CN101423823A - Porcine reproductive and respiratory syndrome recombinant adenovirus rAd-GF35 - Google Patents

Abstract

The invention discloses a virus (PRRSV) recombinant adenovirus rAd-GF35 of porcine reproduction and respiratory syndrome, which belongs to the technical field of high-tech biotechnology. Highly pathogenic PRRSV SY0608 separation strains of GP3 and GP5 protein genes and porcine colony sell stimulating factor (GMCSF) whole gene sequences are amplified, connected in series and cloned into pShuttle-CMV to be transformed into escherichia coli BJ5183 together with pAdEasy-1 so as to obtain recombinant plasmids; and HEK293A cells are transfected to obtain the recombinant adenovirus so as to successfully express PRRSV SY0608 strains GP3 and GP5 and a GMCSF protein. The recombinant virus can express the GMCSF correctly, and play a role of an adjuvant so as to improve the immune activity of the PRRSV GP3 and GP5 proteins and stimulate the immune protective reaction of an organism more effectively. The recombinant adenovirus vaccine has the safety of a subunit vaccine and the antigen proliferating ability of an attenuated vaccine, thereby having wide development and application prospect.

Description

The porcine reproductive and respiratory syndrome recombinant adenovirus rAd-GF 35

One, technical field

The present invention relates to porcine reproductive and respiratory syndrome virus (PRRSV) recombinant adenovirus rAd-GF 35 and vaccine; belong to the biotechnology high-tech area, be exclusively used in and strengthen pig the immanoprotection action of porcine reproductive and respiratory syndrome virus and the financial loss on minimizing pig farm.

Two, background technology

The recombinant adenovirus genetic engineering technique is obtaining application comparatively widely aspect the mankind's the gene therapy, the recombinant adenovirus that much is applied to the human gene therapy has entered three stages phase of clinical experiment.Because it is wide that recombinant adenovirus has host range, the cells infected kind is many, can expressing human source and inhuman source protein, and can not insert host chromosome, reverse mutation rate is low, can copy to very high characteristics such as titre.And GP3 GP5 albumen is by about 41kD of the ORF3 ORF5 of porcine reproductive and respiratory syndrome virus PRRSV coding and the glycoprotein of 25kD, and it is active to have a neutralization by its inductive antibody.GP5 albumen with baculovirus expression can provide 50% protection to farrowing sow, the piglet that the GP5 of this external application baculovirus expression, GP3 immunity sow gives birth to PRRSV seronegativity when wean.Behind the GP5 plasmid immune animal under the cytomegalovirus promoter control, produce the specificity neutralizing antibody of anti-GP5 pig and mouse.The monoclonal antibody evidence is better than monoclonal antibody at GP4 at the neutralization activity of the monoclonal antibody of GP5, and GP5 albumen can cell death inducing.GMCSF has good adjuvant effect, is widely used in antiviral and antineoplastic research, also GMCSF is not applied to the recombinant adenovirus report that is built into porcine reproductive and respiratory syndrome virus PRRSV at present.

Three, summary of the invention

Technical problem the objective of the invention is to develop a kind of PRRSV recombinant adenovirus vaccine that can effectively control porcine reproductive and respiratory syndrome (PRRS), and making under the situation that current PRRS is difficult to prevent and treat has new breakthrough.

Technical scheme embodiment of the present invention are as follows:

The porcine reproductive and respiratory syndrome recombinant adenovirus rAd-GF 35, it is characterized in that, recombinant adenovirus rAd-GF 35 belongs in classification: Adenoviridae (Adenoviridae), mastadenovirus (Mastadenovirus), adenovirus hominis kind (Human adenovirus), the classification name: adenovirus (reorganization), this recombinant adenovirus can carry out effectively expressing to target protein, with clinical symptom not occurring behind this recombinant adenovirus virus infection animal, therefore can be used for antigen expressed albumen.This recombinant adenovirus carries out preservation, preservation date at China Committee for Culture Collection of Microorganisms common micro-organisms center: on March 4th, 2008, preserving number is CGMCC No.2391.。

Above-mentioned porcine reproductive and respiratory syndrome recombinant adenovirus makes up by the following method and forms:

(1) amplification of GMCSF and PRRSV GP3 GP5 gene, clone have designed three primers that can increase and lack terminator codon and insert the pig GMCSF of foot and mouth disease 2A gene according to pig GMCSF sequence (the GenBank accession number is U67175), and primer sequence is as follows:

GMCSF-P1：5-GCT?GGTACCATGGCTCCTACCCGCCCA-3

GMCSF-2AP2.1s：

5-GACGTCGCCGGCCAACT?TGAGAAGGTCAAAGTTCT?TTTTG?ACTGGCCCC-3

GMCSF-2AP2.2s：

5-GCACTCGAGGGGCCCTGGGTTGGACTCGACGTCGCCGGCCAACTTGAG-3

The total RNA reverse transcription product of pig peripheral blood lymphocyte that stimulates with ConA is a template, uses the RT-PCR technology and has amplified the pig GMCSF gene of terminator codon that contained 2A genetically deficient.

