CN1265841C - Swing breeding and respiratory syndrome suicide DNA vaccine and use - Google Patents
Swing breeding and respiratory syndrome suicide DNA vaccine and use Download PDFInfo
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Abstract
The present invention discloses a swine breeding and respiratory syndrome suicide DNA vaccine and application of the vaccine to the prevention and the treatment of swine breeding and respiratory syndromes. The present invention is characterized in that main immunogenicity genes ORF5 of swine breeding and respiratory syndrome viruses are inserted into the BamHI site of an expression carrier pSCA of the suicide DNA vaccine to construct the swine breeding and respiratory syndrome suicide DNA vaccine; simultaneously, the new expression carrier pSCA which is suitable for preparing the suicide DNA vaccine is constructed; the carrier is characterized in that an immediate early promoter of human cytomegaloviruses is introduced into the upper stream of non-structural protein of a son plasmid expression carrier pSFVl replicated by semliki forest viruses; an SV40 poly(A) sequence is introduced into the down stream of a polyclonal site. The suicide DNA vaccine of the present invention combines the advantages of a conventional DNA vaccine and an autonomously replicating RNA vaccine. The present invention has the advantages of simple preparation technology, convenient transportation and storage, safety, good immune effect, etc., and can be used for preventing and treating swine breeding and respiratory syndromes.
Description
Technical field
The present invention relates to animal virology and epizootiology technical field.Relate in particular to a kind of Porcine reproductive and respiratory syndrome " Suicidal DNA Vaccine also relates to the application of this vaccine in anti-system Porcine reproductive and respiratory syndrome.
Background technology
Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, be called for short PRRS, hereinafter referred PRRS) be a kind of new viral infectious of finding in recent years, with breeding difficultys such as sow heating, anorexia, premature labor, miscarriage, stillborn fetus, weak son and various age pig respiratory system disease and high mortality be feature.This disease was reported in southern US early than 1987, had promptly propagated into the Midwest soon, and at the whole America rapid spread.Subsequently some countries such as Canada, Germany, Holland also successively broken out should disease (Bilodeau R etc., Porcine reproductive and respiratory syndrome in Quebec.Vet Rec, 1991,129:102-103; Dea S, Bilodeau R, Athanassious R et al.Swine reproductive and respiratory syndrome virus in Quebec:isolation of an enveloped virus serologically-related to lelystad virus.Can Vet J.1992,33:801-808); The area, Asia reports that this sick time is later relatively, and this disease (Zhang Zhi's one-tenth etc., breeding of Taiwan pig and the evaluation of respiratory tract syndrome I virus, Chinese veterinary's magazine, 1993,19 (4): 268-276) appearred in 1991 in the Taiwan; Japan broke out PRRS (Hiroyoshi Kuwaahara.An outbreak of PRRS in Japan.J Vet Med Sci, 1994,56 (50): 901-909) in 1994; The China's Mainland reported that this disease came into vogue in 1996, and (Guo Baoqing etc. are from the research of doubtful PRRS aborted fetus separation pig reproduction and breath syndrome virus, Chinese poultry infectious disease, 1996,87 (2): 1-5) to be separated to virus.Above-mentioned document shows that Porcine reproductive and respiratory syndrome caused enormous economic loss for global pig industry, and only European PRRS's in 1991 breaks out the death that has just caused 1,000,000 pigs.
Anti-system with the virosiss of other many pigs is the same, and the anti-system of PRRS also mainly is immunoprophylaxis.What be used at present to prevent PRRS mainly is weak malicious Seedling and inactivated vaccine.Although attenuated vaccine can provide immunoprotection preferably, exist virulence to return strong danger, and it is quite high to return strong probability, this point also several years ago state such as Denmark cause this disease to be confirmed in breaking out greatly because of being extensive use of weak malicious Seedling.Compare with weak malicious Seedling, though inactivated vaccine safety often needs the immunity inoculation of repeated multiple times, and the effect instability, also often cause immuning failure.We can say, at present unsatisfactory always to the anti-system of this disease, press for more safe and effective vaccine and prevent and control the generation of this disease and popular.
