CN109762792A - A kind of porcine reproductive and respiratory syndrome virus is fitted into strain and its application - Google Patents

Abstract

The invention discloses a kind of porcine reproductive and respiratory syndrome virus to be fitted into strain and its application, the present invention is based on the full-length infectious CDNA clones of highly pathogenic PRRSV TJM-F92 vaccine low virulent strain and class NADC30 hypotype strain FJ1402, using Reverse Genetics, building has obtained porcine reproductive and respiratory syndrome virus and has been fitted into strain.The chimeric strain experiment proves that; viral growth speed and virus titer are lower than its original strain TJM-F92 on Marc-145 cell; virulence is low; there is preferable immanoprotection action to HP-PRRSV strain BB0907 and class NADC30 strain FJ1402; effective Cross immunogenicity can be provided to highly pathogenic strain and class NADC30 virus strain infection, important foundation has been established in the development for the disease New-type wide-spectrum vaccine.

Description

A kind of porcine reproductive and respiratory syndrome virus is fitted into strain and its application

Technical field

The present invention relates to a kind of new porcine reproductive and respiratory syndrome virus to be fitted into strain, further relates to the chimeric strain strain Construction method and the application in the candidate vaccine of preparation prevention porcine reproductive and respiratory syndrome prevalence strain.

Background technique

Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, It PRRS is) by porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome Virus, PRRSV) caused by a kind of viral blight for seriously endangering pig breeding industry, the complicated multiplicity of clinical symptoms, feature is mother The respiratory disease of pig breeding dysfunction or each age in days swinery.The virus in 1996 is split into for the first time in China, after gradually spread The whole nation.Summer and autumn in 2006 are even more to break out highly pathogenic PRRSV (HP-PRRSV), and main clinical characteristics are high fever, high-incidence Sick rate and the death rate cause huge economic loss to pig breeding industry.HP-PRRSV strain can cause piglet compared to classics PRRSV More serious clinical symptoms cause higher morbidity and mortality.PRRSV genome is easy to happen variation and recombination, and giving should Sick prevention and control bring bigger difficulty.

In recent years, domestic to be separated to successively and the higher Strain of U.S. PRRSV NADC30 strain homology, referred to as class NADC30 strain.Epidemic status has been presented in China in Molecule Epidemiology Investigation studies have shown that class NADC30 strain.The U.S. NADC30 strain is pathogenic to piglet weaker, but obvious with the class NADC30 strains of pathogenic after HP-PRRSV generation genetic recombination Enhancing.PRRSV is single strand plus RNA virus, belongs to Arteriviridae, Arterivirus.The full length viral genome is about 15kb includes at least 11 open reading frame (ORFs), and wherein ORF1a and ORF1b encodes non-structural protein, the end of genome 3 ' 8 The structural proteins of a ORFs main code virus.The oligomer that wherein GP2, GP3, GP4 and M albumen are formed is to induction neutralizing antibody There is certain effect.There are t cell epitopes for these albumen, can be immune with inducing T cell.GP5 is encoded by ORF5, is virus envelope sugar Albumen, and be the main host protective antigen of PRRSV, include the closely related neutralizing epitope of induction neutralizing antibody, GP5 amino The variation of acid sequence will directly reduce the cross protection rate of PRRS vaccine strain.M albumen is the non-glycosylated film egg encoded by ORF6 It is white, it is capable of absorption and the inducing cellular immune of mediate retroviral.GP5 and M albumen can exist in the form of heterodimer, and this Kind heterodimer can promote GP5 albumen from endoplasmic reticulum to Golgi transport, be induced to further enhance GP5 albumen Immune response.N protein is then a kind of highly conserved nucleocapsid protein, has extremely strong immunogenicity, but its induction is anti- Body does not have the ability for neutralizing virus.Since virus genome RNA mutation, recombination etc. cause strain that variation diversity and something lost is presented Diversified trend is passed, brings severe challenge for PRRS prevention and control.Currently, attenuated vaccine is commercialized to homologous strain gene in PRRSV Preferable immanoprotection action, but it is lower to the cross-protection of special-shaped strain.Currently, the commercialized vaccine of PRRS mainly have it is weak Virus live vaccine and inactivated vaccine, attenuated vaccine is preferable than inactivated vaccine immune efficacy, but to different subtype strain intersecting protective It is poor.Such as clinic caused by HP-PRRSV can be effectively relieved in the attenuated vaccines such as TJM-F92, HuN4-F112, JXA1-R and R98 Symptom reduces the piglet death rate, but limited to class NADC30 strain immanoprotection action, therefore studies New-type wide-spectrum vaccine very It is necessary.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of chimeric poison of porcine reproductive and respiratory syndrome virus Strain.The Novel chimeric strain virulence is low, has good immunogenicity, can be to highly pathogenic PRRSV strain and PRRSV class NADC30 virus strain infection provides effective Cross immunogenicity, has established important foundation for the disease new generation vaccine development.

Present invention separation obtains PRRSV class NADC30 strain FJ1402 (abbreviation class NADC30 strain FJ1402) and high cause Characteristic of disease PRRSV strain BB0907 (abbreviation HP-PRRSV BB0907), class NADC30 strain FJ1402 and HP-PRRSV BB0907 Cause a disease similar temperament.

The chimeric strain of PRRSV provided by the invention is the expression on the Nsp2 gene by highly pathogenic PRRSV vaccine strain The genetic fragment of 323-433 amino acids (aa) deletes, simultaneously replaces with its ORF5-6 (open reading frame 5 and 6) The ORF5-6 of PRRSV class NADC30 strain is constructed and is obtained.

The PRRSV vaccine strain that the present invention selects is highly pathogenic PRRSV TJM-F92 vaccine low virulent strain, selection PRRSV class NADC30 strain is class NADC30 strain FJ1402.

The present invention is when building PRRSV is fitted into strain, in the overall length sense of highly pathogenic PRRSV TJM-F92 vaccine low virulent strain On the basis of metachromia cDNA clone and class NADC30 strain FJ1402, using Reverse Genetics, by the Nsp2 gene of TJM-F92 The genetic fragment of 323-433 aa of loss of expression (lacking 111 aa altogether), and Nsp2 gene delection is expressed into 323-433 Position aa after highly pathogenic PRRSV TJM-F92 strain structural protein gene ORF2-7 (ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7) and the region ORF5-6 (ORF5 and ORF6) be substituted for class NADC30 hypotype strain FJ1402 respectively and encode accordingly Region (ORF2-7 and ORF5-6), constructs three infection clones, and the recombinant plasmid containing these three infection clones is distinguished Mare-145 cell is transfected, rescue obtains three and recombinates chimeric strain, is named as rTd111 (TJM-F92 strain Nsp2 missing 111 aa), rTd111/F2-7 (rTd111 strain ORF2-7 is replaced into FJ1402 strain ORF2-7) and rTd111/F5-6 (rTd111 strain ORF5-6 is replaced into FJ1402 strain ORF5-6).

PRRSV of the present invention is fitted into the Constructed wetlands of strain specifically:

(1) infectious CDNA clones for constructing highly pathogenic PRRSV TJM-F92 vaccine low virulent strain, are named as pCMV- TJM；

(2) segment that 323-433 aa (totally 111 aa) are expressed on the Nsp2 gene of pCMV-TJM is deleted, building Infectious CDNA clones are obtained, pTd111 is named as；

(3) using pTd111 as skeleton, its ORF2-7 and ORF5-6 is replaced with into porcine reproductive and respiratory syndrome class respectively The ORF2-7 and ORF5-6 of NADC30 strain FJ1402, building obtain infectious CDNA clones, be named as pTd111/F2-7, pTd111/F5-6；

(4) pTd111, pTd111/F2-7, pTd111/F5-6 are subjected to virus rescue respectively, obtain three kinds of chimeric strains, It is named as rTd111, rTd111/F2-7 and rTd111/F5-6.

RTd111, rTd111/F2-7 and rTd111/F5-6 are passed on respectively 5 times, gene inheritance is stable.Further To rTd111, rTd111/F2-7 and rTd111/F5-6 be fitted into strain carry out verification experimental verification, as the result is shown: rTd111/F2-7 and RTd111/F5-6 is fitted into the speed of growth and virus titer of the strain on Marc-145 cell and is slightly below its original TJM-F92 poison Strain, but the speed of growth on PAM cell and virus titer are similar to original TJM-F92 strain.After infecting PAMs cell, The proinflammatory cytokines of rTd111/F5-6 induction are similar to Td111, slightly above TJM-F92, but rTd111/F2-7 is induced Proinflammatory cytokines be apparently higher than Td111, rTd111/F5-6 and TJM-F92.By rTd111, rTd111/F2-7, RTd111/F5-6, TJM-F92 and FJ1402 are inoculated with 30~35 age in days piglets respectively, through to pig body clinical symptoms, antibody test, Dissect lesion and pathological observation, the results showed that, rTd111/F5-6 virulence is similar to TJM-F92, hence it is evident that it is lower than FJ1402, And rTd111/F2-7 virulence is higher than rTd111/F5-6, illustrates that rTd111/F5-6 has the potentiality as vaccine candidate strain.It chooses 30-35 age in days sodium selenite further studies the piglet immunological Vaccine effectiveness of chimeric vaccine candidates rTd111/F5-6, is immunized 28d is utilized respectively HP-PRRSV strain BB0907 and class NADC30 strain FJ1402 and attacks poison, animal experiment knot through intramuscular injection afterwards Fruit shows that BB0907 and FJ1402 virus strain infection level is substantially reduced in rTd111/F5-6 immune group, and clinical symptoms and pathology become Change substantially reduced.Show that the chimeric strain has good immunogenicity, it can be to highly pathogenic strain and class NADC30 strain Infection provides effective Cross immunogenicity, has established important foundation for the disease new generation vaccine development.

