CN102154365A

CN102154365A - Swine influenza virus H1N1 subtype hemagglutinin (HA)-1 protein recombinant suipoxvirus and preparation method thereof

Info

Publication number: CN102154365A
Application number: CN 201110026879
Authority: CN
Inventors: 许家荣; 陆承平; 黄冬艳
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2011-01-25
Filing date: 2011-01-25
Publication date: 2011-08-17
Anticipated expiration: 2031-01-25
Also published as: CN102154365B

Abstract

The invention belongs to the technical field of biology and discloses swine influenza virus H1N1 subtype HA-1 protein recombinant suipoxvirus and a preparation method thereof. An HA1 gene is designed and synthesized according to the gene sequence of HA1 of an A/swine/Shanghai/1/2005(H1N 1) strain of the swine influenza virus, and the gene is cloned in a suipoxvirus vector pUSZ11 to obtain a transfer vector pUSZ11/H1. The recombinant virus is a positive clone obtained by infecting and transfecting PK15 cells with the suipoxvirus and the transfer vector pUSZ11/H1 and performing homologous recombination. The recombinant suipoxvirus can express HA1 protein stably, and after infection with the virus, the cytopathy becomes regular, the toxic effect of the breeding virus is stabilized at 107TCID50/mL, the breeding virus can effectively stimulate the immune protect reaction of organisms and can be used in the preparation of medicines for preventing and/or treating infection with swine influenza virus H1N1 subtype.

Description

Swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus and preparation method thereof

Technical field

The invention belongs to biological technical field, relate to swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus and preparation method thereof.

Background technology

Poxvirus genome group structure is huge, big genome provides the space for the insertion of a lot of alien genes, can admit a large amount of foreign genes as carrier, and make its effective expression (Barbara E.Straw, Jeffery J.Zimmerman, Sylvie D ' Allaire, David J.Taylor..DISEASES OF SWINE.9 ^THEDITION.[M]).Poxvirus also has good immunogenicity, can excite the host to produce effective immune response.Pig pox virus does not infect other animals, and an infected pigs only produces the mildness reaction at the pig body, uses safer.Therefore, pig pox virus is to the former a kind of comparatively ideal carrier of pig premunition.The recombinant swinepox virus vaccine has the antigen multiplication capacity of attenuated vaccine, and is easy to use, with low cost.So pig pox virus is candidate's carrier that the proprietary genetically engineered live vector vaccine of pig has much glamour.(Swine influenza SI) is the acute respiratory transmissible disease of the boar that caused by A type influenza virus to porcine influenza.Though infecting, simple swine influenza virus shows as sickness rate height (100%), mortality ratio low (about 5%), but swine influenza virus can infect pulmonary alveolar macrophage (Jung T in vivo, Chol C, Chae C.Localization of Swine Influenza Virus in Naturally Infected Pigs[J] Vet Pathol, 2002,39:10-16.).The impaired lung's immune defence mechanism that has a strong impact on of pulmonary alveolar macrophage causes the secondary infection of other pathogenic bacteria and virus easily.In addition, because the existing avian influenza virus of porcine respiratory epithelial cell has Mammals and human influenza virus's acceptor, pig to become the crucial place that reorganization takes place influenza virus gene again, in the ecology of influenza virus and epidemiology, occupy consequence.Present American-European market porcine influenza vaccine is the totivirus inactivated vaccine, and the prevention porcine influenza has been brought into play important effect.But a little less than the ability of inactivated vaccine inducing cell immunity, can not provide effective protection, be difficult to effectively changeable infection and the propagation of swine influenza virus in swinery of control antigen allos and special-shaped virus.

Swine influenza virus A/swine/Shanghai/1/2005 (H1N1) strain (the GenBank number of including is EU502884) is popular advantage strain (Qi Xian in China swinery; China's some areas swine influenza virus molecular epidemiology and Study on Pathogenicity; Agricultural University Of Nanjing's Ph D dissertation; 2006.6; 88-108); HA1 albumen is the main surface antigen of swine influenza virus, can induce body to produce the protectiveness neutralizing antibody, can be used to resist the infection of swine influenza virus.Also be not built at present the report of swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus.

Summary of the invention

The objective of the invention is above-mentioned deficiency, a kind of transfer vector pUSZ11/H1 of swine influenza virus H1N1 hypotype HA1 gene is provided at prior art.

Another object of the present invention provides swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus.

Another purpose of the present invention provides the preparation method of this recombinant swinepox virus.

Purpose of the present invention can be achieved through the following technical solutions:

A kind of transfer vector pUSZ11/H1 of swine influenza virus H1N1 hypotype HA1 gene, this recombinant vectors are to be the BamHI restriction enzyme site gained of the swine influenza virus H1N1 strain HA1 gene order insertion pig pox virus carrier pUSZ11 shown in the SEQID NO.1 with sequence.

A kind of swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus, be by pig pox virus and described transfer vector pUSZ11/H1 infect, transfection PK15 cell, carry out the positive colony that homologous recombination obtains.

Wherein, the preferred ATCC preserving number of described pig pox virus is

Number VR-363 ^TMWild-type pig pox virus wtSPV.

Described swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus rSPV/H1 is preserved in Chinese typical culture collection center on January 6th, 2011, and preserving number is CCTCC NO:V201101.This recombinant swinepox virus rSPV/H1 belongs in classification: Poxviridae (Poxviridae), Suipoxvirus (Suipoxvirus), pig pox virus (Swinepox virus).

The preparation method of swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus comprises the steps:

(1) be the gene order of swine influenza virus A/swine/Shanghai/1/2005 (H1N1) the strain HA1 of EU502884 according to the GenBank number of including, designing and synthesizing sequence voluntarily is the HA1 gene shown in the SEQ ID NO.1;

(2) be that the HA1 gene shown in the SEQ ID NO.1 inserts among the pig pox virus carrier pUSZ11 with described sequence, the evaluation of cutting and check order by PCR, enzyme then obtains the transfer vector pUSZ11/H1 of described swine influenza virus H1N1 hypotype HA1 gene;

(3) with transfer vector pUSZ11/H1 infection, the transfection PK15 cell of pig pox virus and described HA1 gene, carry out homologous recombination and obtain described swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus.