" Suicidal DNA Vaccine (Suicidal DNA vaccine) is a kind of new vaccine design that proposed in recent years; grow up on conventional dna vaccination and " self-replicating type " RNA vaccine (Self-replicating RNA vaccine) basis; not only have the safety and the effectiveness of " self-replicating type " RNA vaccine; and combine the advantage of aspects such as conventional dna vaccination easily prepares, transportation, preservation, can be rated as the important breakthrough of gene vaccine development in recent years.At present, it is early stage that the research of this new generation vaccine still is in research, but the influenza virus that existing report makes up in this way, human herpes simplex vicus " can induce humoral immunization and the cellular immunization that is higher than conventional dna vaccination behind the Suicidal DNA Vaccine immunization experiment animal white mice; and low 100 ~ 1000 times of the immunizing dose of more conventional dna vaccination, " potentiality to be exploited that Suicidal DNA Vaccine is huge and good prospects for application showed.
Porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus; PRRSV; hereinafter referred PRRSV) the glycoprotein GP5 of ORF5 gene code is a topmost protective antigen among the PRRSV; can induce humoral immunization and cellular immunization; and the topmost neutralizing epitope of determining at present also is positioned at its N end extracellular region; therefore be preferred object gene (the Dea S of design PRRS new generation vaccine; Gagnon CA; Mardassi H.Current knowledge on the structural proteins of porcinereproductive and respiratory syndrome (PRRS) virus:comparison of the North American andEuropean isolates.Arch Virol; 2000a, 145:659-688).
Summary of the invention
An object of the present invention is to obtain a kind of " Suicidal DNA Vaccine that can prevent making Porcine reproductive and respiratory syndrome.
That another object of the present invention provides is a kind of, and " Suicidal DNA Vaccine expression vector pSCA, this carrier not only are fit to " the application of Suicidal DNA Vaccine, and might being applied on other similar vaccines of preparation Porcine reproductive and respiratory syndrome.
Another object of the present invention provides the " application of Suicidal DNA Vaccine in anti-system Porcine reproductive and respiratory syndrome of a kind of Porcine reproductive and respiratory syndrome.
The present invention implements by the following technical programs:
A kind of " Suicidal DNA Vaccine; be that " the BamHI site of Suicidal DNA Vaccine expression vector pSCA makes up and forms with porcine reproductive and respiratory syndrome virus main immunogenic gene ORF5 insertion of Porcine reproductive and respiratory syndrome, the escherichia coli Escherichiacoli DH5 α/pSCA-ORF5 that contains this plasmid, be deposited in CCTCC, preserving number is M203073.
Described " the expression vector pSCA of Suicidal DNA Vaccine; it is characterized in that introducing the immediate early promoter sequence of human cytomegalic inclusion disease virus, introduce SV40poly (A) sequence in its multiple clone site downstream in the nonstructural protein gene upstream of Xi Menli gram forest virus replicon plasmid-type expression vector pSFV1.
The present invention also comprises " the application of Suicidal DNA Vaccine in anti-system Porcine reproductive and respiratory syndrome of above-mentioned a kind of Porcine reproductive and respiratory syndrome.
Concrete technical scheme of the present invention is as described in the following technical step:
One, the transformation of pSFV1 carrier
1, primer design:
The primer P1 and the P2 that synthesize SV40 poly in late period (A) the transcription stop signals sequence that to increase according to the complete sequence (seeing the pCI-neo of Promega company " carrier for expression of eukaryon workbook ") of pCI-neo (Promega), the upstream and downstream primer designs SpeI and XvaI site respectively, and the amplification size is 250bp; Design the primer P3 and the P4 of a pair of amplification HCMV IE Enhancer/Promoter element simultaneously, the SphI site is all designed at upstream and downstream primer two ends, and the amplification size is 823bp.Primer sequence is:
P1：5’-CAA
ACTAGTCAAACATGATAAGATAC-3’：
P2：5’-GGC
TCTAGATACCACATTTGTAGAGGT-3’
P3：5’-ACAT
GCATGCTCAATATTGGCCATTAGC-3’
P4：5’-ACAT
GCATGCCTGACTGCGTTAGCAATT-3’
2, pcr amplification
With pCI-neo is template, and P1, P2 are primer amplification SV40poly (A) sequence.Amplification condition is to enter circulation behind 95 ℃ of degeneration 5min, and loop parameter is 95 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, and 35 circulations are extended 10min for back 72 ℃.Reaction finishes the back and carry out electrophoresis detection in 1.0% agarose gel.
