CN101104641A - Fusion protein of PRRS virus used as swine procreation and respiratory tract syndrome vaccine - Google Patents

Abstract

The invention provides a sub-unit PRRS vaccine PE-PQGAB-K3 which is composed of counteragent value fusion proteins. The invention is a PQGAB structure peptide which is formed by a pseudomonas exotoxin site I (binding site-translocation site (II)) frame-shifting with PRRSV ORF5 and an ORF6N terminal combining with a cell receptor, and a code of KDEL-RDEL-KDEL(KEDL3) is added at a carboxyl terminal of fusion proteins. Serum obtained after mouse or pigs are immunized by the invention PE-PQGAB-K3 can inhibit the PRRSV growing and propagating in MAC-19cells, wherein the counteragent value can reach to 16 to 32.

Description

Fusion rotein as the PRRS virus of pig reproduction and respiratory tract syndrome vaccine

Technical field

The invention relates to possess and bring out the fusion rotein time unit vaccine that pig only produces PRRSV counteragent covalent reaction.

Background technology

Pig reproduction and respiratory tract syndrome (porcine reproductive and respiratory syndrome, PRRS) be a kind of by pig reproduction and respiratory tract syndrome virus (PRRSV) infection, it causes the sow breeding difficulty for a kind of meeting, and the respiratory tract infection of all ages pig is master's a pig transmissible disease.PRRSV is regarded as extremely indomitable virus, and it is based on be difficult for bringing out the antibody with counteragent valency in pin main body.And PRRSV is a RNA viruses, can breed via the gene replication system of simplifying in host cell, and also therefore the transgenation probability is very high.In addition, PRRSV infects and can be divided into for two stages: (A) infect respiratory tract and lower respiratory tract epithelial cell system; And (B) infect the monocyte of respiratory tract peripheral organization and hugely have a liking for cell.Therefore, the host need possess body fluid or the mucosa-immune reaction mechanism and the cellular immune mechanism of counteragent valency simultaneously, is beneficial to remove infected cells to be on the defensive.Yet the valency of generation counteragent in animal body that occurring in nature PRRSV can be real promptly is not easy, and therefore general antibody is little to defence PRRSV effect, even can stimulate the sudden change of virus.Therefore, in antibody-dependent enhancement mechanism, the use of non-counteragent valency antibody may cause more serious infection.

In TaiWan, China I-2289933 number (U.S. Patent Publication No. US 20040247617), provide a kind of to the narrow spectrum fusion rotein of target cell tool, this fusion rotein utilizes a Pseudomonas aeruginosa extracellular toxin position and a functional zone framework PRRSV ORF7 nucleoprotein fragment, and adds KDEL news sign indicating number in carboxyl terminal.This fusion rotein can be produced in intestinal bacteria in a large number, immune swine only after, can effectively reduce and remove the generation that pig is only attacked the viremia behind the poison.The record that quotes in full of this patent is incorporated among the application.

In the ORF5 and the assorted two polymerizations reactions of ORF6 (heterodimerization) of PRRSV, the Cys-34 epitope zone of ORF5, and the Cys-8 epitope zone of ORF6, to virus infection and the key effect of mantle assembling (envelope assembly) tool (Snijder Eric J. thereof, Jessica C.et al., Journal ofVirology, January 2003, Vol.77, No.1:97-104).In addition, the reservation queue of PRRSV ORF5 (consensus sequence)-YKNTHLDLIYNA, be the epitope between Amino acid 38 to 44, its N-that is positioned at PRRSV ORF5 holds external district, differentiates to be in one and epitope (Ostrowski M. already, J.A.Galeota, et al., Journal of Virology, May 2002, Vol.76, No.9:424 1-4250).

Known technology also once with ORF5 or the whole albumen framework of ORF6 in the middle of PE and KDEL, immunity only finds the back that to pig the pig of attacking poison only in lung more serious inflammation phenomenon takes place on the contrary, obvious ORF5 or ORF6 have more immunotoxicity than itself.Therefore, desire effectively to prevent and treat pig and only infect PRRS, still be necessary to develop vaccine with high counteragent valency and low immunotoxicity.

Summary of the invention

One of the object of the invention provides a kind of fusion rotein, and array mode.

Another object of the present invention is about utilizing these fusion roteins to prepare the inferior PRRS of the unit vaccine that tool counteragent valency fusion rotein is formed.

A further object of the present invention provides medical composition, and it comprises that fusion rotein of the present invention reaches at pharmaceutically acceptable supporting agent.

For achieving the above object, PE-PQGAB-K3 fusion rotein provided by the invention, it comprises:

One contains the ORF6 partial sequence of pig reproduction and respiratory tract syndrome virus (PRRSV), with the N end protein district of PRRSV ORF5 partial sequence;

One Pseudomonas aeruginosa exotoxin A shift function position; And

One contains the carboxyl terminal portion of KDEL-KDEL-KDEL (K3) sequence.

Described fusion rotein, wherein this PRRSV is the America strain.

Described fusion rotein, wherein this PRRSV is European strain.

Described fusion rotein, wherein this ORF6 partial sequence of this PRRSV is SEQ ID NO.13.

Described fusion rotein, wherein this PRRSV ORF5 partial sequence is SEQ ID NO.12.

Described fusion rotein, wherein the nucleotide sequence in the N of the part of O RF6 of this pig reproduction and respiratory tract syndrome virus (PRRSV) and part of O RF5 end fusion rotein district is SEQ ID NO.1.

Described fusion rotein, wherein this part of O RF6 of this PRRSV is SEQ ID NO.15.

Described fusion rotein, wherein this part of O RF5 of this PRRSV is SEQ ID NO.14.

Described fusion rotein, wherein the nucleotide sequence in the N of the part of O RF6 of this pig reproduction and respiratory tract syndrome virus (PRRSV) and part of O RF5 end fusion rotein district is SEQ ID NO.10.