The concrete grammar that the described HA1 gene of step (2) inserts pig pox virus carrier pUSZ11 is that HA1 gene shown in the SEQ ID NO.1 and pig pox virus carrier pUSZ11 carry out single endonuclease digestion to the described sequence of step (1) respectively for utilizing BamHI, reclaiming size is the HA1 gene target fragment of 1031bp and the big fragment of pig pox virus carrier pUSZ11 linearity that size is 8391bp, connect two fragments, obtain recombinant plasmid, through order-checking with measure forward and reversely, it is that the plasmid of forward is named and is pUSZ11/H1 that sequencing result is met purpose fragment and direction.

Described preparation method also comprises the plaque purification step of described swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus.

Described preparation method also comprises the step of utilizing IFA and Western-blot technology described swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus to be expressed evaluation.

Described swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus prevents and/or treats application in the medicine that swine influenza virus H1N1 hypotype infects in preparation.

Beneficial effect:

The present invention is inserted into the HA1 gene first among the pig pox virus carrier pUSZ11 and obtains recombinant vectors pUSZ11/H1, has vaccinia virus promotor P11 before this plasmid screening sign LacZ gene, carry out homologous recombination after locus coeruleus very obvious, be beneficial to screening and purifying.

The present invention has proposed first with pig pox virus vector construction swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus.Swine influenza virus (H1N1) recombinant swinepox virus that evidence, the present invention make up can stable expression of exogenous albumen, and it tires stablely, and the malicious valency of planting poison is stabilized in 10 ⁷TCID ₅₀/ mL.RSPV/H1 can produce significant humoral immunization and cell immune response by inducing mouse by the animal experiment proof, not only protects cavy and pig to resist the attack of homology SIV H1N1, can also partly resist the attack of special-shaped SIVH3N2.

Recombinant swinepox virus does not infect other animals, and an infected pigs only produces the mildness reaction at the pig body, uses safer.The recombinant swinepox virus vaccine has the antigen multiplication capacity of attenuated vaccine, and is easy to use, with low cost.So porcine influenza recombinant swinepox virus rSPV/H1 is having broad application prospects aspect the anti-system of porcine influenza (H1N1 hypotype).

Biomaterial preservation information

Swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus rSPV/H1 is preserved in Chinese typical culture collection center on January 6th, 2011, and the preservation address is a Wuhan City Wuhan University, and preserving number is CCTCC NO:V201101.

Description of drawings

Fig. 1 pSP1 plasmid map.

The structure synoptic diagram of Fig. 2 pig pox virus transfer vector pUSZ11/H1.

Fig. 3 synthetic HA1 gene BamHI single endonuclease digestion,

M:Marker, 1:HA1 gene BamHI single endonuclease digestion product.

Fig. 4 pig pox virus carrier pUSZ11BamHI single endonuclease digestion,

M:Marker, 1: pig pox virus carrier pUSZ 11BamHI single endonuclease digestion product.

The PCR of Fig. 5 recombinant plasmid identifies, M:Marker, 1: the PCR product of recombinant plasmid.

The enzyme of Fig. 6 recombinant plasmid is cut evaluation; M:Marker, 1: recombinant plasmid BamH I enzyme is cut product.

The locus coeruleus screening of Fig. 7 recombinant swinepox virus rSPV/H1, purifying photo.

Fig. 8 IFA identifies; A: infect the PK15 cell of recombinant swinepox virus, tangible fluorescence is arranged; B: infect the PK15 cell of wild pig pox virus, do not have fluorescence; C: the PK15 cell of virus inoculation does not have fluorescence.

Fig. 9 Western-blot identifies; M:Marker, 1: infect the PK15 cell of recombinant swinepox virus, 2: infect the PK15 cell of wild pig pox virus, 3: the PK15 cell of virus inoculation not.

Mice serum SIV (H1N1) neutralizing antibody changes after Figure 10 immunity.

The special lymphopoiesis of mouse SIV (H1N1) detects after Figure 11 immunity.

Figure 12 SIV (H1N1) stimulates the mouse spleen lymphocyte secrete cytokines to detect.

Figure 13 SIV (H1N1) stimulates cavy peripheral blood lymphocyte secrete cytokines to detect.

Figure 14 cavy lungs histopathology changes.

The A:rSPV/H1 immunity, H1N1 attacks poison; The B:rSPV/H1 immunity, H3N2 attacks poison; The C:wtSPV immunity, H1N1 attacks poison; The D:wtSPV immunity, H3N2 attacks poison; The E:PK15 immunity, H1N1 attacks poison; The F:PK15 immunity, H3N2 attacks poison.

Figure 15 pig is attacked poison (H1N1) postnaris toxin expelling situation measuring result.

Figure 16 pig is attacked poison (H3N2) postnaris toxin expelling situation measuring result.

Figure 17 SIV (H1N1) stimulates pig peripheral blood lymphocyte secrete cytokines to detect.

Figure 18 pig lungs histopathology changes,

Embodiment

The structure of embodiment 1 pig pox virus transfer vector pUSZ11/H1

1.1 the structure of pig pox virus carrier pUSZ11

PUSZ11 plasmid (Huang Dongyan structure) collection of illustrative plates as shown in Figure 2, total length is 8391bp; LF in the pUSZ11 plasmid is the pig pox virus homologous recombination arm on the left side and the 12121-13268bp place homology of pig pox virus Kasza strain.(available from ATCC, preserving number is with wild-type pig pox virus wtSPV Number VR-363 ^TM, down with) genomic dna is template, carries out PCR with a pair of primer SEQ ID NO.2 and SEQ ID NO.3, cuts amplified production with EcoR I and Kpn I enzyme then, (SEQ ID NO.4 1148bp) is cloned among the pUC19 LF; RF in the pUSZ11 plasmid is the pig pox virus homologous recombination arm on the right and the 13457-14831bp place homology of pig pox virus Kasza strain.Be template with the wtSPV genomic dna equally, carry out PCR with primer SEQ ID NO.5 and SEQ ID NO.6, (SEQ ID NO.7,1375bp) clone advances among the above-mentioned pUC19, forms the pUS01 plasmid RF to cut amplified production with Sal I and Hind III enzyme then.With pSP1 (Fig. 1) plasmid (Junghyun Hahn, Se-Hoon Park, Jae-Young Song, et al.Construction of recombinant swinepox viruses and expression of the classical swine fever virus E2 protein Journal of Virological Methods93 (2001) 49-56) be template, with primer to P11ZS:SEQ ID NO.8 and P11ZX:SEQ ID NO.9 (the promotor P11 that wherein has vaccinia virus), to between the BamH I and Sal I site of carrier pUS01, all the other sequences are pUC19 framework sequence the LacZ gene clone.Form pig pox virus carrier pUSZ11 (Fig. 2) thus.