According to the gene design of the highly pathogenic strain SY0608 of accession number EU144079PRRS at the Auele Specific Primer of the domestic strain isolated SY0608 of PRRSV strain GP3, GP5 gene, primer sequence is as follows:

SY-GP3.1：5-GACCTCGAGATGGCTAATAGCTGTACATT-3

SY-GP3.2：5-ACAAAGCTTTCGCCGTGCGGCACT-3

SY-GP5.1：5-ACGAAGCTTATGTTGGGGAAGTGCT-3

SYGP5.2：5-CAGATATCCTAGAGACGACCCCATTG-3

Use the RT-PCR technology amplified the domestic strain isolated SY0608 of PRRSV strain removal the GP3 gene and the full gene of GP5 of terminator codon, on primer, introduce restriction enzyme digestion sites.Cut by enzyme, GMCSF, GP3 and GP5 gene clone are gone among the shuttle vectors pShuttle-CMV (available from STRATAGENE company), cut with PCR by enzyme then and identify, acquisition contains the shuttle vectors pShuttle-CMV-GMCSF-GP3-GP5 of GMCSF, GP3 and GP5 gene, is called for short pSh-GF35;

(2) contain GMCSF, the shuttle vectors pSh-GF35 of PRRSV GP3 and GP5 gene and adenovirus skeleton carrier pAdEasy-1 homologous recombination are identified contains GMCSF, the shuttle vectors pSh-GF35 of GP3 and GP5 gene is transformed among the coli strain BJ5183 (available from STRATAGENE company) by electric method for transformation with adenovirus skeleton carrier pAdEasy-1 (available from STRATAGENE company) behind linearization for enzyme restriction, under the effect of intestinal bacteria recombinase, homologous recombination takes place between shuttle vectors pSh-GF35 and skeleton carrier pAdEasy-1, realized foreign gene GMCSF, GP3 and GP5 gene change the adenovirus skeleton carrier over to, have obtained to contain the recombinant adenovirus plasmid of foreign gene through the screening of 50 μ g/ml kantlex;

(3) recombinant adenovirus plasmid identified of the acquisition of recombinant adenovirus is by cationic-liposome method transfection HEK293A cell (buying from invitrogen company), and recombinant adenovirus plasmid becomes complete virus in HEK293A cell internal packing.

The vaccine made from above-mentioned porcine reproductive and respiratory syndrome recombinant adenovirus:

With the continuous passage 30 times on the HEK293A cell of the recombinant adenovirus of purifying, testing goal gene transcription and expression prove that target protein expresses stable.The malicious valency of recombinant adenovirus is stabilized in 10 ^8.77TCID ₅₀/ 1.0ml.

The purification of Recombinant adenovirus of taking preservation PRRSV in the enlarged culturing process is by 10 ⁴TCID ₅₀Inoculation grows up to the HEK293A cell of individual layer.When cytopathy reaches 70-90%, receive poison, once can obtain the PRRSV recombinant adenovirus vaccine through multigelation.

Beneficial effect

The present invention has proposed first GMCSF connected with PRRSV structure gene GP3 and GP5 and has been used for adenovirus vector construct, and this recombinant adenovirus has been broken through the limitation of conventional inactivated vaccine of PRRSV and attenuated vaccine.This recombiant vaccine has the security of the duplication characteristic and the inactivated vaccine of attenuated vaccine, and make PRRSV GP3 and GP5 albumen obtain to express in advance, and increased the effect of GMCSF biological activity adjuvant, changed the few and characteristics of waiting a moment of PRRSV infected pigs post neutralization antibody generation.Because what this recombinant adenovirus vaccine adopted is adenovirus hominis serum 5 type carriers, animals such as people, pig are not had pathogenic, pass through safety evaluation easily.Proved that by pig body immunity challenge test and neutralization test this recombinant adenovirus can produce protection to animal, the neutralizing antibody of inducing specific PRRSV produces, in it and antibody titer be 1:32.

The PRRSV recombinant adenovirus that evidence, the present invention make up is stable to exogenous protein expression, and it tires stablely, and the malicious valency of planting poison is stabilized in 10 ^8.77TCID ₅₀/ 1.0ml.Proved that by mouse immuning test and neutralization test this recombinant adenovirus has produced the neutralizing antibody at PRRSV, in it and antibody titer be 1:64.

Recombinant adenovirus is not pathogenic to animal, has the security of height.Utilize this recombinant adenovirus immune mouse to obtain specificity neutralizing antibody at PRRSV, this antibody external can in and PRRSV.

Compare with inactivated vaccine with the attenuated vaccine of the PRRSV of present application; this virus can be duplicated propagation and once and constantly be expressed PRRSV GP3 and GP5 albumen in the pig body; make body continue to be subjected to GP3 and the proteic stimulation of GP5; constantly produce at GP3 and the proteic neutralizing antibody of GP5; simultaneously the GMCSF of Biao Daing can control agent in immune response; from humoral immunization and two aspects of cellular immunization; regulate organism immune response; therefore so just can provide persistent protection, aspect the preventing and treating of PRRS, have broad application prospects the pig body.

Four, description of drawings

The shuttle vectors synoptic diagram is gone in Fig. 1 pig GMCSF and PRRSV GP3 and GP5 gene clone

Fig. 2 contain the shuttle vectors of goal gene and adenovirus skeleton carrier homologous recombination and obtain the synoptic diagram of recombinant adenovirus

Fig. 3 PRRSV-SY0608 GP3 GP5 gene and pig GMCSF RT-PCR product

M:DL15000Marker 1:SY-GP3PCR product; The 2:SY-GP5PCR product; 3: lack signal peptide

GMCSF PCR product

Fig. 4 recombinant plasmid Kpn I/EcoR V enzyme is cut qualification result

M, 1kb DNA Ladder Marker; 1, identify GMCSF-GP3-GP5.

The PacI enzyme of Fig. 5 recombinant plasmid is cut evaluation

M：1kb?plus?DNA?Marker；1：pAd-GF35/Pac?I.