The present invention can be used as the medical composition of vaccine, and it comprises:

The part of O RF6 of one pig reproduction and respiratory tract syndrome virus (PRRSV) and the N of part of O RF5 end fusion rotein district;

One Pseudomonas aeruginosa exotoxin A shift function position;

One contains the carboxyl terminal portion of KDEL-KDEL-KDEL (KDEL3) sequence; And

One pharmaceutically acceptable carrier.

Described medical composition, wherein this PRRSV is the America strain.

Described medical composition, wherein this PRRSV is European strain.

Described medical composition, wherein this part of O RF6 of this PRRSV is SEQ ID NO.13.

Described medical composition, wherein this part of O RF5 of this PRRSV is SEQ ID NO.12.

Described medical composition, wherein the nucleotide sequence in the N of the part of O RF6 of this pig reproduction and respiratory tract syndrome virus (PRRSV) and part of O RF5 end fusion rotein district is SEQ ID NO.1.

Described medical composition, wherein this part of O RF6 of this PRRSV is SEQ ID NO.15.

Described medical composition, wherein this part of O RF5 of this PRRSV is SEQ ID NO.14.

Described medical composition, wherein the nucleotide sequence in the N of the part of O RF6 of this pig reproduction and respiratory tract syndrome virus (PRRSV) and part of O RF5 end fusion rotein district is SEQ ID NO.10.

Described medical composition, wherein this carrier comprises a freund adjuvant, emulsifying agent mannide list oleate (mannide mono-oleate emulsifier, ISA720, ISA206) adjuvant.

Described medical composition, wherein this carrier comprises an ISA206 adjuvant.

Say further that again PE-PQGAB-K3 fusion rotein of the present invention comprises: one contains the ORF6 partial sequence of pig reproduction and respiratory tract syndrome virus (PRRSV), with the N end protein district of PRRSV ORF5 partial sequence; One Pseudomonas aeruginosa exotoxin A shift function position; An and carboxyl terminal portion that contains KDEL-KDEL-KDEL (K3) sequence.

The present invention also comprises a kind of medical composition that can be used as vaccine, and it comprises: the part of O RF6 of a pig reproduction and respiratory tract syndrome virus (PRRSV) and the N of part of O RF5 end fusion rotein district; One Pseudomonas aeruginosa exotoxin A shift function position; One contains the carboxyl terminal portion of KDEL-KDEL-KDEL (KDEL3) sequence; An and pharmaceutically acceptable carrier.

The PRRSV virus strain kind of indication is not limit among the present invention, can be american type virus strain, Europe class virus strain or Australia C-type virus C strain, and in preferred embodiment of the present invention, the PRRSV virus strain is american type virus strain or Europe class virus strain.

Among the present invention, the fragment that the N end protein district of fusion rotein can comprise does not limit, and is preferably arbitrary PRRSV fragment that possesses antigenic characteristic, as PRRSV ORF5, ORF6 or ORF7.With the american type virus strain is example, and the ORF6 partial sequence of preferable PRRSV can be SEQ ID NO.13.PRRSV ORF5 partial sequence then can be SEQ ID NO.12.With the Europe class virus strain, the part of O RF6 of preferable PRRSV can be SEQ ID NO.15, the preferable SEQ ID NO.14 that then can be of the part of O RF5 of PRRSV.

Among the present invention, the nucleotide sequence that fusion rotein contains part PRRSV ORF6 and part PRRSV ORF5 is through upgrading, and its sequence does not limit, be preferably to show in intestinal bacteria system camber, and the protein structure that the shows nucleotide sequence identical with wild-type.With the american type virus strain is example, and nucleotide sequence is SEQ ID NO.1 behind the better upgrading.And be example with the Europe class virus strain, nucleotide sequence is SEQ ID NO.10 behind the better upgrading.

Medical composition of the present invention can comprise the adjuvant that use is fit to according to known technology; Dispersion agent or wetting Agent for Printing Inks (as Tween 80) and suspension agent allotment aseptic injection, for example, the aseptic injection aqueous solution or oiliness suspension.Aseptic injection preparation also can be used for thinner or the aseptic parenteral solution in the solvent or the suspension of nontoxicity injection, for example, and the solution in 1,3 butylene glycol.In spendable acceptable carrier and solvent, have N.F,USP MANNITOL, water, Ringer solution, with etc. open sodium chloride solution.In addition, commonly used going up uses sterilization, fixed oil as solvent or suspension medium (for example acid of synthetic list or Diglyceride class).Lipid acid (for example oleic acid and its glyceride derivative), and acceptable oils (for example sweet oil or Viscotrol C, the especially ethylating kenel of its polyoxy) on the natural medicaments can be used in injectable formulation.These oil solutions or suspension also can contain long-chain alcohol thinner or dispersion agent or carboxymethyl cellulose or similar dispersion agent.Other general interfacial agent that uses for example Tweens or Spans or other similar emulsifying agent or bioavailability toughener (generally being used to make pharmaceutically acceptable solid, liquid or other dosage form) also can be used for the purpose of allocating.

The composition that oral administration medicine supplying is used can be any oral acceptable dosage form of, including but not limited to capsule, ingot, emulsion, and waterborne suspension, dispersion agent and solution.In the lozenge example of oral use, the general carrier that uses comprises newborn candy and W-Gum.Generally also often add lubricant, for example, Magnesium Stearate.For for the capsule form oral administration medicine supplying, spendable thinner comprises lactose and W-Gum.When oral dispensing aqueous dispersant or emulsion, can make active ingredient and emulsifying agent or suspension agent combined suspension or be scattered in the oil phase.If need, can add specific sweeting agent, flavour agent or tinting material.Nasal cavity spray or inhalation composition can be according to known technology preparations in the technology of medicine prescription, and can be made into normal saline solution, use phenylcarbinol or other sanitas that is fit to, increase known dissolving agent or dispersion agent in absorption enhancer, fluorocarbon and/or other technology of bioavailability.Contain the suppository form dispensing that the composition of benzazolyl compounds can also rectal administration.