Based on the above experimental result, the present invention selects rTd111/F5-6 to need the chimeric poison of PRRSV to be protected as the present invention Strain.In the rTd111/F5-6 the 5th generation for being fitted into strain, further cultivate and has carried out preservation, deposit number by the present invention For CGMCC No.14762, the entitled porcine reproductive and respiratory syndrome virus of preservation (porcine reproductive and Respiratory syndrome virus) rTd111/F5-6F5, depositary institution is Chinese microorganism strain preservation conservator Meeting common micro-organisms center (CGMCC), depositary institution address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the deposit date is On May 25th, 2018.

Further, the porcine reproductive and respiratory syndrome virus (porcine reproductive and Respiratory syndrome virus) in rTd111/F5-6 F5 CGMCC No.14762 strain, the core of ORF5 and 6 Thuja acid is identical as the ORF5-6 of class NADC30 strain FJ1402, other open reading frames and highly pathogenic PRRSV TJM-F92 Vaccine low virulent strain is identical, but on Nsp2 gene 111 aa (323-433) of loss of expression segment.

Further, the porcine reproductive and respiratory syndrome virus (porcine reproductive and Respiratory syndrome virus) rTd111/F5-6 F5 CGMCC No.14762 strain nucleotide sequence such as SEQ Shown in ID NO.1, the nucleotide sequence of ORF5 is as shown in SEQ ID NO.2, the nucleotide sequence of ORF6 such as SEQ ID Shown in NO.3.

Further, the chimeric strain that the present invention constructs is to HP-PRRSV strain BB0907 and class NADC30 strain FJ1402 There is preferable immanoprotection action, therefore it also exists as the application of the candidate vaccine of porcine reproductive and respiratory syndrome prevalence strain Within the scope of the present invention, it is preferred that the PRRSV be fitted into strain be rTd111/F5-6 F5 CGMCC No.14762 or rTd111/F5-6。

Further, strain is fitted into the present invention also provides porcine reproductive and respiratory syndrome virus to breed and exhale in preparation pig Inhale the application in syndrome vaccine, it is preferred that it is rTd111/F5-6 F5 that the porcine reproductive and respiratory syndrome virus, which is fitted into strain, CGMCC No.14762 or rTd111/F5-6.

Further, the present invention also provides a kind of vaccine, which includes that the pig of immune amount breeds and exhales Syndrome virus chimeric strain is inhaled, which is rTd111/F5-6 F5 CGMCC No.14762 or rTd111/F5-6. Preferably, the immune amount of strain is fitted into vaccine are as follows: 10^5.0TCID₅₀/ ml virus liquid.

Further, the present invention also provides above-mentioned vaccines in the drug of preparation prevention porcine reproductive and respiratory syndrome Using.Further, the porcine reproductive and respiratory syndrome be caused by classical porcine reproductive and respiratory syndrome virus strain infection, or It is to be caused by highly pathogenic PRRSV virus strain infection, or caused by PRRSV class NADC30 virus strain infection, or by above-mentioned three kinds Strain cross-infection causes.

The present invention is with highly pathogenic PRRSV TJM-F92 vaccine low virulent strain (i.e. high-pathogenicity porcine reproductive and respiration syndrome Strain TJM-F92 vaccine low virulent strain) full-length infectious CDNA clones and class NADC30 hypotype strain FJ1402 based on, utilize Reverse Genetics, building have obtained porcine reproductive and respiratory syndrome virus and have been fitted into strain.The chimeric strain experiment proves that, Viral growth speed and virus titer are lower than its original strain TJM-F92 on Marc-145 cell, and virulence is low, to HP-PRRSV poison Strain BB0907 and class NADC30 strain FJ1402 has preferable immanoprotection action, can be to highly pathogenic strain and class NADC30 virus strain infection provides effective Cross immunogenicity, and important foundation has been established in the development for the disease New-type wide-spectrum vaccine.

Preservation information

Porcine reproductive and respiratory syndrome virus rTd111/F5-6 F5 has been preserved in the micro- life of China on May 25th, 2018 Object culture presevation administration committee common micro-organisms center (CGMCC), depositary institution address are BeiChen West Road, Chaoyang District, BeiJing City 1 Number institute 3, deposit number are CGMCC No.14762.

Detailed description of the invention

Fig. 1 PRRSV is fitted into virus strain infection's property clone's building schematic diagram, and (A) lacks Nsp2 table using pCMV-TJM as skeleton Up to the segment of 111 aa, pTd111 is obtained, what is marked on the figure gene is the position of corresponding expression 323-433 amino acids； (B) constructs chimeric cDNA clone pTd111/F2-7 and pTd111/F5-6 using pTd111 as skeleton respectively.

Fig. 2 PRRSV is fitted into plaque form (A) and growth curve (B) of the strain on Marc-145 cell.

Fig. 3 PRRSV is fitted into lesion (A) and growth curve (B) of the strain on PAMs cell.

Fig. 4 virus infection PAMs Cellular inflammatory cell factor quantitative fluorescent PCR measurement result.

Piglet body temperature (A) and average daily gain (B) measurement result after Fig. 5 virus inoculation.

Fig. 6 PRRSV is fitted into after strain inoculation virus load (B) measurement knot in piglet viremia virusemia (A) and lung tissue Fruit.

14 days piglet lung tissue pathological observation results (200 ×) after Fig. 7 inoculation.

PRRSV detection of specific antibody result after Fig. 8 piglet immunological.

Fig. 9 HP-PRRSV BB0907 strain attacks piglet Temperature changing (A) and survivorship curve (B) after poison.

Figure 10 HP-PRRSV BB0907 strain attacks after poison virus in piglet viremia virusemia (A) and lungs and lymph node tissue Carrying capacity (B) measurement result.

Figure 11 HP-PRRSV BB0907 strain attacks piglet lungs naked eyes pathological change and changes in histopathology after poison.

Figure 12 PRRSV class NADC30 strain FJ1402 attacks piglet Temperature changing (A) and survivorship curve (B) after poison.

Figure 13 PRRSV class NADC30 strain FJ1402 is attacked after poison in piglet viremia virusemia (A) and lungs and lymph node tissue Virus load (B) measurement result.

Figure 14 PRRSV class NADC30 strain FJ1402 attacks piglet lungs naked eyes pathological change and histopathology change after poison Change.

Specific embodiment

The present invention is using high-pathogenicity porcine reproductive and respiration syndrome strain (abbreviation highly pathogenic PRRSV or HP-PRRSV) TJM-F92 vaccine low virulent strain infectious CDNA clones pCMV-TJM, (referred to as by porcine reproductive and respiratory syndrome class NADC30 strain Class NADC30 strain) Nsp2 gene expression characteristics, it is deleted on the Nsp2 gene of the vaccine strain and expresses 323-433 aa (totally 111 A aa) segment, construct infectious CDNA clones pTd111, then as skeleton, by its ORF2-7 (ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7) and ORF5-6 (ORF5 and ORF6) replace with the corresponding of class NADC30 strain FJ1402 respectively ORFs (open reading frame), constructs infectious CDNA clones pTd111/F2-7 and pTd111/F5-6, and rescue is recombinated Viral rTd111 (TJM-F92 strain Nsp2 lacks 111 aa), (rTd111 strain ORF2-7 is set chimeric strain rTd111/F2-7 It is changed to FJ1402 strain ORF2-7) and rTd111/F5-6 (rTd111 strain ORF5-6 is replaced into FJ1402 strain ORF5-6), point In supplementary biography generation 5 times, gene inheritance is stablized.RTd111/F2-7 and rTd111/F5-6 is fitted into strain and grows on Marc-145 cell Speed and virus titer are lower than its original TJM-F92 strain, and the congenital immunity of infection PAMs cell rTd111/F5-6 induction is thin Intracellular cytokine expression is similar to its original vaccine strain TJM-F92, the congenital immunity cell factor of rTd111/F2-7 induction Expression is apparently higher than its original vaccine strain TJM-F92.By rTd111, rTd111/F2-7, rTd111/F5-6, TJM- F92 and FJ1402 is inoculated with 30~35 age in days piglets respectively, through to pig body clinical symptoms, antibody test, dissect lesion and pathology Learn observation, the results showed that, rTd111/F5-6 virulence is similar to TJM-F92, hence it is evident that lower than FJ1402, and rTd111/F2-7 virulence Higher than rTd111/F5-6.For the piglet immunological Vaccine effectiveness for further studying chimeric vaccine candidates rTd111/F5-6, choose 30-35 age in days sodium selenite, immune rear 28d are utilized respectively HP-PRRSV strain BB0907 and class NADC30 strain FJ1402 through flesh Poison is attacked in meat injection.Animal test results show that BB0907 and FJ1402 virus strain infection is horizontal obvious in rTd111/F5-6 experimental group It reduces.The above results show that the chimeric strain has good immunogenicity, can be to highly pathogenic strain and class NADC30 poison Strain infection provides effective Cross immunogenicity, has established important foundation for the disease new generation vaccine development.

