CN104593336B - Porcine reproductive and respiratory syndrome embedded virus RvHBJX and its application - Google Patents

Abstract

The present invention provides a kind of porcine reproductive and respiratory syndrome embedded virus RvHBJX and its applications, belong to viral genetic engineering technical field.Porcine reproductive and respiratory syndrome embedded virus RvHBJX of the invention is the embedded virus using low pathogenicity PRRSV strain HB-1/3.9 as skeleton comprising the structural protein coding region highly pathogenic PRRSV strain JXwn06, the embedded virus is stablized through MARC-145 cells in vitro continuous passage 80 times, viral genetic property performance；Virus gradually increases the adaptability of host cell with the increase of passage number；Embedded virus, which passes on 60 times (P60) or more, has preferable safety to ontology animal; and it can be to the immunoprotection of highly pathogenic PRRSV virus strain infection offer 100% after being inoculated with; it can be used as the vaccine candidate strain of porcine reproductive and respiratory syndrome; for preventing porcine reproductive and respiratory syndrome, have a good application prospect.

Description

Porcine reproductive and respiratory syndrome embedded virus RvHBJX and its application

Technical field

The present invention relates to viral genetic engineering technical fields, and in particular, to a kind of porcine reproductive and respiratory syndrome is chimeric Viral RvHBJX and its application.

Background technique

Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, It PRRS is) a kind of highly contagious disease as caused by porcine reproductive and respiratory syndrome virus (PRRSV), mainly with farrowing sow Miscarriage, premature labor, the production breeding difficulty diseases such as stillborn foetus and the mummification of fetus and piglet occurs and respiratory tract disease occurs in growing-finishing pig Shape is characterized (Rossow et al., 1994).Since early summer in 2006, Severe PRRS is broken out, and it is main to sweep across China rapidly Hog area causes large quantities of live pigs dead, causes huge economic losses to China's pig breeding industry.Vaccine is considered as prevention and control and purification The important tool of PRRSV.But PRRSV infection can cause immunosupress and persistent infection, the long-time service of attenuated live vaccine There are virulence to return the potential risks such as strong (Madsen et al., 1998).Infection clones technology provides for the research of new generation vaccine Technical guarantee.Since the infection clones of first plant of PRRSV successfully construct (Meulenberg et al., 1998), instead It has been used for analyzing and studying regulatory mechanism, the cause that the gene structure of PRRSV is synthesized with function, viral RNA to Genetic Manipulative Technology The research of interpretation of the cause, onset and process of an illness system and new generation vaccine.

It is, in general, that carrying out immunity energy with the high strain of homology obtains preferable resistance.Therefore by popular strain Perhaps, certain gene substitutions can improve the immune efficacy of vaccine into traditional vaccine.Ellingson et al. utilization MLV vaccine virus, MN184 strain is that parental virus constructs 5 embedded viruses, and two of them are MLV vaccine virus, 5 ' UTR/ORF1 of MN184 strain The virus of phase double replacement, one is the embedded virus that MN184ORF5-6 has been replaced using MLV as skeleton, and one is using MLV as skeleton The embedded virus of MN184ORF7-3 ' UTR is replaced, one is the chimeric disease that 3 ' UTR of MN184 strain has been replaced using MLV as skeleton Poison.Challenge viral dosage after these embedded viruses are immune shows the disease of MLV vaccine virus, 5 ' UTR/ORF1 phase double replacement of MN184 strain Poison and generation can be induced similar to conventional cell culture vaccine by the embedded virus that skeleton has replaced MN184ORF5-6 of MLV Resistance.Lee et al. has been replaced in infectious CDNA clones plasmid with the structural protein coding region of South Korea PRRSV prevalence strain Corresponding region and save out corresponding PRRSV embedded virus, which can be neutralized by the serum of natural infection pig, accordingly In vivo studies not yet carry out.These results suggest that come development of new vaccine being feasible by embedded virus.

Previous isolated strain PRRSV HB-1 (sh)/2002 (GenBank indexed number AY150312) (the Gao et in China Al., but pathogenic low 2004), with the homology highest (97.2%) of highly pathogenic strain on whole genome sequence.The poison Strain obtains the clonal strain HB-1/3.9 that can be well proliferated in MARC-145 cell through MARC-145 cell continuous passage (GenBank indexed number EU360130) (Ran Zhi light etc., 2006), the whole genome sequence homology with highly pathogenic strain are 97.4%.The two strain genome very high homologies and biological characteristics, such as virulence, pathogenic, growth characteristics, immunogenicity It differs greatly.

Summary of the invention

The purpose of the present invention is to provide a kind of porcine reproductive and respiratory syndrome embedded viruses；Another object of the present invention exists In providing purposes of the embedded virus as chimeric vaccine candidates.

Present invention firstly provides a kind of porcine reproductive and respiratory syndrome embedded viruses, are named as RvHBJX, are with low cause Characteristic of disease PRRSV strain HB-1/3.9 is skeleton, the chimeric disease containing the structural protein coding region highly pathogenic PRRSV strain JXwn06 Poison.

Wherein, the gene order of the structural protein coding region highly pathogenic PRRSV strain JXwn06 such as SEQ ID NO.1 institute Show.

The present invention provides porcine reproductive and respiratory syndrome embedded virus RvHBJX full length cDNA clone plasmid, be by with Lower method constructs to obtain:

(1) using pWSK-JXwn as template, PCR amplification obtains the total protein-coding region of JXwn06, amplified production life Entitled HJSP-S2；

(2) using pWSK-HB-1/3.9 as template, the structural protein coding region PCR amplification HB-1/3.9 two sides sequence, divides respectively HJSP-S1, HJSP-S3 are not named as it；

(3) by after step (1) and the amplified production purification and recovery of (2), with HJSP-S1, HJSP-S2, HJSP-S33 piece Duan Zuowei template, PCR amplification obtain HJSP-S123；

(4) pWSK-HB-1/ of identical double digestion is connected to through Rsr II, Asc I double digestion after HJSP-S123 purifying In 3.9 plasmids, the building of PRRSV chimeric plasmid is completed, pW-HJSP is named as.