The indirect immunofluorescence antibody coloration result of the HEK293A cell that Fig. 6 recombinant adenovirus infects

A: identify GMCSF B: identify GP3C: identify that GP5 expresses D: wild poison contrast

Fig. 7 Western bLot identifies the expression of target protein

1: do not infect the 293-A cell of recombinant adenovirus, do not have band 2: identify the expression 3 of GMCSF: identify the expression 4 of GP3: identify the expression of GP5

Five, embodiment:

(1) structure of engineering body-PRRSV recombinant adenovirus (rAd-GF35)

The amplification of 1 goal gene, clone, evaluation and sequencing analysis

1.1 design of primers

Seven primers of pig GMCSF gene order design with reference to PRRSV S Y0608 pnca gene sequence (GenBank Accession Number EU144079) and accession number U67175:

SY-GP3.1：5-GACCTCGAGATGGCTAATAGCTGTACATT-3；

SY-GP3.2：5-ACAAAGCTTTCGCCGTGGGCACT-3；

SY-GP5.1：5-ACGAAGCTTATGTTGGGGAAGTGCT-3；

SYGP5.2：5-ACGAAGCTTATGTTGGGGAAGTGCT-3；

GMCSF-P1：5-ACGAAGCTTATGTTGGGGAAGTGCT-3；

GMCSF-2AP2.1s：5-GACGTCGCCGGCCAACTTGAGAAGGTCAAAGTTCTTTTTGACTGGCCCC-3；

GMCSF-2AP2.2s：5-GCACTCGAGGGGCCCTGGGTTGGACTCGACGTCGCCGGCCAACTTGAG-3

After canavailn ConA (5ug/ml) stimulation pig peripheral blood lymphocytes, according to TRIzol (Invitrogen) reagentSpecification sheets extracts the total RNA of post-stimulatory peripheral blood lymphocytes and carries out reverse transcription, according to Promage ThermoScript II mMLV specification sheets with the RNA reverse transcription, with the reverse transcription product is template, application RT-PCR technology has amplified and has contained the pig GMCSF gene that foot and mouth disease 2A gene (have lytic activity, expression product is decomposed into GM-CSF and GP3-GP5) has lacked terminator codon.

According to PRRS strain SY0608 (EU144079) GP3, two pairs of Auele Specific Primers of GP5 gene order design.Total RNA of the monkey-kidney cells MARC-145 cell after the extraction PRRS virus SY0608 virus strain infection, make template with the product after the reverse transcription, use the RT-PCR technology amplified the domestic strain isolated SY0608 of PRRSV strain disappearance the GP3 gene and the full gene of GP5 of terminator codon.

1.2 pcr amplification PRRSV SY0608 strain GP3, GP5 gene and pig GMCSF gene

The mRNA reverse transcription product that extracts of the pig peripheral blood lymphocyte that stimulates with ConA and be that template is carried out the pcr amplification goal gene respectively with the mRNA reverse transcription product cDNA of the MARC-145 cell extraction of SY0608 virus strain infection.

With the cDNA product is template, is the GP3 gene that the upstream and downstream primer amplification does not contain terminator codon with SY-GP3.1 and SY-GP3.2, amplifies the GP3 gene of the about 780bp of size; Above downstream primer SY-GP5.1 and SYGP5.2 amplification contain the GP5 gene of terminator codon, amplify the GP5 gene of the about 620bp of size; With GMCSF-P1 and GMCSF-2AP2.1s is the purpose fragment that the upstream and downstream primer amplification goes out 448bp, and doing template with first round PCR product 448bp again is the GMCSF gene that the upstream and downstream primer amplification goes out 475bp with GMCSF-P1 and GMCSF-2AP2.2s.The PCR reaction system is as follows: get 0.5 μ l plasmid template, add PCR mixed solution 24.5 μ l, comprise 2.5 μ l, 10 * PCR-Buffer, 1.5 μ l 25mmoL/L MgCL ₂, 2.0 μ l 2.5mmoL/L dNTPs, 1.0 μ l 10mmoL/L upstream and downstream primers, 1.0U Taqpolymerase adds distilled water to 24.5 μ l.

The PCR reaction parameter is as follows: 94 ℃ of pre-sex change 3min, carry out 35 PCR circulations (94 ℃ of 45s, 62 ℃ of 45s, 72 ℃ of 60s) again, and last 72 ℃ are extended 10min.

1.3 the recovery of goal gene and purifying

Reclaim test kit with DNA glue and reclaim target gene fragment, concrete operations are undertaken by the specification sheets that the TaKaRa gel reclaims test kit.Enzyme cut and ligase enzyme all available from TaKaRa company, using method is carried out according to specification sheets.

1.4 the clone of recombinant shuttle vector

1.4.1 the enzyme of pcr product and carrier cuts back to close Restriction Enzyme all available from TaKaRa company, using method is carried out according to specification sheets.The GMCSF gene is cut and reclaimed through Kpn I, Xho I enzyme; The GP3 gene is cut and is reclaimed through Xho I, Hind III enzyme; The GP5 gene is cut and is reclaimed through Hind III, EcoR V enzyme.Simultaneously carrier pShuttle-CMV (stratagene company) is cut and reclaims through Kpn I, EcoR V enzyme.The product that obtains is used for following ligation.It is as follows that enzyme is cut system:

3.0 μ l M Buffer, 20.0 μ l gene PCRs reclaim product or carrier, and each 10.0U of restriction enzyme mends to cumulative volume 30.0 μ l with aseptic double-distilled water.

1.4.2 target gene fragment is connected ligase enzyme all available from TaKaRa company with carrier, using method is carried out according to specification sheets.

The ligation volume is 10.0 μ l.System is as follows: 1.0 μ l, 10 * Ligase Buffer, and the pShuttle-CMV that 1.0 μ l enzymes cut back to close, the enzyme of each 1.0 μ l is cut the GMCSF that reclaim the back, GP3 and GP5 gene, 100U T4 DNA Ligase mends to cumulative volume 10.0 μ l with aseptic double-distilled water.4 ℃ connect 8～10h behind the mixing.Set carrier self simultaneously and connect contrast (do not add and connect gene).