In the following, enumerating the acquisition methods of the chimeric strain of the present invention, specifically so that those skilled in the art are more detailed to the present invention Thin understanding.In following methods, unless otherwise stated, Science and Technology noun used is logical with those skilled in the art The meaning understood.Also, infectious CDNA clones used herein, cell culture, cell transformation, virus transfection, virus The technologies such as rescue, immunological experiment, recombinant plasmid, recombinant virus are all the technologies that this field has had been reported that, step can be with It is carried out using conventional steps disclosed in the prior art.

Embodiment

1 material and method

1.1 viruses, cell and plasmid

PRRSV highly pathogenic strain BB0907 and class NADC30 strain FJ1402 is separated by this laboratory and is identified and protect It deposits, PRRSV vaccine (TJM-F92 plants) is given by force China, Hua Weite (Taizhou) bioengineering Co., Ltd, TJM-F92 vaccine strain Infectious CDNA clones plasmid pCMV-TJM, constructed and saved by this laboratory.The primary pulmonary alveolar macrophage of pig (PAMs) ginseng It is prepared according to literature method；Marc-145 cell is by this laboratory liquid nitrogen cryopreservation, and from the U.S., ATCC is purchased.

1.2 other reagents and test animal

RNA extracts kitTotal RNA Kit I is purchased from OMEGA；SuperScript^TMIII Reverse Transcriptase is purchased from Invitrogen；Restriction enzyme A scI and SpeI are purchased from NEB；DMEM/F12 training It supports base and is purchased from Vicente；Low melting point agar (Agarose) is purchased from SIGMA；IDEXX HerdChek PRRS X3ELISA kit purchase From IDEXX.30-35 age in days PRRSV, PCV2, PRV and CSFV antibody and antigen negative piglet cultivate purchased from Nantong City Family.

The building of 1.3 chimeric virus strain infection's property cDNA clones

1.3.1 design of primers

According to the gene order design primer of TJM-F92 strain and FJ1402 strain, primer sequence is shown in Table 1.Primer is by south The synthesis of Jing Jinsirui Bioisystech Co., Ltd, way of purification RPC.

1 infectious CDNA clones of table construct primer

In upper table, italic and the sequence underlined indicate restriction enzyme site, and the sequence of overstriking is the sequence of FJ1402 strain.

In upper table, each primer effect is as follows:

T-A-F and Td111aa-R is the primer for expanding TJM plants of A segment 111aa deletion segment leading portions；

Td111aa-F and T-A-R is the primer for expanding TJM plants of A segment 111aa deletion segment back segments；

T-A-F and T-A-R is by the primer of two sections of progress fusion DNA vaccines before and after TJM plants of A segment 111aa deletion segments；

T-D-F and T-1b-R is the primer for expanding TJM plants of D segment ORF2 leading portions；

FJ-2-F and FJ-7-R is the primer for expanding FJ1402 strain ORF2-ORF7；

T-3'UTR-F, T-D-SwaI and T-D-SpeI are the primers for expanding TJM plants of D segment ORF7 back segments；

T-D-F and T-4-R is the primer for expanding TJM plants of D segment ORF5 leading portions；

FJ-5-F and FJ-6-R is the primer for expanding FJ1402 strain ORF5-ORF6；

T-7-F, T-D-SwaI and T-D-SpeI are the primers for expanding TJM plants of D segment ORF6 back segments.

1.3.2 it is fitted into the building of virus strain infection's property cDNA clone plasmid

The specific steps of the TJM-F92 vaccine strain laboratory infectious CDNA clones pCMV-TJM PRRSV building are referring to text Offer " building and identification of the recombination porcine reproductive and respiratory syndrome virus of expression porcine circovirus 2 type ORF2 gene " (Zhang Tingjie Deng " viral journal " the 1st phase in 2015).

Using the PRRSV TJM-F92 vaccine strain infectious CDNA clones pCMV-TJM of laboratory building as skeleton, press PRRSV class NADC30 strain Nsp2 gene expression characteristics delete expression 323-433 altogether on the Nsp2 gene of pCMV-TJM The genetic fragment of 111 aa, building obtain infectious CDNA clones pTd111 (Fig. 1-A).Steps are as follows for specific experiment:

Using pCMV-TJM as template, TJM plants of A segments are expanded with T-A-F, Td111aa-R and Td111aa-F, T-A-R respectively 111aa deletion segment front and rear sections.It then, is primer with T-A-F and T-A-R using spread sheet section as template, it will by SOE PCR Forward and backward two sections are merged, and the A (d111) of missing 111aa is obtained.With PacI and XhoI respectively to segment A (d111) and pCMV- TJM carries out double digestion, to the segment and carrier progress recovery purifying after digestion and connects, finally by the A after missing 111aa (111aa) is successfully substituted into pCMV-TJM, obtains the pTd111 that Nsp2 has lacked 111aa.

PTd111 respective segments and the respective segments of FJ1402 strain are fused together by SOE PCR, are respectively obtained The recombination D segment (Td111/F2-7) of chimeric FJ1402 strain ORF2-7 and the recombination D segment of chimeric FJ1402 strain ORF5-6 (Td111/F5-6), the D segment being transformed is substituted into pTd111, the sense of access respectively eventually by double digestion (AscI and SpeI) Metachromia cDNA clone pTd111/F2-7 and pTd111/F5-6 (Fig. 1-B).Steps are as follows for specific experiment:

PTd111/F2-7 building: expanding ORF2 leading portion and ORF7 back segment in D segment using pTd111 as template respectively, with The cDNA of FJ1402 strain is template amplification ORF2-ORF7 sections, is fused together, is fitted by three sections by fusion DNA vaccine The D (Td111/F2-7) of FJ1402 strain ORF2-7；D (Td111/F2-7) and pTd111 are carried out with AscI and SpeI respectively double Digestion, by after digestion segment and carrier be attached, obtain pTd111/F2-7.

PTd111/F5-6 building: expanding ORF5 leading portion and ORF6 back segment in D segment using pTd111 as template respectively, with The cDNA of FJ1402 strain is template amplification ORF5-ORF6 sections, is fused together, is fitted by three sections by fusion DNA vaccine The D (Td111/F5-6) of FJ1402 strain ORF5-6；D (Td111/F5-6) and pTd111 are carried out with AscI and SpeI respectively double Digestion, by after digestion segment and carrier be attached, obtain pTd111/F5-6.

1.3.3 the identification and sequence for being fitted into virus strain infection's property cloned plasmids are analyzed

WithEndo-free Plasmid DNA Mini Kit I extracts infectious CDNA clones plasmid PTd111, pTd111/F2-7 and pTd111/F5-6, and chimeric place's nucleotide sequence is measured, it is analysed whether through BioEdit software In the presence of missing and mutation.

The rescue of 1.4 chimeric strains and biological property analysis

1.4.1 it is fitted into the rescue of strain

According to the method for 3000 specification of lipofectamine, respectively by infectious CDNA clones plasmid pTd111, Marc-145 cell of the pTd111/F2-7 and pTd111/F5-6 transfection in cell density up to 70~90%, be placed in 37 DEG C, 5%CO₂Culture is observed in incubator.When hole, inner cell lesion CPE reaches 80% or so, harvest transfection cell, multigelation, Centrifugation takes the single layer Marc-145 cell continuous passage culture of vial supernatant access culture 48h.To being passaged to for the 5th generation (F5) Viral extraction RNA, send by template amplification ORF5 gene and Nsp2 gene of the cDNA after reverse transcription to Jin Sirui biotechnology Co., Ltd carries out sequence verification.Correct recombinant virus will be sequenced respectively and be named as rTd111, rTd111/F2-7 and rTd111/F5-6。

1.4.2 virus plaque experiment and growth curve measurement

Marc-145 cellular products multigelation 3 times for generating 80%CPE are collected, are diluted to 10 with DMEM^-2To 10^-4It is dilute Degree of releasing.It takes 100 holes μ l/ to be seeded to 12 orifice plates of confluent monolayers Marc-145 cell respectively, carries out plaque select according to a conventional method Test.Plaque to appear, picking plaque be connected to it is new cover in cell monolayer hole, purified virus 3~4 times, stabilization can be obtained With the recombinant virus of purifying.