Carrier in the medical composition is necessary for " can accept ", mean with fill a prescription in active ingredient compatible (and be preferably can make prescription stable) and to dock subject sufferer harmless.The example of other carrier comprises colloidal silica, Magnesium Stearate, Mierocrystalline cellulose, sodium lauryl sulphate, reaches D﹠amp; Yellow No. 10 of C.

And contain in the medical composition of fusion rotein of the present invention, preferably also comprise an immunological adjuvant; The immunological adjuvant that is suitable for is not limit, it can be any adjuvant that is usually used in vaccine, comprise that aluminium glue and oily adjuvant are as: freund adjuvant (Freund ' s FCA or FIA) or emulsifying agent mannide list oleate (mannide mono-oleate emulsifier, ISA720 or ISA206, SEPPIC ^, France), be preferably the ISA206 adjuvant.

Detailed description of the present invention is set forth in the following explanation.In explanation of the present invention and claim with apparent further feature of the present invention, purpose and advantage.

Description of drawings

Fig. 1 represents the sketch according to the PE-PQGAB-K3 fusion rotein of example 1.

Fig. 2 represents to construct schema according to the PE of example 1 (Δ III)-PQGAB plastid.

Fig. 3 represents according to 1 synthetic nucleic acid fragment of example electrophorogram, totally 4 segment DNA fragments (a section 70bp, b section 129bp, c section 186bp, d section 204bp).

Fig. 4 represents PE (Δ III)-PQGAB plastid collection of illustrative plates.

Fig. 5 is for utilizing the PE-PQGAB-K3 antigen protein fragment quantitative results figure in extraction of 8M Wyler's process and the stripping inclusion body (inclusion bodies).

Embodiment

The present invention is characterised in that, ORF5 and the most antigen of ORF6 are removed in discovery, only stay tens Amino acids of N terminal number of ORF5 and ORF6, win peptide chain PQGAB with heterozygosis of this framework, in the middle of the embedding PE and KDEL3 sequence that then makes, this fusion rotein PE-PQGAB-KDEL3 confirms to have serum counteragent valency ability through mouse and pig immunity test only.

For explaining the object of the invention, following example is provided, only, it is not in order to limit the scope of the invention.

Embodiment 1: the PQGAB of America strain PRRSV merges the victory peptide

The ORF5 of America strain PRRSV and ORF6 marismortui structural protein sequence are the databases that derives from the U.S. state-run biotechnology data center (NCBI).And change known according to above-mentioned virus infection machine, the zone that PRRSV virus has a counteragent valency is respectively the N end of ORF6 and ORF5.Be the 2nd to No. 26 Amino acid of ORF6 structural protein and win peptide district (SEQ ID NO.13), and the 30th to No. 63 Amino acid of ORF5 structural protein wins peptide district (SEQ ID NO.12).List merging these the two sections Amino acid sequences that win the peptide district, as follows:

GSSLDDFCYDSTAPQKVLLAFSITYASNDSSSHLQLIYNLTLCELNGTDWLANKFDWA

That is, the victory peptide is merged in PRRSV-ORF6-2～26-ORF5-31～63, and it is by (ORF6)-G ₂SSLDDFCYDSTAPQKVLLAFSITY ₂₆(SEQ ID NO.13) reaches (ORF5)-A ₃₁SNDSSSHLQLIYNLTLCELNGTDWLANKFD WA ₆₃(SEQ IDNO.12) two victory peptide fragment are formed, wherein, and the A that the Q that the P that the GSSLDDFC fragment claims, YDSTAPQKVLLAFSITY fragment claim, ASNDSSSHLQLIYNLTLC fragment claim, and the B that claims of ELNGTDWLANKFDWA fragment.The PQ fragment belongs to a part of ORF6 fragment, and the AB fragment belongs to a part of ORF5 fragment.

This example utilizes PQGAB to win the peptide corresponding circle of sensation, constructs one and has the key protein matter (being epitope) of bringing out counteragent valency immune protective efficiency, brings out the live body immune effect to reach.PE-PQGAB-K3 fusion rotein sketch and the PE schema that (Δ III)-the PQGAB plastid is constructed is respectively shown in Fig. 1 and 2.

Embodiment 2

The below establishment of explanation PQGAB nucleotide sequence.Because an Amino acid can correspondingly be organized different nucleotide sequences more, therefore can be earlier by academic data (as http://www.kazusa.or.jp/codon), find relatively to be adapted at the corresponding sequence of the nucleic acid that shows in intestinal bacteria (E.coli) the host system, and avoid the corresponding sequence of nucleic acid that intestinal bacteria are not easy identification, performance.In like manner, if this sequence will show, then also to select to be suitable for the sequence that yeast system (Saccharomyces or Pichia spp.) is expressed in the yeast system.

According to the Amino acid sequence in PQGAB fusion rotein district, find out and be suitable for the corresponding nucleotide sequence that shows in the intestinal bacteria host system, and in 5 of nucleotide sequence ' and 3 '-end connects the Restriction enzyme sequence respectively, is beneficial to gene fragment montage step backward.For cutting efficiency that increases ferment and the design that is beneficial to the PCR introduction, these gene two ends can add that some repeat the nucleic acid of base, as CCC, AAA, GGG or TTT.The visible SEQID NO.1 of PQGAB fusion rotein district nucleotide sequence in this example behind the upgrading.

