CN104419687B - Restructuring PRRS virus HV nsp29 and its application - Google Patents

Abstract

The invention discloses a kind of restructuring PRRS virus HV nsp29, the viral genome cDNA sequences of restructuring PRRS are that sequence 1 is replaced with sequence of the sequence 2 from 5 ' end the 814th 1395 from the sequence of 5 ' end the 1474th 2055；Sequence 1 is replaced with what sequence 3 was obtained from the sequence of 5 ' end the 158th 2002 from the sequence of 5 ' end the 7683rd 9527.Compared with the high pathogenic strain HV of wild type PRRSV, the speed that restructuring PRRS viruses are grown on PAM is decreased obviously, and the pathogenicity in pig body substantially weakens.Infected after different PRRS viruses again through the piggy after its immunity, the state of mind is good, and appetite is normal, and PRRS virus quantities are substantially less than non-immunized piggy in vivo, illustrate that restructuring PRRS viruses can be developed as attenuated vaccine strain.

Description

Restructuring PRRS virus HV-nsp29 and its application

Technical field

The present invention relates to a kind of restructuring PRRS virus HV-nsp29 and its application.

Background technology

In organism, every kind of amino acid at least corresponds to a codon, and most has six kinds of corresponding codons, coding The codon of same amino acid is referred to as synonym.Research shows, biological generally existing synonym lack of balance in vivo The phenomenon for using, for example:A certain species or a certain gene are normally tended to using one or more specific synonym, this A little codons are referred to as optimal codon (optimal codon), and this phenomenon is referred to as codon preference (codon bias). Likewise, the use of codon pair also has Preference, but the Preference of codon pair is not rely on the Preference of codon. For example, arginine (arginine, Arg) can be encoded by six kinds of codons, and glutamic acid (glutamic acid, Glu) can be by Two kinds of codon codings, so amino acid can be by 12 kinds of synonym to coding, if according to codon to Arg-Glu Preference calculate each synonym to the occurrence number in human genome, then codon is to CGC- in theory The occurrence number of GAA is 2,397 times, but reality is only occurred in that 268 times, so CGC-GAA is non-preference in human genome Property codon pair；And the theoretical number of times that codon encodes Arg-Glu to AGA-GAA is 2,644 times, but its actual occurrence number is then It it is 4,195 times, therefore AGA-GAA is the Preference codon pair in human genome.

Although up to the present people do not know Crack cause of the codon to Preference, the use of codon pair Translation efficiency can but be affected.2008, nucleocapsids of the Coleman et al. poliovirus (poliovirus) (capsid) gene is mutated, and on the premise of amino acid sequence and RNA secondary structures is not changed, is carried out same in gene Adopted codon substitutions so that the codon of Preference to being replaced into the codon pair of non-Preference, after chemical synthesis mutation Then the fragment of synthesis is connected into virus full length cDNA clone, the side of this attenuated virus using reverse genetics means by fragment Method is referred to as synthesizing virus weakening engineering (synthetic attenuated virus engineering, SAVE).Disease after displacement The translation rate of malicious capsid genes is remarkably decreased, and the duplication energy in vitro on cell of the recombinant virus containing mutant nucleotide sequence Power and the pathogenicity in mouse all substantially reduce, and in challenge test, recombinant virus can be effectively protected wild type strains Subinfection again for immune mouse.The method of this virus reduction is with a wide range of applications, 2010 and 2013, has respectively People has transformed influenza virus with same method, obtains the obvious influenza virus low virulent strain of the weak effect of cause.Most of all, In the engineering of this attenuated virus, the reduction of virus is caused by hundreds and thousands of point mutation, therefore its return strong can Energy property is extremely low.

Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syn drome, letter Claim PRRS), blue otopathy is commonly called as, is by porcine reproductive and respiratory syndrome virus (porcinereproductive and Respiratory syndrome viruse, PRRSV) cause a kind of high incidence, high mortality, the swine disease of low cure rate. At present, the PRRS vaccines for widely using commercialization in the world include attenuated vaccine and inactivated vaccine.The PRRSV vaccines of inactivation do not have Effect or protection that can only be limited to the infection offer of homologous strain；Attenuated vaccine persistently disseminates virus, especially has reply So that virulence returns strong possibility, attenuated vaccine returns the epistasis safety problem that always fears are entertained that for mutation.

Content of the invention

It is an object of the invention to provide a kind of can be used as the restructuring PRRS of attenuated vaccine strain viruses.

Claimed restructuring PRRS virus HV-nsp29, the corresponding cDNA sequence of its genome is by sequence table Sequence 1 replaces with sequence of the sequence 2 from 5 ' end the 814th 1395 from the sequence of 5 ' end the 1474th 2055 Row；Sequence 1 is replaced with sequence 3 from 5 ' ends the 158th from the sequence of 5 ' end the 7683rd 9527 The sequence obtained by the sequence of 2002.