1.4.3 the preparation of bacillus coli DH 5 alpha competent cell and conversion

In 3.0ml LB liquid nutrient medium, the 200rpm overnight shaking is cultivated at the single colony inoculation of picking DH5 α on the LB flat board; Next day, the volume by 2% was inoculated in the new LB substratum, and 3-4h to OD600=0.3～0.5 is cultivated in the 200rpm concussion; Bacterium is transferred in the sterilization centrifuge tube of 1.5ml every pipe 1.0ml, ice bath 30min; 4 ℃ of centrifugal 30s of 12000rpm; Supernatant discarded is used the ice-cold CaCL of 1.0ml0.1moL/L fully ₂Resuspended bacterium, ice bath 10min; 4 ℃ of centrifugal 30s of 12000rpm; Abandon supernatant fully, with the ice-cold CaCL of 100 μ l 0.1moL/L ₂Resuspended bacterium is placed in 4 ℃ of refrigerator 24h standby.

Get and connect product 10.0 μ l and join in the competent escherichia coli cell of 100 μ l prepared fresh, mixing gently, ice bath 30min; Put heat-shocked 90s in 42 ℃ of water-baths then, put rapidly and cool off 1～2min in the ice; Every pipe adds 800 μ lLB substratum of preheating, and 45min is cultivated in 37 ℃ of 200rpm concussions; The centrifugal 30s precipitum of 12000rpm with the resuspended thalline of 100 μ l supernatants, is coated on the 100 μ g/ml kalamycin resistance flat boards then, cultivates 16～24h for 37 ℃.

1.4.4 a small amount of of recombinant plasmid is extracted and is identified

Alkaline lysis adopts Kpn I, EcoR V double digestion authentication method to identify after extracting plasmid in a small amount.The endonuclease reaction system is 30.0 μ l.System is as follows: 3.0 μ l, 10 * M Buffer, and 10 μ l plasmids, 10U Kpn I, EcoR V mend to cumulative volume 30.0 μ l with aseptic double-distilled water.

37 ℃ of effect 1.0～3.0h carry out agarose gel electrophoresis then.

1.4.5 the acquisition of recombinant plasmid and name

The plasmid called after pShuttle-CMV-GMCSF-GP3-GP5 that the restriction enzyme digestion and electrophoresis collection of illustrative plates is correct abbreviates pSh-GF35 as.And be sent to precious biotechnology (Dalian) company limited and check order.

1.5 cDNA sequential analysis

Synthesized a pair of sequencing primer according to pShuttle-CMV carrier sequencing primer sequence by the Shanghai cellular biochemical, primer sequence is

Adseq1：5-GGT?ATA?TAT?AAG?CAG?AGC?TG-3；

Adseq2：5-GTG?GTA?TGG?CTG?ATT?ATG?ATC?AG-3。

Examining order is assisted to finish by precious biotechnology (Dalian) company limited.(ABIPRISMTM377XL DNA Sequencer) carries out two-way order-checking at the dna sequencing instrument.And the Nucleotide of the gene in measured sequence and aminoacid sequence that pushes over and the GenBank database and protein are carried out homology relatively with NCBI BLAST, carry out the restriction enzyme and the reading frame of gene again with Vector NTI and DNAStar software.Proved that the gene that we were cloned into is correct, met carrier fully and read the frame requirement.

2 shuttle vectors pSh-GF35 and adenovirus skeleton carrier pAdEasy-1 homologous recombination

2.1 pSh-GF35 PmeI linearizing and purifying be with 10 * NE buffer4,5.0 μ l, 100 * BSA, 0.5 μ l, and pShuttle-CMV-GF35 30.0 μ l, PmeI 1.0 μ l add to cumulative volume 50.0 μ l with distilled water.37 ℃ of effect 3.0h get 2.0 μ l then and carry out 1% (g/ml) agarose gel electrophoresis.Utilizing in a small amount, glue recovery test kit reclaims purifying.Purification process is the same.

2.2 fresh BJ5183 colony inoculation of picking is in the 10ml LB substratum that contains 30 μ g/ml Streptomycin sulphates on Streptomycin sulphate (30 μ g/ml) resistant panel in the preparation of intestinal bacteria BJ5183 electricity transformed competence colibacillus bacterium, 37 ℃ of 200rpm overnight shakings are cultivated; Be inoculated in the new LB substratum that contains Streptomycin sulphate by 1/1000 volume in second day, shaking culture makes its A550 ≈ 0.8, and culture is put 1.0h on the ice bath; 4 ℃ of centrifugal 10min of 3000rpm; With the long-pending aseptic ice-cold WB liquid re-suspended cell of initial bacteria liquid; (the ultrapure glycerine of WB=10%, 90% distilled water v/v); The centrifugal 30min of 3000rpm; Use behind the resuspended thalline of WB liquid of original volume centrifugal again; 3000rpm is centrifugal, and unnecessary supernatant is removed in the 30min hypsokinesis, remains the resuspended thalline of WB liquid of 1/500 volume, the every pipe of packing 40 μ l ,-70 ℃ of preservations.