1.2HA1 synthetic, the clone of gene and the structure of pig pox virus transfer vector pUSZ11/H1

Gene order (the GenBank number of including is EU502884) according to swine influenza virus A/swine/Shanghai/1/2005 (H1N1) strain HA1, implementation sequence is HA1 gene (the preceding BamHI of the adding restriction enzyme site, vaccinia virus P11 promoter sequence, atg start codon and with 534 HA1 genes that replaced to T by A of SEQ ID NO.1 voluntarily, after add terminator TAATAA and BamHI restriction enzyme site), this HA1 gene fragment length is 1037bp, entrust Shanghai Invitrogen company synthetic, and insert pUC19 BamHI restriction enzyme site.

The pUC19 carrier and the pig pox virus carrier pUSZ11 that will contain synthetic HA1 gene use the BamHI single endonuclease digestion respectively.It is as follows that enzyme is cut system: 7 μ L BamHI, 10 μ L, 10 * K Buffer, 50 μ L plasmids, 33 μ L aseptic double-distilled waters, totally 100 μ L.30 ℃ of effect 4h.

After enzyme cuts and finishes, the agarose gel electrophoresis with 1%, the HA1 clip size is that 1031bp (Fig. 3), pUSZ11 clip size are 8391bp (Fig. 4), the product that reclaims after test kit is cut enzyme with the DNA sepharose reclaims then.Method is pressed the test kit explanation.

Connect with ligase enzyme again, be inserted among the pig pox virus carrier pUSZ11 (Fig. 2).Linked system is as follows: 10.0 μ LHA1 enzymes cut back to close product, and 1.0 μ L carrier pUSZ11 enzymes cut back to close product, 1.0 μ L, 10 * T4DNALigase, and 2.0 μ L, 10 * DNALigase Buffer supplies cumulative volume 20.0 μ L with aseptic double-distilled water.16 ℃ connect 16h behind the mixing.Set carrier self simultaneously and connect contrast.

1.2.1 the conversion of bacillus coli DH 5 alpha competent cell and a small amount of of recombinant plasmid are extracted

Get connection product transformed into escherichia coli DH5 α competent cell (available from TIANGEN BIOTECH, down together) according to a conventional method, coat then on the 100 μ g/mL amicillin resistance flat boards, cultivate about 24h for 37 ℃.The picking foreign gene is connected product and transforms institute and be coated with single bacterium colony on the resistant panel with carrier, be inoculated in the LB substratum that contains 50 μ g/mL penbritins of 3.0mL the cultivation of 180rpm overnight shaking.High-Speed Plasmid Mini Kit according to Geneaid Biotech extracts recombinant plasmid in a small amount, is stored in-20 ℃.

1.2.2 the evaluation of recombinant plasmid

PCR identifies that the recombinant plasmid that extracts with 1.2.1 is a template, carries out PCR with upstream primer P1SEQ ID NO.10 (containing vaccinia virus P11 promotor) and downstream primer P2 SEQ ID NO.11.System is as follows: template 0.5 μ L, P11 μ L, P21 μ L, Mix 12.5 μ L, aseptic double-distilled water 10 μ L, totally 25 μ L.The PCR parameter is: 94 ℃ of 4min, 94 ℃ of 45s, 51 ℃ of 45s, 72 ℃ of 1min; 30 circulations; 72 ℃ of 7min.Product is with 1% agarose gel electrophoresis analysis (Fig. 5).

Enzyme is cut the method for identifying the employing single endonuclease digestion and is identified.The endonuclease reaction system is 20 μ L:1 μ LBamHI, 2 μ L, 10 * K Buffer, 10 μ L plasmids, 7 μ L aseptic double-distilled waters.30 ℃ of water-bath 4h.Product is with 1% agarose gel electrophoresis analysis (Fig. 6).1% agarose gel electrophoresis proof PCR fragment is cut big or small consistent with expected results with enzyme.

Order-checking is identified PCR and enzyme cut and is identified that being the male recombinant plasmid send Invitrogen company two-way order-checking, and the Nucleotide of swine influenza virus (H1N1) the HA1 gene in measured sequence and deduced amino acid and the GenBank database and protein are carried out homology relatively with NCBIBLAST, carry out the restriction enzyme of gene and read the frame analysis with Vector NTI and DNAstar software again, prove that the gene that we are cloned into and the homology of goal gene are 100%, and the gene that is cloned into meets carrier fully and reads the frame requirement.It is that the plasmid of forward is named into pUSZ11/H1 (Fig. 2) that sequencing result is met purpose fragment and direction.

The structure and the evaluation of embodiment 2 swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus

2.1 infect and transfection

Usefulness antibiotic-free MEM in 6 orifice plates (available from TIANGEN BIOTECH, down together) nutritive medium cultivation PK15 cell (available from ATCC,

Number:CCL-33 ^TM, down together) and to forming individual layer, abandon nutritive medium; Wild-type pig pox virus wtSPV with 0.02MOI infects, and 37 ℃ of effect 2h shake 3 times therebetween, abandon infection liquid, wash 1 time with washing lotion, and every hole adds the 2mL antibiotic-free and keeps liquid.Get 10 μ L liposomes (Invitrogen company product) and be added among the 250 μ L serum-free MEM, mixing is put room temperature 5min gently; The plasmid that 4 μ g are identified is added to 250 μ L serum-free MEM, mixings gently.With two pipe mixings, put under the room temperature and act on 20min, mixture is added in 6 orifice plates, rock mixing back and forth.Abandon transfection liquid behind 37 ℃ of effect 6h, add 2.5mL/ hole antibiotic-free and keep liquid, 37 ℃ are cultured to the tangible cytopathy of appearance, receive poison and are used for screening.

