6

<212>DNA

＜213〉artificial sequence

<220>

＜223〉L of synthetic wins the nucleotide sequence of peptide

 

<400>7

gggccc 6

 

<210>8

<211>12

<212>DNA

＜213〉artificial sequence

<220>

＜223〉L of synthetic wins the nucleotide sequence of peptide

 

<400>8

gggcccggac?ct 12

 

<210>9

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉L of synthetic wins the nucleotide sequence of peptide

 

<400>9

gggcccggac?ctggtcca 18

 

<210>10

<211>124

<212>PRT

＜213〉enterotoxigenic Escherichia coli (enterotoxigenic Escherichia coli)

<220>

＜223〉LTB wins the aminoacid sequence of peptide

 

<400>10

 

Met?Asn?Lys?Val?Lys?Cys?Tyr?Val?Leu?Phe?Thr?Ala?Leu?Leu?Ser?Ser

1 5 10 15

Leu?Tyr?Ala?His?Gly?Ala?Pro?Gln?Thr?Ile?Thr?Glu?Leu?Cys?Ser?Glu

20 25 30

Tyr?Arg?Asn?Thr?Gln?Ile?Tyr?Thr?Ile?Asn?Asp?Lys?Ile?Leu?Ser?Tyr

35 40 45

Thr?Glu?Ser?Met?Ala?Gly?Lys?Arg?Glu?Met?Val?Ile?Ile?Thr?Phe?Lys

50 55 60

Ser?Gly?Glu?Thr?Phe?Gln?Val?Glu?Val?Pro?Gly?Ser?Gln?His?Ile?Asp

65 70 75 80

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

85 90 95

Tyr?Leu?Thr?Glu?Thr?Lys?Ile?Asp?Lys?Leu?Cys?Val?Trp?Asn?Asn?Lys

100 105 110

Thr?Pro?Asn?Ser?Ile?Ala?Ala?Ile?Ser?Met?Lys?Asn?***

115 120

 

<210>11

<211>2

<212>PRT

＜213〉artificial sequence

<220>

＜223〉L wins the aminoacid sequence of peptide

 

<400>11

Gly?Pro?***

1

 

<210>12

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉L wins the aminoacid sequence of peptide

 

<400>12

Gly?Pro?Gly?Pro?***

1

 

<210>13

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉L wins the aminoacid sequence of peptide

<400>6

Gly?Pro?Gly?Pro?Gly?Pro?***

1 5

 

<210>14

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉HDEL of synthetic wins peptide

 

<400>14

His?Asp?G1u?Leu?***

1

 

<210>15

<211>1146

<212>DNA

＜213〉banana (Musa spp.cv.Hsien Jin Chiao (AAA group))

 

<220>

<221>CDS

<222>(1)..(1146)

 

<300>

<308>Genbank/AF502575

<309>2005-12-31

 