Sequence 1 is sequence 2 from 5 ' ends the 814th from the sequence of 5 ' end the 1474th 2055 The sequence of 1395；Sequence 1 is sequence 3 from 5 ' ends the from the sequence of 5 ' ends the 7683rd 9527 The DNA fragmentation of the sequence of 158 2002, and the present invention need to be to be protected；

Meanwhile, recombinant plasmid, recombinant bacterium or recombinant cell containing the fragment, and the present invention need to be to be protected.Specifically Says that the recombinant plasmid is sequence 1 will to be corresponded in plasmid pcDNA3.1-HV from 5 ' end the 1474th 2055 in ground DNA fragmentation replace with DNA fragmentation of the sequence 2 from 5 ' end the 814th 1395, by plasmid pcDNA3.1-HV Sequence 3 is replaced with from 5 ' ends the 158th corresponding to sequence 1 from the sequence of 5 ' end the 7683rd 9527 The plasmid that the sequence of 2002 is obtained.Plasmid pcDNA3.1-HV is by the genome of the HV strains shown in the sequence 1 in sequence table Full length sequence is cloned into and obtains in eukaryotic expression vector pcDNA3.1.

Application of the above-mentioned recombinant plasmid in Prepare restructuring PRRS virus HV-nsp29 or PRRS vaccines, and restructuring PRRS Applications of the viral HV-nsp29 in PRRS vaccines are prepared, and the present invention need to content to be protected.

It is demonstrated experimentally that sequence table will be corresponded in the full length cDNA clone of high for wild type PRRSV pathogenic strain HV genomes Sequence 1 is sequence 2 from 5 ' end the 814th 1395 from the sequence alterations of 5 ' end the 1474th 2055, by open country Corresponding to sequence 1 from 5 ' ends the 7683rd in the full length cDNA clone of the high strain HV genomes that cause a disease of raw type PRRSV The sequence alterations of 9527 are the recombinant virus HV-nsp29 that sequence 3 is obtained from after 5 ' end the 158th 2002, with The high pathogenic strain HV of wild type PRRSV is compared, in PAM（Porcine alveolar macrophage）The speed of upper growth is decreased obviously.Restructuring PRRS virus pathogenicities of the HV-nsp29 in pig body substantially weakens, and infects difference again through the piggy after HV-nsp29 immunity After PRRS viruses, the state of mind of piggy is good, and appetite is normal, health, and PRRS virus quantities are substantially less than without immunity in vivo Piggy, illustrate that restructuring PRRS viruses provided by the present invention can be developed as attenuated vaccine strain.

Description of the drawings

Fig. 1 is the virus titer result of different time after restructuring PRRS viruses infected pigs PAM cells.

Fig. 2 is the Cytopathic effect of induction in 84 hours after restructuring PRRS viruses infected pigs PAM cells.

Fig. 3 is the rectal temperature change of piggy after inoculation HV-wt or HV-nsp29.

Fig. 4 is the survival rate change of piggy after inoculation HV-wt or HV-nsp29.

Fig. 5 is PRRS virus titers change in little Swine serum after inoculation HV-wt or HV-nsp29.

Fig. 6 is that real-time fluorescence quantitative PCR detects the inoculation HV-wt or HV-nsp29 middle PRRS diseases that piggy is respectively organized after two weeks Malicious titre change.

Fig. 7 is the hematoxylin eosin stain result for being inoculated with HV-wt or HV-nsp29 piggy some of tissue slice after two weeks.

The survival rate change that Fig. 8 is immune HV-nsp29 with non-immune piggy after the different PRRS viruses of inoculation.

The rectal temperature change that Fig. 9 is immune HV-nsp29 with non-immune piggy after the different PRRS viruses of inoculation.

Figure 10 is immunity HV-nsp29 and non-immune piggy in serum, PRRS is viral after the different PRRS of inoculation are viral to drip Degree change.

It is viral in the different PRRS of inoculation that Figure 11 is real-time fluorescence quantitative PCR detection immunity HV-nsp29 and non-immune piggy The middle PRRS virus titers change that piggy is respectively organized after two weeks.

Figure 12 is immunity HV-nsp29 and non-immune little Swine serum NAT change.

Figure 13 be flow cytomery difference PRRS strains to immune HV-nsp29 and non-immune little pig lymphocyte Carry out again post-stimulatory INF γ and DC8⁺Level.

Figure 14 be on PAM cells to HV-nsp29 continuous passages during observation of cell pathology partial results.Wherein, P1, p10 and p20 represent the generation of the 1st, 10 and 20 respectively.

CPB values of the Figure 15 for nsp2 the and nsp9 genes in the HV-nsp29 of 1st generation, the 10th generation and the 20th generation.Wherein, P1, p10 and p20 represent the generation of the 1st, 10 and 20 respectively.

Specific embodiment

Experimental technique used in following embodiments if no special instructions, is conventional method.

In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.

The high pathogenic strain HV of wild type PRRSV used in following embodiments：No. Genbank is JX317648, Gong Zhongke Obtain from China Agricultural University.

Experimental data in following embodiments if no special instructions, is the mean value of three repetitions, and is carried out with t detections Statistical analysis, work as P<Significant difference when 0.05.

In following embodiments, the CPB values of gene are equal to the arithmetic mean of instantaneous value of the CPS values of each codon pair in gene, formula For：.

Formula I

The CPS values of the codon pair are codon to index (codon pair score, CPS), represent single password Son is equal to its actual occurrence number in genome to the use Preference in genome, the CPS values of a codon pair The natural logrithm of ratio between theoretical occurrence number, formula is：

Codon is obtained by directly statistics to the actual occurrence number in the genome of pig, and its theoretical occurrence number Then need to calculate, be above formula I）, apply this algorithm exclude the frequency of occurrences of amino acid and password in genome The impact of the Preference of son.More than zero, the CPS values of one codon pair represent which is Preference codon in the genome of pig Right, and the minus codon of CPS values is to being then non-Preference codon pair.3,721 in the genome of application formula I calculating pigs Individual codon pair（Terminator codon pair is not included）CPS values.