Wherein, the primer that step (1) PCR amplification uses is JX-S2F and JX-S2R, nucleotide sequence such as SEQ ID NO.2, shown in 3；

Step (2) PCR amplification is respectively using HB-S1F, HB-S1R and HB-S3F, HB-S3R as primer, nucleotide sequence point Not as shown in SEQ ID NO.4-7；

The primer of step (3) PCR amplification is HJ-fusionF and HJ-fusionR, nucleotide sequence such as SEQ ID Shown in NO.8-9.

The present invention also provides the method for preparing porcine reproductive and respiratory syndrome virus embedded virus RvHBJX, including it is following Step:

(1) porcine reproductive and respiratory syndrome embedded virus RvHBJX full length cDNA clone plasmid is constructed；

(2) rescue of embedded virus RvHBJX.

In the above method, the cloned plasmids of step (1) construct by the following method to be obtained:

1) using pWSK-JXwn as template, PCR amplification obtains the total protein-coding region of JXwn06, amplified production life Entitled HJSP-S2；

2) using pWSK-HB-1/3.9 as template, the structural protein coding region PCR amplification HB-1/3.9 two sides sequence, divides respectively HJSP-S1, HJSP-S3 are not named as it；

3) after the amplified production purification and recovery by step 1) and 2), with HJSP-S1, HJSP-S2, HJSP-S33 segment is made For template, PCR amplification obtains HJSP-S123；

4) pWSK-HB-1/3.9 of identical double digestion is connected to through Rsr II, Asc I double digestion after HJSP-S123 purifying In plasmid, the building of PRRSV chimeric plasmid is completed, pW-HJSP is named as.

Whole genome sequence measurement shows that structural protein coding region is consistent with JXwn06 in pW-HJSP plasmid, and remaining bone Frame is consistent with HB-1/3.9, while two genetic marker Mlu I of HB-1/3.9 parent's Revive virus and Sfi I restriction enzyme site are still So exist.

Further, the primer that PCR amplification uses in step 1) is JX-S2F and JX-S2R, nucleotide sequence such as SEQ ID NO.2, shown in 3；PCR amplification is respectively using HB-S1F, HB-S1R and HB-S3F, HB-S3R as primer in step 2), nucleosides Acid sequence is respectively as shown in SEQ ID NO.4-7；The primer of PCR amplification is HJ-fusionF and HJ-fusionR in step 3), Its nucleotide sequence is as shown in SEQ ID NO.8-9.

In the method for preparing porcine reproductive and respiratory syndrome virus embedded virus RvHBJX of the invention, step (2) it is embedding The rescue of combination of syndromes poison RvHBJX is realized by the following method:

1) Rsr II, Pac I endonuclease digestion pW-HJSP full length cDNA clone plasmid are used, plasmid linearization is made；It obtains For linearization plasmid after phenol chloroform and dehydrated alcohol precipitating, drying, ultraviolet specrophotometer measures the dense of purified plasmid Degree；

2) it is transcribed in vitro immediately；

3) transcription product is after TURBO DNase I digestion, with DMRIE-C liposome transfection MARC-145 cell, daily Observe and record the production of cytopathy CPE；The cells and supernatant for obvious CPE occur is enterprising in MARC-145 cell Row continuous passage, per generation 3-5d.The Revive virus of acquisition is RvHBJX.

In an embodiment of the present invention, above-mentioned steps 2) in-vitro transcription be according to mMessage What High Yield Capped RNA Transcription kit (Ambion Inc., Texas, USA) operating instruction carried out.

The present invention provides a kind of porcine reproductive and respiratory syndrome chimerics, breed and exhale containing pig provided by the invention Virus after inhaling syndrome embedded virus RvHBJX or its secondary culture.

The present invention provides the viruses after porcine reproductive and respiratory syndrome embedded virus or its secondary culture to prepare vaccine In purposes.

Present invention discover that embedded virus RvHBJX, on MARC-145 cell after continuous passage 80 times, virus titer is by the 5th The 10 of generation^4_.5TCID₅₀/ mL is increased to 10^7_.51TCID₅₀/mL.By multiple continuous subculture in vitro separately, embedded virus it is cell adapted Ability significantly improves.

The present invention compares it to highly pathogenic by selection RvHBJXP40, P60, P80 strain piglet infected experimentally, analysis The immune protective of PRRSV infection, as a result, it has been found that PRRSV is fitted into strain RvHBJX P40, P60 and P80 cell passage strain is exempted from The clinical symptoms that infection piglet can be mitigated after epidemic disease piglet reduce the virus load in infection piglet serum, significantly reduce piglet The dead quantity, mitigate virus infection caused by lung injury.The embedded virus is through MARC-145 cells in vitro continuous passage 80 Secondary, viral genetic property performance is stablized；Virus gradually increases the adaptability of host cell with the increase of passage number； PRRSV, which is fitted into strain RvHBJX P60 passage 60 times (P60) or more, has preferable safety to ontology animal, and after inoculation The immunoprotection that 100% can be provided highly pathogenic PRRSV virus strain infection, can be used as the vaccine of porcine reproductive and respiratory syndrome Candidate Strain exploitation.