<400>15

atgcagatct?ttgttaaaac?tctcactggc?aagaccatca?cccttgaggt?tgaatcctct 60

gacaccatcg?acaatgtcaa?ggctaagatc?caggacaaag?agggaatccc?tccagaccag 120

caaaggctga?tctttgccgg?taagcaactt?gaggatggcc?ggacccttgc?ggattacaac 180

atccagaagg?agtccaccct?ccacctcgta?cttcgccttc?gtggtggcat?gcaaatcttt 240

gtcaagacct?tgactggcaa?gaccatcacc?ctcgaggtgg?agagttctga?caccatcgac 300

aatgtcaagg?ctaagattca?ggataaggag?ggcattcctc?cagaccagca?aaggctcatc 360

tttgccggca?agcagcttga?ggatggccgc?accctggctg?attacaacat?ccagaaggag 420

tccaccctcc?accttgtgct?tcgacttcgg?ggtggcatgc?aaatctttgt?caagaccttg 480

actggcaaga?ccatcaccct?cgaggtggag?agttctgaca?ccatcgacaa?tgtcaaggcc 540

aagattcagg?ataaggaggg?cattccaccg?gaccagcaga?ggctcatctt?tgccggcaag 600

cagctggagg?acggccgcac?cttggctgat?tacaacatcc?agaaggagtc?caccctccac 660

cttgtcctcc?gcctccgtgg?tggcatgcaa?atcttcgtca?agactttgac?tgggaagacc 720

atcaccctta?aggtggagag?ctcggacacc?atcgacaatg?taaaggccaa?gattcaggac 780

aaggagggta?ttcccccgga?ccagcaaagg?ctcatctttg?ccggcaagca?gcttgaggat 840

ggccgcaccc?tggcagatta?caacattcag?aaggagtcta?cccttcacct?tgtgctgaga 900

cttaggggtg?gcatgcagat?ctttgttaag?acgcttacag?ggaagaccat?taccttggag 960

gtggagagct?cggacacgat?tgataatgtc?aaggcaaaga?tccaggacaa?ggaggggatt 1020

ccaccggatc?agcagaggct?gatctttgct?gggaagcagc?tggaggacgg?gcgcaccctg 1080

gcagattaca?acattcagaa?ggaatccacc?cttcacctgg?tgctccgcct?ccgcgggggt 1140

cattaa 1146

 

<210>16

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

 

<400>16

cagatgcatt?ggggaactgc?ttg 23

 

<210>17

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

 

<400>17

tgcgagctct?caaagctcat?cgtggggacg?accccatcg 39

 

<210>18

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

 

<400>18

taagccatgg?ggtcgtccct?a 21

 

<210>19

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

 

<400>19

ggcgagctct?tacaattcat?catgtttggc?atatttgac 39

 

<210>20

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

 

<400>20

aatccgcggc?aacccctgta 20

 

<210>21

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

 

<400>21

taaccatggg?acgaccc 17

 

<210>22

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

 

<400>22

ttagggccca?tgttggggaa?ct 22

 

<210>23

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>23

ttagggcccg?gacctatgtt?ggggaact 28

 

<210>24

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>24

ttagggcccg?gacctggtcc?aatgttgggg?aact 34

 

<210>25

<211>993

<212>DNA

＜213〉artificial sequence

<220>

＜223〉LTB-L2-ORF5 merges the nucleotide sequence that wins peptide

 

<400>25

atgaataaag?taaaatgtta?tgttttattt?acggcgttac?tatcctctct?atatgcacac 60

ggagctcccc?agactattac?agaactatgt?tcggaatatc?gcaacacaca?aatatatacg 120

ataaatgaca?agatactatc?atatacggaa?tcgatggcag?gcaaaagaga?aatggttatc 180

attacattta?agagcggcga?aacatttcag?gtcgaagtcc?cgggcagtca?acatatagac 240

tcccagaaaa?aagccattga?aaggatgaag?gacacattaa?gaatcacata?tctgaccgag 300

accaaaattg?ataaattatg?tgtatggaat?aataaaaccc?ccaattcaat?tgcggcaatc 360

agtatgaaaa?acgggcccat?gttggggaac?tgcttgaccg?tgggctgttg?ctcgcggtcg 420

ctttttttgt?ggtgtattgt?gccgttctgt?ttagctgcgc?tcgtcagcgc?caacggaaac 480

agcagctctt?attcccagtt?gatttataac?ctgacgctat?gtgagctgaa?tggcacagat 540

tggctggcag?acagatttga?ttgggcagtg?gaaacttttg?tcatctttcc?cgtgattact 600

cacatcgttt?cctacggtgc?ccttactacc?agccacctcc?ttgatacagt?cggtctggtc 660

actgtatcca?ccgccgggtt?ctatcacggg?cggtatgtct?tgagcagcat?ctacgcggtc 720

tgtgccctgg?ctgcgttaat?ttgcttcgtc?attagactgg?cgaagaattg?catgtcctgg 780

cgctactcat?gtaccaggta?caccaacttt?cttctagaca?ctaagggcag?aatttatcgt 840

tggcgatcgc?ccgtcatcat?agagaaaggg?ggtaaagttg?aggtcgaagg?ccatttgatc 900

gacctcaaga?gagttgtgct?tgatggttcc?gcggcaaccc?ctgtaaccag?aattccagcg 960

gaacgatggg?gtcgtcccca?cgatgagctt?tga 993

 