Embodiment 1, the acquisition of restructuring PRRS viruses

First, the selection and transformation of target zone

In PRRSV reproduction processes, the RNA polymerase that the RNA of virus is relied on is that nsp9 plays central role；Nsp2 is Viral maximum replication protein, and difference is maximum between highly pathogenic PRRSV and America classical strainses；The present invention have chosen The gene of the nsp2 and nsp9 of PRRSV carries out codon and Preference is weakened, due to containing some and gene in each gene Group replicates relevant regulating and controlling sequence, and therefore, we choose the piece that tolerable is mutated on a large scale and lacks in above-mentioned 2 genes respectively Duan Jinhang is transformed（As shown in table 1）, that is, keep the amino acid sequence of albumen constant, carry out the displacement of synonym, often replace Codon, calculates the CPB values for once replacing post-fragment, to guarantee that the CPB values for replacing restrovirus fragment decline, applies this The CPB values of nsp2 and nsp9 specified segments are dropped to -0.2 or -0.4 by program respectively, with less than gene in pig genome CPB values（It is calculated between -0.2～0.3 being in normal distribution）.As the CPB values of gene are bigger, represent in this gene partially The codon of good property is bigger to shared proportion, conversely, the proportion of the codon pair of non-Preference is bigger；CPB values when gene Drop below gene in pig genome CPB Distribution value scopes when, then the expression of the gene can reduce, so as to reach reduction The purpose of virus.RNA structure software analysis PRRSV original segments are applied after completing all displacements with mutation post-fragment Secondary structure and sequence free energy, determine that the secondary structure of mutant fragments RNA does not occur larger change compared with original series.

The genetic fragment that table 1, PRRSV codons are weakened to Preference

2nd, the structure and virus rescue of infectious CDNA clones

1st, DNA fragmentation is artificial synthesized

It is to connect DNA fragmentation digestion into containing the high pathogenic strain HV full-length cDNAs of wild type PRRSV（No. Genbank is JX317648）Plasmid pcDNA3.1-HV（PcDNA3.1 is purchased from invitrogen companies, and the construction method of pcDNA3.1-HV is shown in Hereafter）Between corresponding restriction enzyme site, wild virus sequence is contained and including appropriate digestion identification sequence in artificial synthesized two ends respectively DNA sections shown in the sequence 24 of row, the position of the restriction endonuclease recognition sequence at its two ends are as follows：

Restriction endonuclease recognition sequence of the 3rd 8 of sequence 2 for HpaI（GTTAAC）, the of sequence 4 1683 1688 restriction endonuclease recognition sequences for MfeI（CAATTG）；

The 10th 21 of sequence 3 and the 10th 21 of sequence 4 restriction endonuclease recognition sequence for BstXI （CCANNNNNNTGG）, the 2238th 2243 of sequence 3 and the 2238th 2243 of sequence 4 is BclI Restriction endonuclease recognition sequence（TGATCA）；

2nd, the structure of infectious CDNA clones

By HpaI the and MfeI double digestions of the DNA fragmentation shown in sequence 2, DNA fragmentation 1-nsp2 is obtained；

By BstXI the and BclI double digestions of the DNA fragmentation shown in sequence 3, DNA fragmentation 2-1-nsp9 is obtained；

By BstXI the and BclI double digestions of the DNA fragmentation shown in sequence 4, DNA fragmentation 2-2-nsp9 is obtained；

DNA fragmentation 1-nsp2 is connected with the skeleton fragment of the plasmid pcDNA3.1-HV through HpaI and MfeI double digestions, Obtain recombinant plasmid P-nsp2；

Skeleton fragment phase by the DNA fragmentation 2-1-nsp9 and plasmid pcDNA3.1-HV through BstXI and BclI double digestions Even, recombinant plasmid P-nsp9 is obtained；

Skeleton fragment phase by the DNA fragmentation 2-2-nsp9 and plasmid pcDNA3.1-HV through BstXI and BclI double digestions Even, recombinant plasmid P-nsp9-2 is obtained；

Fragment 2-1-nsp9 is connected with the skeleton fragment of the recombinant plasmid P-nsp2 through BstXI and BclI double digestions, Obtain recombinant plasmid P-nsp29.

The construction method of pcDNA3.1-HV is：Full-length genome sequence to HV strains（Such as 1 institute of sequence in sequence table Show）It is analyzed, has picked out 6 restriction enzyme sites for sectionally smooth join full-length genome sequence（Respectively Smi I, Xho I, Afl II, Cla I, EcoR V and Not I；Wherein Xho I, Afl II, Cla I and EcoR V be original in genome, It is included in the sequence for amplifying；Smi I and Not I restriction enzyme sites are to separately design the piece of interpolation at viral genome two ends Section）.Full-length genome is divided into 5 fragments by 6 restriction enzyme sites, by the gene of the HV strains shown in the sequence 1 in sequence table Group full length sequence is cloned into eukaryotic expression vector pcDNA3.1（It is purchased from invitrogen companies）In, obtain pcDNA3.1- HV.Wherein, primer pair HP1F, HP1R for expanding（For amplified fragments 1）, HP2F, HP2R（For amplified fragments 2）, HP3F、HP3R（For amplified fragments 3）, HP4F, HP4R（For amplified fragments 4）With HP5F, HP5R（For amplified fragments 5）'s Sequence is as follows：