2.2 the screening of porcine influenza recombinant swinepox virus rSPV/H1, purifying and expression are identified

The locus coeruleus of recombinant virus screens above-mentioned culture multigelation 3 times, and 10 ¹, 10 ², 10 ³, 10 ⁴, 10 ⁵, 10 ⁶Doubly individual layer PK15 is inoculated in the dilution back.Replace serum free medium with the nutrient agar medium that contains 200 μ g/mL X-gal (available from TIANGEN BIOTECH), 1% low melting-point agarose (available from TIANGEN BIOTECH) behind the 16h, continue to cultivate 3～5d, the blue plaque that occurs is the porcine influenza recombinant swinepox virus, called after rSPV/H1.

The plaque of recombinant virus is cloned single blue plaque with 10 ², 10 ³, 10 ⁴, 10 ⁵, 10 ⁶Doubly dilution back inoculation individual layer PK15 replaces serum free medium with the nutrient agar medium that contains 200 μ g/mL X-gal, 1% low melting-point agarose behind the 16h, continues to cultivate 3～5d, the single plaque of picking (Fig. 7).Repeat 5 times.

Porcine influenza recombinant swinepox virus rSPV/H1 expresses evaluation:

IFA inclines and keeps liquid after identifying that obvious CPE (4-5d) appears in the PK15 cell wait to inoculate rSPV/H1 and wtSPV, after the PBS washing 2 times, with the fixing 30-45min of 4 ℃ of cold dehydrated alcohols, abandons stationary liquid, and 96 porocyte plates are dried naturally.Connecing porcine influenza H1N1 positive serum (the clinical serum of using the prosperous bio tech ltd porcine influenza test kit of Beijing Ai Deshi unit to detect that the poison cell hole adds 50 μ L dilution in 1: 50, down together), and in not connecing the poison cell hole, add equivalent porcine influenza H1N1 negative serum (with the clinical serum of the prosperous bio tech ltd porcine influenza test kit detection of Beijing Ai Deshi unit, down together) in contrast, put in 37 ℃ of wet boxes and act on 45min, PBS washing 3 times, each 5min; (observations under the inverted fluorescence microscope is put in PBS washing 3 times for (available from doctor's moral biotechnology company limited, with 100 times of 0.01M pH 7.4PBS dilutions), 37 ℃ of wet box effect 45min to add 50 μ L SPA-FITC markers.RSPV/H1 infects and the cell hole (A) that adds porcine influenza H1N1 positive serum is observed positive cell typical specificity bright green fluorescence is arranged, and wtSPV infects the interior no specificity green fluorescence (Fig. 8) of the cell hole (C) of hole (B) and contrast, as seen has only recombinant virus rSPV/H1 can express HA1 albumen.

The Western-blot evaluation is got and is infected the supernatant inoculation PK-15 cell that contains recombinant swinepox virus of collecting, preserving behind the 96h, collects the cell that infects recombinant swinepox virus behind the cultivation 72h, adds in the centrifuge tube of 1.5mL, 2000rpm, centrifugal 5min abandons supernatant, with PBS washing 2 times, abandon supernatant, add cell pyrolysis liquid 100 μ L, ice bath 30min, add 2 * sample-loading buffer, 100 μ L, boil 3min, centrifugal 5min gets the supernatant electrophoretic analysis.Use the same method and handle the PK15 cell that infects with wtSPV and do not connect malicious PK15 cell as negative control.Carry out the SDS-PAGE electrophoresis with 12% polyacrylamide gel behind the cell boiling lysis.

Electrophoresis is pressed document (Du Yijun, porcine alpha-IFN and granulocyte-macrophage colony stimutaing factor are to the antigenic immuno-potentiation research of foot and mouth disease virus VP 1, Agricultural University Of Nanjing's Ph D dissertation, 2008.6,38) method transfer printing after finishing.Pvdf membrane after the transfer printing is transferred in the confining liquid (PBST that contains 5% skimming milk), and 4 ℃ of sealings are spent the night behind room temperature jog 3～5h; Deblocking liquid inclines, wash film 3 times with washings PBST, each 10min adds an anti-solution then (with the rabbit H1 hypotype porcine influenza polyvalent antibody of confining liquid dilution in 1: 1000, with swine influenza virus H1N1 immune rabbit gained according to a conventional method), 1.5～2h vibrates under the room temperature; Wash film 3 times with PBST again, each 10min adds the goat anti-rabbit igg-HRP marker (available from doctor's moral biotechnology company limited) with confining liquid dilution in 1: 30000 then, room temperature vibration 1.5～2h; Wash film 3 times with PBST again, each 10min.Use HRP-DAB substrate colouring reagents box (available from TIANGEN BIOTECH) to develop the color at last.Concrete steps are: add 1xHRP reaction buffer 1mL, reagent A 50 μ L, reagent B50 μ L successively before transfer printing finishes in the colour developing box, mixing keeps in Dark Place, and uses in the 30min.After transfer printing finishes pvdf membrane is taken off, put into the colour developing box colour developing liquid of mixing in advance, stop colour developing after waiting to observe tangible purpose band.Pvdf membrane is dried, observe.The result shows that the cell that infects recombinant swinepox virus has its intended purposes band, and the PK15 cell that wtSPV infects and do not connect malicious PK15 cell and do not have purpose band appearance (Fig. 9) as seen has only recombinant virus rSPV/H1 can express the HA1 target protein.

Recombinant virus rSPV/H1 through identifying is preserved in Chinese typical culture collection center, is called for short CCTCC, the preservation address is a Wuhan City Wuhan University, and preserving number is CCTCC NO:V201101, and preservation day is on January 6th, 2011.

2.3 the titer determination of porcine influenza recombinant swinepox virus rSPV/H1

In 96 orifice plates, individual layer to be grown up to recombinant swinepox virus rSPV/H1 (CCTCC NO:V201101) freeze thawing 3 times, is made 10 doubling dilutions with keeping liquid with virus with the PK15 cell seeding; Discard the nutritive medium in 96 orifice plates that cover with individual layer PK15 cell, the viral liquid that the inoculation dilution is good, 4 holes of each extent of dilution, 100 μ L/ holes; 37 ℃, 5%CO ₂Cultivate 5d under the saturated humidity, observe CPE, accumulate each dilution CPE hole count, press the TCID that the Reed-Muench method is calculated virus ₅₀The TCID of recombinant swinepox virus rSPV/H1 (CCTCC NO:V201101) ₅₀Be 10 ^7.0/ mL.