2.3 pSh-GF35 and pAdEasy-1 cotransformation BJ5183 bacterium are taken out BJ5183 electricity transformed competence colibacillus bacterium two pipes of-70 ℃ of preservations, slowly melt on ice bath; PAdEasy-1 carrier and the linearizing pShuttle-CMV-G35 plasmid of getting purifying mix in the centrifuge tube of a sterilization 500 μ l; Add the mixture of linearizing pShuttle-CMV-GF35 plasmid and pAdEasy-1 carrier and linearizing pShuttle-CMV-GF35 respectively in contrast on two pipe BJ5183 electricity transformed competence colibacillus bacteriums; BJ5183 bacterium behind the adding plasmid is changed in the ice-cold pole cup, make drop be suspended in pole cup metal sheet central authorities; Carry out electricity immediately and transform, the parameter of electric conversion instrument is set to: 2.5kV, 25 μ F, 200 Ω; The intact pipe of revolutionization adds the room temperature LB solution of 1.0ml immediately, and fully resuspended bacterium; 37 ℃ of shaking culture 1.0h of bacterium after resuspended; Get the reorganization bacterium respectively and coat three kalamycin resistance flat boards, contrast thalline coating one flat plate, incubated overnight obtains recombinant bacteria.

2.4 recombinant bacteria select and recombinant adenovirus plasmid extracts through 24h at least and cultivates, the minimum recombinant bacteria colony inoculation on the picking kalamycin resistance flat board contains in the LB substratum of kantlex in 3.0ml, incubated overnight; Extract recombinant adenovirus plasmid rAd-GF35, be dissolved in the 20 μ l aseptic double-distilled waters, carry out the agarose gel electrophoresis of 0.8% (g/ml) then; Suspicious recombinant plasmid transformed DH5 α competence bacterium, coating kantlex flat board; Choosing colony extracts plasmid, carries out the agarose gel electrophoresis of 0.8% (g/ml).

2.5. the evaluation of recombinant adenovirus plasmid rAd-GF35

2.5.1 agarose gel electrophoresis

The less colony inoculation of picking about 37 ℃ of shaking culture 15h, adopts the alkaline lysis method of extracting plasmid in the LB of kalamycin resistance liquid nutrient medium from the incubated overnight flat board; Agarose gel electrophoresis through 0.8% is set the contrast of shuttle vectors and skeleton carrier simultaneously; The shuttle plasmid that can preliminary judgement positive recombinant plasmid is connected with self.

2.5.2 Pac I enzyme is cut evaluation

With preliminary judgement male recombinant plasmid transformed DH5 α competent cell, extract in a small amount and carry out enzyme behind the plasmid and cut evaluation.It totally is 20.0 μ l that enzyme is cut.System is as follows: 2.0 μ l, 10 * NE Buffer, 1,0.2 μ l, 100 * BSA, and 5 μ g Plasmid DNA, 0.2 μ l Pac I mends to cumulative volume 20.0 μ l with aseptic double-distilled water.

37 ℃ act on 1.0～3.0h, carry out 0.8% agarose gel electrophoresis then, if obtain two bands, one is 30kb, and another is that 4.5kb or 3.0kb are then positive.

2.6 the packing of recombinant adenovirus rAd-GF 35

2.6.1 the linearizing of recombinant adenovirus plasmid

2.6.1.1 the purifying of recombinant adenovirus plasmid

With the positive recombinant adenovirus plasmid of plasmid purification test kit purifying.With spectrophotometric determination OD260 and OD280, calculate its content and purity.Dna content (μ g/ml)=extension rate * 50 * OD260.

2.6.1.2 the linearizing of recombinant adenovirus plasmid rAd-GF35 and recovery

The enzyme system of cutting is 100.0 μ l.System is as follows: 10.0 μ l, 10 * NE Buffer, 1,1.0 μ l, 100 * BSA, and 30 μ g recombinant adenovirus plasmid DNA, 1.0 μ l Pac I mend the l to cumulative volume to 100.0 μ with aseptic double-distilled water.37 ℃ act on 3.0h, carry out 0.8% agarose gel electrophoresis then, and the confirmation enzyme reclaims DNA after cutting entirely, and method is the same.Measure its OD260 and OD280 with ultraviolet spectrophotometer, calculate its content and purity.

2.6.2 transfection

Concrete operations are undertaken by the operational manual of TransFast Transfection Reagent (Promega).Transfection is carried out on 24 porocyte plates, adds the complete growth media (or making serum-concentration drop to 2～6%) of 1.0ml37 ℃ of preheating after the transfection gently, and the HEK293A cell is put into CO2gas incubator, cultivates 7～14d for 37 ℃.

2.6.3 the results of recombinant adenovirus rAd-GF 35 and propagation

When cells transfected feature pathology (local cells become circle, reticulate) or cell state occurred and is not enough to keep ,-20 ℃ of frozen results were viral.6 porocyte plates of HEK293A cell are covered with in inoculation after the freeze thawing, 37 ℃ of absorption 1.0h, and every hole adds 2.0ml and keeps liquid (DMEM that contains 2% serum), virus of proliferation.

2.7 the evaluation of recombinant adenovirus

2.7.1 indirect immunofluorescence assay (IFA)

Cover with on the 96 porocyte plates of individual layer HEK293A cell one and to inoculate recombinant adenovirus rAd-GF 35 (1000MOI), repeat 6 holes; At 37 ℃, 5% CO ₂Cultivate 48～72h (CPE not occurring) in the incubator; Discard nutritive medium, use PBS washed cell 1 time, discard PBS then; 45min in 37 ℃ of incubators makes moisture evaporation; Put-20 ℃ of freezing 45min then; Every hole adds the dehydrated alcohol of 4 ℃ of precoolings of 150 μ l, puts 4 ℃ of fixedly 45min; Discard ethanol, use PBS washed cell 1 time; Add mouse anti GMCSF respectively, the serum of mouse anti PRRSV GP3 and GP5,100 μ l/ holes, 3 holes of every kind of monoclonal antibody inoculation; 37 ℃ of incubation 30min～60min in wet box; Discard antibody, with the PBS in 200 μ l/ holes washing 6 times; FITC-the sheep anti-mouse igg that adds dilution in 1: 100,50 μ l/ holes, 37 ℃ of incubation 30min in wet box; Discard liquid in the hole, with PBS washing 4 times; Observe down in fluorescent microscope.Simultaneously 293 cells of cell that infects with wild-type adenovirus wtAd and not infection in contrast.