<210>26

<211>999

<212>DNA

＜213〉artificial sequence

<220>

＜223〉LTB-L4-ORF5 merges the nucleotide sequence that wins peptide

 

<400>26

atgaataaag?taaaatgtta?tgttttattt?acggcgttac?tatcctctct?atatgcacac 60

ggagctcccc?agactattac?agaactatgt?tcggaatatc?gcaacacaca?aatatatacg 120

ataaatgaca?agatactatc?atatacggaa?tcgatggcag?gcaaaagaga?aatggttatc 180

attacattta?agagcggcga?aacatttcag?gtcgaagtcc?cgggcagtca?acatatagac 240

tcccagaaaa?aagccattga?aaggatgaag?gacacattaa?gaatcacata?tctgaccgag 300

accaaaattg?ataaattatg?tgtatggaat?aataaaaccc?ccaattcaat?tgcggcaatc 360

agtatgaaaa?acgggcccgg?acctatgttg?gggaactgct?tgaccgtggg?ctgttgctcg 420

cggtcgcttt?ttttgtggtg?tattgtgccg?ttctgtttag?ctgcgctcgt?cagcgccaac 480

ggaaacagca?gctcttattc?ccagttgatt?tataacctga?cgctatgtga?gctgaatggc 540

acagattggc?tggcagacag?atttgattgg?gcagtggaaa?cttttgtcat?ctttcccgtg 600

attactcaca?tcgtttccta?cggtgccctt?actaccagcc?acctccttga?tacagtcggt 660

ctggtcactg?tatccaccgc?cgggttctat?cacgggcggt?atgtcttgag?cagcatctac 720

gcggtctgtg?ccctggctgc?gttaatttgc?ttcgtcatta?gactggcgaa?gaattgcatg 780

tcctggcgct?actcatgtac?caggtacacc?aactttcttc?tagacactaa?gggcagaatt 840

tatcgttggc?gatcgcccgt?catcatagag?aaagggggta?aagttgaggt?cgaaggccat 900

ttgatcgacc?tcaagagagt?tgtgcttgat?ggttccgcgg?caacccctgt?aaccagaatt 960

ccagcggaac?gatggggtcg?tccccacgat?gagctttga 999

 

<210>27

<211>1005

<212>DNA

＜213〉artificial sequence

<220>

＜223〉LTB-L6-ORF5 merges the nucleotide sequence that wins peptide

 