By TJM-F92 vaccine strain and 3 kinds of chimeric strains of rescue respectively with the dose inoculation Marc-145 of MOI=0.1 Cell, 12h after infection, for 24 hours, 36h, 48h and 72h collect cell supernatant measurement TCID respectively₅₀, according to different time points Virus titer draws viral one step growth curve (3 parallel holes are arranged in every kind of viral each time point).

Inducing action of the 1.5 chimeric strains to primary PAMs congenital immunity cell factor

To understand the variation that porcine alveolar macrophage PAMs infects generation major cytokine after chimeric strain, further explain The infection mechanism of bright chimeric strain.It chooses PRRSV, PCV2 and PRV antigen and antibody is negative common weanling pig, separation With culture PAMs, by each viral (TJM-F92, rTd111, rTd111/F2-7 and rTd111/F5-6) respectively with MOI=0.1's Dose inoculation cell (3 parallel holes are arranged in every kind of viral each time point), while setting does not connect malicious Mock control wells.In virus Different time points after inoculation (6h, 12h, for 24 hours, 36h, 48h and 72h) collect cell conditioned medium respectively, are detected with Real-time PCR Various cytokine mRNA levels and the titre for measuring virus draw viral one step growth curve.

Pathogenicity of the 1.6 chimeric strain vaccine Candidate Strains to piglet

30 PRRSV feminine gender piglets are divided into six groups (n=5) at random.1-5 group injects rTd111, rTd111/ respectively The virus liquid of F2-7, rTd111/F5-6, TJM-F92, FJ1402, injection volume are as follows: every pig muscle injection 3mL 10⁵TCID₅₀/ ML virus liquid, the interior observation piglet body temperature of the 6th group of injection isometric PBS, 14d and clinical symptoms.And 7d, 14d are adopted after immune Blood, serum is for measuring viremia virusemia.14d after immune, carries out all piglets to slaughter dissect, carries out general pathology change to lungs Change observation, takes lung tissue to freeze and measured for virus load；Separately part lung tissue is taken to be fixed with 4% paraformaldehyde and carries out disease Neo-Confucianism observation.

The immune protective of 1.7 chimeric strain vaccine Candidate Strains is tested

30 PRRSV feminine gender piglets are divided into 3 groups (n=10), isolated rearing at random.Wherein one group of every pig muscle note Penetrate 3mL TJM-F92 (10⁵TCID₅₀/ mL), another group of every pig muscle injection 3mL rTd111/F5-6 (10⁵TCID₅₀/ mL), Every pig muscle injection 3mL PBS of last group, observes the clinical symptoms of pig body daily.7d, 14d, 21d and 28d are adopted after immune Blood carries out Study On Immunogenicity.28d after immune, every group 5 use class using the highly pathogenic strain BB0907 attack of PRRSV, 5 NADC30 hypotype strain FJ1402 attack, 4mL/ head.After attacking poison, pig body state is observed daily, is measured rectal temperature, is being attacked respectively 7d, 14d and 21d blood sampling measurement viremia virusemia after poison.21d after poison is attacked, all piglets are carried out to slaughter dissect, lungs are carried out General pathology variation observation, takes lung tissue and tonsillotome to be used for the measurement and pathological study of virus load.

2 results

2.1 the Identification of Biological Characteristics of chimeric strain

As shown in Fig. 2, the plaque form and size of three kinds of recombinant viruses rTd111, rTd111/F2-7 and rTd111/F5-6 It is similar, with parent's strain TJM-F92 no significant difference.Growth curve the results show that rTd111 growth characteristics and parent's strain It is only variant in individual time point compared to almost the same.But strain rTd111/F2-7 and rTd111/F5-6 are fitted into Marc- Growth titre peak value in 145 cells is located at 48h after infection, is below parent's strain TJM-F92's and recombinant virus rTd111 Virus infection titer.

2.2 chimeric virus strain infection PAMs characteristics

There is obvious lesion after PAMs inoculation TJM-F92, rTd111, rTd111/F2-7 and rTd111/F5-6 strain, It is coarse to show as cell edges, there is the poly- heap (Fig. 3-A) in part.One step growth curve the results show that recombinant virus rTd111, RTd111/F2-7 and rTd111/F5-6 and parent plant TJM-F92 are also almost the same (Fig. 3-B) in the growth characteristics of PAMs cell.

Meet 6h, 12h after poison, for 24 hours, using fluorescent quantitation Real-time PCR method detection IL-6, TNF-α, IFN-β and The mRNA expression of IFN-γ, the results show that the inflammation inducing factor of recombinant strain rTd111 level with TJM-F92 strain without Notable difference (P > 0.05), and rTd111/F2-7 is the most significant (P < 0.05) to the up-regulation effect of inflammatory factor mRNA level in-site, RTd111/F5-6 to the induced levels of cell factor then between rTd111 and rTd111/F2-7 (Fig. 4).

Pathogenicity of the 2.3 embedded virus vaccine candidate strains to piglet

2.3.1 clinical change

Piglet is inoculated with 3ml (10⁵TCID₅₀/ml)rTd111、rTd111/F2-7、rTd111/F5-6、TJM-F92、FJ1402 After virus liquid, test group piglet does not occur obvious clinical abnormal symptom, and mean body temperature maintains between 39.5~40 DEG C or so. Further immune rear piglet 0d and 14d weight data is counted, as the result is shown embedded virus immune group and PBS group piglet Average daily gain also no significant difference (P > 0.05), see Fig. 5.

2.3.2 viremia virusemia and virus load measurement

PRRSV carrying capacity in serum is detected after each virus inoculation with fluorescence quantifying PCR method, as a result such as Fig. 6-A, rTd111/ F5-6, rTd111 (P > 0.05) similar with TJM-F92 immune group piglet PRRSV viremia, and rTd111/F2-7 is immune Group is slightly above viremia caused by above-mentioned three kinds of virus.Virus load testing result such as Fig. 6-B in lung tissue, Virus load and FJ1402 attack poison in each immune group piglet lungs of rTd111/F2-7, rTd111/F5-6, rTd111 and TJM-F92 Control group is compared and is substantially reduced (P < 0.05).

2.3.3 pathological study

14d slaughters all piglets after inoculation, acquires lung tissue, prepares histotomy, observes changes in histopathology, knot Fruit are as follows: rTd111/F5-6, rTd111 and TJM-F92 group only show slight lesion, and alveolar structure is complete, and alveolar septum slightly increases Thickness, and rTd111/F2-7 and FJ1402 group obviously show interstitial pneumonia lesion, inflammatory cells increased or red blood cell leaching Profit, alveolar wall thickening are more obvious (Fig. 7).Show that rTd111/F5-6 is pathogenic very low, and rTd111/F2-7 has certain cause a disease Property.

The immune protection effectiveness of 2.4 chimeric vaccine candidates

2.4.1 immune antibody assay

Swine serum is immunized to the different time points of acquisition and carries out antibody test with IDEXX PRRS X3ELISA kit.Knot Fruit shows the immune effect of chimeric strain rTd111/F5-6 and parent's vaccine strain TJM-F92 quite (p > 0.05), can be effective Excite the antibody mediated immunity response of piglet.PBS group piglet blood-serum P RRSV antibody is then always negative (S/P < 0.4) (Fig. 8).

2.4.2 HP-PRRSV BB0907 strain challenge test result

2.4.2.1 clinical symptoms

After HP-PRRSV BB0907 strain attacks poison, there is obvious fervescence (>=40 DEG C), duration in not immune piglet Relatively long, apathetic, loss of appetite, and it is dead 2 final.Chimeric strain rTd111/F5-6 and parent's vaccine strain TJM-F92 exempts from Piglet body temperature is without significantly raised and clinical symptoms after epidemic disease, all strong (Fig. 9) living.Evaluation result shows: chimeric strain rTd111/ F5-6 has good protective effect to the attack of PRRSV velogen strain BB0907.

2.4.2.2 viremia virusemia and tissue virus load

PRRSV carrying capacity after poison is attacked in fluorescence quantifying PCR method detection, as a result such as Figure 10-A, rTd111/F5-6 immune group and parent This vaccine strain TJM-F92 immune group piglet PRRSV viremia is close (P>0.05), be substantially less than non-immune group (P< 0.05)。

Virus load testing result such as Figure 10-B, rTd111/F5-6 immune group piglet lungs in lung tissue and lymph node And virus load is similar to parent's vaccine strain TJM-F92 (P>0.05) in lymph node, be substantially less than non-immunized controls group (P< 0.05), show the immune infection level that can reduce HP-PRRSV BB0907 strain of rTd111/F5-6.