Testing and attack malicious protectiveness test through animal safety proves, present invention porcine reproductive and respiratory syndrome obtained It will not cause apparent clinical symptoms after chimeric HBJX inoculation piglet, the safety used as vaccine is preferable；And it can Preferable immunoprotection is provided for infection piglet, mitigates highly pathogenic PRRSV infection bring harm.The virus of the chimeric Skeleton derives from the natural low pathogenicity PRRSV HB-1/3.9 strain being clinically separated, and avoids attenuated vaccine strain virulence and easily returns by force Risk, meanwhile, the highly pathogenic PRRSV strain structural protein coding region being placed in strain can also stimulate body after inoculation The neutralizing antibody for popular strain is generated, the immune protection effectiveness of the vaccine is improved.In addition, in chimeric HBJX sequence Antidiastole of 2 genetic markers being artificially introduced also for vaccine strain and natural infection strain provides clear target spot, this is chimeric The application of vaccine is beneficial to the purification of China swinery PRRS.What the construction method of embedded virus provided by the present invention obtained saves Rescue virus can continuous passage in vitro, genetics characteristic is relatively stable.

Detailed description of the invention

Fig. 1 is that embedded virus RvHBJX full length cDNA clone plasmid pW-HJSP constructs schematic diagram, and wherein white box represents The gene of JXwn06 strain；Grey box represents the gene of HB-1/3.9 strain；Restriction enzyme site with " * " is genetic marker, and 1 is The structural protein coding region JXwn06,2 be the structural protein coding region HB-1/3.9 flank.

Fig. 2 is the CPE (200 ×) that embedded virus RvHBJX is generated on MARC-145 cell, and wherein A figure is RvHB-1/ 3.9；B figure is RvJXwn；C figure is RvHBJX；D figure is negative control.

The IFA that Fig. 3 is embedded virus RvHBJX identifies (200 ×), and wherein A figure is RvHBJX；B figure is RvHB-1/3.9；C Figure is RvJXwn；D figure is negative control.

Fig. 4 is proliferation dynamics figure of the embedded virus RvHBJX on cell, and wherein A figure is on MARC-145 cell；B figure For on PAM cell.

Fig. 5 is body temperature and the daily gain variation for infecting piglet, and Fig. 5 A is infection piglet Temperature changing；Fig. 5 B infects piglet day Weight gain variation.

Fig. 6 is virus antigen detection result immunohistochemistry figure in embedded virus infection piglet lungs, lymphoodi mandibulares, wherein A is P5 infected group lungs；B is P20 infected group lungs；C is P40 infected group lungs；D is control group lungs；E is under P5 infected group Jaw lymph node；F is P20 infected group lymphoodi mandibulares；G is P40 infected group lymphoodi mandibulares；H is control group lymphoodi mandibulares.

Fig. 7 is piglet Temperature changing and symptom scores situation map after attacking poison, and wherein Fig. 7 A is to attack piglet body temperature after poison Variation diagram；Fig. 7 B is to attack piglet symptom scores after poison.

Fig. 8 is the survival rate that piglet is tested after attacking poison.

Fig. 9 is the multigraph that increases day by day of piglet before and after attacking poison.

Figure 10 is the virus titer after attacking poison in piglet serum.

Specific embodiment

Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to Range.

MARC-145 cell of the present invention DMEM culture medium culture.CDNA grams of wild type full-length of HB-1/3.9 strain Grand carrier pWSK-HB-1/3.9, JXwn06 strain wild type full-length cDNA clone carrier pWSK-JXwn, clonal strain RvHB-1/ 3.9 and RvJXwn be disclosed in document (Zhou L, Zhang J, Zeng J, Yin S, Li Y, Zheng L, Guo X, Ge X, Yang HC.2009.The 30-amino-acid deletion in the Nsp2 of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China is not related to its virulence.J Virol.83(10):5156-5167)；PEASY Blunt cloned plasmids and large intestine Bacillus Trans5 α is purchased from Beijing Quanshijin Biotechnology Co., Ltd；PRRSV N protein monoclonal antibody N35 deposit number For CGMCC NO.3238, it is disclosed in Chinese patent CN101661042B.

The building of 1 embedded virus full length cDNA clone plasmid of embodiment

It is complete with PRRSV JXwn06 with reference to PRRSV HB-1/3.9 whole genome sequence (GenBank indexed number EU360130) Genome sequence (GenBank indexed number EF641008), design primer are fitted into strain RvHBJX full length cDNA clone for PRRSV Building (table 1), primer closes by Invitrogen hero's life technology Co., Ltd, the U.S. (Invitrogen, Beijing, China) At being made into the water of nuclease free using concentration.

Primer is used in the building of 1 HBJX full length cDNA clone of table

Note: (it is used to increase the protectiveness alkali of digesting efficiency including two sides for restriction endonuclease recognition sequence at horizontal line Base)；Primer sequence is write with capitalization, indicates to carry out PCR amplification by template of HB-1/3.9, corresponding position is represented in HB- Position in 1/3.9 genome；Primer sequence is write with lowercase, indicates to carry out PCR amplification by template of JXwn06, accordingly Position represents the position in JXwn06 genome.

The construction strategy of embedded virus RvHBJX full length cDNA clone plasmid is shown in Fig. 1.Use PfuUltra^TMII fusion HS DNA Polymerase (high-fidelity DNA polymerase) (Stratagene, La Jolla, CA) expands target fragment, according to product Specification constructs 50 μ L reaction systems, amplification condition are as follows: 95 DEG C of 2min, 95 DEG C of 30s, 56-58 DEG C of 30s, 72 DEG C of 2min.Wherein, Using pWSK-JXwn as template, JX-S2F/JX-S2R is primer, expands the total protein-coding region of JXwn06, is named as HJSP-S2；Using pWSK-HB-1/3.9 as template, HB-S1F/HB-S1R, HB-S3F/HB-S3R are primer, expand HB-1/ respectively 3.9 structural protein coding region two sides sequences, are named as HJSP-S1, HJSP-S3.Amplified production is through 0.8% agarose gel electrophoresis After inspection, with EasyPure Quick Gel Extraction Kit (Beijing Quanshijin Biotechnology Co., Ltd, Beijing, in State) kit progress purification and recovery.With HJSP-S1 after the recovery, HJSP-S2, HJSP-S33 segment is as template, HJ- FusionF/HJ-fusionR is primer, expands HJSP-S123.Through Rsr II, Asc I double digestion after HJSP-S123 purifying, even It is connected in the pWSK-HB-1/3.9 plasmid of identical double digestion, connection product is transformed into Trans5 α competent cell, bacterium colony PCR mirror Order clone bacterium, and serve the handsome Bioisystech Co., Ltd in sea and carry out sequencing.Sequencing verifies correct plasmid life Entitled pW-HJSP.The extraction of plasmid uses EasyPure Plasmid MiniPrep Kit (Quan Shijin, Beijing, China) reagent The recombinant plasmid of box, acquisition measures concentration with ultraviolet specrophotometer after PCR and digestion identification, sets -20 DEG C of preservations.