2.4 swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus rSPV/H1 stability test

In recombinant swinepox virus rSPV/H1 (CCTCC NO:V201101) 30 generations of continuous passage on the PK15 cell, detect that HA1 is proteic among the rSPV/H1 transcribes and express per 5 generations respectively, measure its histocyte median infective dose simultaneously.The result shows that HA1 albumen is expressed stable in recombinant swinepox virus rSPV/H1 (CCTCC NO:V201101) continuous passage process, its histocyte median infective dose does not change yet.Connect poison back cytopathy occurrence law, the malicious valency of planting poison is stabilized in 10 ⁷TCID ₅₀/ mL.

The immunological testing of embodiment 3 mouse

Get 45 of female BALB/c mouse in age in 6-8 week, be divided into 3 groups at random, 15 every group.The 1st group of every muscle immunity rSPV/H1 (CCTCC NO:V201101), dosage is 0.2 * 10 ^7.0TCID ₅₀The 2nd group of every intramuscular injection wtSPV 0.2 * 10 ^7.0TCID ₅₀, the 3rd group every intramuscular injection PK15 lysis supernatant 0.2mL.Behind 21d, the 35d respectively with identical dosage booster immunization 2 times.Respectively at after the first immunisation 21,35, the 42d blood sampling measures neutralizing antibody; After first immunisation 21,35,42d gets spleen, isolated lymphocytes is measured lymphproliferation response (5/group/times); 35d gets spleen after the first immunisation, isolated lymphocytes, and (doctor Qi Xian separates with SIVH1N1 (A/swine/Shanghai/1/2005 (H1N1)).The GenBank number of including is EU502884) stimulate the amount of IFN-γ and IL-4 in the mensuration culture supernatant.

3.1 neutralizing antibody changes

Virus neutralization tests-fixed virus dilute serum method.At first, measure swine influenza virus H1N1 (A/swine/Shanghai/1/2005 (the H1N1)) TCID of (doctor Qi Xian separates, and the GenBank number of including is EU502884) with reference to animal virology (second edition) ₅₀The serum sample of every mouse is detected H1N1 neutralizing antibody (down together) respectively.

36h plants mdck cell (available from the female willing biochemical industry in Shanghai company limited, down with) in 96 porocyte plates before virus neutralization tests.56 ℃ of deactivation 30min of serum to be checked, after the centrifugal 5min degerming of 12000rpm, careful sucking-off supernatant (is pressed serum 1: 4,1: 8,1: 16,1: 32 respectively with keeping liquid (DMEM (available from TIANGENBIOTECH) that contains 2%FCS),, make 2 continuous doubling dilution; Viral H1N1 (A/swine/Shanghai/1/2005 (H1N1)) is diluted to respectively and contains 200TCID with keeping liquid ₅₀/ 100 μ L, serum and the isopyknic viral liquid with dilution (contains 200TCID then ₅₀/ 100 μ L) mix, in 37 ℃ of water-bath effect 1h.Discard the nutritive medium in 96 well culture plates that cover with the individual layer mdck cell, the serum-virus mixed solution is joined in the cell hole, 100 μ L/ holes, each serum dilution is done 4 multiple hole, cultivates 2～3d for 37 ℃.Set following contrast simultaneously: (i) porcine influenza H1N1 negative serum contrast; (ii) viral porcine influenza H1N1 negative serum contrast; A (iii) viral porcine influenza H1N1 positive serum (NAT is 1: 32) is as positive control; (iv) blank.The result judges, when CPE appears in a viral porcine influenza H1N1 negative serum control wells, and the CPE hole count of each serum dilution is write down in a viral porcine influenza H1N1 positive serum, negative serum and blank hole when all CPE not occurring.Tire as the neutralization of serum to be checked with the serum greatest dilution that can suppress CPE fully.

Serum neutralization test result (Figure 10) shows: two control groups (being wtSPV immune group and PK15 lysis supernatant group) mice serum does not have among the porcine influenza H1N1 and activity; It is active that rSPV/H1 immune group mice serum has SIV (H1N1) neutralization, and two to exempt from the back in rising trend.

3.2SIV (H1N1) special lymphopoiesis detects

3.2.1 the preparation of mouse spleen lymphocyte

Reference literature (Du Yijun, porcine alpha-IFN and granulocyte-macrophage colony stimutaing factor be to the antigenic immuno-potentiation research of foot and mouth disease virus VP 1, Agricultural University Of Nanjing's Ph D dissertation, and 2008.6,66-67) carry out.Carry out cell counting after lymphocyte prepares, regulate cell density to 5 * 10 with RPMI-1640 (available from TIANGEN BIOTECH) perfect medium ⁶Individual/mL; Bed board is added to cell suspension in the 96 porocyte plates, 100 μ l/ holes.

3.2.2 lymphocyte proliferation assay

In the good lymphocytic hole of plantation, the swine influenza virus (A/swine/Shanghai/1/2005 (H1N1)) that adds 20 μ g/ml is as stimulating former 100 μ l, and control wells adds 100ul RPMI-1640 perfect medium, and each makes 3 repeating holes.37 ℃ of 5%CO ₂Cultivate 66h in the incubator, the sucking-off supernatant adds the fresh substratum of 160ul, and every hole adds 40ulMTT (5mg/ml) (available from TIANGEN BIOTECH) more simultaneously, and 37 ℃ are continued to cultivate 4h down.Nutrient solution is abandoned in suction, and every hole adds 100 μ l DMSO (available from TIANGEN BIOTECH), and crystallization is melted in vibration, measures the value of OD570nm.Calculate stimulation index:

Stimulation index SI=stimulates the OD value in hole/the do not stimulate OD value in hole

Detected result (Figure 11) shows: special cell activation propagation appears in recombinant swinepox virus immune group mouse spleen lymphocyte after SIV (H1N1) stimulates.Compare between group: immunity back 21d, 35d and 42d, recombinant swinepox virus immune group and two control group lymphopoiesis significant differences (P＜0.05).

3.3SIV (H1N1) stimulate the mouse spleen lymphocyte secrete cytokines to detect

Stimulate isolating lymphocyte with H1N1 virus (A/swine/Shanghai/1/2005 (H1N1)) as stimulator antigen (dense 10 μ g/mL eventually), cultivate 66h down for 37 ℃.Measure the amount of cytokine IFN-γ and IL-4 in the culture supernatant with ELISA test kit (available from Groundwork Biotechnology Diagnosticate), by specification is operated.