With P3, P4 is primer, and pCI-neo is a template amplification HCMV IE Enhancer/Promoter fragment.Amplification condition is to enter circulation behind 95 ℃ of degeneration 5min, and loop parameter is 95 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, and 35 circulations are extended 10min for back 72 ℃.Reaction finishes the back and carry out electrophoresis detection in 1.0% agarose gel.
3, the transformation of pSFV1 carrier
With using SpeI and XbaI enzyme cutting behind SV40poly (A) fragment purification of amplification, reclaim the purpose fragment, insert in SpeI enzyme action and dephosphorylized carrier pSFV1, recombiant plasmid is identified called after pSFVA through enzyme action and PCR.To use the SphI enzyme action behind the segmental amplified production purification of HCMV IE Enhancer/Promoter then, insert the SphI site of pSFVA, recombiant plasmid is also identified through enzyme action and PCR, called after pSCA, this carrier be used for Porcine reproductive and respiratory syndrome " structure of Suicidal DNA Vaccine; according to thinking of the present invention, the applicant thinks that this carrier might be applied on the structure of similar vaccine.
Two, the PRRSV ORF5 gene " structure of Suicidal DNA Vaccine expression plasmid
Design the primer P52S and the P52R (same P51R) of a pair of ORF5 complete coding region that increases, upstream and downstream primer two ends have been designed BamHI and XbaI site respectively.Primer sequence is as follows:
P52S 5’-GAA
GGATCCTCAGTATGTTGGGGAAATGCTTGACC-3’
P52R 5’-TTT
TCTAGAGACGACCCCATTGTTCCGC-3’
With pMD-ORF5 is template, carries out pcr amplification, and the amplification size is 607bp.The PCR reaction condition is: 94 ℃ of 5min; Enter the PCR circulation, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, after 35 circulations, 72 ℃ are extended 10min.After reaction finishes, detect amplification with 1.0% agarose gel electrophoresis.Purification reclaims the purpose fragment, is cloned among the carrier pMD18-T, and the selection two ends all contain the plasmid in BamHI site, called after pMD-ORF5-1, and sequencing analysis confirms that no base mismatches.
BamHI digested plasmid pMD-ORF5-1 reclaims the purpose fragment, inserts the corresponding site of pSCA, the positive recombiant plasmid called after pSCA-ORF5 of acquisition.
Three, a large amount of preparations of plasmid pSCA-ORF5
(1) picking contains the colony inoculation of plasmid pSCA-ORF5 in 75mL LB culture fluid, 37 ℃ of 300r/min overnight incubation, 6000r/min5min, the collecting cell precipitation, with 3mL solution I (50mmol/L glucose, 10mmol/L EDTA, 25mmol/L Tris-Cl (pH8.0), the rearmounted 4 ℃ of preservations of autoclaving are standby) (dispelling or vortex) suspends.
(2) add 6mL solution II (0.2mol/L NaOH, 1% SDS, now with the current), ice bath 7-10min.
(3) add 4.5mL solution III (3mol/L potassium acetate, glacial acetic acid adjust pH to 4.8), ice bath 7-10min.4℃ 10000r/min10-15min。
(4) get supernatant, add the isopropyl alcohol of 0.6 times of volume, mixing ,-20 ℃ of 30min or room temperature 5min.Room temperature, 10000r/min 6-15min.
(5) abandon supernatant, with 75% ethanol rinsing once, vacuum is drained or natural drying, adds 1.5mL TE (10.O mmol/L Tris-HCl, 1.O mmol/L EDTA) suspension (washing about wall 20min).
(6) add the 5mol/L NH that 1.5ml is the pre-cooling of 1 times of volume ice
4Ac, mixing.4℃ 10000r/min 10min。
(7) supernatant is transferred to the centrifuge tube of 7mL, adds the isopropyl alcohol mixing of 1 times of volume (3mL) ,-20 ℃ of effect 30min.12000r/m 15min。
(8) abandon supernatant, with 75% ethanol rinsing once, vacuum is drained or natural drying, adds 500 μ L TE and suspends (washing about wall 20min), and be transferred to the centrifuge tube of 1.5mL.
(9) add an amount of RNase, 37 ℃ of 1h remove RNA.
(10) add 13% PEG8000 (containing 1.6mol/LNaCl) of 1 times of volume, mixing ,-20 ℃ of 30min (can spend the night).Centrifugal, abandon supernatant, heavy molten with 400 μ L TE.