Whole genome sequence measurement shows that structural protein coding region is consistent with JXwn06 in pW-HJSP plasmid, and remaining bone Frame is consistent with HB-1/3.9, while two genetic marker Mlu I of HB-1/3.9 parent's Revive virus and Sfi I restriction enzyme site are still So exist, illustrates that structural protein coding region is successfully replaced by JXwn06 corresponding region in HB-1/3.9 genome.

The rescue of 2 embedded virus RvHBJX of embodiment

Take pW-HJSP full length cDNA clone plasmid made from 15 μ g embodiments 1, with Rsr II, Pac I (NEB, Beijing, in State) endonuclease digestion, 37 DEG C of water-baths are overnight, make closed circular form plasmid total Linearization.It is added phenol chloroform 2-3 times, dehydrated alcohol It precipitates, after centrifugal drying, adds 10 μ L to dissolve without RNase water (Ambion, Texas, USA) and precipitate, ultraviolet specrophotometer measurement Plasmid concentration after purification.Immediately with Ambion company High Yield Capped RNA Transcription kit is transcribed in vitro.After 37 DEG C of water-bath 2h, 1 μ L TURBO DNase I is added and mixes, instantaneously 37 DEG C of incubation 15min after centrifugation, to digest template plasmid DNA.

Select that form is normal, eugonic MARC-145 cell, after pancreatin digestion, with about 10⁶The cell density of a/mL It is transferred to six porocyte culture plates, every hole 2mL.37 DEG C, 5%CO₂, overnight incubation under saturated humidity, until forming 80% or so Cell monolayer.Careful that cells and supernatant is sucked out, every hole adds 2mL to balance to room temperature I Reduced Serum Medium (Invitrogen Corporation, NY, USA) is washed 1-2 times；To the 1.5mL of no RNase 1mL is added in eppendorf pipe to balance to room temperatureI Reduced Serum Medium；By DMRIE-C Liposome (Invitrogen Corporation, NY, USA), which is vortexed, to be mixed, and takes 10 μ L to be added in each pipe, and the mixing that is vortexed；To It is separately added into 10 μ L full length mRNAs in pipe, is slightly briefly vortexed and mixes；Liposome-RNA mixture is transferred to immediately and is covered with 80% or so cell monolayer, uniform fold；Two wild types conducts pair of pWSK-HB-1/3.9 and pWSK-JXwn are set up simultaneously According to.CO₂After 37 DEG C of culture 4h of incubator, transfection liquid is carefully sucked out, the growth medium of 10% serum content of 2mL is added；Daily Cell is observed, after there is obvious cytopathy (CPE), takes culture supernatant to be inoculated with the MARC-145 cell covered with, continuous passage is extremely 12 generations, per generation 3-5d；And CPE is observed daily.

After transfecting 2-3d, there is the typical CPE of PRRSV in 3 groups of cells.By rescue using HB-1/3.9 as skeleton, The chimeric strain for having replaced JXwn06 structural proteins is named as RvHBJX, i.e. chimeric strain.Embedded virus is thin in MARC-145 The CPE situation occurred after the upper continuous passage of born of the same parents 3 times, is shown in Fig. 2.

Expression of the 3 JXwn06 structural proteins of embodiment in chimeric strain RvHBJX

To the 3rd generation virus of embedded virus RvHBJX carry out gene sequencing, the results show that its structural protein coding region with JXwn06's is consistent, and gene delection or mutation do not occur.

N protein is the non-glycosylated structural proteins that PRRSV genome ORF 7 encodes.By RvHBJX, RvHB-1/3.9, RvJXwn P3 generation virus is inoculated with MARC-145 cell respectively, carries out immunofluorescent test with PRRSV N protein monoclonal antibody, observation is set Change expression of the albumen in embedded virus.Visible bright yellow-green fluorescence in the cytoplasm of embedded virus RvHBJX infection, Identical as parent's Revive virus RvHB-1/3.9, RvJXwn, control cell is without visible fluorescence (Fig. 3).Show the structure of insertion Albumen can be expressed correctly in chimeric strain.

The drafting of 4 embedded virus RvHBJX one step growth curve of embodiment

By RvHBJXP3 strain and parent's Revive virus RvHB-1/3.9, RvJXwn with 0.01MOI dose inoculation MARC- 145 and PAM cell takes supernatant to measure its TCID every 12h₅₀Value draws virus in the one step growth curve of host cell.

RvHBJX 96h after infecting MARC-145 cell reaches peak of proliferation, saves strain RvJXwn (72hpi) compared with parent Delay for 24 hours, is shown in the A figure of Fig. 4, but the virus titer difference of each phase is not significant (P > 0.05)；In infection 36h, virus drop Degree is significantly higher than always RvHB-1/3.9 (P < 0.05).

Each Revive virus 48h after infecting PAM cell reaches peak of proliferation, sees the B figure of Fig. 4.RvHBJX is early in infection The growth rate ratio RvHB-1/3.9 of phase is fast, and is in significant difference (P < 0.05) in pi for 24 hours.Each Revive virus is infecting The proliferative capacity of early stage is RvJXwn > RvHBJX > RvHB-1/3.9 after PAM.The above result shows that chimeric strain RvHBJX exists There is proliferation dynamics curve identical with parent's strain, virus multiplication capacity variance is little on different host cells.