207 nucleic acid altogether among the SEQ ID NO.1, when utilizing ferment to carry out montage with plastid, it is cut that the nucleic acid of above-mentioned continuous trigram has part, enters in the plastid and have 186 to 180 nucleic acid at least.

After the target nucleotide sequence is determined, before beginning to synthesize, need utilize computer software (as DNAstrider) to analyze the collection of illustrative plates that has the Restriction enzyme point of contact on the target nucleotide sequence, and according to the collection of illustrative plates result, the sequence of Restriction enzyme is installed in target nucleotide sequence two ends, uses for carry out montage backward.And because the subject matter that generates must carry out the montage of Restriction enzyme, therefore be preferably in when avoiding in the target nucleotide sequence occurring with montage identical Restriction enzyme and cut the position.For this reason, must utilize software commonly used again, whether have these to cut position (restriction map) in the analyzing DNA sequence.If identical Restriction enzyme is cut bit sequence when having with montage in the target nucleotide sequence, just must change nucleotide sequence again, look for the different IPs acid sequence of identical Amino acid correspondence, make the Restriction enzyme in the target nucleic acid sequence fragments cut the position disappearance.

Then utilize the disclosed method of aforementioned TaiWan, China I-2289933 number (U.S. Patent number US20040247617) patent, carry out the upgrading of corresponding nucleotide sequence according to the wild-type Amino acid, make above-mentioned wild-type Amino acid to show in a large number by the intestinal bacteria system.The emphasis of upgrading is mainly at the nucleic acid fragment with the wild virus strain, not influencing the Amino acid that it shows originally, and under the situation that can effectively show, carries out the upgrading of nucleotide sequence in the escherichia coli host system.Nucleotide sequence behind this upgrading can utilize manyly to introduction respectively, synthesizes with polymerase chain reaction, and the right reference numeral of all introductions is asked for an interview table 1.

Table 1: the right reference numeral of America strain PRRSV target antigen PQGAB introduction

Target antigen	The forward introduction	Sequence numbering (Seq.ID No.)	Reverse introduction	Sequence numbering (Seq.ID No.)
					PQGAB	F1		2	R1	6
PQGAB	F2		3	R2							7
					PQGAB	F3		4	R3	8
PQGAB	F4	5

The sequence of forward introduction and reverse introduction is as follows respectively:

Forward introduction F1 (SEQ ID No.2) is the 81st to No. 124 Amino acid sequence of SEQ ID No.1, that is,

5′-GCT TTC TCC ATC ACC TAC GCT TCC AAC GAC TCC TCCTCC CAC CT-3′；

Forward introduction F2 (SEQ ID No.3) is the 48th to No. 96 Amino acid sequence of SEQ ID No.1, that is,

5′-C GAC TCC ACC GCT CCC CAG AAA GTT CTG CTG GCT TTCTCC ATC ACC TA-3′；

Forward introduction F3 (SEQ ID No.4) is the 22nd to No. 65 Amino acid sequence of SEQ ID No.1, that is,

5′-GGT TCC TCC CTG GAC GAC TTC TGC TAC GAC TCC ACCGCT CCC CA-3′；

Forward introduction F4 (SEQ ID No.5) is the 1st to No. 41 Amino acid sequence of SEQ ID No.1, that is,

5′-CCC AAA CCC CAT ATG GAA TTC GGT TCC TCC CTG GACGAC T-3′；

Oppositely introduction R1 (SEQ ID No.6) is the 148th to the 1 No. 06 Amino acid sequence of SEQ ID No.1, that is,

5′-A CAG GGT CAG GTT GTA GAT CAG TTG CAG GTG GGA GGAGGA GTC-3′；

Oppositely introduction R2 (SEQ ID No.7) is the 176th to No. 133 Amino acid sequence of SEQ ID No.1, that is,

5′-GC CAG CCA GTC GGT ACC GTT CAG TTC GCA CAG GGTCAG GTT GTA-3′；

Oppositely introduction R3 (SEQ ID No.8) is the 204th to the 1 No. 64 Amino acid sequence of SEQ ID No.1, that is,

5′-TTT TTT CTC GAG AGC CCA GTC GAA TTT GTT AGC CAGCCA GTC GG-3′；

Wherein R1, R2 and R3 are the reverse complementary gene sequence of table 1 gene preface sign indicating number.

At first utilize the polymerization methods of no dna profiling, utilize forward introduction F1 and reverse introduction R1 to carry out the polymerization of nucleic acid fragment, wherein 10-18 base respectively arranged is to be designed to combination complimentary to one another to 3 ' end parts of each introduction, with via the read-write of polymerizing enzyme and supply and become a bifilar dna profiling polymerisate.

After finishing the PCR first time, get the template DNA of the polymerisate of 0.01～4 μ l as the PCR second time, add second group of introduction simultaneously to forward introduction F2 and each 0.01～4 μ l of reverse introduction R2,, begin to carry out the PCR second time together with required dNTPs, reaction reagent and Pfu polysaccharase etc.; Then according to same way as, gradation adds after different introductions carry out PCR to F3 and R3, carries out the PCR step of introduction to F4 and R3 again, can synthesize the 207bp nucleotide sequence behind the PQGAB upgrading.

The nucleic acid fragment that is synthesized respectively can confirm that the clip size of its product conforms to expection after the electrophoresis test.PQGAB-1 section (207bp) is as Fig. 3; PQGAB-produces a, b, c and d totally 4 segment DNA fragments (a section 70bp, b section 129bp, c section 186bp, d section 204bp).

The PQGAB of embodiment 3, Europe class virus strain PRRSV

Embodiment 1～2 is the fusion rotein design of PRRSV american type, but PRRSV at the infection prevalence rate in the whole world except PRRSV american type virus strain, prevailing of Europe class virus strain, Australia C-type virus C strain is also quite general, because these virus strain are not high in the similarity of structural protein Amino acid, only 60～80%, therefore design for the ORF5 of other type and the fusion rotein of ORF6, also can be respectively according to similar fashion as embodiment 1～2, the design of carrying out sequence is with synthetic.