<400>27

atgaataaag?taaaatgtta?tgttttattt?acggcgttac?tatcctctct?atatgcacac 60

ggagctcccc?agactattac?agaactatgt?tcggaatatc?gcaacacaca?aatatatacg 120

ataaatgaca?agatactatc?atatacggaa?tcgatggcag?gcaaaagaga?aatggttatc 180

attacattta?agagcggcga?aacatttcag?gtcgaagtcc?cgggcagtca?acatatagac 240

tcccagaaaa?aagccattga?aaggatgaag?gacacattaa?gaatcacata?tctgaccgag 300

accaaaattg?ataaattatg?tgtatggaat?aataaaaccc?ccaattcaat?tgcggcaatc 350

agtatgaaaa?acgggcccgg?acctggtcca?atgttgggga?actgcttgac?cgtgggctgt 420

tgctcgcggt?cgcttttttt?gtggtgtatt?gtgccgttct?gtttagctgc?gctcgtcagc 480

gccaacggaa?acagcagctc?ttattcccag?ttgatttata?acctgacgct?atgtgagctg 540

aatggcacag?attggctggc?agacagattt?gattgggcag?tggaaacttt?tgtcatcttt 600

cccgtgatta?ctcacatcgt?ttcctacggt?gcccttacta?ccagccacct?ccttgataca 660

gtcggtctgg?tcactgtatc?caccgccggg?ttctatcacg?ggcggtatgt?cttgagcagc 720

atctacgcgg?tctgtgccct?ggctgcgtta?atttgcttcg?tcattagact?ggcgaagaat 780

tgcatgtcct?ggcgctactc?atgtaccagg?tacaccaact?ttcttctaga?cactaagggc 840

agaatttatc?gttggcgatc?gcccgtcatc?atagagaaag?ggggtaaagt?tgaggtcgaa 900

ggccatttga?tcgacctcaa?gagagttgtg?cttgatggtt?ccgcggcaac?ccctgtaacc 960

agaattccag?cggaacgatg?gggtcgtccc?cacgatgagc?tttga 1005

 

<210>28

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>28

actgaattcg?tccgctgctt?tgctgt 26

 

<210>29

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>29

gcgcgtcgac?caaatttttg?ttagattgca?tg 32

Claims (13)

1. a genetic expression composition is characterized in that, comprises:

Promotor;

Coding pig reproduction and breathing syndrome virus ORF5 win the polynucleotide of peptide;

NOS terminates sub; And

The polynucleotide of the heavy ubiquitin gene 3 ' MAR of banana, it has the nucleotide sequence as SEQ ID No:2;

Wherein 5 ' end of the polynucleotide of this coding pig reproduction and breathing syndrome virus ORF5 victory peptide is connected in 3 ' end of this promotor, and this coding pig reproduction and breathing syndrome virus ORF5 win the 5 ' end that 3 ' end of the polynucleotide of peptide is connected in this NOS termination, and 3 ' end of this NOS termination is connected with 5 ' end of the polynucleotide of the heavy ubiquitin gene 3 ' MAR of banana again; This promotor drives the Transcription that the downstream coding wins the polynucleotide of peptide in the organism that contains this genetic expression composition.

2. genetic expression composition as claimed in claim 1 is characterized in that, described coding PRRSVORF5 wins the polynucleotide of peptide, and it has the nucleotide sequence as SEQ ID No:1.

3. genetic expression composition as claimed in claim 1, it is characterized in that, described promotor comprises CaMV35S, the heavy ubiquitin gene promotor of banana, and it has can be in the suitable promotor of this biology expression in vivo target gene as the nucleotide sequence of SEQ ID No:3 or other.

4. genetic expression composition as claimed in claim 1 is characterized in that, the pig reproduction of described coding and breathing syndrome virus ORF5 win the polynucleotide of peptide, and its 3 ' end can be further keeps 5 ' the holding and be connected of polynucleotide that message wins peptide with the coding endoplasmic reticulum; 3 ' the end that this coding endoplasmic reticulum keeps the polynucleotide of message victory peptide is connected with 5 ' end of NOS termination as claimed in claim 1.

5. genetic expression composition as claimed in claim 4 is characterized in that, described coding endoplasmic reticulum keeps the polynucleotide that message wins peptide, and its sequence is selected from SEQ ID No:4 or SEQ ID No:5.

6. genetic expression composition as claimed in claim 1, it is characterized in that, pig reproduction of described coding and breathing syndrome virus ORF5 win the polynucleotide of peptide, and its 3 ' end can further be connected with the 5 ' end that breathing syndrome virus ORF6 wins the polynucleotide of peptide with the reproduction of coding pig; This coding pig reproduction and breathing syndrome virus ORF6 win the polynucleotide of peptide, and its 3 ' end can be further keeps 5 ' the holding and be connected of polynucleotide that message wins peptide with the coding endoplasmic reticulum; 3 ' the end that this coding endoplasmic reticulum keeps the polynucleotide of message victory peptide is connected with 5 ' end of NOS termination as claimed in claim 1.