HP1F TAAATTTAA ATGACGTATAGGTGTTGGCTCT

HP1R GCGTGCGAGGTAACATCA

HP2F TTCTCCCAAAGATGATTCTCG

HP2R GGCAGCGGTACTTAAACAAG

HP3F GCCCTTAACAGAAACAGATGG

HP3R TACGACGGTAGATGCTCCTC

HP4F GGGAAGAAGAAGACTAGGACAAT

HP4R TCAGGGTGAACGGTAGAGC

HP5F GCCCTGTCATTGAACCAACT

HP5R TTATTAGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAATTAC

GGCCGCATGGTT

3rd, virus rescue

Recombinant plasmid P-nsp2, P-nsp9, P-nsp9-2 and P-nsp29 are transfected 293FT cells respectively（It is purchased from the U.S. ATCC cell banks）, after transfection, 72 hours multigelation cells collect cell pyrolysis liquid, will be thin with pig alveolar macrophage for cell pyrolysis liquid Born of the same parents（Abbreviation PAM）Nutrient solution mixing and continuously pass 3-5 generations, acquisition culture supernatant.The supernatant is taken, using the anti-PRRSV of mouse N protein specific antibody SDOW-17（It is purchased from Rural Technologies）To recombinating, PRRS viruses carry out indirect immunofluorescence Detection.As a result, recombinant plasmid P-nsp2, P-nsp9 and P-nsp29 is the positive, can successfully save out PRRSV in PAM； Recombinant plasmid P-nsp9-2, for feminine gender, i.e., can not save out PRRSV in PAM.

3rd, the measure of restructuring PRRS viruses one step growth curve

By high for wild type PRRSV pathogenic strain HV（Hereinafter referred to as HV-wt）, restructuring PRRS virus HV-nsp2, HV- Nsp9 and HV-nsp29 are respectively with MOI=0.01（When infecting, virus is 0.01 with the ratio of PAM cell quantities）Infection PAM, sense 12h after dye, 24h, 36h, 48h, 60h, 72h and 84h take a small amount of cells and supernatant and determine virus titer（As a result such as Fig. 1 and Biao 2 Shown）.Simultaneously observation of cell pathology take pictures under white light（Partial results are as shown in Figure 2）.

Above-mentioned virus titer method for measuring is specific as follows：

In 96 porocyte culture plates（Nutrient solution containing RPMI1640）Middle inoculation PAM cells, 37 DEG C, 5%CO₂Train in incubator Foster 24h；Virus liquid is made continuous 10 times of gradient dilution with RPMI1640 nutrient solutions, from 10 in 1.5ml EP pipes^-1To 10^-7； By the poison disease vaccination for having diluted in Tissue Culture Plate, each dilution factor is inoculated with 8 holes, is inoculated with 100 μ l per hole；If not being inoculated with disease 8 hole of normal cell controls of poison；12h, 24h, 36h, 48h, 60h, 72h and 84h after virus inoculation, takes in a small amount of cell culture Clearly, using the anti-PRRSV N proteins specific antibody SDOW-17 of mouse（It is purchased from Rural Technologies）PRRS viruses are carried out Indirect immunofluorescene assay, counts the quantity in virus-positive hole, calculates viral 50tissue infection dose in every milliliter of virus liquid (TCID₅₀/ml).

The method of above-mentioned virus breeding is as follows：

PAM is inoculated into Tissue Culture Flask（Nutrient solution containing RPMI1640）Middle adhere-wall culture 24h, sucks supernatant, accesses Virus liquid；37 DEG C adsorb 60 minutes, and period gently rocked blake bottle every 15 minutes, so that virus liquid is fully connect with PAM cells Touch；Then RPMI1640 nutrient solutions are added, and after 37 DEG C of culture 36h, frozen-thawed cell three times between -80 DEG C and room temperature, to crack Cell；Cell pyrolysis liquid is transferred to 50ml centrifuge tubes, 4 DEG C, 5000g be centrifuged 10 minutes, supernatant is virus liquid, by supernatant Liquid dispense to 1.5ml EP manage, be stored in -80 DEG C standby.

The virus titer result of different time after table 2, each restructuring PRRS viruses infection PAM（log₁₀（TCID₅₀/ml））

PRRSV	12h	24h	36h	48h	72h	84h
							HV-wt	0	4.5	5.83	6.94	7.11	7.11
HV-nsp2	0	4.61	5.61	6.39	6.72	6.5
							HV-nsp9	0	2.17*	3.17*	3.56*	3.72*	3.83*
HV-nsp29	0	2.55*	2.61*	3.17*	3.72*	3.89*

P<0.05 is significant difference

Table 2 and Fig. 1 show that the high pathogenic strain HV of wild type PRRSV is grown on PAM rapidly, 48 hours virus after infection Titre reaches peak value 10⁷TCID₅₀/ ml, restructuring PRRS virus replications speed are significantly lower than the high pathogenic strain of wild type PRRSV HV, 48 hours after infection PAM, recombinate PRRS virus HV-nsp2（That is the mutation of nsp2）Virus titer than wild type PRRSV High pathogenic strain HV is about low 3-5 times, and the PRRS virus HV-nsp9 that recombinate（That is the mutation of nsp9）With restructuring PRRS virus HV- nsp29（That is the mutation of nsp2 and nsp9）Higher than the wild type PRRSV pathogenic strain HV of virus titer have dropped 1,000-10, 000 times.