The Amino acid sequence in the PQGAB fusion rotein district of Europe class virus strain PRRSV is shown in SEQ IDNO.11.Wherein comprise ORF6-M1～I28 (SEQ ID NO.1 5) fragment, and the fragment sequence of ORF5-F31～A64 (SEQ ID NO.14).With SEQ ID NO.11 serves as according to the designed synthetic introduction of using, and is respectively four groups, as shown in table 2 below.

Table 2: the right reference numeral of European strain PRRSV target antigen PQGAB introduction

Target antigen	The forward introduction	Sequence numbering (Seq.ID No.)	Reverse introduction	Sequence numbering (Seq.ID No.)
					PQGAB-EP	F1	16	R1	20
PQGAB-EP	F2	17	R2	21
					PQGAB-EP	F3	18	R3	22
PQGAB-EP	F4	19	R4	23

Through as embodiment 2 described modes,, can externally synthesize the European strain PQGAB nucleotide sequence behind the upgrading by introduction sequence as table 2.For cutting efficiency that increases ferment and the design that is beneficial to the PCR introduction,, can add that equally some tools repeat the nucleic acid of base, as CCC, AAA, GGG or TTT in the two ends of European strain PQGAB nucleotide sequence.European strain PQGAB nucleotide sequence in this example behind the upgrading is SEQ ID NO.10.

Embodiment 4: the plastid that contains the target sequence is constructed

The upgrading nucleic acid fragment of preparing with embodiment 2 is an example, with the above-mentioned 207bp dna fragmentation that synthesizes, respectively with after Restriction enzyme EcoRl, the Xhol cutting, insert had one in conjunction with the victory peptide sequence of shift function, and in the intestinal bacteria plastid of a carboxyl terminal victory peptide, the gained plastid is the pPE-PQGAB-K3 plastid.

Construct in containing T7 promotor and the segmental pET15 plastid of tool microbiotic (ampicilin) resistance system, can show the fusion rotein that contains PRRSV PQGAB and remove toxic Pseudomonas aeruginosa exotoxin A (Pseudomonasexotoxin A without domain III), plastid figure pedigree as shown in Figure 4.

At last respectively with above-mentioned plastid transfection in bacterial strain that can show this fused protein or cell.

Embodiment 5, the proteinic performance of target and analysis

Through determining to contain the bacterial classification of above plastid, 90% above bacterium all contains plastid and PQGAB gene, is stored in the 2ml sub-bottling under-70 ℃ of temperature with the glycerine preservation method individually.In sterilisable chamber, above preservation vaccine is got the 500ml triangular flask that 2ml is inoculated in sterilization, it contains 200ml LB (+500 μ g/ml Amp), cultivates 10～12 hours down with the 150rpm rotating speed in 37 ℃ of rotary type incubators, finishes kind of bacterium and cultivates.The OD600 of this vaccine should reach 1.0 ± 0.4.

In sterilisable chamber, the 50ml strain liquid distinctly is inoculated in totally eight bottles of the 3000ml triangular flasks of sterilization, this triangular flask contains 1250ml LB (+500 μ g/ml Amp+50ml 10%Glucose), cultivated 2～3 hours down with the 150rpm rotating speed in 37 ℃ of rotary type incubators, detect OD600 at 0.3 ± 0.1 o'clock, after the injection ultimate density is the IPTG protein induce agent of 50 ppm, cultivated 2 hours down with the 150rpm rotating speed, promptly finish the protein production step in 37 ℃ of rotary type incubators.

Then, utilize the PE-PQGAB-K3 antigen protein fragment in extraction of 8M Wyler's process and the stripping inclusion body (inclusion bodies), (short run, lot) extraction can obtain 300～400mg antigen amount with ten liters of bacterium liquid for each.Utilize west ink dot method (Western-blotting method), coomasie blue staining, SDS-PAGE electrophoretic analysis, and measure the density of electrophoretic band with densometer (densitometer), with the quantification of protein (result such as Fig. 5) that carries out each antigenic solution.Above-mentioned antigen protein is got 0.2 ± 0.02 mg, as the main composition of injection high dosage.Get the main composition of 0.02 ± 0.002 mg, carry out a pig immunity and attack malicious effect test as the injection low dosage.

Only immunity and attack the effect test of poison of embodiment 6, pig

In the SPF farm, pig only is divided into three groups at random, every group of five pigs, and every group of pig only raised respectively in the insulated chamber that is equiped with air-conditioning and air regenerating device.The pig of PE-PQGAB-E3 vaccination group is injected respectively by intramuscular and to contain 1ml PE-PQGAB-E3 (containing 200 μ g protein/dosage), at 1ml ISA206 (SEPPIC only when 14 and 28 ages in days ^France) emulsive 2ml vaccine in the adjuvant, twice of immunity.GP5﹠amp; The M immune group then with PE-ORF5-K3 and PE-ORF6-K3 (respectively containing 200 μ g protein/dosage) immunity.Control group is then brought up under no immunization.

Two weeks after last vaccine inoculation, pig is only given 100mg ketamine (ketamine) solution to reach sedative effect in the intramuscular throwing, 2% lignocaine (Lidocaine) of 1ml of then dripping in nose suppresses after the cough-reflex action, with nose inside fire attack poison pig.Use the PRRSV culture of the MD-1 strain of fresh 1ml, with about 1 * 10 ⁷TCID ₅₀The dosage of/ml is attacked five pigs in malicious every group.