The external continuous reception and registration and its genome mutation analysis of 5 embedded virus RvHBJX of embodiment

Chimeric strain RvHBJX P3 kind poison 1mL is inoculated in well-grown MARC-145 cell monolayer, 37 DEG C of incubation 1h 5%DMEM culture solution is added afterwards, cultivates 3-5d, until about 80% cell occurs, harvest is viral when CPE, multigelation 3 times；Take cell Freeze thawing liquid 1mL repeats above step, is passed on, until reaching 80 generations (P80).

The TCID of chimeric strain P5-P80 is measured respectively₅₀, and choose P5, P10, P20, P30, P40, P60 and P80 and carry out entirely Genome sequence determination, genome sequencing are shown in Table 2 with primer sequence, and primer is closed by Shanghai Ying Jun Bioisystech Co., Ltd At.The target gene that amplification obtains send Hua Da gene sequencing.With DNASTAR, the biological informations such as Clustaxl, Genedoc are soft Part analyzes the genomic sequence of each passages strain of embedded virus and the amino acid sequence of derivation.

2 HBJX whole genome sequence amplimer of table

Virus titer measurement result is shown in Table 3.Embedded virus RvHBJX is on MARC-145 cell after continuous passage 80 times, disease Malicious titre is by the 10 of the 5th generation^4.5TCID₅₀/ mL is increased to 10^7.51TCID₅₀/mL.By multiple continuous subculture in vitro separately, it is fitted into disease The cell adapted ability of poison significantly improves.

3 PRRSV of table is fitted into the titer determination (TCID of strain RvHBJX difference passages strain₅₀/mL)

The PRRSV amino acid sequence for being fitted into each generation the genomic sequence of strain HBJX and its derivation is compared It is right.The structural protein coding region P3 is consistent with JXwn06, in addition to two genetic markers containing HB-1/3.9, remaining gene with HB-1/3.9 is identical；Point mutation occurs for 12258 nucleotide of the P5 in ORF2a, is same sense mutation；Base of the P10 in P5 point mutation There are 4 nucleotide that point mutation occurs again on plinth, wherein 2 are same sense mutation；P20 has 6 cores again on the basis of P10 point mutation Point mutation occurs for thuja acid, wherein 3 are same sense mutation；P30 has 1 point that point mutation, P40 occurs on the basis of P20 point mutation There are 9 points to mutate again on the basis of P30 point mutation, wherein 6 are same sense mutation.

4 PRRSV embedded virus RvHBJX P1-P40 whole genome sequence mutation analysis of table

Further RvHBJX P40, P60 and P80 whole genome sequence are compared, the results show that P60 and P40 It compares, increases the point mutation of 10 nucleotide sites, wherein 8 are same sense mutation and Nsp2 the 279th amino acid V The 116th amino acids V → L of → A and 3 '-UTR.P80 increases the mutation of 8 nucleotide sites newly on the basis of P60, wherein 5 For same sense mutation and Nsp2 the 543rd amino acid L → P, the 139th the 76th amino acids of amino acids A → L and Nsp12 of Nsp5 C → A (table 5).

5 PRRSV embedded virus HBJX P40-P80 whole genome sequence mutation analysis of table

6 embedded virus RvHBJX P5 of embodiment, P20, P40 infect the pathogenicity of piglet

20 28 age in days SPF pigs are randomly divided into 4 groups, every group 5.Respectively embedded virus P5 infected group is fitted into strain P20 infected group, chimeric strain P40 infected group and control group.4 test group isolated rearings in different animal houses.Infected group 2mL 2 × 10 is inoculated with through collunarium⁵TCID₅₀Virus liquid, control group are inoculated with the 5%DMEM culture medium of 2mL, daily observation experiment piglet Clinical symptoms, record body temperature and changes of weight, be observed continuously to off-test.Meanwhile 0 after infection, 3,5,7,10, 14,21, each experiment pig blood of 28d acquisition and infection pig Nasal swabs.The 10 times of gradient dilutions of serum sample being separated to are followed by Kind MARC-145 cell, observes CPE, records the hole count of each dilution CPE, calculates infection piglet according to Reed-M ü ench method The TCID of virus in blood₅₀；Treated Nasal swabs detect the viral nucleic acid in sample with RT-PCR method, and analysis infection is young The toxin expelling situation of pig.It is cutd open when off-test and kills all pigs, observe the substantially lesion of the tissues such as its lungs, lymph node, brain, and Paraffin section is made in selection lungs, lymphoodi mandibulares tissue, and Immunohistochemical Staining analyzes the distribution of the viral antigen in each tissue.

1, clinical symptoms, body temperature and the daily gain variation of piglet are infected

Chimeric strain P5 infected group piglet performance ochrodermia, apparent abdominal respiration, anorexia, syntexis, sitting position of dog gesture, stream Tears；Body temperature starts to increase after infection for 24 hours, reaches as high as 41 DEG C；Die off after infecting 15d.P20 infected group pig goes out occasionally The depressed, diarrhea of existing spirit；Body temperature after infection 6,10,11d be increased to 40 DEG C, pig death is had no during test.P40 infection Group piglet does not observe manifest symptom during the test；Only 6d and 13d is increased to 40 DEG C or more to body temperature after infection, and infection is young Pig all survives.It is uninfected by control pig to off-test clinic and body temperature and is showed no exception.Test pig Temperature changing is shown in Fig. 5 A.Sense Contaminate the variation of piglet daily gain as shown in Figure 5 B, negative growth is presented in weight loss, daily gain to RvHBJX P5 infected group before death. Weight gradually rises after P20 infected group and P40 infected group Infection in Piglets, every pig average daily gain about 300g, with control group There is no difference.