2.4.2.3 lung pathology is observed

21d slaughters piglet after attacking poison, and Figure 11 is shown in lungs naked eyes pathological change, compared with non-Immunization control group, RTd111/F5-6 and TJM-F92 immune group piglet lungs are without obvious naked eyes lesion.Lung histology observation the results show that RTd111/F5-6 and TJM-F92 immune group alveolar structure is complete, and alveolar septum thickens unobvious.The above results show Piglet, which is immunized, in rTd111/F5-6 can be effectively reduced clinical symptoms and pathological change caused by HP-PRRSV BB0907 strain.

2.4.3 PRRSV class NADC30 strain FJ1402 challenge test result

2.4.3.1 clinical symptoms

After FJ1402 strain attacks poison, non-immune group piglet body temperature is significantly raised (>=40 DEG C), and dead 1, TJM-F92 is immune Generating heat (>=40 DEG C) also occurs in piglet, no phenomena of mortality, and piglet is immunized without obvious clinical symptoms (Figure 12), table in rTd111/F5-6 The immune attack that can be effective against class NADC30 strain FJ1402 of bright rTd111/F5-6.

2.4.3.2 viremia virusemia and tissue virus load

PRRSV carrying capacity after poison is attacked in fluorescence quantifying PCR method detection, as a result such as Figure 13-A, TJM-F92 immune group and is not immunized Group piglet PRRSV viremia is similar (P > 0.05), and rTd111/F5-6 immune group viremia virusemia is then substantially less than and is not immunized Group (P < 0.05).

Virus load testing result is shown in lung tissue and lymph node, and virus carries in TJM-F92 immune group piglet lungs Amount is not significant (P > 0.05) with non-Immunization control group difference, and rTd111/F5-6 immune group virus load is substantially less than not Immunized controls group (P < 0.05) (Figure 13-B) illustrates the immune infection level that can reduce FJ1402 strain of rTd111/F5-6, and The protecting effect of TJM-F92 vaccine strain is relatively poor.

2.4.3.3 lung pathology is observed

As shown in figure 14,21d slaughters piglet, TJM-F92 immune group and non-Immunization control group piglet lungs after attacking poison There is obvious enlargement, diffusivity and focal sample consolidation, lungs color are dark red.Histological examination shows interstitial pneumonia, alveolar Wall thickening, inflammatory cells increased or red blood cell infiltration.RTd111/F5-6 immune group piglet lungs are without obvious naked eyes lesion, tissue It learns and checks the slight lesion of performance, alveolar structure is complete, and alveolar septum slightly thickens.

The above results show that rTd111/F5-6 is immunized piglet and can be effectively reduced PRRSV class NADC30 strain FJ1402's Attack, and TJM-F92 cannot then provide the clinical protection effect to FJ1402 virus strain infection completely.

Sequence table

<110>Agricultural University Of Nanjing

<120>a kind of porcine reproductive and respiratory syndrome virus is fitted into strain and its application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 14631

<212> DNA

<213>porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virusrTd111/F5-6 F5 CGMCC No.14762)