Tangible fluorescence appears in the recombinant adenovirus cells infected in endochylema, any fluorescence does not then appear in control cells.

2.7.2?Western?bLot

2.7.2.1 antigenic preparation

The inoculation recombinant adenovirus rAd-GF 35 is in 293 cells that cover with individual layer, the viral liquid of results to the complete pathology ,-20 ℃ of multigelations 3 times; In 4 ℃, the centrifugal 30min of 6000rpm, remove cell debris; Slowly adding PEG-6000 and NaCL, is that 8% and 3%, 4 ℃ of slow stirring is spent the night to final concentration respectively; 8000rpm centrifugation 30min obtains viral crude extract; Add the PBS be equivalent to initial volume 1/100～1/1000 (0.1moL/L, pH7.2), resuspended precipitation is put 4 ℃ and is spent the night; The centrifugal 10min of 1000rpm collects supernatant and is spissated viral liquid.Concentrate wild-type adenovirus wtAd in contrast simultaneously.

2.7.2.2 SDS-PAGE and Western bLot

To add 5 * Loading Buffer in the sample, 100 ℃ are boiled 5min; Sample is joined in the well, carry out 12% SDS-PAGE, protein isolate.In kind the wild-type adenovirus wtAd of Chu Liing in contrast.

2.7.3.3 transfer printing

SDS-PAGE glue is switched to suitable size, put in the transfer printing damping fluid and shake 15～20min; Cut a slice size NC film identical, put in the transfer printing damping fluid and soak 15～20min with glue; Cut 2 Whatman filter paper identical simultaneously, put in the transfer printing damping fluid and soak into glue size; Make in the following order " sandwich ": negative pole-Whatman filter paper-glue-NC film-Whatman filter paper-positive pole, note not producing bubble between film and the glue; Adopt half-dried transfer printing to carry out transfer printing, 15V transfer printing 20～30min; Transfer printing finishes, and the ponceau dyeing NC film with 5% is to determine the transfer printing effect; Use rinsed with deionized water, the wash-out ponceau.

2.7.3.4?Western?bLot

NC film (nitrocellulose filter) is placed 10% skimming milk (PBST), shake sealing and spend the night; With PBST washing 4 times, each 10min; Add the anti-of 10% skimming milk dilution, 37 ℃ are shaken and hatch 2h; With PBST washing 4 times, each 10min; With 10% skimming milk dilution goat anti-mouse igg-HRP (1: 10000), 37 ℃ are shaken and hatch 2h; With PBST washing 4 times, each 10min; Dry NC film, A liquid and the B liquid of equivalent mixing chemical luminous substrate ECL directly are added drop-wise to mixed solution on the NC film, hatch 2～10min; Wrap with preservative film, exposure on x-ray film is observed after development, photographic fixing.Occur obviously in purpose band position, band clearly, any band is not then seen in contrast, proves that recombinant adenovirus can carry out specific expressed to GP3 and GP5 albumen.

2.8 the titre of recombinant adenovirus

Before virus titer is measured about 48h with HEK293A cell seeding 96 porocyte plates, with recombinant adenovirus freeze thawing 3 times, usefulness is kept liquid (DMEM that contains 2% serum) virus is made 10 doubling dilutions; Discard the nutritive medium in the 96 porocyte plates that cover with individual layer HEK293A, the viral liquid that the inoculation dilution is good, each 4 hole of extent of dilution inoculation, 100 μ l/ holes; 37 ℃, 5%CO ₂Cultivate 5d under the saturated humidity, observe CPE, each dilution CPE hole count that counts is pressed the TCID that the Reed-Muench method is calculated virus ₅₀The titre of recombinant adenovirus is 10 ^8.77TCID ₅₀/ 1.0ml.

2.9 granulocyte colony factor biological activity is identified

Granulocyte colony factor preparation:, be inoculated on PK-15 (a kind of kidney cell line of pig is preserved by this laboratory) cell of long face individual layer with rAd-GF35, gather in the crops cultivate 24h in 37 ℃ of incubators that contain 5%CO2 after, multigelation three, 6000 leaves the heart, after removing cell debris, be used for following experiment.Connect malicious PK-15 cell in contrast with wild-type adenovirus wtAd

Medullary cell preparation: get 3-5 piglet shin bone in age in week, after cutting with saw, medullary cell Hank ' s is gone out, be put in the 10ml centrifuge tube, 2000 leave heart 15min after, supernatant discarded adds Hank ' s, to survey carefully that cell sweeps away on the wall, recentrifuge is behind the triplicate, with containing the 1640 resuspended of 10%FCS, after the cell counting, cell density is adjusted to 5 * 10 ⁶Individual/ml is used for following experiment.

2.9.1 mtt assay is surveyed the granulocyte colony factor, stimulation index (SI)

100 μ l are contained 5 * 10 ⁶Individual/ml medullary cell is added in 96 orifice plates with the DMEM of different dilution rAd-GF35 inoculation PK-15 cell conditioned mediums and 10% FCS.Cultivated 5 days, careful supernatant discarded adds the MTT fresh culture 200 μ l that contain 0.5mg/ml, cultivates 4h again, discards, and adds the dimethyl sulfoxide (DMSO) (DMSO) of 50 μ l, and 37 ℃ of vibration 10min survey OD ₅₇₀Set up the contrast of wtAd inoculation PK-15 supernatant simultaneously, SI=(granulocyte colony factor irritation cell OD ₅₇₀)/(control cells OD ₅₇₀).Through the medullary cell that rAd-GF35 inoculation PK-15 cell conditioned medium stimulates, can breed SI is 2.67, and the medullary cell that wtAd inoculation PK-15 supernatant is hatched can not to breed SI be 1.034.The GMCSF biologically active that proof is expressed with recombinant adenovirus, propagation that can irritation cell.