One exempts from back 35d, and IFN-γ and IL-4 content detection result (Figure 12) show in the mouse spleen lymphocyte culture supernatant: each is organized all cytokine secretion in the mouse spleen lymphocyte culture supernatant.Compare between group: IFN-γ and IL-4 content are significantly higher than other two control groups (P＜0.05) in the rSPV/H1 immune group mouse spleen lymphocyte culture supernatant.

The immunological testing of embodiment 4 cavys and attack malicious protection test

Get 200-300 and restrain 36 of female Hartly cavys, be divided into 6 groups at random, 6 every group.1st, 2 groups of every immune rSPV/H1, dosage is 0.4 * 10 ^7.0TCID ₅₀3rd, 4 groups of every immune wtSPV, dosage is 0.4 * 10 ^7.0TCID ₅₀5th, 6 groups every injection PK15 lysis supernatant 0.4mL.Immunization ways is the top intramuscular injection of two back leg shin bones.21d is respectively with identical dosage booster immunization 1 time.28d blood sampling after the first immunisation, isolated lymphocytes stimulates with swine influenza virus H1N1, the amount of IFN-γ and IL-4 in the mensuration culture supernatant.NAT is measured in the 35d blood sampling.35d attacks poison, uses every nasal cavity injection 0.2 * 10 of 1mL needleless injector for the 1st, 3,5 group ^5.0TCID ₅₀HN1; 2,4,6 groups of every nasal cavity injections 0.2 * 10 ^5.0TCID ₅₀H3N2.Attack the poison back and observe 7d.The clinical manifestation record: every day is during feeding, the appearance situation of record observation item, at last according to 7 days record results, average, the concrete grammar reference (Wang Xinglong, variation of porcine reproductive and respiratory syndrome virus epidemic isolates NSP2 gene genetic and GM-CSF are to the immuno-potentiation research of GP3/GP5, Agricultural University Of Nanjing's Ph D dissertation, 2010.6,61) carry out.The observation item spiritedness is depressed, appetite decline, palpebral edema, cough, have difficulty in breathing and 6 indexs of having a running nose, according to every 0-3 branch of severity; Lung is scoring reference literature (the Wesley R D of pathology substantially, Tang M, Lager K M.Protection of Weaned Pigs by Vaccination with Human Adenovirus 5Recombinant Viruses Expressing the Hemagglutin in and the Nucleoprotein of H3N2Swine Influenza Virus[J] Vaccine, 2004,22 (25/26): 3427-3434.) carry out.42d gets lung tissue processing, inoculated into chick embryo (2 generations of blind passage) with whole cavy euthanasia, and HA, HI test detects influenza virus, gets lung sample simultaneously and does pathological section with formaldehyde fixed, observes pathological change.

4.1SIV (H1N1) stimulate cavy peripheral blood lymphocyte secrete cytokines to detect

The aseptic peripheral blood of getting cavy is used anticoagulant heparin, with 1 times of physiological saline dilution, is added on the lymphocyte separation medium face of 4mL 2000r/min, centrifugal 20min gently; Get the interface buffy coat, wash 3 times, be suspended from the RPMI RPMI-1640, calculate and adjust cell concn to 2 * 10 with PBS ⁶/ ml.Cell suspension is added in 96 orifice plates every hole 100 μ l.

In the good lymphocytic hole of plantation, the swine influenza virus H1N1 antigen that adds 20 μ g/ml is as stimulating former 100 μ l, and control wells adds 100ul RPMI-1640 perfect medium, and each makes 3 repeating holes.37 ℃ of 5%CO ₂Cultivate 66h in the incubator, the sucking-off supernatant.The detection method of cytokine is the same.

One exempts from back 28d, and IFN-γ and IL-4 content detection result (Figure 13) show in the cavy peripheral blood lymphocyte culture supernatant: each is organized in the cavy peripheral blood lymphocyte culture supernatant all cytokine secretion.Compare between group: IFN-γ and IL-4 content are significantly higher than other two control groups (P＜0.05) in the rSPV/H1 immune group cavy peripheral blood lymphocyte culture supernatant.

4.2 changing, observes cavy lungs histopathology

Carry out the lungs histopathology and change observation, preparation process reference literature (the Wang Xinglong of histopathologic slide, variation of porcine reproductive and respiratory syndrome virus epidemic isolates NSP2 gene genetic and GM-CSF are to the immuno-potentiation research of GP3/GP5, Agricultural University Of Nanjing's Ph D dissertation, 2010.6,62-63) carry out.

Attack poison back 7d, the 1st group of (A) guinea pig lung tissue no change, the 2nd group (B) has slight pathological change, and tangible pathological change has all taken place in control group, shows as serious interstitial proliferation, the hyperemia that has, lymphocytic infiltration and alveolar wall destruction (Figure 14).

4.3 cavy immunoprotection test (table 1)

The serum neutralization test result shows that immunity back 35d all can detect the special neutralizing antibody of SIV (H1N1) in the rSPV/H1 immune group guinea pig serum, but between the different cavy antibody titers low be 1: 8, high reaches 1: 64.WtSPV, PK15 immune group serum do not neutralize active to SIV (H1N1).And after testing, all test guinea pig serum are to the no neutralizing effect of SIV (H3N2).

One immunity back 35d attacks poison.After attacking malicious 7d, all there is not the flu-like symptom appearance with the cavy that SIV (H1N1) attacks still with the immune rSPV/H1 of SIV (H3N2) attack no matter be.Control group then shows as fervescence, depressed, the appetite of spirit descends, has a running nose, has difficulty in breathing, coughs, and especially have a running nose clearly, and other symptoms is slight.

Have only the 2/6 visible pathological change of slight naked eyes of having merely hit of attacking in the rSPV/H1 immune group, and all control group cavy lungs there are all large-scale purple, meat to become the district to occur with SIV (H3N2).

The cavy of immunity rSPV/H1 is only from being separated to SIV (H3N2) with 1/6 of SIV (H3N2) attack, and control group all is separated to SIV.

Embodiment 5 pig bodies are attacked malicious protection test

This part test is finished in the Jiangsu Province Agriculture Science Institute.