2, infection piglet substantially dissect changes

It is the visible oedema of embedded virus RvHBJX P5 infected group piglet dissect and interstitial pneumonia etc. and hydropericardium, big Cerebral hemorrhage, lymph node bleeding and enlargement etc.；It is cutd open when off-test and kills survival piglet, the visible lungs dispersivity necrosis of P20 infected group, Lower jaw/inguinal lymph nodes hyperemia enlargement；P40 infected group piglet lungs, lymph node etc. are without obvious naked eyes lesions visible.Control group Piglet lungs, lymph node etc. are organized without lesions visible.

3, the viral distribu-tion in piglet viremia virusemia, nasal cavity toxin expelling and its tissue is infected

Piglet serum sample is acquired in different time points, using the viral nucleic acid in RT-PCR detection sample, analyzes piglet Viremia virusemia continued case, the results are shown in Table 6.P5 infected group piglet switchs to the positive in 5DPI viremia virusemia, all before death 21d still has 1 swine disease toxaemia not turn out cloudy；Viremia virusemia all switchs to the positive to P20 and P40 infected group since 7DPI, 28DPI all switchs to feminine gender.Control group piglet viremia virusemia is feminine gender.

The RT-PCR testing result of viral nucleic acid in 6 piglet serum of table

Note: denominator indicates that test sample number, molecule indicate number positive, and "-" expression does not detect

Using RT-PCR, viral nucleic acid detection, analysis infection piglet nasal cavity toxin expelling feelings are carried out to the nose swab of acquisition simultaneously Condition (table 7).P5 infected group begins with piglet nasal cavity toxin expelling in 3DPI and is positive, and toxin expelling is continued until that 14DPI switchs to feminine gender； P20 infected group piglet starts nasal cavity toxin expelling in 5DPI and is positive, same as P5 to be continued until 14DPI；P40 infected group piglet exists Nasal cavity toxin expelling is not detected during test.

7 piglet nasal cavity toxin expelling testing result of table

Note: denominator indicates that test sample number, molecule indicate number positive, and "-" expression does not detect

Piglet lungs and lymphoodi mandibulares are acquired, paraffin section is made, carry out immunohistochemical staining, observe PRRSV antigen Distribution in infection piglets, is as a result shown in Fig. 6.

It scores referring to the method for Halbur etc. ImmunohistochemistryResults Results, that is, 5 visuals field is randomly selected, according to PRRSV Positive cell number meter average value: no PRRSV positive cell is 0 point；1-10 positive cell is calculated as 1 point；11-30 positive cell It is calculated as 2 points；31-100 positive cell is calculated as 3 points；More than 100 positive cells are calculated as 4 points.Embedded virus difference passages strain The immunohistochemistry marking situation of infection piglet is shown in Table 8.As embedded virus passage number increases it can be seen from table, infection Viral antigen in piglets is in reduction trend.

Table 8 infects the immunohistochemistry appraisal result of piglet lungs and lymphoodi mandibulares PRRSV antigen

As embedded virus RvHBJX is passaged to 20 generations or more on cell, clinical symptoms caused by virus infection and group The substantially lesion for knitting organ gradually mitigates；The viremia virusemia duration is also gradually shortened；Virus is in lungs and lymph node tissue In content be gradually lowered；Apparent nasal cavity toxin expelling is can't detect to 40 generations；In addition to P5 infection piglet can cause pig Dead outer, P20 and P40 do not have found piglet death, and do not influence normal growth and the feeding of piglet.Piglet pathogenicity knot Fruit shows the increase with cell passage number, and embedded virus also gradually increases the safety of piglet.

Embodiment 7 is fitted into the immune protective effect assessment of strain RvHBJX P40, P60, P80

On the basis of pathogenicity, the present invention selects RvHBJX virus P40, P60, P80 strain piglet infected experimentally, Its immune protective to highly pathogenic PRRSV infection is compared in analysis, to assess PRRSV RvHBJX as gene chimeric Feasibility, provide reference data to screen safety is good, protectiveness is high PRRSV chimeric vaccine candidates.By 2mL 2 × 10⁵TCID₅₀PRRSV RvHBJX P40, P60, P80 collunarium is inoculated with 4 week old piglets, and control group test pig is inoculated with 2mL DMEM training Nutrient solution, all survival pigs of dissect after the highly pathogenic PRRSV JXwn06P8,21d of 4 weeks postoperative infection same doses.Observation piglet faces Bed symptom and lungs pathological change record its body temperature and daily gain situation, the viremia virusemia and nasal cavity toxin expelling of detection infection piglet Situation, immune protective of the analysis PRRSV RvHBJX cell passage strain to infection piglet.

1, piglet is immunized and attacks Temperature changing, clinical symptoms and death condition after poison

Before attacking poison to JXwn06 after immune, the appetite and the state of mind of all pigs are normal, do not observe obvious clinical condition Shape, body temperature are maintained between 39.5-40.4 DEG C.4-11d and 13d after poison is attacked, the mean body temperature of control group test pig is higher than 41.0 DEG C, wherein having 2 temperature of pig body 5d, 7d and 9d after attacking poison is more than 42.0 DEG C；6-12d has after RvHBJX P40 immune group attacks poison 2 temperature of pig body are higher than 41.0 DEG C, wherein 1 pig body temperature in 6d and 11d after attacking poison is more than 42.0 DEG C, all temperature of pig body after 20d It is down to 40.0 DEG C or less.6-10d has 2 temperature of pig body to be higher than 41.0 DEG C after RvHBJX P60 immune group attacks poison, all pigs after 17d Temperature decline is to 40.0 DEG C or less.RvHBJX P80 immune group has 3 temperature of pig body to be increased to 41.0 DEG C or more after attacking malicious 8d, 16d All temperature of pig body are down to 40.0 DEG C or less (Fig. 7 A) afterwards.