2.9.2 medullary cell culture experiment

In 50ml cell bottle, add the medullary cell that 5ml collects respectively, add the PK-15 cell conditioned medium of 0.1% rAd-GF35 and wtAd inoculation more respectively.After cell being blown down in 5 days, centrifugally change fresh substratum.Cultivated compare and experimental group cell number 20 days.The experimental group cell has obvious unnecessary control group as can be seen, proves the GMCSF biologically active that recombinant adenovirus is expressed, and can keep the external survival of medullary cell.

2.10 the small white mouse immunity test of recombinant adenovirus

Get 40 8 age in week a healthy cleaning level small white mouse be divided into four groups at random, 10 every group.One group is set at contrast, takes oral, subcutaneous respectively and three kinds of immunization wayses of intramuscular injection for all the other 3 groups.Immunizing dose is 10 ⁴TCID ₅₀/ 0.2ml in immunity 2 weeks of pitch time, gathers mouse serum and does virus neutralization tests behind the continuous immunity four times.The NAT difference of three kinds of immunization wayses is not remarkable, and NAT is 1:64.

2.11 the pig body of recombinant adenovirus immunity with attack poison protection experiment

Get 10 healthy piglets (the PRRSV antigen-antibody detects negative), be divided into two groups at random.One group is made as contrast, and another group is the recombinant adenovirus immune group.Respectively at musculi colli injection DMEM and recombinant adenovirus.The recombinant adenovirus dosage of inoculation is 5 * 10 ^8.77TCID ₅₀, immunity is 3 weeks at interval, twice of continuous immunity.Two exempt from two weeks of back, attack poison with the highly pathogenic strain SY0608 of PRRSV, and attacking the toxic agent amount is 100 TCID ₅₀Cellular immunization and humoral immunization detected result show that recombinant adenovirus can be induced the very strong specific cellular immunization and the generation of humoral immunization.Wherein be to induce IFN-γ to produce particularly significantly, two to exempt from back two all serum I FN-γ secretory volumes be 500pg/ml, and to compare difference extremely remarkable with control group 50pg/ml.The generation that recombinant adenovirus can the inducing specific neutralizing antibody simultaneously, attack poison before the neutralizing antibody level can reach 1:32.Attack malicious result and show, in pathological change, immune group and control group comprehensive grading result difference be (35:85) significantly; It is not remarkable that body temperature changes difference; Viral level difference extremely significantly (10 in the serum ⁴: 10 ²); After slaughtering, viral level significant difference (1:100) in the tissue.Prove that constructed recombinant adenovirus has good immunization, will alleviate the clinical symptom of attacking malicious pig, reduce the pathological change of histoorgan and reduce the viremia level.

2.11 the stability test of recombinant adenovirus

In recombinant adenovirus rAd-GF 35 30 generations of continuous passage on 293 cells, detect that GP5 is proteic among the rAd-GF35 transcribes and express per 5 generations respectively, measure its histocyte median infective dose simultaneously.The result shows that GP5 albumen is expressed stable in recombinant adenovirus rAd-GF 35 continuous passage process, its histocyte median infective dose does not change yet.

In sum, with the continuous passage 30 times on the HEK293A cell of the recombinant adenovirus rAd-GF 35 of purifying, in per 5 generations, check that recombinant adenovirus to the transcribing and expression of rAd-GF35 protein gene, measures its TCID simultaneously ₅₀The result proves that recombinant adenovirus is stable to the rAd-GF35 protein gene expression, and the histocyte median infective dose is stable, connects poison back cytopathy occurrence law.The malicious valency of planting poison is stabilized in 10 ^8.77TCID ₅₀/ 1.0ml.

Prepare vaccine with the porcine reproductive and respiratory syndrome recombinant adenovirus, with the continuous passage 30 times on the HEK293A cell of the recombinant adenovirus of purifying, detect its porcine reproductive and respiratory syndrome GP5 gene transcription and expression, prove that the expression of PRRSV target protein is stable.The malicious valency of recombinant adenovirus is stabilized in 10 ^8.77TCID ₅₀/ 1.0ml.

The PRRSV recombinant adenovirus of taking purification storage in the enlarged culturing process is by 10 ⁴The TCID50 inoculation grows up to the HEK293A cell of individual layer.When cytopathy reaches 70-90%, receive poison, once can obtain the PRRSV recombinant adenovirus vaccine through multigelation.

Recombinant adenovirus of the present invention is not pathogenic to animal, and it can be propagated by number of ways, and the pig that lives together is infected, and this is very important in the reorganization seedling.This specific character can make the pig that does not have immunity or leakage to exempt from also obtain immunity, makes the antibody horizontal of colony consistent, the attack that can resist exogenous virus preferably.Utilize this recombinant adenovirus immune mouse to obtain specificity neutralizing antibody at PRRSV, this antibody external can in and PRRSV.

Compare with inactivated vaccine with the attenuated vaccine of the PRRSV of present application, this virus can in the pig body, copy propagation and Constantly express PRRSV GP3 and GP5 albumen, make body continue to be subjected to the stimulation of GP3 and GP5 albumen, constantly produce For the neutralizing antibody of GP3 and GP5 albumen, the GMCSF that expresses simultaneously can control agent in immune response, from body fluid Organism immune response is regulated in immunity and two aspects of cellular immunity, so just can provide lasting protection to the pig body, because of This has broad application prospects aspect the preventing and treating of PRRS.