<400> 1

atgacgtata ggtgttggct ctatgccacg gcatttgtat tgtcaggagc tgtgaccata 60

ggcacagccc aaaacttgct gcacgggaac accctcctgt gacagccctc ttcaggggga 120

ttaggggtct gtccctaaca ccttgcttcc ggagttgcac tgctttacgg tctctccacc 180

cctttaacca tgtctgggat acttgatcgg tgcacgtgta cccccaatgc cagggtgttt 240

gtggcggagg gccaggtcta ctgcacacga tgtctcagtg cacggtctct ccttcctctg 300

aatctccaag ttcctgagct tggggtgctg ggtctattct ataggcccga agagccactc 360

cggtggacgt tgccacgtgc attccccact gtcgagtgct cccccgccgg ggcctgctgg 420

ctttctgcga tctttccgat tgcacgaatg actagtggaa acctgaactt tcaacaaaga 480

atggtgcggg tcgcagctga aatctacaga gccggccaac tcacccctac agttctaaag 540

actctacaag tttatgaacg gggttgtcgc tggtacccca ttgtcgggcc cgtccctggg 600

gtgggcgttt acgccaactc cctgcatgtg agtgacaaac ctttcccggg agcaactcat 660

gtgttaacca acttgccgct cccgcagagg cccaaacctg aggacttttg cccttttgag 720

tgtgctatgg ctgacgtcta tgacattggt cgtggcgccg tcatgtatgt ggccggagga 780

aaggtctctt gggcccctcg tggtgggaat gaagtgaaat ttgaacctgt ccccaaggag 840

ttgaagttgg ttgcgaaccg actccacacc tccttcccgc cccatcacgt agtggacatg 900

tccaggttta ccttcatgac ccctgggagt ggtgtctcca tgcgggttga gtaccaatac 960

ggctgcctcc ccgctgacac tgtccctgaa ggaaactgct ggtggcgctt gtttgactcg 1020

ctcccaccgg aagttcagta caaagaaatt cgccacgcta accaatttgg ctatcaaacc 1080

aagcatggtg tccctggcaa gtacctacag cggaggctgc aagttaatgg tcttcggaca 1140

gtgaccgaca cacatggacc tatcgtcata cagtacttct ctgttaagga gagttggatc 1200

cgccacctga agttggtgga agaacccagc ctccccgggt ttgaggatct cctcagaatc 1260

agggttgagc ccaatacgtc accactggct ggaaaggatg agaagatttt ccggtttggc 1320

agtcataagt ggtacggtgc cggaaagaga gcaaggaaaa cacgctctgg tgcgactact 1380

atggtcgctc atcacgcttc gtccgctcat aaaatccggc aggccacgaa gcacgagggt 1440

gccggcgcta acaaggctga gcatctcaag cgctactctc cgcctgccga agggaactgt 1500

ggttggcact gcatttccgc catcgccaac cggatggtga attccaactt tgagaccacc 1560

cttcctgaaa gagtaaggcc ttcagatgac tgggccactg acgaggatct tgtgaatacc 1620

atccaaatcc tcaggctccc tgcggccttg gacaggaacg gcgcttgcgg tagcgccaag 1680

tacgtgctta aactggaggg tgagcattgg actgtctctg tgatccctgg gatgtcccct 1740

actttgctcc cccttgaatg tgttcagggt tgttgtgaac ataagggcgg tcttgtttcc 1800

ccggatgcgg tcgaaatttc cggatttgat cctgcctgcc ttgaccgact ggctaaggta 1860

atgcacttgc ctagcagtac catcccagcc gctctggccg aattgtccga cgactccaac 1920

cgtccggttt ccccggccgc tactacgtgg actgtttcgc aattctatgc tcgtcataga 1980

ggaggagatc atcatgacca ggtgtgctta gggaaaatca tcagcctttg tcaagttatt 2040

gaggactgct gctgccatca gaataaaacc aaccgggcta ctccggaaga ggtcgcggca 2100

aagattgatc agtacctccg tgacgcaaca agtcttgagg aatgcttggc caaacttgag 2160

agagtttccc cgccgagcgc agcggacacc tcctttgatt ggaatgttgt gcttcctggg 2220

gttgaggcga cgaatcagac aaccgaacaa cctcacgtca actcatgctg caccccggtc 2280

cctcccgtga ctcaagagcc tttgccaaga gttcaacctc gaagaacaaa gtctgtcaaa 2340

agtttgccag aggacaagcc tgtccctgct ccgcgcagga aggtcagatc cgattgcggc 2400

agcccggttt tgatgggcga caatgtccct aacggttcgg aagaaactgt cggtggtctc 2460

ctcaattttc cgacaccatc cgagccgatg acacctatga gtgagcccgt acttgtgccc 2520

gcgtcgcgac gtgtccccaa gctgatgaca cctttgagtg ggtcggcacc agttcctgca 2580

ccgcgtagaa ctgtgacaac aacgctgacg caccaggatg agcctctgga tttgtctgcg 2640

tcctcacaaa cggaatatga ggcttccccc ctaacaccat cgcagaacat gggcatcctg 2700

gaggcggggg ggcaagaagc tgagggagtc ctgagtgaaa tctcggatat actaaatgac 2760

accaaccctg cacctgtgtc atcaagcagc tccctgggtt cagtggccac cgaggatgtt 2820

ccacgcatcc tcgggaaaat aggagacact gacgagctgc ttgaccgagg tccctcggca 2880

ccctccaagg gagaaccggt ctgtgaccaa cctgccaaag atccccggat gtcgccgcgg 2940

gagtctgacg agagcataat agttccgccc gcagatacag gtggtgtcgg ctcattcact 3000

ggtttgccgt cttcagatgg tgtggatgtg gacggggggg ggccgttaag aacggtaaaa 3060

acaaaagcag aaaggctctt agatcaactg agctgccagg tttttagcct cgtttcccat 3120

ctccctattt tcttctcaca cctcttcaaa tctgacagtg gttattctcc gggtgattgg 3180

ggttttgcag cttttactct attttgcctc tttttatgtt acagttaccc attcttcggt 3240

tttgctcccc tcttgggtgt attttctggg tcttctcggc gtgtgcgaat gggggttttt 3300

ggctgctggt tggcttttgc tgttggtctg ttcaagcctg tgtccgaccc agtcggcact 3360

gcttgtgagt ttgactcgcc agagtgtagg aacgtccttc attcttttga gcttctcaaa 3420

ccttgggacc ctgtccgcag ccttgttgtg ggccccgtcg gtctcggcct tgccattctt 3480

ggcaggttac tgggcggggc acgctacatc tggcactttt tgcttaggct tggcattgtt 3540

gcagactgtg tcttggctgg agcttatgtg ctttctcaag gtaggtgtaa aaagtgctgg 3600

ggatcttgtg taagaactgc tcctaatgag atcgccttca acgtgttccc ttttacacgt 3660

gcgaccaggt cgtcactcat cgacctgtgc gatcggtttt gcgcaccaaa aggcatggac 3720

cccatttttc tcgccactgg gtggcgtggg tgctggaccg gccggagtcc cattgagcaa 3780

ccttctgaaa aacccatcgc gttcgcccag ctggatgaga agaggattac ggctagaact 3840

gtggtcgctc agccttatga tcccaaccag gccgtaaagt gcttgcgggt attacaggcg 3900

ggtggggcga tggtggccga ggcagtccca aaagtggtca aagtttccgc tattccattc 3960

cgagctcctt tctttcccgc tggagtgaaa gttgatcctg agtgcagaat cgtggttgat 4020

cccgatactt ttactacagc cctccggtct ggctattcca ccgcgaacct cgtccttggt 4080

acgggggact ttgcccagct gaatggacta aagatcaggc aaatttccaa gccttcaggg 4140

ggaggcccac acctcattgc tgccttgcat gttgcctgct cgatggcgtt acacatgctt 4200

gctggtgttt atgtaactgc agtggggtcc tgcggtgccg gtaccaacga tccgtggtgc 4260

actaacccgt ttgctgtccc tggctatgga cctggctctc tttgcacgtc tagattgtgc 4320

atctcccaac acggcctcac cttgcccttg acagcacttg tggcgggatt cggccttcaa 4380

gagattgcct tggtcgtttt gatctttgtc tccatcggag gcatggctca taggttgagt 4440

tgtaaggctg acatgttgtg catcttactc gcaatcgcta gttatgtttg ggtacctctt 4500

acctggttgc tttgtgtgtt tccttgttgg ttgcgctggt tctctttgca ccccctcacc 4560

atcctgtggt tggtgttttt cttgatttct gtaaatatac cctcgggaat cttggccgtg 4620

gtgttattgg tttctctctg gcttttaggt cgttatacta acattgctgg tctcgtcacc 4680

ccttatgaca ttcatcatta caccagtggc ccccgcggtg tcgccgcctt ggccaccgcg 4740

ccagatggaa cctacttggc tgccgtccgc cgtgctgcgc tgactggtcg taccatgctg 4800

ttcaccccgt ctcagcttgg gtccctcctt gagggcgctt tcagaactca aaaaccctca 4860

ctgaacaccg tcaatgtggt cgggtcctcc atgggctctg gcggagtgtt cactattgac 4920

gggaaaatca agtgcgtgac tgccgcacat gtccttacgg gtaactcagc tagggtttcc 4980

ggggtcggct tcaatcaaat gcttgacttt gatgtaaaag gggacttcgc catagctgat 5040

tgcccgaatt ggcaaggggt tgctcccaag gcccagttct gcgaggatgg gtggactggt 5100

cgcgcctatt ggctgacatc ctctggcgtt gaacccggtg ttattgggaa tgggttcgcc 5160

ttctgcttca ccgcgtgtgg cgattctgga tccccagtga ttaccgaagc cggtgagctt 5220

gtcggcgttc acacaggatc aaacaaacaa ggaggaggca ttgtcacgcg cccctcaggc 5280

cagttttgta atgtgaagcc catcaagctg agcgagttga gtgaattctt cgctggacct 5340

aaggtcccgc tcggtgatgt gaaaattggc agtcacataa ttaatgacac atgcgaggtg 5400

ccttcagatc tttgtgccct gcttgctgcc aaacccgaac tggaaggagg cctttccaca 5460

gttcaacttc tgtgtgtgtt tttcctcctg tggagaatga tggggcatgc ctggacgccc 5520

ttggttgctg tggggttttt catcctgaat gagattctcc cagctgttct ggtccggagt 5580

gttttctcct ttgggatgtt tgtgctatct tggctcacac catggtctgc gcaagtcctg 5640

atgatcaggc ttctgacagc agcccttaac agaaacagat ggtctcttgg tttttacagc 5700

cttggtgcaa taaccagttt tgtcgcagat cttgcggtaa ctcaagggca tccgttacag 5760

gtggtaatga acttaagcac ctatgccttc ctgccccgga tgatggttgt gacctcgcca 5820

gtcccagtga tcgcgtgtgg tgttgtgcac ctccttgcca taattttgta cttgtttaag 5880

taccgctgcc ttcacaatgt ccttgttggc gatggggtgt tctcttcggc tttcttcttg 5940

cgatactttg ccgagggaaa gttgagggaa ggggtgtcgc aatcctgtgg gatgagtcat 6000

gagtcgctga ctggtgccct cgccatgaga ctcactgacg aggacttgga tttccttacg 6060

aaatggactg attttaagtg ctttgtttct gcgtccaaca tgaggaatgc agcgggccaa 6120

tttatcgagg ctgcttatgc aaaagcacta agaattgaac ttgctcagtt ggtgcaggtt 6180

gataaggtcc gaggcaccat ggccaaactc gaggctttcg ccgataccgt ggcaccccaa 6240