(11) add equal-volume phenol: imitative: isoamyl alcohol extracting 2 times, chloroform: isoamyl alcohol extracting 1 time.
(12) add 100 μ l 10mol/L NH
4Ac fully behind the mixing, adds the dehydrated alcohol of 2 times of volumes ,-20 ℃ of precipitation 30min.4 ℃ of centrifugal 5-10min of 12000r/min.
(13) abandon supernatant, with 75% ethanol rinsing once, vacuum is drained or natural drying, adds 50 μ L TE or H
2O is heavy molten, put-20 ℃ standby.Four, the structure that is used for the conventional dna vaccination expression plasmid pCI-ORF5 of contrast test
Design the primer P51S and the P51R of a pair of ORF5 complete coding region that increases, upstream and downstream primer two ends have been designed XhoI and XbaI site respectively.Primer sequence is as follows:
P51S 5’-GAA
CTCGAGAGTATGTTGGGGAAATGCTTGACC-3’
P51R 5’-TTTT
CTAGAGACGACCCCATTGTTCCGC-3’
With pMD-ORF5 be template (Fang Liurong etc., the clone and the sequence analysis of pig breeding and breathing syndrome virus YA strain ORF5 gene, Hua Zhong Agriculture University's journal, 2002,21 (6): 501-505), carry out pcr amplification, the amplification size is 619bp.The PCR reaction condition is: 94 ℃ of 5min; Enter the PCR circulation, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, after 35 circulations, 72 ℃ are extended 10min.After reaction finishes, detect amplification with 1.0% agarose gel electrophoresis.Purification reclaims the purpose fragment, the amplified production of purification is behind XhoI, XbaI enzyme cutting, directly be cloned into the corresponding site of carrier for expression of eukaryon pCI-neo (Promega), the recombiant plasmid pCI-ORF5 that obtains identifies through XhoI, XbaI and XhoI+XbaI enzyme action and PCR and confirms to make up correct that the no base of order-checking confirmation mismatches.
Five, " Suicidal DNA Vaccine and the production technology that is used for the conventional dna vaccination of comparison
The main flow process of production of vaccine technology: a large amount of extractions of the conversion of plasmid, plasmid, the mensuration and the dilution of plasmid concentration.
1, the conversion of plasmid
Will " Suicidal DNA Vaccine expression plasmid pSCA-ORF5 (ammonia benzyl resistance) and conventional dna vaccination expression plasmid pCI-ORF5 (ammonia benzyl resistance) difference transformed into escherichia coli DH5 α competent cell.Concrete operations are: 1) get 100 μ l competent cell suspensions and transfer in the aseptic 1.5ml EP pipe, add 10 μ l and connect product, rotate gently with the mixed content thing, place 30min on ice.2) centrifuge tube was put in the circulator bath that is warmed to 42 ℃ in advance thermal shock 90 seconds.3) fast centrifuge tube is transferred in the ice bath, made cell cooling 1 ~ 2min.4) every pipe adds 400 μ l LB culture medium.With water-bath culture medium is heated to 37 ℃, centrifuge tube is transferred on 37 ℃ of shaking tables then, incubation 45min makes bacteria resuscitation.For reaching effective conversion, rotating speed should not be above 225 rev/mins during recovery.5) get competent cell that 100 μ l transform and transfer to and contain on the corresponding antibiotic LB agar plate, cell transformed is uniformly applied to agar plate surface with an aseptic elbow glass rod.6) plate is put 37 ℃ of cultivations, be absorbed until liquid, be inverted plate then and cultivate, bacterium colony can appear in 12 ~ 16h.
2, a large amount of extractions of plasmid are with the method (this place omits this detailed preparation process) of above-mentioned " three, a large amount of preparations of the plasmid pSCA-ORF5 ".
3, the mensuration of plasmid concentration, dilution
Utilize the concentration of the big upgrading grain of spectrophotometric determination, it is diluted to 1 μ g/ μ l or 0.2 μ g/ μ l, promptly can be used for the animal injection with phosphate buffer (PBS pH7.4).