In addition, different time observation of cell pathology, the high pathogenic strain HV infection of discovery wild type PRRSV after infection PAM After PAM can be induced rapidly to crack, HV-nsp2 can also inducing moiety PAM cracking, but the HV-nsp9 and HV- of same amount Until 84 hours after nsp29 infection, there is not obvious pathology yet in cell（Fig. 2）.

The result of step 3 shows, compared with the high pathogenic strain HV of wild type PRRSV, password in the gene of nsp2 and nsp9 Son makes restructuring PRRS viruses to Preference reduction, and replication capacity all there occurs and be decreased obviously in vitro.

The pathogenicity of embodiment 2, restructuring PRRS viruses in pig body

The common great Bai of 16 4 healthy week old is buied from the negative pig farm of PRRSV and 2 type pig circular ring virus (PCV2) By ELISA and fluorescence real-time quantitative PCR, pig, further confirms that all pigs are PRRSV and PCV2 negative antibodies, PRRSV, pig Pestivirus (classical swine fever virus, CSFV), PCV2 and Pseudorabies virus (pseudorabies virus,PRV）Nucleic acid is negative.All pigs are randomly divided into 2 groups (8 per group) and collunarium inoculation poison amount is 10⁵TCID₅₀/ ml's Virus liquid 2ml, each nostril 1ml.The high pathogenic strain HV of wherein 1 group inoculation wild type PRRSV, another 1 group of pig are inoculated with recombinant virus HV-nsp29.

1st, after virus inoculation, daily whether observation piggy occurs coughing, has difficulty in breathing, apocleisis, diarrhoea, astasia, trembling Etc. clinical symptoms；Rectal temperature was monitored per two days, as a result as follows：

There is typical indigo plant otopathy symptom in the piggy of the high pathogenic strain HV of inoculation wild type PRRSV, sent out quickly including skin Dark purple, hyperpyrexia, expiratory dyspnea, cough, apocleisis, chemosis, tremble, astasia etc..High pathogenic in inoculation wild type PRRSV 6 days after strain HV, the rectal temperature of piggy all rises to 40 DEG C or so, 8 days after inoculation, rectal temperature continuously rise to 41 DEG C with On（Fig. 3）, their all death in 12 days after virus inoculation（Fig. 4）；

Inoculation HV-nsp29 virus piggy in, have 2 after infection 8-12 days rectal temperatures rise to 39.5 DEG C or so, Subsequently normal temperature range is dropped to（Between 37-39 DEG C）, fervescence is not occurred, in addition 3 in whole experiment process In all do not generate heat（Fig. 3）, all piggys all health survival terminate to experiment（Refer to postvaccinal 34 days）, postvaccinal during The state of mind is good, and fur is normal, and appetite is good, without death（Fig. 4）.

2nd, 4,8,12,16,24 and 34 days collection pig vena cava anterior blood after virus inoculation, collects serum, detects therein Virus titer, as a result as shown in Figure 5.4 days after virus inoculation, high pathogenic titres of the strain HV in serum of wild type PRRSV is Rise to 10⁶TCID₅₀/ ml, and in serum, only have a small amount of HV-nsp29 viruses, 12 days after virus inoculation, wild type PRRSV is high The serum-virus titre of pathogenic strain HV is further up, reaches 10^7.4TCID₅₀The titre of/ml, HV-nsp29 in serum is to the greatest extent Pipe also reaches peak value after inoculation for 12-14 days, but which only has 10^3.4TCID₅₀/ ml, pathogenic strain HV's higher than wild type PRRSV Titre have dropped about 10,000 times；The titre of HV-nsp29 is gradually reduced virus inoculation afterwards within 14 days, to after inoculation 34 days, The presence of viral HV-nsp29 has been nearly no detectable in serum.

In above-mentioned detection serum, the method for virus titer is as follows：

Every 250 μ l add 750 μ l Trizol (invitrogen), are blown and beaten with rifle repeatedly, are stored at room temperature 5 minutes；In above-mentioned EP Guan Zhong, adds 0.2ml chloroforms, covers EP lids, firmly shakes 15 seconds, be stored at room temperature 10 minutes, 12000g (4 DEG C) in hand Centrifugation 15 minutes；Take upper strata aqueous phase to be placed in new EP pipes, add 0.5ml isopropanols, be stored at room temperature 10 minutes, 12000g (4 DEG C) Centrifugation 10 minutes；Supernatant is carefully abandoned, plus 1ml75% ethanol is washed, (4 DEG C) of 7500g is centrifuged 5 minutes, abandons supernatant；Allow precipitation RNA spontaneously dry at room temperature；20 μ l DEPC water dissolving RNAs are eventually adding, with Nanodrop1000 (Thermo Scientific) quantitative RNA.