Sequence table

＜110〉Agricultural University Of Nanjing

＜120〉porcine reproductive and respiratory syndrome recombinant adenovirus rAd-GF 35

＜130〉specification sheets

<140>00

<141>2008-06-12

<160>9

<170>PatentIn?version?3.5

<210>1

<211>27

<212>DNA

＜213〉synthetic

<220>

<221>GMCSF-P1

<222>(1)..(27)

<400>1

<210>2

<211>49

<212>DNA

＜213〉synthetic

<220>

<221>GMCSF-2AP2.1s

<222>(1)..(49)

<400>2

<210>3

<211>48

<212>DNA

＜213〉synthetic

<220>

<221>GMCSF-2AP2.2s

<222>(1)..(48)

<400>3

<210>4

<211>29

<212>DNA

＜213〉synthetic

<220>

<221>SY-GP3.1

<222>(1)..(29)

<400>4

<210>5

<211>24

<212>DNA

＜213〉synthetic

<220>

<221>SY-GP3.2

<222>(1)..(24)

<400>5

<210>6

<211>25

<212>DNA

＜213〉synthetic

<220>

<221>SY-GP5.1

<222>(1)..(25)

<400>6

<210>7

<211>26

<212>DNA

＜213〉synthetic

<220>

<221>SYGP5.2

<222>(1)..(26)

<400>7

<210>8

<211>20

<212>DNA

＜213〉synthetic

<220>

＜221〉primer Adseq1

<222>(1)..(20)

<400>8

<210>9

<211>23

<212>DNA

＜213〉synthetic

<220>

＜221〉primer Adseq2

<222>(1)..(23)

<400>9

Claims (4)

1. porcine reproductive and respiratory syndrome recombinant adenovirus rAd-GF 35, it is characterized in that: recombinant adenovirus rAd-GF 35 belongs in classification: Adenoviridae (Adenoviridae), mastadenovirus (Mastadenovirus), adenovirus hominis kind (Human adenovirus), classification name: adenovirus (reorganization), this recombinant adenovirus carries out preservation at China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date: on March 4th, 2008, preserving number is CGMCC No.2391.

2. porcine reproductive and respiratory syndrome recombinant adenovirus according to claim 1 makes up by the following method and forms:

(1) amplification of GMCSF and PRRSV GP3, GP5 gene, clone are according to three primers of GenBank accession number U67175 sequences Design of pig rHuGM-CSF:

GMCSF-P1：5-GCT?GGTACCATGGCTCCTACCCGCCCA-3

GMCSF-2AP2.1s：5-GACGTCGCCGGCCAACTTGAGAAGGTCAAAGTTCTTTTTGACTGGCCCC-3

GMCSF-2AP2.2s：5-GCACTCGAGGGGCCCTGGGTTGGACTCGACGTCGCCGGCCAACTTGAG-3

With canavailn ConA isolating pig peripheral blood lymphocyte is stimulated, extract lymphocytic total RNA then, with the product after total RNA reverse transcription is template, uses the RT-PCR technology and amplifies the pig rHuGM-CSF GMCSF gene that comprises foot and mouth disease virus 2A gene and disappearance terminator codon;

Design is at the Auele Specific Primer of the domestic strain isolated SY0608 of PRRSV strain GP3 albumen and GP5 protein gene:

SY-GP3.1：5-GACCTCGAGATGGCTAATAGCTGTACATT-3

SY-GP3.2：5-ACAAAGCTTTCGCCGTGCGGCACT-3

SY-GP5.1：5-ACGAAGCTTATGTTGGGGAAGTGCT-3

SYGP5.2：5-CAGATATCCTAGAGACGACCCCATTG-3

CDNA product with the monkey-kidney cells MARC-145 cell of SY0608 virus strain infection is a template, uses GP3 gene and the full gene of GP5 that the RT-PCR technology has amplified domestic PRRSV strain isolated SY0608 virus strain disappearance terminator codon;

Introduce restriction enzyme digestion sites at upstream and downstream primer 5 ' end, cut by enzyme, GMCSF, GP3, GP5 gene clone are gone among the shuttle vectors pShuttle-CMV, cut with PCR by enzyme then and identify, acquisition contains the shuttle vectors pShuttle-CMV-GMCSF-GP3-GP5 of GMCSF, GP3 and GP5 gene, is called for short pSh-GF35;

(2) the shuttle vectors pSh-GF35 that contains GMCSF, GP3 and GP5 gene that identifies of shuttle vectors pSh-GF35 and adenovirus skeleton carrier pAdEasy-1 homologous recombination is transformed among the coli strain BJ5183 by electric method for transformation with adenovirus skeleton carrier pAdEasy-1 behind linearization for enzyme restriction, under the effect of intestinal bacteria recombinase, between shuttle vectors pSh-GF35 and skeleton carrier pAdEasy-1 homologous recombination takes place, obtained to contain the recombinant adenovirus plasmid of GMCSF, GP3 and GP5 gene through the screening of 50 μ g/ml kantlex;

(3) recombinant adenovirus plasmid identified of the acquisition of recombinant adenovirus is by cationic-liposome method transfection HEK293A cell, and recombinant adenovirus plasmid becomes complete recombinant adenovirus rAd-GF 35 in HEK293A cell internal packing.

3. use the vaccine of claim 1 or 2 described porcine reproductive and respiratory syndrome recombinant adenovirus rAd-GF 35s preparations.

4. prepare the method for vaccine with claim 1 or 2 described recombinant adenovirus rAd-GF 35s, it is characterized in that:

The PRRSV recombinant adenovirus of taking cryopreservation is by 10 ⁴TCID ₅₀Inoculation grows up to the HEK293A cell of individual layer, receives poison when cytopathy reaches 70-90%, once can obtain PRRSV recombinant adenovirus rAd-GF 35 vaccine through multigelation.

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