Six, the immune efficacy of vaccine check
1, to the immuning effect test of Balb/c white mice
With " Suicidal DNA Vaccine pSCA-ORF5 and conventional dna vaccination pCI-ORF5 are through the back leg intramuscular injection Balb/c mice in 6 ages in week, 6 every group, every mice 100 μ l (containing 100 μ g plasmids), immunity twice altogether, 4 weeks at interval; Use blank plasmid vector pCI-neo, pSCA negative control simultaneously as nucleic acid immunization.Each immunity 4 weeks of back, separation of serum detected ELISA antibody through the blood sampling of tail vein negative pressure.Two exempt from back 4 all eyeball blood-letting, and separation of serum detects ELISA antibody and neutralizing antibody." inductive ELISA antibody of Suicidal DNA Vaccine pSCA-ORF5 and neutralizing antibody are all apparently higher than conventional dna vaccination pCI-ORF5 for the result.
2, vaccine is to the immuning effect test of ablactational baby pig
Will " Suicidal DNA Vaccine pSCA-ORF5, conventional dna vaccination pCI-ORF5 inject ablactational baby pig through musculi colli, and 4 every group, every pig 500 μ l (containing 100 μ g plasmids), immunity twice altogether, 4 weeks at interval; Use blank plasmid vector pCI-neo, pSCA negative control simultaneously as nucleic acid immunization.Took a blood sample through vena cava anterior in 4 weeks in each immunity back, separation of serum detects ELISA antibody and neutralizing antibody level, gathers anticoagulation simultaneously, and isolated lymphocytes detects the cellullar immunologic response level." inductive humoral immunization of Suicidal DNA Vaccine pSCA-ORF5 and cell immune response are all apparently higher than conventional dna vaccination pCI-ORF5 for the result.
Description of drawings
Biological material specimens of the present invention is that bacterial strain DH5 α/pSCA-ORF5 was in preservation on October 22 in 2003, deposit number is: CCTCCNO:M203073, classification called after escherichia coli (Escherichia coli), be deposited in Chinese typical culture collection center (CCTCC), be positioned at Wuhan City, Hubei Province Wuhan University.
Fig. 1 has shown the structure flow chart of the conventional dna vaccination pCI-ORF5 of PRRSV YA strain ORF5 gene
Fig. 2 has shown " the structure flow chart of Suicidal DNA Vaccine expression vector pSCA
Fig. 3 has shown PRRSV YA strain ORF5 gene " the structure flow chart of Suicidal DNA Vaccine pSCA-ORF5
Fig. 4 has shown " the ELISA antibody horizontal behind Suicidal DNA Vaccine pSCA-ORF5 and the conventional dna vaccination pCI-ORF5 immunity Balb/c mice
Fig. 5 has shown " the ELISA antibody horizontal behind Suicidal DNA Vaccine pSCA-ORF5 and the conventional dna vaccination pCI-ORF5 immunity ablactational baby pig
Fig. 6 has shown " inductive cell immune response level behind Suicidal DNA Vaccine pSCA-ORF5 and the conventional dna vaccination pCI-ORF5 immunity ablactational baby pig
The specific embodiment
The present invention is further illustrated below in conjunction with Figure of description:
1, the structure of conventional dna vaccination expression plasmid pCI-ORF5
With pMD-ORF5 is template, and P52S and P52R are primer amplification ORF5 gene, and the amplified production of purification directly is cloned into the corresponding site of carrier for expression of eukaryon pCI-neo behind XhoI, XbaI enzyme cutting, obtains recombiant plasmid pCI-ORF5.It makes up flow process and sees Fig. 1.
2, " the structure of Suicidal DNA Vaccine expression vector pSCA
With pCI-neo is template, and P1, P2 are primer amplification SV40poly (A) sequence, with SpeI and XbaI enzyme cutting, reclaims the fragment of the about 250bp of size behind the purification, is inserted into the SpeI site of carrier pSFV1, obtains recombiant plasmid pSFVA.And then be template with pCI-neo, P3, P4 are primer amplification HCMV IE Enhancer/Promoter fragment, use the SphI enzyme action behind the amplified production purification, reclaim the fragment of the about 823bp of size, are inserted into the SphI site of pSFVA, acquisition recombiant plasmid pSCA.It makes up flow process and sees Fig. 2.
3, " the structure of Suicidal DNA Vaccine pSCA-ORF5
BamHI digested plasmid pMD-ORF51 reclaims the purpose fragment, inserts the BamHI site of pSCA, the positive recombiant plasmid called after pSCA-ORF5 of acquisition.It makes up flow process and sees Fig. 3.