Take 5 μ l RNA and be RT-PCR.Add in 250 μ l PCR pipes 5 μ l RNA and 1 μ l Random Primer (10 μm, TaKaRa), mix；70 DEG C 5 minutes, put rapidly cooled on ice 2 minutes；Following components is added in said mixture：M-MLV5 × Reaction Buffer5 μ l, dNTP (2.5mM each) 5 μ l, Recombinant RNasin Ribonuclease Inhibitor25units, M-MLV RT (Promega) 200units, finally plus DEPC water polishings volume is to 25 μ l；Gently mix Even, 37 DEG C 60 minutes；PCR pipe is transferred to 70 DEG C, 15 minutes；Cooled on ice is placed in, cDNA products are stored in -20 DEG C.

Real-Time PCR are anti-using SYBR Premix Ex TaqTM II (Perfect Real Time) of TaKaRa Answer system.Premix Ex TaqTM (2 ×) 10 μ l, PCR primer ORF7F (AATAACAACGGCAAGCAGCA), The each 0.8 μ l of ORF7R (GCACAGTATGATGCGTCGGC) (10 μm), ROX Reference Dye II (50 ×) 0.4 μ l, cDNA 2 μ l of template, 6 μ l of aseptic double-distilled water, 20 μ l of cumulative volume.React on ABI7900Real-Time PCR System, take two Footwork PCR expands standardization program：95℃30s；95 DEG C of 5s, 60 DEG C of 30s (40 circulations).Experiment all contains one with gradient every time The 10 of dilution⁰–10⁷The calibration curve that TCID50/ml PRRSV particles are made, processes number with SDS2.2.2for7900 software analysis According to.

3rd, after 14 days, 3 piggys of per group of each cut open inspection wherein, are inoculated with the high pathogenic strain HV's of wild type PRRSV to virus inoculation Piggy occurs standing at which and stops the symptoms such as diet（Etc. referring to skin cyanosis, hyperpyrexia, expiratory dyspnea, cough, detest Food, chemosis, tremble, astasia）Shi Jinhang cut open inspections.The pathological change of each organ is observed during cut open inspection and is sampled, 1 part 10% neutral formalin of sample is fixed rear histotomy and carries out hematoxylin eosin stain analysis lesion tissue, another 1 part of sample Product be stored in after liquid nitrogen flash freezer -80 DEG C for extract total tissue RNA carry out real-time fluorescence quantitative PCR determine tissue in PRRS Viral RNA content（As a result as shown in fig. 6, wherein ordinate is the right of contained PRRSV RNA copy numbers in every μ g total serum IgEs Numerical value）.

The assay method of above-mentioned PRRS viral RNAs content is as follows：

Per 5mg, tissue plus 1ml Trizol (invitrogen) grindings, are blown and beaten with rifle repeatedly, are stored at room temperature 5 minutes；Upper State in EP pipes, add 0.2ml chloroforms, cover EP lids, firmly shake 15 seconds in hand, be stored at room temperature 10 minutes, 12000g (4 DEG C) are centrifuged 15 minutes；Take upper strata aqueous phase to be placed in new EP pipes, add 0.5ml isopropanols, be stored at room temperature 10 minutes, 12000g (4 DEG C) are centrifuged 10 minutes；Supernatant is carefully abandoned, plus 1ml75% ethanol is washed, (4 DEG C) of 7500g is centrifuged 5 minutes, abandons supernatant；Allow The RNA of precipitation is spontaneously dried at room temperature；20 μ l DEPC water dissolving RNAs are eventually adding, with Nanodrop1000 (Thermo Scientific) quantitative RNA.

Take 500ng RNA and be RT-PCR.500ng RNA and 1 μ l Random Primer are added in 250 μ l PCR pipes (10 μm, TaKaRa), mix；70 DEG C 5 minutes, put rapidly cooled on ice 2 minutes；Following components is added in said mixture： M-MLV5 × Reaction Buffer5 μ l, dNTP (2.5mM each) 5 μ l, Recombinant RNasin Ribonuclease Inhibitor25units, M-MLV RT (Promega) 200units, finally plus DEPC water polishing volumes To 25 μ l；Gently mix, 37 DEG C 60 minutes；PCR pipe is transferred to 70 DEG C, 15 minutes；Cooled on ice is placed in, cDNA products are preserved In -20 DEG C.

Real-Time PCR are anti-using SYBR Premix Ex TaqTM II (Perfect Real Time) of TaKaRa Answer system.Premix Ex TaqTM (2 ×) 10 μ l, PCR primer ORF7F (AATAACAACGGCAAGCAGCA), The each 0.8 μ l of ORF7R (GCACAGTATGATGCGTCGGC) (10 μm), ROX Reference Dye II (50 ×) 0.4 μ l, cDNA 2 μ l of template, 6 μ l of aseptic double-distilled water, 20 μ l of cumulative volume.React on ABI7900Real-Time PCR System, take two Footwork PCR expands standardization program：95℃30s；95 DEG C of 5s, 60 DEG C of 30s (40 circulations).Experiment all contains one with gradient every time The calibration curve that 100 107TCID50/ml PRRSV particles of dilution are made, processes number with SDS2.2.2for7900 software analysis According to.

Fig. 6 results show, in the heart, liver,spleen,kidney, brain, thymus gland, tonsillotome, bronchial lymph nodes, submandibular lymph nodes, abdomen stock In ditch lymph node and lymphonodi mesenterici, the viral RNA amount of HV-nsp29 is substantially lower than the high pathogenic strain of wild type PRRSV HV, especially in first target organs' lung of virus, the viral RNA amount of the high pathogenic strain HV of wild type PRRSV compares HV-nsp29 High 1,000 times.