Inoculation back is in the time of the 14th day, and the pig of all sacrifices is only carried out complete dissection.Collect liver sample (partly reaching each centre and appendage), and utilize 10% neutral buffered formalin to be fixed, to carry out the follow-up pathological examination of organizing from caudate lobe of liver from two of epicranial lobe.This check system is to adopt blind pattern (blind fashion) and appraised through comparison (Opriessnig T according to the interstitial pneumonia severity, P.G.Halbur, et al., Journal of Virology, 76 (2002): 11837-11844 and Halbur, P.G., P.S.Paul, et al., 1996.J.Vet.Diagn.Investig.8:11-20), distinguish with 0 to 6, the bigger expression lesion degree of numeral is more serious.

Experimental result

For the second time back two weeks of immunity, a pig blood white cell sample detects PRRSV with RT-PCR, and the result is presented at PRRSV and attacks before the poison, and all pigs only all do not cause viremia.After 3,7 and 14 days, utilize RT-PCR to detect pig blood white cell sample only once more after attacking poison, the result is as shown in table 3.

After table 3:PRRSV attacks poison, a pig PRRSV viremia ratio

Fate	Control group	PE-PQGAB-K3	PE-ORF5-K3 PE-ORF6-K3
				3	3/5	3/5	2/5
7	3/4 (dead 1 *)	2/5	2/4 (dead 2 *)
				14	3/4 (dead 1 *)	2/5	2/4 (dead 2 *)

^*The dead pig of expression only before detects the PRRSV viremia by RT-PCR

All pigs only (are comprised that the pig of having sacrificed only only reaches the pig that also survived when research finishes in two weeks) dissect that macroscopic test shows, attacks a poison back pig lungs and shows control group and ORF5﹠amp; The ORF6 vaccine group observe more widely pathology and serious between matter lung film inflammation, PE-PQGAB-K3 vaccine group of the present invention then do not find these widely pathology and between matter lung film inflammation.

Table 4: attack poison comparison for the macroscopic lung lesion that PRRSV-was brought out in back 14 days with PRRSV

Pig is only numbered	Control group pathology index	PE-PQGAB-K3 vaccine group pathology index	PE-ORF5-K3, PE-ORF6-K3 vaccine group pathology index
				1	6 *	5	6
2	6	3	5
				3	6	4	6
4	6	4	6
				5	5	3	6
Average index	5.75 +0.50	3.80 ±0.84	5.80 ±0.45

^*: interstitial pneumonia pathology index.

As shown in table 4, the macroscopic lung lesion severity that PE-PQGAB-K3 vaccine group of the present invention is showed is significantly than control group and ORF5﹠amp; The ORF6 vaccine group is little.

The macroscopic lung lesion index that table 5, PE-PQGAB-K3 vaccine group are showed is with biometrics tool utmost point significant difference level, than control group and ORF5﹠amp; ORF6 vaccine group low (p＜0.01)

Group	Number	Summation	On average	Variance
					Control group PE-PQGAB-K3 group PE-ORF5﹠ORF6-K3 group	5 5 5	29 19 29	5.8 3.8 5.8	0.2 0.7 0.2

ANOVA
							The change source	SS	Degree of freedom	MS	F	The P-value	Threshold value
Summation in the group between group	13.33333 4.4 17.73333	2 12 14	6.666667 0.366667	18.18182	0.000233	3.885294

Compare group and ORF5﹠amp; ORF6 vaccine group low (p＜0.01)

Show according to above experimental result is clear; PE-PQGAB-K3 of the present invention not only can effectively protect pig only to avoid the PRRSV infection really; and according to vaccine of the present invention, (PE-ORF5-K3 for example, PE-ORF6-K3) interstitial pneumonia that is showed is more slight with respect to other vaccine for it.

Table 6, serum power valency summary table

Group	coating antigen		IFA titers	NT titers
					PE(ΔIII) IgG-ELISA	PQGAB titers(S/BK)
	A PE-ABCF-K3 PE-PQGF-K3	12					80	8-16	8-16
B Negative CTL			1	1	＜8	＜8
	C PE-PQG1AB-K3	17					30	32-64	16-64

Attacking a poison back pig antibody power valency changes as shown in Figure 6.

Sequence table

＜110〉Healthbanks Biotech Co., Ltd.

＜120〉as the fusion rotein of the PRRS virus of pig reproduction and respiratory tract syndrome vaccine

<130>S0160/0613

<160>23

<170>PatentIn version 3.2

<210>1

<211>207

<212>DNA

＜213〉pig reproduction and respiratory tract syndrome virus

<400>1

cccaaacccc atatggaatt cggttcctcc ctggacgact tctgctacga ctccaccgct 60

ccccagaaag ttctgctggc tttctccatc acctacgctt ccaacgactc ctcctcccac 120

ctgcaactga tctacaacct gaccctgtgc gaactgaacg gtaccgactg gctggctaac 180

aaattcgact gggctctcga gaaaaaa 207

<210>2

<211>44

<212>DNA

＜213〉synthetic

<220>

＜223〉forward introduction PQGAB-US.

<400>2

Gctttctcca tcacctacgc ttccaacgac tcctcctccc acct 44

<210>3

<211>48

<212>DNA

＜213〉synthetic

<220>

＜223〉the forward introduction of PQGAB-US.

<400>3

cgactccacc gctccccaga aagttctgct ggctttctcc atcaccta 48

<210>4

<211>44

<212>DNA

＜213〉synthetic

<220>

＜223〉the forward introduction of PQGAB-US.

<400>4

ggttcctccc tggacgactt ctgctacgac tccacegctc ccca 44

<210>5

<211>44

<212>DNA

＜213〉synthetic

<220>

＜223〉the forward introduction of PQGAB-US

<400>5

ggttcctccc tggacgactt ctgctacgac tccaccgctc ccca 44

<210>6

<211>43

<212>DNA

＜213〉synthetic

<220>

＜223〉the reverse introduction of PQGAB-US.