The symptoms such as after PRRSV JXwn06 infects 4d, control group pig starts to occur breathing, spiritual depressed, appetite stimulator.It attacks After malicious 9d, start death, extremely preceding performance appetite abolish, spirit is depressed, lie prone sleeping, skin cyanosis, coat are slightly random, has difficulty in breathing, shake It quivers.RvHBJX P40 immune group has 3 pigs to be short of breath after attacking malicious 11d, appetite stimulator, restores normal after 18d； RvHBJX P60 immune group has 2 pigs rhinorrheas, abdominal respiration, feed intakes reductions occur after attacking malicious 7d, and symptom is gradually after 13d Mitigate, restores normal after 15d.RvHBJX P80 immune group 8-12d after attacking poison only has 1 pig to occur having a running nose, spirit is depressed, The symptoms such as appetite stimulator.

It is experiment piglet marking (Li Y, et al.2014.Nsp9and according to the symptom scores standard that Li et al. is formulated Nsp10 contribute to the fatal virulence of highly pathogenic porcine Reproductive and respiratory syndrome virus emerging in China.PLoS Pathog.), Wherein pig death adds 25 points, and specific appraisal result is shown in Fig. 7 B.11d is until off-test, control group clinical score after attacking poison There is significant difference (P < 0.05) compared with RvHBJX P40, P60 and P80 immune group.Attack 18-21d, RvHBJX after poison P40 immune group symptom scores are significantly higher than RvHBJX P60, P80 immune group (P < 0.05).

Control group 9d, 12d, 13d after attacking poison respectively have 1 pig death, and when 16d is all dead；RvHBJX P40 immune group 12d and 19d respectively has 1 pig death after attacking poison；To when off-test, P60 immune group and P80 immune group test pig are all survived (Fig. 8).

Test daily gain in pigs situation is shown in Fig. 9 before and after attacking poison.Before attacking poison, 4 groups of test pig average daily gains are in 0.19-0.29kg Between, difference is little.After attacking poison, there is appetite stimulator even abolish in control group test pig, leads to weight loss, until before dead Average daily gain is only 0.04kg；And P40 immune group, the average daily gain of P60 immune group and P80 immune group are respectively 0.14kg, 0.22kg and 0.26kg, compared with the control group equal significant difference (P < 0.05).

2, the detection that piglet attacks malicious posterula toxin expelling and viremia virusemia is immunized

Nasal cavity toxin expelling can be detected in all pigs of 3-7d after attacking poison.10d after poison is attacked, control group piglet nasal cavity toxin expelling is still complete Portion is the positive, and RvHBJX P40 immune group, P60 immune group and P80 immune group nasal cavity toxin expelling Positive rate are followed successively by 1/4, 2/4 and 2/5.14d after poison is attacked, P80 immune group nasal cavity toxin expelling all switchs to feminine gender；Control group, P40 immune group and P60 immune group Respectively there is 1 pig testing result to be positive.21d after poison is attacked, 3 immune group piglet nasal cavity toxin expellings turn out cloudy (table 9).

Table 9 attacks after poison viral nucleic acid testing result in piglet nose swab

All piglets can detect viremia virusemia after attacking malicious 3d.Control group baby pig disease toxaemia state be continued for Before death.10d after poison is attacked, 1 swine disease toxaemia of P80 immune group disappears；Attack 14d after poison, P40, P60 and P80 immune group pig blood Viral nucleic acid recall rate in clear is 33% (1/3), 50% (2/4) and 40% (2/5) respectively.To when 21d off-test, own The viremia virusemia of immune group piglet disappears (table 10).

Table 10 attacks piglet viremia virusemia testing result after poison

The positive serum of above-mentioned viral nucleic acid detection is inoculated on MARC-145 cell, the virus titer in serum is measured (Figure 10).5d after poison is attacked, the virus titer in control group piglet serum is up to 10^5.13TCID₅₀/ mL, gradually decreases later, 14d When be reduced to 10^2.07TCID₅₀/mL.Virus titer in P40, P60 and P80 immune group piglet serum is consistently lower than control group, and Significant difference (the P < 0.05) in 5d, 10d, 14d.There is no notable difference between 3 immune groups.

3, piglet is immunized and attacks the Tissue distribution of lungs pathological change and viral nucleic acid after poison

Attack the dissect immediately of dead pig after poison.21d dissect whole survival test pig after poison is attacked, lungs pathological change is observed, And (Halbur et al., 1995) is given a mark referring to the Halbur lung lesion standards of grading formulated.Control group death pig lung Popular name for becomes more serious, and large area consolidation, necrosis, hyperemia, interstitial pneumonia etc. can be observed.The lung lesion phase of each immune group To relatively gently, only consolidation occurs for lung great Ye and lobulus pulmonis edge, and lesion score is also significantly lower than control group (P < 0.05) (table 11).

11 each group lung lesion appraisal result of table

^aSignificant difference (P < 0.05) compared with the control group

Acquire the tissue such as piglet lungs, lymphoodi mandibulares, inguinal lymph nodes, tonsillotome, kidney, spleen, brain, RT-PCR Method detects the PRRSV nucleic acid in each tissue.P40, P60, P80 immune group piglets band poison rate are respectively 36.14% (9/28), 17.86% (5/28) 14.29% (5/35), significant difference (P < 0.05) compared with the control group.Wherein, P40 immune group and P80 Also significant difference (P < 0.05) (table 12) between immune group.

Viral nucleic acid detection case in 12 histoorgan of table

^a,bSignificant difference is indicated between different letters

In conclusion PRRSV is fitted into strain RvHBJX P40, P60 and P80 cell passage strain can subtract after piglet is immunized The clinical symptoms of subinfection piglet reduce the virus load in infection piglet serum, significantly reduce the The dead quantity of piglet, mitigate Lung injury caused by virus infection.Wherein, PRRSV, which is fitted into strain RvHBJX P60, to be highly pathogenic PRRSV infection son Pig provides 100% immunoprotection, and the Candidate Strain that can be used as PRRSV gene chimeric carries out subsequent vaccine development.