In the piggy body of the high pathogenic strain HV of inoculation wild type PRRSV, obvious interstitial pneumonia is occurred in that, thymus gland is serious There are the pathology damages such as blood point in atrophy, enlargement of lymph nodes, and kidney.And piggy has no that any pathology becomes after being inoculated with HV-nsp29 Change.Hematoxylin eosin stain test is further characterized by the infection of the high pathogenic strain HV of wild type PRRSV and result in serious group Pathology damage is knitted, the alveolar membranes that cause of lymphocytic infiltration including lung are thickened（Hypertrophic interstitial pneumonia）；Brain, lung, kidney Deng the Perivascular cuffing that the lymphocytic infiltration of organ is caused（Vasculitis）；Kidney cell density becomes big, Destructurized, between cortex and medullary substance, boundary is obscured（Partial results are as shown in Figure 7）.But the piggy in HV-nsp29 infection In only observed slight Hypertrophic interstitial pneumonia, there is not serious inflammatory reaction in brain and thymus gland（Partial results are such as Shown in Fig. 7）.

Above-mentioned test result indicate that, pathogenic in piggy body of PRRS virus HV-nsp29 of recombinating is obviously reduced.

Embodiment 3, the protective effect of restructuring PRRS virus HV-nsp29

1st, the immunity of pig

The common great Bai of 12 4 healthy week old is buied from the negative pig farm of PRRSV and 2 type pig circular ring virus (PCV2) By ELISA and fluorescence real-time quantitative PCR, pig, further confirms that all pigs are PRRSV and PCV2 negative antibodies, PRRSV, pig Pestivirus (classical swine fever virus, CSFV), PCV2 and Pseudorabies virus (pseudorabies virus,PRV）Nucleic acid is negative.All pigs are randomly divided into 3 groups (4 per group), wherein two groups are taken as immune group（Name respectively For A groups and B groups）Nasal immunization poison amount is 10 respectively⁵TCID₅₀The recombinant virus HV-nsp29 virus liquid 2ml of/ml, each nostril 1ml, remaining one group is control group（It is named as CK groups）Not immune, cultivate under equal conditions.

2nd, poison is attacked

42 days after immunity, it is 2 × 10 by A groups and each piggy collunarium inoculation poison amount of CK groups⁶TCID₅₀Wild type PRRSV of/ml The virus liquid 2ml, each nostril 1ml of high pathogenic strain HV；It is 2 × 10 by each for B groups piggy collunarium inoculation poison amount⁶TCID₅₀/ ml's (No. Genbank is JX317649 to the high pathogenic strain JX of wild type PRRSV, and the public can be obtained from China Agricultural University）Virus liquid 2ml, each nostril 1ml.

3rd, the identification that attacks after poison

1）From the beginning of the attacking after poison of step 2, whether observation piggy occurs coughing, has difficulty in breathing, apocleisis, diarrhoea, standing daily The vertical unstable, clinical symptoms such as tremble；Rectal temperature was monitored per two days；As a result as follows：

In CK groups, after the high pathogenic strain HV of inoculation wild type PRRSV, still there is serious blue otopathy symptom；Immune group In, no matter attack with homologous strain HV, or heterologous strain JX, well, appetite is normal for piggy all state of mind, the work of health Terminate to experiment（Fig. 8）, do not occur generating heat in experimentation（Fig. 9）, skin cyanosis, the clinical symptoms such as apocleisis；

2）Attacking 4,8,12 days after poison from step 2, gathers vena cava anterior blood, separates serum, detects the virus titer of serum （Method is identical with the step 2 in embodiment 2）, as a result as shown in Figure 10, after recombinant virus HV-nsp29 immunity, homologous poison The titre of strain and heterologous strain is all very low, and only about 10³TCID₅₀/ ml, lower than in CK group by about 1,000 times.

3）Attacking after poison from step 2, when standing occurs in piggy and stops the symptoms such as diet（Etc. refer to skin send out Dark purple, hyperpyrexia, expiratory dyspnea, cough, apocleisis, chemosis, tremble, astasia）Shi Jinhang cut open inspections, remaining health pig are supported To attacking 14 days cut open inspections after poison.Observe the pathological change of each organ during cut open inspection and sample, be stored in after liquid nitrogen flash freezer -80 DEG C with The PRRS viral RNA contents in real-time fluorescence quantitative PCR measure tissue are carried out in extraction total tissue RNA, method is with embodiment 2 Step 3, as a result as shown in figure 11（Wherein ordinate is the logarithm value of contained PRRSV RNA copy numbers in every μ g total serum IgEs）, In each tissue, the virus quantity in A groups and B groups after recombinant virus HV-nsp29 immunity is substantially lower than CK groups.

4th, the identification that attacks before poison

1）Serum NAT is determined

After immunity 8,16,24,34 days, vena cava anterior blood is gathered, separates serum, detect serum NAT, As a result as shown in figure 12,8 days after immunity, a small amount of neutralizing antibody in serum, occurs, prolongation over time, neutralizing antibody Titre gradually rise.