<400>6

acagggtcag gttgtagatc agttgcaggt gggaggagga gtc 43

<210>7

<211>44

<212>DNA

＜213〉synthetic

<220>

＜223〉the reverse introduction of PQGAB-US.

<400>7

gccagccagt cggtaccgtt cagttcgcac agggtcaggt tgta 44

<210>8

<211>44

<212>DNA

＜213〉synthetic

<220>

＜223〉the reverse introduction of PQGAB-US.

<400>8

ttttttctcg agagcccagt cgaatttgtt agccagccag tcgg 44

<210>9

<211>207

<212>PRT

＜213〉pig reproduction and respiratory tract syndrome virus

<400>9

Cys Cys Cys Ala Ala Ala Cys Cys Cys Cys Ala Thr Ala Thr Gly Gly

1 5 10 15

Ala Ala Thr Cys Gly Gly Thr Thr Cys Cys Thr Cys Cys Cys Thr

20 25 30

Gly Gly Ala Cys Gly Ala Cys Thr Thr Cys Thr Gly Cys Thr Ala Cys

35 40 45

Gly Ala Cys Thr Cys Cys Ala Cys Cys Gly Cys Thr Cys Cys Cys Cys

50 55 60

Ala Gly Ala Ala Ala Gly Thr Thr Cys Thr Gly Cys Thr Gly Gly Cys

65 70 75 80

Thr Thr Thr Cys Thr Cys Cys Ala Thr Cys Ala Cys Cys Thr Ala Cys

85 90 95

Gly Cys Thr Thr Cys Cys Ala Ala Cys Gly Ala Cys Thr Cys Cys Thr

100 105 110

Cys Cys Thr Cys Cys Cys Ala Cys Cys Thr Gly Cys Ala Ala Cys Thr

115 120 125

Gly Ala Thr Cys Thr Ala Cys Ala Ala Cys Cys Thr Gly Ala Cys Cys

130 135 140

Cys Thr Gly Thr Gly Cys Gly Ala Ala Cys Thr Gly Ala Ala Cys Gly

145 150 155 160

Gly Thr Ala Cys Cys Gly Ala Cys Thr Gly Gly Cys Thr Gly Gly Cys

165 170 175

Thr Ala Ala Cys Ala Ala Ala Thr Thr Cys Gly Ala Cys Thr Gly Gly

180 185 190

Gly Cys Thr Cys Thr Cys Gly Ala Gly Ala Ala Ala Ala Ala Ala

195 200 205

<210>10

<211>219

<212>PRT

＜213〉pig reproduction and respiratory tract syndrome virus

<400>10

Cys Cys Cys Gly Ala Ala Thr Thr Cys Cys Ala Thr Ala Thr Gly Gly

1 5 10 15

Thr Cys Gly Ala Cys Ala Thr Gly Gly Gly Thr Thr Cys Thr Cys Thr

20 25 30

Cys Gly Ala Cys Gly Ala Cys Thr Thr Thr Thr Gly Thr Ala Ala Cys

35 40 45

Gly Ala Cys Thr Cys Thr Ala Cys Cys Gly Cys Thr Gly Cys Thr Cys

50 55 60

Ala Gly Ala Ala Ala Cys Thr Gly Gly Thr Thr Cys Thr Gly Gly Cys

65 70 75 80

Thr Thr Thr Thr Thr Cys Thr Ala Thr Cys Ala Cys Cys Thr Ala Cys

85 90 95

Ala Cys Cys Cys Cys Ala Ala Thr Cys Thr Thr Thr Gly Thr Thr Gly

100 105 110

Cys Thr Gly Gly Thr Gly Gly Thr Thr Cys Thr Thr Cys Thr Thr Cys

115 120 125

Thr Ala Cys Cys Thr Ala Cys Cys Ala Gly Thr Ala Cys Ala Thr Cys

130 135 140

Thr Ala Cys Ala Ala Cys Cys Thr Cys Ala Cys Cys Ala Thr Cys Thr

145 150 155 160

Gly Thr Gly Ala Ala Cys Thr Cys Ala Ala Cys Gly Gly Thr Ala Cys

165 170 175

Cys Gly Ala Cys Thr Gly Gly Cys Thr Gly Thr Cys Thr Ala Ala Cys

180 185 190

Cys Ala Cys Thr Thr Thr Gly Ala Cys Thr Gly Gly Gly Cys Thr Cys

195 200 205

Thr Cys Gly Ala Gly Ala Ala Ala Ala Ala Ala

210 215

<210>11

<211>219

<212>DNA

＜213〉pig reproduction and respiratory tract syndrome virus

<400>11

cccgaattcc atatggtcga catgggttct ctcgacgact tttgtaacga ctctaccgct 60

gctcagaaac tggttctggc tttttctatc acctacaccc caatctttgt tgctggtggt 120

tcttcttcta cctaccagta catctacaac ctcaccatct gtgaactcaa cggtaccgac 180

tggctgtcta accactttga ctgggctctc gagaaaaaa 219

<210>12

<211>33

<212>PRT

＜213〉pig reproduction and respiratory tract syndrome virus

<400>12

Ala Ser Asn Asp Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr

1 5 10 15

Leu Cys Glu Leu Asn Gly Thr Asp Thp LeuAlaAsn Lys Phe Asp Thp

20 25 30

Ala

<210>13

<211>25

<212>PRT

＜213〉pig reproduction and respiratory tract syndrome virus

<400>13

Gly Ser Ser Leu Asp Asp Phe Cys Tyr Asp Ser Thr Ala Pro Gln Lys

1 5 10 15

Val Leu Leu Ala Phe Ser Ile Thr Tyr

20 25

<210>14

<211>28

<212>PRT

＜213〉pig reproduction and respiratory tract syndrome virus

<400>14

Met Gly Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Ala Gln Lys

1 5 10 15

Leu Val Leu Ala Phe Ser Ile Thr Tyr Thr Pro Ile

20 25

<210>15

<211>34

<212>PRT

＜213〉pig reproduction and respiratory tract syndrome virus

<400>15

Phe Val Ala Gly Gly Ser Ser Ser Thr Tyr Gln Tyr Ile Tyr Asn Leu

1 5 10 15

Thr Ile Cys Glu Leu Asn Gly Thr Asp Trp Leu Ser Asn His Phe Asp

20 25 30

Trp Ala

<210>16

<211>42

<212>DNA

＜213〉synthetic

<220>

＜223〉the forward introduction of PQGAB-EP.