All pigs are all raised in the environmental facility of no SIV.30 6 ages in week greatly enhance binary pig (SIV, PRRSV, PCV, SPV feminine gender), be divided into 6 groups at random, 5 every group, each group is raised at independent colony house.The 1st group and the 2nd group of immunity rSPV/H1, dosage is 1 * 10 ⁷TCID ₅₀/ 1mL/ head; 3rd, 4 groups of injection wtSPV, dosage is 1 * 10 ⁷TCID ₅₀/ 1mL/ head; 5th, 6 groups every injection PK15 lysis supernatant 1mL.Immunization ways is intramuscular injection.21d after immunity, the precaval vein blood sampling detects the specificity neutralizing antibody at SIV; Separate peripheral blood lymphocyte simultaneously, stimulate, the amount of cytokine IFN-γ and IL-4 in the mensuration culture supernatant with SIV (H1N1).21d after immunity, all pigs are attacked poison, and attacking malicious mode is that nasal cavity injects (syringe needle of choosing).1st, 3,5 groups of every nasal cavities inject 1 * 10 ^5.0TCID ₅₀SIV (H1N1); 2,4,6 groups of every nasal cavities inject 1 * 10 ^5.0TCID ₅₀SIV (H3N2).Attack poison back 5d continuously, observe clinical manifestation, survey body temperature, adopt nose swab and check nasal cavity toxin expelling situation, attack poison back 5d, pig carries out euthanasia handles, and observes the lungs pathological change.The scoring reference literature of influenza virus content detection and lung cardinal principle pathology carries out (Wesley R D in the nose swab, Tang M, Lager K M.Protection of Weaned Pigs by Vaccination with Human Adenovirus 5Recombinant VirusesExpressing the HemagGlutin in and the Nucleoprotein of H3N2 Swine Influenza Virus[J] .Vaccine, 2004,22 (25/26): 3427-3434.).Get lung tissue and handle, inoculate 10 age in days SPF chicken embryos (2 generations of blind passage), HA, HI test detects SIV; Get the lung tissue formaldehyde fixed simultaneously, do pathology section examination.

Clinical manifestation record: when give feeding animal every day (early, middle and late 3 times), observe animal, the appearance situation of record observation item according to 5 days record results, averages at last, obtain animal clinical manifestation marking value, the concrete grammar reference (Wang Xinglong, variation of porcine reproductive and respiratory syndrome virus epidemic isolates NSP2 gene genetic and GM-CSF are to the immuno-potentiation research of GP3/GP5, Agricultural University Of Nanjing's Ph D dissertation, 2010.6,61) carry out.Clinical manifestation record comprises that spirit is depressed, appetite decline, palpebral edema, have a running nose, expiratory dyspnea, cough, skin rubefaction and stiff 8 indexs of ight soil, every has 1 fen, does not have 0 fen.

5.1 attack poison back measurement of bldy temperature result

The rectal temperature of attacking back first day the 2nd, 3,4,5,6 group of pig of poison all has in various degree rising, but the amplitude that rises is limited, and one is 40 ℃, and body temperature is replied normal after 2 days.The rectal temperature of the 1st group of pig remains on normal range 38.7-39.3 ℃ always after attacking poison.

5.2 attack malicious postnaris toxin expelling situation measuring result

From attacking the nose swab that begins to gather every pig before the poison, until off-test.Press Wesley (Wesley R D, Tang M, Lager K M.Protection of Weaned Pigs by Vaccination with Human Adenovirus 5 Recombinant Viruses Expressing the Hemagglutin in and the Nucleoprotein of H3N2 Swine Influenza Virus[J] Vaccine, 2004,22 (25/26): 3427-3434.) detect SIV amount in the nose swab, result such as Figure 15,16.After attacking poison with homology virus H1N1, in the nasal cavity of PK15 and wtSPV group pig the SIV secretion is arranged all, and 5 pigs of malicious immune group of recombinating do not detect virus.After attacking poison with the viral H3N2 of abnormal shape, in the nasal cavity of PK15 and wtSPV group pig the SIV secretion is arranged all also, 3 have the SIV secretion in 5 pigs of the malicious immune group of recombinating, and virus quantity is lower than control group.

5.3SIV (H1N1) stimulate pig peripheral blood lymphocyte secrete cytokines to detect

Pig peripheral blood medium size lymphocyte stimulates special cytokine content detected result (Figure 17) to show through SIV (H1N1): 21d after immunity, each is organized in the pig peripheral blood lymphocyte culture supernatant all cytokine secretion.Compare between group: IFN-γ and IL-4 content are significantly higher than other two control groups (P＜0.05) in the rSPV/H1 immune group pig peripheral blood lymphocyte culture supernatant.

5.4 changing, pig lung tissue pathology observes (Figure 18)

Attack the 1st group of (A) piglet lungs no change in poison back, the 2nd group (B) gets the part of observing pathology, and slight pathological change is arranged, and 3,4,5,6 groups all have tangible pathological change.Mainly be alveolar endolymph cellular infiltration, alveolar wall destruction, epithelial cell shedding, be full of lymphocyte in the bronchial lumen.

5.5 the pig body is attacked malicious protection test (table 2)

The serum neutralization test result shows that immunity back 21d all can detect the special neutralizing antibody of SIV (H1N1) in the rSPV/H1 immune group porcine blood serum, but antibody titers changes greatly between the different pig, and low is 1: 8, and high reaches 1: 32.WtSPV, PK15 immune group porcine blood serum do not neutralize active to SIV (H1N1).And after testing, all test pig serum are to the no neutralizing effect of SIV (H3N2).

Immunity back 21d attacks poison, during 26d, does not all have the appearance of SI symptom no matter be with the pig that SIV (H1N1) attacks still with the immune rSPV/H1 of SIV (H3N2) attack.And the control group pig shows as fervescence, spirit depressed, appetite decline, palpebral edema, has a running nose, like for sleeping in, expiratory dyspnea, cough and skin rubefaction, but most of symptom is slight, and the time length is shorter.

There is 3/5 the visible pathological change of slight naked eyes is arranged with what SIV (H3N2) attacked in the rSPV/H1 immune group pig, the only about 1.5cm of diseased region * 1.5cm size.And all control group pig lungs all have purple, solid district to occur, yet the diseased region of about 80% pig is about 3cm * 3cm, often is confined to a certain zone.