The concrete grammar that above-mentioned serum NAT is determined is as follows：

By test serum in 56 DEG C of heat inactivations, 2 times of gradient dilutions are carried out with RPMI1640 nutrient solutions, the volume after dilution is 50 μ l, then respectively with 50 μ l containing 100TCID₅₀The nutrient solution of the high pathogenic strain HV of wild type PRRSV be incubated 60 at 37 DEG C Minute, the 100 μ l of mixed liquor after this is incubated are seeded in 96 orifice plates containing pig PAM, after 48 hours, using the anti-PRRSV of mouse N protein specific antibody SDOW-17（It is purchased from Rural Technologies）Indirect immunofluorescene assay is carried out to PRRS viruses, The quantity in statistics virus-positive hole, calculates virus 50tissue infection dose (TCID in every milliliter of virus liquid₅₀/ml).When virus half Array is knitted infective dose and is compareed（Without the cell culture well that increase serum only connects same amount of virus）Compare minimizing 90% when be considered as by Neutralization, NAT take log₂Value is represented.

2）Specific C D8 in Flow cytometry serum⁺The reaction of positive T cell

34 days after immunity, take 3 pigs from CK groups and A groups at random, extract peripheral blood respectively, separate lymphocyte, Lymphocyte is stimulated again 6 hours at 37 DEG C as antigen with strain HV or JX (10 μ g/ml) respectively again, be subsequently adding Concentration is that the Protein transport inhibitor Brefeldin A of 10 μ g/ml act on the secretion with closing cell's factor in 2 hours.400g from The heart collects lymphocyte cell in 5 minutes, and the antibody or isotype control Ab of the anti-CD8 α marked with FITC dye 30 points at 4 DEG C Clock.PBS washes cell and once fixes cell 20 minutes with 4% paraformaldehyde room temperature afterwards, and 400g is centrifuged 5 minutes and collects cell.Add The antibody or isotype control Ab of the anti-IFN-γ of the RPE marks diluted with 0.1% saponin is in 4 DEG C of stained over night.Will after washing Cell is resuspended in 0.5% paraformaldehyde and uses flow cytomery, as a result uses FlowJo software analysis, as a result as shown in figure 13（Refer to that blood sampling detached cell HV and JX stimulates in the never immune pig body that crosses.The percent value in the upper right corner is represented IFN-γ and the double positive cells of CD8+ account for the percentage of total cell.）.

Compared with control group, after immunity, stimulating again for homologous and heterologous antigen can induce obvious IFN-γ reaction. This explanation, the immunity of HV-nsp29 can induce piggy to produce protective immune response.

The result explanation of step 14, the immunity of HV-nsp29 can protect animal for homologous and heterologous highly pathogenic The subinfection again of PRRSV.

Embodiment 4, restructuring PRRS viruses have genetic stability and extremely low return strong possibility

As the greatest problem of existing PRRSV vaccines is which has and returns strong possibility, therefore exploitation has heredity steady Qualitative and be easy to street strain distinguish PRRSV attenuated vaccines of new generation extremely urgent.Our applied cryptographies are sub to weakening hand Section causes weak highly pathogenic PRRSV, its cause weak during have hundreds of point mutation, this allow its by the method for sequencing with Wild type strains make a distinction.Simultaneously as HV-nsp29 cause weak caused by hundreds of point mutation, in theory for it It is extremely low to return strong possibility, and is confirmed in further experiment, and specific experiment method and result are as follows：

To HV-nsp29 continuous passages on PAM cells, keep connecing poison amount every time unanimously in succeeding generations, in virus breeding During observation of cell pathology, continuously passed to for the 20th generation.In succeeding generations, the replication capacity of HV-nsp29 viruses and induction are thin There is no significant change in the ability of born of the same parents' pathology（Partial results are as shown in figure 14）；

Nsp2 and nsp9 genes in the HV-nsp29 in the 10th generation and the 20th generation are sequenced, with first generation HV-nsp29 Sequence alignment find that nsp2 the and nsp9 genes in the 10th generation and the 20th generation do not occur back mutation, calculate theirs respectively CPB values find that, compared with the first generation sequence, CPB values almost do not change（Figure 15）.

As a result show, HV-nsp29 have genetic stability and extremely low return strong possibility, weak with PRRSV is developed into The potentiality of malicious vaccine.

Claims (8)

1. a kind of restructuring PRRS is viral, it is characterised in that：The genome cDNA sequence of restructuring PRRS viruses is by sequence table sequence Row 1 replace with sequence of the sequence 2 from 5 ' end the 814th 1395 from the sequence of 5 ' end the 1474th 2055； And sequence 1 is replaced with sequence 3 from 5 ' ends the 158th from the sequence of 5 ' end the 7683rd 9527 The sequence obtained by the sequence of 2002.

2. application of the virus in PRRS vaccines are prepared described in claim 1.

3.DNA fragments, its sequence are that sequence 1 is replaced with sequence table sequence from the sequence of 5 ' end the 1474th 2055 Sequence of the row 2 from 5 ' end the 814th 1395；And sequence 1 is replaced from the sequence of 5 ' end the 7683rd 9527 It is changed to what sequence 3 was obtained from the sequence of 5 ' end the 158th 2002.

4. application of the DNA fragmentation described in claim 3 in restructuring PRRS viruses described in claim 1 are prepared.

5. application of the DNA fragmentation described in claim 3 in PRRS vaccines are prepared.

6. the recombinant plasmid, recombinant bacterium or recombinant cell containing DNA fragmentation described in claim 3.

7. application of the recombinant plasmid described in claim 6 in restructuring PRRS viruses described in claim 1 are prepared.

8. application of the recombinant plasmid described in claim 6 in PRRS vaccines are prepared.