Testing and attack malicious protectiveness test through animal safety proves, present invention porcine reproductive and respiratory syndrome obtained It will not cause apparent clinical symptoms after chimeric HBJX inoculation piglet, the safety used as vaccine is preferable；And it can Preferable immunoprotection is provided for infection piglet, mitigates highly pathogenic PRRSV infection bring harm.The virus of the chimeric Skeleton derives from the natural low pathogenicity PRRSV HB-1/3.9 strain being clinically separated, and avoids attenuated vaccine strain virulence and easily returns by force Risk, meanwhile, the highly pathogenic PRRSV strain structural protein coding region being placed in strain can also stimulate body after inoculation The neutralizing antibody for popular strain is generated, the immune protection effectiveness of the vaccine is improved.In addition, in chimeric HBJX sequence Antidiastole of 2 genetic markers being artificially introduced also for vaccine strain and natural infection strain provides clear target spot, this is chimeric The application of vaccine is beneficial to the purification of China swinery PRRS.

The Revive virus that the construction method of embedded virus provided by the present invention obtains can continuous passage in vitro, lose It is relatively stable to pass characteristic.So this Revive virus can carry out and PRRSV structural proteins function as effective scientific research material Relevant scientific research.

Claims (7)

1. porcine reproductive and respiratory syndrome embedded virus, which is characterized in that it is RvHBJX, is with low pathogenicity PRRSV virus Strain HB-1/3.9 is skeleton, and highly pathogenic PRRSV strain JXwn06 directly replaces low pathogenicity PRRSV in structural protein coding region The embedded virus obtained after virus stain HB-1/3.9 structural proteins；

The gene order of highly pathogenic PRRSV strain structural protein coding region is as shown in SEQ ID NO.1.

2. porcine reproductive and respiratory syndrome embedded virus RvHBJX full length cDNA clone plasmid, which is characterized in that by with lower section Method constructs to obtain:

(1) using pWSK-JXwn as template, PCR amplification obtains the total protein-coding region of JXwn06, and amplified production is named as HJSP-S2；

(2) using pWSK-HB-1/3.9 as template, the structural protein coding region PCR amplification HB-1/3.9 two sides sequence, is ordered respectively respectively Entitled HJSP-S1, HJSP-S3；

(3) by after step (1) and the amplified production purification and recovery of (2), with HJSP-S1,3 segments of HJSP-S2, HJSP-S3 are made For template, PCR amplification obtains HJSP-S123；

(4) the pWSK-HB-1/3.9 matter of identical double digestion is connected to through Rsr II, Asc I double digestion after HJSP-S123 purifying In grain, the building of PRRSV chimeric plasmid is completed, pW-HJSP is named as；

Wherein, the primer that step (1) PCR amplification uses is JX-S2F and JX-S2R, nucleotide sequence such as SEQ ID NO.2,3 It is shown；

Wherein, step (2) PCR amplification is respectively using HB-S1F, HB-S1R and HB-S3F, HB-S3R as primer, nucleotide sequence Respectively as shown in SEQ ID NO.4-7；

Wherein, the primer of step (3) PCR amplification is HJ-fusionF and HJ-fusionR, nucleotide sequence such as SEQ ID Shown in NO.8-9.

3. the method for preparing porcine reproductive and respiratory syndrome virus embedded virus described in claim 1, which is characterized in that including Following steps:

(1) porcine reproductive and respiratory syndrome embedded virus RvHBJX full length cDNA clone plasmid is constructed；

(2) rescue of embedded virus RvHBJX.

4. method as claimed in claim 3, which is characterized in that the cloned plasmids of step (1) construct by the following method to be obtained:

1) using pWSK-JXwn as template, PCR amplification obtains the total protein-coding region of JXwn06, and amplified production is named as HJSP-S2；

2) using pWSK-HB-1/3.9 as template, the structural protein coding region PCR amplification HB-1/3.9 two sides sequence, is ordered respectively respectively Entitled HJSP-S1, HJSP-S3；

3) after the amplified production purification and recovery by step 1) and 2), with HJSP-S1,3 segment conducts of HJSP-S2, HJSP-S3 Template, PCR amplification obtain HJSP-S123；

4) the pWSK-HB-1/3.9 plasmid of identical double digestion is connected to through RsrII, AscI double digestion after HJSP-S123 purifying In, the building of PRRSV chimeric plasmid is completed, pW-HJSP is named as；

The primer that PCR amplification uses in step 1) is JX-S2F and JX-S2R, nucleotide sequence such as SEQ ID NO.2,3 institutes Show；PCR amplification is respectively using HB-S1F, HB-S1R and HB-S3F, HB-S3R as primer in step 2), and nucleotide sequence is respectively such as Shown in SEQ ID NO.4-7；The primer of PCR amplification is HJ-fusionF and HJ-fusionR, nucleotide sequence in step 3) As shown in SEQ ID NO.8-9.

5. method as claimed in claim 3, which is characterized in that the rescue of step (2) embedded virus RvHBJX passes through with lower section Method is realized:

1) Rsr II, Pac I endonuclease digestion pW-HJSP full length cDNA clone plasmid are used, plasmid linearization is made；What is obtained is linear Change plasmid after phenol chloroform and dehydrated alcohol precipitating, drying, ultraviolet specrophotometer measures the concentration of purified plasmid；

2) it is transcribed in vitro；

3) transcription product, with DMRIE-C liposome transfection MARC-145 cell, is observed daily after TURBO DNase I digestion And the production of cytopathy CPE is recorded, the cells and supernatant for obvious CPE occur is connected on MARC-145 cell Generation is resumed, per generation 3-5d, the Revive virus of acquisition is RvHBJX.

6. the vaccine containing porcine reproductive and respiratory syndrome embedded virus described in claim 1 or its secondary culture virus.

7. porcine reproductive and respiratory syndrome embedded virus described in claim 1 or its secondary culture virus are preparing the use in vaccine On the way.