ctctcgcccg gtgacattgt tgttgccctt ggccacacgc ctgttggcag catcttcgac 6300

ctaaaggttg gtagcaccaa gcatactctc caagctattg agactagagt ccttgccggg 6360

tccaaaatga ctgtggcgcg tgtcgttgac ccaacccccg cacccccgcc cgtacctgtg 6420

cccatccctc tcccaccgaa agttctggag aacggtccca atgcctgggg ggatgaggac 6480

cgtttgaaca agaagaagag gcgcaggatg gaagccgtcg gcatttttgt catggacggg 6540

aaaaagtacc agaaattttg ggacaagaat tccggtgatg tgttttatga ggaggtccat 6600

attagcacag acgagtggga gtgccttaga actggcgacc ctgtcgactt tgatcctgag 6660

acagggattc agtgtgggca tatcaccatt gaagataagg tttacaatgt cttcacctcc 6720

ccatctggta ggagattctt ggtccccgcc aaccccgaga atagaagagc tcagtgggaa 6780

gccgccaagc tttccgtgga gcaagccctt ggtatgatga acgtcgacgg cgaactgact 6840

gccaaagaac tggagaaact gaaaagaata attgacaaac tccagggcct gactaaggag 6900

cagtgtttaa actgctagcc gccagcggcc tgacccgctg tggtcgcggc ggcttagttg 6960

ttactgagac agcggtaaaa atagtcaaat ttcacaaccg gaccttcacc ctaggacctg 7020

tgaacttaaa agtggccagt gaggttgagc taaaagacgc ggttgagcac aaccaacatc 7080

cggttgccag accggttgat ggtggtgtcg tgctcctgcg ctctgcagtt ccttcgctta 7140

tagatgtctt gatctccggc gctgatacat ctcctaagtt actcgcccgc cacgggccgg 7200

gaaacactgg gattgatggc acgctttggg attttgaggc cgaggctact aaagaggaag 7260

ttgcactcag tgcgcaaata atacaggctt gtgatattag gcgcggcgac gcacctgaaa 7320

ttggtctccc ttataagttg taccctgtta ggggcaaccc tgagcgggta aaaggagttt 7380

tacagaacac aaggtttgga gacatacctt acaaaacccc cagtgacact ggaagcccgg 7440

tgcacgcggc tgcctgcctc acgcctaatg ctactccggt gactgatggg cgctccgtct 7500

tggctacaac catgccctct ggctttgagt tgtatgtgcc gaccattcca gcgtccgtcc 7560

ttgattatct tgattctagg cctgactgcc ctaaacagtt aacagagcac ggttgtgagg 7620

atgctgcatt aagagacctc tccaagtatg atttgtccac ccaaggcttt gttttgcctg 7680

gagttcttcg ccttgtgcgg aagtacctgt tcgcccacgt gggtaagtgc ccgcccgttc 7740

atcggccttc cacttaccct gctaagaatt ctatggctgg aataaatggg aacaggtttc 7800

caaccaagga cattcagagc gtccctgaaa tcgacgttct gtgcgcacag gctgtgcgag 7860

aaaactggca aactgttacc ccttgtaccc tcaagaaaca gtactgtggg aagaagaaga 7920

ctaggacaat acttggcacc aataacttca ttgcgttggc ccatcgggca gcgttgagtg 7980

gtgttaccca gggcttcatg aaaaaagcgt tcaactcgcc catcgccctc gggaaaaaca 8040

aatttaagga gctacaagcc ccggtcctag gcaggtgcct tgaagctgat cttgcgtcct 8100

gcgatcgatc cacacctgca attgtccgct ggtttgccgc caatcttctt tatgaactcg 8160

cctgtgctga ggagcatcta ccgtcgtacg tgctgaactg ctgccacgac ttactggtca 8220

cgcagtccgg cgcggtgact aagagaggtg gcctgtcgtc tggcgacccg attacctctg 8280

tgtcaaacac catttacagc ttagtgatat atgcacagca catggtgctc agttacttca 8340

aaagtgctca ccctcatggc cttctgtttc tgcaagacca gctgaagttt gaggacatgc 8400

tcaaggttca acccctgacc gtctattcgg acgaccttgt gctgtatgcc gagtctccct 8460

ccatgccaaa ctaccactgg tgggttgaac atctgaacct tatgctgggt ttccagacgg 8520

acccaaagaa gacaaccatc acagactcac catcattcct aggttgcagg ataataaatg 8580

ggcgccagct ggtccctaac cgtgacagga tcctcgcggc cctcgcctac cacatgaagg 8640

cgagcaatgt ttctgaatac tacgcctcgg cggctgcaat actcatggac agctgtgctt 8700

gtttggagta tgatcctgaa tggtttgaag agctcgtggt tgggatagcg cagtgcgccc 8760

gcaaggacgg ctacagcttt cctggcccac cgttcttctt gtccatgtgg gaaaaactca 8820

ggtccaatca tgaggggaag aagtccagaa tgtgcgggta ctgcggggcc ccggctccgt 8880

acgccactgc ctgtggtctc gatgtctgtg tttaccacac ccacttccac cagcattgtc 8940

ctgttataat ctggtgtggc cacccggcgg gttctggttc ttgtagtgag tgcgaacccc 9000

ccctaggaaa aggcacaagc cctctagatg aggtgttaga acaagttccg tacaagcctc 9060

cgcggactgt gatcatgcat gtggagcagg gtctcacccc tcttgaccca ggtagatacc 9120

agactcgccg cggattggtc tccgttaggc gtggcatcag gggaaatgaa gtcgacctac 9180

cagacggtga ttacgccagt accgccttgc tccctacttg taaagagatc aacatggtcg 9240

ctgtcgcctc taacgtgttg cgcagcaggt ttatcatcgg cccacccggt gctgggaaaa 9300

cacactggct tcttcaacaa gtccaggatg gtgatgtcat ttacacgcca actcaccaga 9360

ccatgctcga catgattagg gctttgggga cgtgccggtt caacgttcca gcaggtacaa 9420

cgctgcaatt ccctgccccc tcccgtaccg gcccatgggt tcgcatcttg gccggcggtt 9480

ggtgtcctgg caagaactcc ttcctggatg aagcggcgta ttgcaatcac cttgacgtct 9540

tgaggcttct cagtaaaaca actctcactt gcctagggga cttcaaacaa ctccaccctg 9600

tgggttttga ctcccattgc tatgtatttg acatcatgcc tcagacccaa ttaaagacca 9660

tctggaggtt cgggcagaat atctgtgatg ccattcaacc agattacagg gacaaactta 9720

tggccatggt caacacgacc cgtgtgacct acgtggaaaa acctgtcagg tacgggcaag 9780

tcctcacccc ctaccacagg gaccgagagg acggcgccat tactatcgac tccagtcaag 9840

gcgccacatt tgatgtggtt acactgcatt tgcccactaa agattcactc aacaggcaaa 9900

gagctcttgt tgctatcacc agggcaaggc atgctatctt cgtgtatgac ccacacaggc 9960

aattgcagag catgtttgat cttcccgcga gaggcacacc cgtcaacctc gcagtgcacc 10020

gtgacgaaca gctgatcgta ttagacagaa acaacagaga aatcacggtt gctcaggctc 10080

taggcaatgg agataaattc agggccacag ataagcgcgt tgtagattct ctccgcgcta 10140

tttgcgcaga cctggaaggg tcgagctccc cgctccccaa ggtcgcgcat aacttgggat 10200

tccatttctc acctgatttg actcagtttg ctaaactccc ggcagaactt gcaccccact 10260

ggcccgtggt gacaacccag aacaatgaaa ggtggccaga tcggctggta gccagcctcc 10320

gccctatcca taaatatagc cgcgcgtgca ttggtgccgg ctatatggtg ggcccctcgg 10380

tgtttttagg cacccctggg gttgtgtcat actatctcac aaaatttgtt agaggcgagg 10440

ctcaagtgct tccggagaca gtcttcagca ccggccgaat tgaggtagat tgtcgagagt 10500

atcttgatga tcgggagcga gaagttgctg agtccctccc acatgccttc atcggcgatg 10560

tcaaaggtac caccgttggg ggatgtcatc acgttacctc caaatacctt ccgcgcttcc 10620

ttcccaagga atcagttgcg gtggtcgggg tttcgagccc cgggaaagcc gcgaaagcag 10680

tttgcacatt gacggatgtg tacctcccag accttgaagc gtacctccac ccagagaccc 10740

agtccaggtg ctggaaagtg atgttggact ttaaggaggt tcgactgatg gtatggaaag 10800

acaagacggc ctattttcaa cttgaaggcc gccattttac ctggtatcaa cttgcaagct 10860

acgcctcata catccgagtt cctgttaatt ctactgtgta cttggacccc tgcatgggcc 10920

ctgctctttg caacaggagg gttgtcgggt ccacccattg gggagctgac ctcgcagtca 10980

ccccttatga ttacggtgcc aaaattattc tgtctagtgc ataccatggt gaaatgcctc 11040

caggttacaa aattctggcg tgcgcggagt tctcgcttga tgatccagta aggtacaaac 11100

acacctgggg atttgaatcg gatacagcgt atctgtacga gtttactgga aatggtgagg 11160

actgggagga ttacaatgat gcgtttcggg cgcgccagaa agggaaaatt tataaagcta 11220

atgccaccag catgaggttt cattttcccc cgggccctgt cattgaacca actttaggcc 11280

tgaattgaaa tgaaatgggg tctatgcaaa gcctctttga caaaattggc caactttttg 11340

tggatgcttt cacggaattt ctggtgtcca ttgttgatat catcatattt ttggccattt 11400

tgtttggctt cacaatcgcc ggttggctgg tggtcttatg catcagactg gtttgctccg 11460

cggtactccg tgcgcgctct accgttcacc ctgagcaatt acagaagatc ttatgaggcc 11520

tttctttctc agtgtcaggt agacattccc acctggggcg tcaaacaccc tttgggggtg 11580

ctttggcacc ataaggtgtc aaccctgatt gatgaaatgg tgtcgcgtcg aatgtaccgc 11640

gtcatggaaa aagcagggca ggctgcctgg aaacaggtgg tgagcgaggc tacattgtct 11700

cgcattagtg gtttggatgt ggtggctcat tttcaacatc ttgccgctat tgaagccgag 11760

acttgtaaat atttggcttc ccggctaccc atgctgcaca acctgcgctt gacagggtca 11820

aatgtaacca tagtgtataa tagtactttg gatcaggtgt ttgccatttt cccaacccct 11880

ggttcccggc caaagcttca tgattttcag caatggctaa tagctgtaca ttcctccata 11940

ttttcctccg ttgcagcttc ttgtactctt tttgttgtgc tgtggttgcg aattccaatg 12000

ctacgttctg tttttggttt ccgctggtta ggggcaactt ttcttttgaa ctcatggtga 12060

attacacggt atgcccgctt tgcccaaccc ggcaggcagc cgccgagatc cttgaacccg 12120

gcaagtcttt ttggtgcagg atagggcatg accgatgtag tgagaacgat catgacgaac 12180

tagggttcat ggttccgcct ggcctctcca gcgaaggcca cttgaccagt gtttacgcct 12240

ggttggcgtt cctgtccttc agctacacgg cccagttcca tcccgagata tttgggatag 12300

ggaatgtgag tcaagtttat gttgacatca agcaccaact catctgcgct gttcatgacg 12360

gggataacgc caccttgcct cgccatgaca atatttcagc cgtatttcag acctactacc 12420

aacaccaggt cgacggcggc aattggtttc acctggaatg gctacgccct ttcttttcct 12480