The pig of immunity rSPV/H1 only is separated to SIV (H3N2) from 1 pig lung tissue of attacking with SIV (H3N2), and the control group pig all is separated to SIV.

This research has successfully made up and has expressed the recombinant swinepox virus rSPV/H1 of SIVH1N1 HA1, and has carried out immunity respectively and attack poison protection experiment in mouse, cavy and pig body.The result shows, immunity produced the special antibody of SIV (HA1) in the animal body of recombinant swinepox virus rSPV/H1, IFN-γ, IL-4 content are significantly higher than control group in the lymphopoiesis exponential sum culture supernatant of immune group.Proof rSPV/H1 can significantly improve the humoral immunization and the cell immune response of body.The cavy of immunity rSPV/H1 and pig are attacked the no flu-like symptom in poison back and occur.Flu-like symptoms such as control group then shows as fervescence, spirit is depressed, appetite descends, have a running nose, expiratory dyspnea, cough.Have only in the rSPV/H1 immune group that 2/6 cavy, 3/5 pig have the visible pathological change of slight naked eyes in the animal that special-shaped viral SIV (H3N2) attacks, and purple, meat that all control group lungs all have range size not wait become district's appearance, lung's pathological change especially severe of cavy, 60% the zone of generally having an appointment presents tangible carnificationization.After attacking poison with homology virus, in the nasal cavity of PK15 and wtSPV group pig the SIV viral secretory is arranged all, and 5 pigs of malicious immune group of recombinating do not detect virus.After attacking poison with special-shaped virus, in the nasal cavity of PK15 and wtSPV group pig the SIV viral secretory is arranged all also, 3 have the SIV viral secretory in 5 pigs of the malicious immune group of recombinating, but virus quantity is lower than control group.

Have only in the animal of immunity rSPV/H1 in animal 1/6 cavy that special-shaped viral SIV (H3N2) attacks, the 1/5 pig lung tissue to be separated to SIV (H3N2), and control group all is separated to SIV.

In sum, this research has obtained porcine influenza H1N1HA1 albumen recombinant swinepox virus rSPV/H1.RSPV/H1 can produce significant humoral immunization and cell immune response by induced animal.Can not only protect cavy and pig to resist the attack of homology SIV H1N1, can also partly resist the attack of special-shaped SIV H3N2.

The immunne response of table 1 cavy and attack poison back result

Annotate: the influenza of cavy (SI) symptom value is to observe 6 projects (every 0-3 branch) every day, observe 7 days scoring summations divided by 7 continuously.

The immunne response of table 2 pig body and attack poison back result

Annotate: the influenza of pig (SI) symptom value is for observe 8 projects (0 or 1 minute every), every day 3 times, observe 5 days scoring summations divided by 15 continuously at every turn.

The all statistic datas of the present invention are carried out analyzing and processing with the method for t check, and P＜0.05 is considered to significant difference.

SIV described in the present patent application file (H1N1) refers to that all (doctor Qi Xian separates swine influenza virus H1N1 (A/swine/Shanghai/1/2005 (H1N1)).The GenBank number of including is EU502884); Described SIV (H3N2) refers to that all (doctor Qi Xian separates swine influenza virus H3N2 (A/swine/Guangxi/1/2004 (H3N2)).The GenBank number of including is FJ157986).

All restriction enzymes, T4DNA Ligase, DNA sepharose used in the present patent application file reclaim test kit all available from TaKaRa, the ELISA test kit that detects zooblast factor IFN-γ and IL-4 is available from Groundwork Biotechnology Diagnosticate, and other not marked reagent, material are all available from TIANGEN BIOTECH company.

BALB/c mouse is available from Nanjing Medical University's Experimental Animal Center, and cavy greatly enhances the binary pig available from Changzhou health and happiness boar factory available from Nanjing Military Command's hospital general's Experimental Animal Center.

Claims

1. the transfer vector pUSZ11/H1 of a swine influenza virus H1N1 hypotype HA1 gene is characterized in that this transfer vector is is the BamHI restriction enzyme site gained of the swine influenza virus H1N1 strain HA1 gene order insertion pig pox virus carrier pUSZ11 shown in the SEQ ID NO.1 with sequence.

2. swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus, it is characterized in that this virus be with pig pox virus and the described transfer vector pUSZ11/H1 of claim 1 infect, transfection PK15 cell, carry out the positive colony that homologous recombination obtains.

3. swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus according to claim 2 is characterized in that described pig pox virus for the ATCC preserving number is Number VR-363 ^TMWild-type pig pox virus wtSPV.

4. swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus according to claim 3, it is characterized in that described swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus is preserved in Chinese typical culture collection center on January 6th, 2011, preserving number is CCTCC NO:V201101.

5. the preparation method of the described swine influenza virus H1N1 of claim 2 hypotype HA1 albumen recombinant swinepox virus is characterized in that comprising the steps:

(2) be that the HA1 gene of SEQ ID NO.1 inserts among the pig pox virus carrier pUSZ11 with described sequence, the evaluation of cutting and check order by PCR, enzyme then obtains the transfer vector pUSZ11/H1 of described swine influenza virus H1N1 hypotype HA1 gene;

6. the preparation method of swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus according to claim 5, it is characterized in that concrete grammar that the described HA1 gene of step (2) inserts pig pox virus carrier pUSZ11 is that HA1 gene and the pig pox virus carrier pUSZ11 of SEQ ID NO.1 carries out single endonuclease digestion to the described sequence of step (1) respectively for utilizing BamHI, reclaiming size is the HA1 gene target fragment of 1031bp and the big fragment of pig pox virus carrier pUSZ11 linearity that size is 8391bp, connect two fragments, obtain recombinant plasmid, through order-checking with measure forward and reversely, it is that the plasmid of forward is named and is pUSZ11/H1 that sequencing result is met purpose fragment and direction.

7. the preparation method of swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus according to claim 5 is characterized in that described preparation method also comprises the plaque purification step of described swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus.

8. the preparation method of swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus according to claim 5 is characterized in that described preparation method also comprises the step of utilizing IFA and Western-blot technology described swine influenza virus H1N1 hypotype HA1 albumen recombinant swinepox virus to be expressed evaluation.

9. the described swine influenza virus H1N1 of claim 2 hypotype HA1 albumen recombinant swinepox virus prevents and/or treats application in the medicine that swine influenza virus H1N1 hypotype infects in preparation.