4, " Suicidal DNA Vaccine pSCA-ORF5 is to the mensuration of Balb/c white mice immune efficacy
(1) immune programme for children of Balb/c mice
The Balb/c mice is divided into 4 groups, and 6 every group, adopt the back leg intramuscular injection, every mice 100 μ l (containing 100 μ g plasmids), immunity twice altogether at interval 4 weeks, is used blank plasmid vector pCI-neo, the pSCA negative control as nucleic acid immunization simultaneously.Each immunity 4 weeks of back, separation of serum detected ELISA antibody through the blood sampling of tail vein negative pressure.Two exempt from back 4 all eyeball blood-letting, and separation of serum detects ELISA antibody and neutralizing antibody.
(2) ELISA antibody test
Utilize the GP5-ELISA method of setting up to detect the ELISA antibody of serum at PRRSV, result such as Fig. 4.
(3) neutralizing antibody detects
Standard according to World Organization for Animal Health (OIE) is recommended, detect sero-fast PRRSV neutralizing antibody level, result of the test such as table 1:
Table 1Balb/c mice two is exempted from the neutralizing antibody level in the 4th week of back
| Group (6/group) | Before the immunity | Head exempts from 8 weeks of back (two exempt from 4 weeks of back) | |||
| ＜1∶4 | ≥1∶4 | ≥1∶8 | ≥1∶16 | ||
| pCI-neo pSCA pCI-ORF5 pSCA-ORF5 | ND ND ND ND | 6/6 6/6 2/6 0/6 | 0/6 0/6 4/6 6/6 | 0/6 0/6 1/6 3/6 | 0/6 0/6 0/6 1/6 |
Annotate: ND represents not do
5, " Suicidal DNA Vaccine pSCA-ORF5 is to the mensuration of ablactational baby pig immune efficacy
(1) immune programme for children of pig
Ablactational baby pig is divided into 5 groups at random, and 4 every group, adopt the musculi colli injection, every pig 500 μ L (containing 100 μ g plasmids), immunity twice altogether at interval 4 weeks, is used blank plasmid vector pCI-neo, the pSCA negative control as nucleic acid immunization simultaneously.Took a blood sample through vena cava anterior in 4 weeks in each immunity back, separation of serum detects the neutralizing antibody level, gathers anticoagulation simultaneously, and isolated lymphocytes detects the cellullar immunologic response level.
(2) neutralizing antibody detects
Standard according to World Organization for Animal Health (OIE) is recommended, detect sero-fast porcine reproductive and respiratory syndrome virus neutralizing antibody level, result of the test such as table 2:
The 4th week after the immunity of table 2 ablactational baby pig, the neutralizing antibody level in the 8th week
| Group (4 pig/groups) | Before the immunity | Head exempts from 4 weeks of back | Head exempts from 8 weeks of back | |||
| ≥1∶4 | ≥1∶8 | ≥1∶4 | ≥1∶8 | ≥1∶16 | ||
| pCI-neo pSCA pCI-ORF5 pSCA- | 0/4 0/4 0/4 1/4 | 0/4 0/4 0/4 0/4 | 0/4 0/4 1/4 4/4 | 0/4 0/4 0/4 2/4 | 0/4 0/4 0/4 0/4 | |
Annotate :-expression neutralizing antibody feminine gender
(3) ELISA antibody test
Utilize the GP5-ELISA method of setting up to detect sero-fast PRRSV ELISA antibody, result such as Fig. 5.
(4) cellular immune level detects
The inducing and measuring of antigen-specific cytotoxic t lymphocytes activity (CTL).Result of the test such as Fig. 6.
Claims (2)
1, a kind of " Suicidal DNA Vaccine; it is characterized in that " the BamHI site of Suicidal DNA Vaccine expression vector pSCA makes up and forms with porcine reproductive and respiratory syndrome virus main immunogenic gene ORF5 insertion of Porcine reproductive and respiratory syndrome, the escherichia coli Escherichia coli DH5 α/pSCA-ORF5 that contains this plasmid, be deposited in CCTCC, preserving number is M203073.
2, " the application of Suicidal DNA Vaccine in preparation Porcine reproductive and respiratory syndrome medicine of the described a kind of Porcine reproductive and respiratory syndrome of claim 1.
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