7. genetic expression composition as claimed in claim 1 is characterized in that, the 5 ' end that described promotor 3 ' end can be further with coding heat-labile toxin B sub-cell wins the polynucleotide of peptide is connected; Its 3 ' end is connected with the 5 ' end that coding L wins the polynucleotide of peptide; 3 ' the end that this coding L wins the polynucleotide of peptide is connected with the 5 ' end that breathing syndrome virus ORF5 wins the polynucleotide of peptide with coding pig as claimed in claim 1 reproduction; This coding pig reproduction is connected with the 5 ' end that coding endoplasmic reticulum reservation message wins the polynucleotide of peptide with the 3 ' end that breathing syndrome virus ORF5 wins the polynucleotide of peptide; 3 ' the end that this coding endoplasmic reticulum keeps the polynucleotide of message victory peptide is connected with 5 ' end of NOS termination as claimed in claim 1;

Wherein the polynucleotide of this coding heat-labile toxin B sub-cell victory peptide has the nucleotide sequence as SEQ ID No:6; This coding L wins the polynucleotide of peptide, and its polynucleotide sequence is selected among SEQ ID No:7, SEQID No:8 and the SEQ ID No:9 at least a.

8. a recombination fusion protein is characterized in that, comprises: heat-labile toxin B sub-cell wins peptide, L wins peptide, pig reproduction and breathing syndrome virus ORF5 victory peptide, pig reproduction and breathing syndrome virus ORF6 victory peptide and endoplasmic reticulum reservation message wins peptide;

Wherein this heat-labile toxin B sub-cell wins the N end that peptide is positioned at recombination fusion protein; The N end that this L wins peptide is connected with the C end that LTB wins peptide; The C end that this L wins peptide is connected with the N end that breathing syndrome virus ORF5 wins peptide with this pig reproduction; This pig reproduction is connected with the N end that this endoplasmic reticulum keeps message victory peptide with the C end that breathing syndrome virus ORF5 wins peptide; This endoplasmic reticulum keeps message and wins the C end that peptide is positioned at recombination fusion protein;

Wherein this heat-labile toxin B sub-cell victory peptide has the aminoacid sequence as SEQ ID No:10; This L wins peptide, and its aminoacid sequence is selected among SEQ ID No:11, SEQ ID No:12 and the SEQ ID No:13 at least a; This endoplasmic reticulum reservation message victory peptide has the aminoacid sequence as SEQ ID No:14.

9. an expression vector comprises genetic expression composition as claimed in claim 1.

10. pig reproduction and respiratory complication oral vaccine with a plant production is characterized in that, comprise genetic expression composition as claimed in claim 1.

11. pig reproduction and respiratory complication oral vaccine with a plant production is characterized in that, get with the following step preparation:

Step 1: constructing the genetic expression that contains genetic expression composition as claimed in claim 1 changes and to grow carrier and change and grow plastid to obtain reorganization;

Step 2: the reorganization of step 1 changeed grow plastid and change and grow, change to obtain containing this reorganization that the plant that grows plastid changes cell colonization or tissue is grown in the plant commentaries on classics into vegetable cell or tissue;

Step 3: the plant of culturing step 2 gained changes cell colonization or tissue is grown in the plant commentaries on classics, part organ, tissue or the cell that plant is grown in plant or commentaries on classics grown in the commentaries on classics that contains genetic expression composition as claimed in claim 1 with generation, and wherein this commentaries on classics is grown part organ, tissue or the cell that plant or commentaries on classics grow plant and is direct for the only oral vaccine of pig.

12. method of preventing pig infected pigs's reproduction and respiratory complication, it is characterized in that, this method comprises: the commentaries on classics that will treat containing of significant quantity genetic expression composition as claimed in claim 1 is only grown part organ, tissue or cell feeding pig that plant or commentaries on classics grow plant, so that it produces antibody to pig reproduction and breathing syndrome virus, with reproduction of preventing infection pig and respiratory complication.

13. a separation is characterized in that from the polynucleotide sequence of the heavy ubiquitin gene 3 ' MAR of banana it has the sequence as SEQ ID No:2.

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