cttggttggt tttaaatgtt tcgtggtttc tcaggcgttc gcctgcaagc catgtttcag 12540

ttcgagtctt tcggacatca aaaccaacac caccgcagca tcaggcttcg ttgtcctcca 12600

ggacatcagc tgccttaggc atggcgactc gtcctctccg acgattcgca aaagttctca 12660

gtgccgcacg gcgataggga cgcccgtgta catcaccatc actgccaatg tcacagatga 12720

aaattatcta cattcttctg atctcctcat gctttcttct tgccttttct atgcttccga 12780

gatgagtgaa aagggattca aagtggtgtt tggcaatgtg tcaggcatcg tggctgtgtg 12840

cgtcaacttt accagctacg tccaacacgt caaggagttt acccaacgct ccttagtggt 12900

cgatcatgtg cgactgcttc atttcatgac acctgagacc atgaggtggg caaccgtttt 12960

agcctgtctt tttgccatcc tactggcaat ttgaatgttc aagtatgttg gggaaatgct 13020

tgaccgcggg ttactactcg caattgcctt ttttgtggta tatcgtgcca ttctgttttg 13080

ctgcgctcgt caacgccagc agcaacagca gctcccattt acagttgatt tataacctga 13140

cgatatgtga gctgaatggc acagattggc tgagcacaaa atttgactgg gcagtggaga 13200

ctttcgttat ctttcctgtg ttgactcata ttgtctctta cggcgccctt accactagcc 13260

attttcttga cacggtcggc ctgatcactg tgtccaccgc cggttattat cacaagcggt 13320

atgtattgag tagcatctac gctgtctgtg ccctggctgc gttggtttgc ttcgccatta 13380

ggttggcgaa aaattgcatg tcctggcgct actcatgcac cagatatacc aattttcttc 13440

tggatactaa gggcaaactc taccgctggc ggtcacccgt catcattgag aaggggggta 13500

aagttgatgt tgaaggtcat ttaatcgacc tcaagacagt tgtgcttgat ggttccgcgg 13560

caacccctgt aaccaagatt tcagcggaac aatggggtcg tccatagacg acttctgcaa 13620

tgacagcacg gctgtacaaa aggtgctctt ggcgttttct atcacctaca cgccaataat 13680

gatatatgcc ttaaaagtaa gtcgtggtcg actgctgggg cttttacacc tcttgatttt 13740

cctgaactgt gctttcactt ttgggtatat gacatttgtc cattttcaga gtacaaacaa 13800

ggtcgcactt accctggggg cagttgtcgc tctcctctgg ggggtgtatt cagccctgga 13860

aacctggaga ttcatcacct ccaggtgccg gttgtgcttg ctaggccgca agtacattct 13920

ggcccctgcc caccacgtag aaagtgccgc aggctttcat ccgataacgg caagtgataa 13980

ccacgcattt gtcgtccggc gtcccggctc cactacggtc aacggcacac tggtgcccgg 14040

gttgaaaagc ctcgtgttgg gtggcagaag agctgtcaaa cgaggagtgg tgaaccttgt 14100

taaatatgcc aaataacaac ggcaagcagc aaaagaaaaa gaaggggaat ggccagccag 14160

tcaatcagct gtgccaaatg ctgggtaaga tcatcgccca acaaaaccag tccagaggca 14220

agggaccggg gaagaaaaat aggaagaaaa acccggggaa gccccatttc cctctagcga 14280

ctgaagatga cgtcaggcat cactttaccc ctagtgagcg gcaattgtgt ctgtcgtcga 14340

tccagactgc cttcaatcag ggtgctggaa cttgtgccct gtcagattca gggaggataa 14400

gttacactgt ggagtttagt ttgccgacgc aacatactgt gcgtctgatc cgcgccacag 14460

catcaccctc agcatgatgg gctggcattc tttggcacct cagtgttaga attgggagaa 14520

tgtgtggtga atggcactga ttgacactgt gcctctaagt cacctattca attagggcga 14580

ccgtgtgggg gtaaagttta attggcgaga accatgcggc cgtaattaaa a 14631

<210> 2

<211> 603

<212> DNA

<213>porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virusrTd111/F5-6 F5 CGMCC No.14762)

<400> 2

atgttgggga aatgcttgac cgcgggttac tactcgcaat tgcctttttt gtggtatatc 60

gtgccattct gttttgctgc gctcgtcaac gccagcagca acagcagctc ccatttacag 120

ttgatttata acctgacgat atgtgagctg aatggcacag attggctgag cacaaaattt 180

gactgggcag tggagacttt cgttatcttt cctgtgttga ctcatattgt ctcttacggc 240

gcccttacca ctagccattt tcttgacacg gtcggcctga tcactgtgtc caccgccggt 300

tattatcaca agcggtatgt attgagtagc atctacgctg tctgtgccct ggctgcgttg 360

gtttgcttcg ccattaggtt ggcgaaaaat tgcatgtcct ggcgctactc atgcaccaga 420

tataccaatt ttcttctgga tactaagggc aaactctacc gctggcggtc acccgtcatc 480

attgagaagg ggggtaaagt tgatgttgaa ggtcatttaa tcgacctcaa gacagttgtg 540

cttgatggtt ccgcggcaac ccctgtaacc aagatttcag cggaacaatg gggtcgtcca 600

tag 603

<210> 3

<211> 525

<212> DNA

<213>porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virusrTd111/F5-6 F5 CGMCC No.14762)

<400> 3

atggggtcgt ccatagacga cttctgcaat gacagcacgg ctgtacaaaa ggtgctcttg 60

gcgttttcta tcacctacac gccaataatg atatatgcct taaaagtaag tcgtggtcga 120

ctgctggggc ttttacacct cttgattttc ctgaactgtg ctttcacttt tgggtatatg 180

acatttgtcc attttcagag tacaaacaag gtcgcactta ccctgggggc agttgtcgct 240

ctcctctggg gggtgtattc agccctggaa acctggagat tcatcacctc caggtgccgg 300

ttgtgcttgc taggccgcaa gtacattctg gcccctgccc accacgtaga aagtgccgca 360

ggctttcatc cgataacggc aagtgataac cacgcatttg tcgtccggcg tcccggctcc 420

actacggtca acggcacact ggtgcccggg ttgaaaagcc tcgtgttggg tggcagaaga 480

gctgtcaaac gaggagtggt gaaccttgtt aaatatgcca aataa 525

Claims (10)

1. a kind of porcine reproductive and respiratory syndrome virus is fitted into strain, it is characterized in that: the porcine reproductive and respiratory syndrome virus Chimeric strain is the expression 323-433 on the Nsp2 gene by highly pathogenic PRRSV vaccine strain The genetic fragment of amino acids deletes while the ORF5-6 of the strain being replaced with to the ORF5-6 structure of PRRSV class NADC30 strain Obtained by building.

2. porcine reproductive and respiratory syndrome virus according to claim 1 is fitted into strain, it is characterized in that: described highly pathogenic Porcine reproductive and respiratory syndrome virus vaccine strain is highly pathogenic PRRSV TJM-F92 vaccine low virulent strain, the PRRSV class NADC30 strain is class NADC30 strain FJ1402.

3. a kind of porcine reproductive and respiratory syndrome virus is fitted into strain, it is characterized in that: entitled porcine reproductive and respiratory syndrome is sick Malicious rTd111/F5-6 F5, deposit number are CGMCC No.14762.

4. porcine reproductive and respiratory syndrome virus according to claim 3 is fitted into strain, it is characterized in that: its nucleotide sequence As shown in SEQ ID NO.1.

5. porcine reproductive and respiratory syndrome virus described in claim 1 or 3 is fitted into the construction method of strain, it is characterized in that including Following steps:

(1) infectious CDNA for constructing highly pathogenic PRRSV TJM-F92 vaccine low virulent strain clones pCMV-TJM；

(2) segment that 323-433 amino acids are expressed on the Nsp2 gene of pCMV-TJM is deleted, building obtains infectivity CDNA clone pTd111；

(3) using pTd111 as skeleton, its ORF5 and ORF6 are replaced with into porcine reproductive and respiratory syndrome class NADC30 strain The ORF5 and ORF6 of FJ1402, building obtain infectious CDNA clones pTd111/F5-6；

(4) pTd111/F5-6 is subjected to virus rescue, obtains porcine reproductive and respiratory syndrome virus rTd111/F5-6, is i.e. pig is numerous It grows and respiratory syndrome virus chimeric strain.

It breeds and breathes 6. porcine reproductive and respiratory syndrome virus of any of claims 1-5 is fitted into strain as pig The application of the candidate vaccine of syndrome prevalence strain.

It breeds and exhales in preparation pig 7. porcine reproductive and respiratory syndrome virus of any of claims 1-5 is fitted into strain Inhale the application in syndrome vaccine.

8. a kind of vaccine, it is characterized in that: effective component include immune amount pig of any of claims 1-5 breeding with Respiratory syndrome virus chimeric strain.

9. application of the vaccine according to any one of claims 8 in the drug of preparation prevention porcine reproductive and respiratory syndrome.

10. application according to claim 9, it is characterized in that: the porcine reproductive and respiratory syndrome is bred by classical pig Virus strain infection causes with respiration syndrome, or is caused by high-pathogenicity porcine reproductive and respiration syndrome virus strain infection, or by Porcine reproductive and respiratory syndrome class NADC30 virus strain infection causes, or is caused by above-mentioned three kinds of strain cross-infections.