<400>16

ctggcttttt ctatcaccta caccccaatc tttgttgctg gt 42

<210>17

<211>41

<212>DNA

＜213〉synthetic

<220>

＜223〉the forward introduction of PQGAB-EP

<400>17

gactctaccg ctgctcagaa actggttctg gctttttcta t 41

<210>18

<211>39

<212>DNA

＜213〉synthetic

<220>

＜223〉the forward introduction of PQGAB-EP.

<400>18

ggttctctcg acgacttttg taacgactct accgctgct 39

<210>19

<211>38

<212>DNA

＜213〉synthetic

<220>

＜223〉the forward introduction of PQGAB-EP.

<400>19

cccgaattcc atatggtcga catgggttct ctcgacga 38

<210>20

<211>42

<212>DNA

＜213〉synthetic

<220>

＜223〉the reverse introduction of PQGAB-EP.

<400>20

gatgtactgg taggtagaag aagaaccacc agcaacaaag at 42

<210>21

<211>42

<212>DNA

＜213〉synthetic

<220>

＜223〉the reverse introduction of PQGAB-EP.

<400>21

gttgagttca cagatggtga ggttgtagat gtactggtag gt 42

<210>22

<211>39

<212>DNA

＜213〉synthetic

<220>

＜223〉the reverse introduction of PQGAB-EP.

<400>22

gtggttagac agccagtcgg taccgttgag ttcacagat 39

<210>23

<211>38

<212>DNA

＜213〉synthetic

<220>

＜223〉the reverse introduction of PQGAB-EP.

<400>23

ttttttctcg agagcccagt caaagtggtt agacagcc 38

Claims (20)

1. PE-PQGAB-K3 fusion rotein, it comprises:

One contains the ORF6 partial sequence of pig reproduction and respiratory tract syndrome virus (PRRSV), with the N end protein district of PRRSV ORF5 partial sequence;

One Pseudomonas aeruginosa exotoxin A shift function position; And

One contains the carboxyl terminal portion of KDEL-KDEL-KDEL (K3) sequence.

2. fusion rotein as claimed in claim 1, wherein this PRRSV is the America strain.

3. fusion rotein as claimed in claim 1, wherein this PRRSV is European strain.

4. fusion rotein as claimed in claim 2, wherein this ORF6 partial sequence of this PRRSV is SEQ ID NO.13.

5. fusion rotein as claimed in claim 2, wherein this PRRSV ORF5 partial sequence is SEQID NO.12.

6. fusion rotein as claimed in claim 2, wherein the nucleotide sequence in the N of the part of O RF6 of this pig reproduction and respiratory tract syndrome virus (PRRSV) and part of O RF5 end fusion rotein district is SEQ IDNO.1.

7. fusion rotein as claimed in claim 3, wherein this part of O RF6 of this PRRSV is SEQID NO.15.

8. fusion rotein as claimed in claim 3, wherein this part of O RF5 of this PRRSV is SEQID NO.14.

9. fusion rotein as claimed in claim 3, wherein the nucleotide sequence in the N of the part of O RF6 of this pig reproduction and respiratory tract syndrome virus (PRRSV) and part of O RF5 end fusion rotein district is SEQ IDNO.10.

10. medical composition that can be used as vaccine, it comprises:

The part of O RF6 of one pig reproduction and respiratory tract syndrome virus (PRRSV) and the N of part of O RF5 end fusion rotein district;

One Pseudomonas aeruginosa exotoxin A shift function position;

One contains the carboxyl terminal portion of KDEL-KDEL-KDEL (KDEL3) sequence; And

One pharmaceutically acceptable carrier.

11. medical composition as claimed in claim 10, wherein this PRRSV is the America strain.

12. medical composition as claimed in claim 10, wherein this PRRSV is European strain.

13. medical composition as claimed in claim 11, wherein this part of O RF6 of this PRRSV is SEQ ID NO.13.

14. medical composition as claimed in claim 11, wherein this part of O RF5 of this PRRSV is SEQ ID NO.12.

15. medical composition as claimed in claim 11, wherein the nucleotide sequence in the N of the part of O RF6 of this pig reproduction and respiratory tract syndrome virus (PRRSV) and part of O RF5 end fusion rotein district is SEQ ID NO.1.

16. medical composition as claimed in claim 12, wherein this part of O RF6 of this PRRSV is SEQ ID NO.15.

17. medical composition as claimed in claim 12, wherein this part of O RF5 of this PRRSV is SEQ ID NO.14.

18. medical composition as claimed in claim 12, wherein the nucleotide sequence in the N of the part of O RF6 of this pig reproduction and respiratory tract syndrome virus (PRRSV) and part of O RF5 end fusion rotein district is SEQ ID NO.10.

19. medical composition as claimed in claim 10, wherein this carrier comprises a freund adjuvant, emulsifying agent mannide list oleate (mannide mono-oleate emulsifier, ISA720, ISA206) adjuvant.

20. medical composition as claimed in claim 15, wherein this carrier comprises an ISA206 adjuvant.

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