CN102417912B - Molecular design of porcine parvovirus-like particle B cell epitope insertion site - Google Patents

Abstract

The invention relates to a molecular design of porcine parvovirus-like particle B cell epitope insertion site and belongs to the field of genetic engineering vaccine. Structure modeling of porcine parvovirus (PPV) capsid protein VP2 is performed by using bioinformatics software and the location of four extrusive Loop structures is determined by three-dimensional structure analysis. Firstly, on the basis of molecular simulation and literature support, we infer that loop 2,4 region can act as insertion sites of exogenous epitope gene. As is shown through experiments, after respective deletion of corresponding genes in PPV VP2 Loop2 (212aa-245aa), Loop (413aa-424aa) and then expression by adenovirus expression system, all recombinant virus with deletion mutations of Loop 2,4 can assemble regular virus-like particles [PPV: delta V LPs]. The invention also relates to an application of recombinant PPV delta V P2 virus-like particles of exogenous gene expressed by the recombinant virus in vaccination and the like.

Description

The molecular designing of pig parvoviral sample particle B cell epitope insertion point

One, technical field

The present invention relates to the molecular designing of pig parvoviral sample particle B cell epitope insertion point, belong to the recombinant vaccine field.

Two, background technology

Pig parvoviral (PPV) virus particle outward appearance is sexangle or circle, and without cyst membrane, the axles such as icosahedron are three-dimensional symmetrical, and capsid is comprised of 32 capsomeres, 2 structural polypeptides of PPV genome encoding, VPl and VP2.Molecular mass is respectively 84ku and 64ku, separately has a structural polypeptide VP3 to produce molecular mass 60ku by the VP2 hydrolysis.Wherein VP2 is the main capsid protein that consists of virus particle, has comprised the main protection antigen of PPV on this albumen.The N terminal amino acid sequence of VPl PROTEIN C terminal amino acid and VP2 is overlapped.The C of VP2 end is exposed to the surface of this albumen, so the integrity of C section is to keep this capsid protein secondary structure necessary, and its N end is positioned at the inside of secondary structure.

Virus-like particle (VLPs) has two outstanding characteristics and advantage as the vaccine delivery instrument: the first, can further modify transformation to its surface with chemically crosslinked or engineered method; The second, VLPs can wrap up molecular weight and suitable nucleic acid or the non-nucleic acid molecule of electric charge.These two characteristics are for providing desirable platform according to different purposes ground versatile and flexible Design and optimization vaccine.In brief, different vaccine forms such as polypeptide, nucleic acid, even adjuvant can carry or load on VLPs alone or in combination, and it is not enough separately thereby overcome.Polypeptide-VLPs chimeric: polypeptide antigen is easy to preparation, but a little less than immunogenicity, is easy to degraded after entering body, and this has limited the practical application of polypeptide vaccine.If polypeptide is integrated in VLPs, may overcome these obstacles.

The VP2 gene can independently be assembled into virus-like particle and have good immunogenicity in mammalian cell or during baculovirus expression.Sedlik etc. are connected the antigenic determinant district of PPV VP2 N-terminal and the 118-132 amino acids that comprises lymphocyte choroid plexus encephalitis (LCMV), then be cloned in rhabdovirus expression vector PACYM, transfection expression VP2-LCMV albumen, utilize this recombinant protein immunized mice can induce strong cytotoxic T cell reaction (ctl response), time length reaches 9 months in vivo, and can resist the LCMV attack of lethal quantity.The use PPV VP2 covalent attachment hepatitis B virus HBSAg determinant such as Richard district polypeptide, then immune mouse is induced the very strong t cell responses for the HBSAg determinant that inserts of generation, and can resist the hepatitis B virus lethal hit.Existing studies show that: can utilize VP2 oneself to be packaged into the characteristic of ghost particle, exogenous genetic fragment is inserted into N-terminal, expression alien gene uses as a kind of antigen vectors, but its foreign gene that carries can only stimulate the immunity of T lymphocyte inducing cell.

Strong antibody response can be induced in VLPs exposes repeating structure territory, and this makes these structural domains be fit to very much to introduce exogenous antigen and forms VLPs, but insertion point, the epitope size often can disturb correct VLPs to form.Alicia Hurtado philosophy disappearance canine parvovirus (CPV) VP2 loop 1,2,3, corresponding gene in 4, express with baculovirus expression system, loop 1,3 as a result again, 4 deletion mutantion strain all can not give expression to virus-like particle, and the deletion mutantion strain of loop 2 (217～234) can be assembled out the virus-like particle of rule.They studies have shown that further loop 2 can hold the exogenous antigen determinant.Rueda once inserted the gene of poliovirus C3B cell antigen determinant respectively in 4 rings of CPV VP2 gene and carried out amalgamation and expression after C-terminal, result only has the fusion rotein that inserts ring 2 the 225th amino acids can produce anti-polypeptide reaction, but can not produce anti-bone marrow poliomyelitis virus neutralization reaction.So change into ring 2 the 226th, 227,228 amino acids place's insertions, these fusion roteins can produce anti-bone marrow poliomyelitis virus neutralization reaction as a result.

There are some researches show, the threefold axis direction on PPV virus-like particle surface has spinal (spike), and it is the higher structure that is formed by loop, is positioned at the most surperficial of capsid, has comprised most of epitope of PPV; Five solid axle directions have cylindrical channel (cylindrical structure), are formed by 5 β-ribbon, during the partial amino-acid residue of N-terminal inside folds and makes and antigenic surface; In addition, around five solid axles, Canyon Area (canyon) is arranged, the twofold axis direction has depression (depression), and they may be the virus receptors of PPV.Think accordingly: although the N of VP2 end can form virus-like particle as insertion point, the N end of empty particle is in the cylindrical channel of five solid axles, and recombinant chou can not stimulate the immunne response that produces the B cell antigen epi-position; The peptide section that consists of five heavy axial Canyon Areas and twofold axis direction depression is not suitable as insertion point equally.

Three, summary of the invention

Technical problem

The object of the invention is the molecular designing of pig parvoviral sample particle B cell epitope insertion point and carries out the development of polyvalent recombinant vaccine with this restructuring PPV Δ VP2 virus-like particle as B cell epitope carrier.

Technical scheme

The molecular designing of pig parvoviral sample particle B cell epitope insertion point is characterized in that,

1) clone of Δ VP2 ( Loop 2,4 disappearances) goal gene

Be template according to NADL-2 in GenBank (NC:001718) gene order, with software PrimerPremier 5.0 design primer Loop 11, Loop 22, Loop 23, Loop 14, Loop 42 and Loop 43, wherein Loop 11 and Loop 14 add respectively restriction enzyme site, and primer sequence is as follows:

Loop 11：5-gtc-gac-atg-agt-gaa-aat-gtg-gaa-caa-c-3(sal I)

Loop 22：5-tgg-att-tag-gtt-tct-gat-g-3

Loop 23：5-cat-cag-aaa-cct-aaa-tcc-aga-ctc-aat-aca-aac-agg-act-3

Loop 14：5-gcc-ctc-gag-cta-gta-taa-ttt-tct-tgg-3(xhoI)

Loop 42：5-ttg-ttg-ctt-tgg-agc-tct-tcc-3

Loop 43：5-gga-aga-gct-cca-aag-caa-caa-ctt-tta-cct-tca-gat-cc-3

By SOE method gene splicing: take plasmid pT VP2 as template, with primer Loop 11, Loop 22 carries out pcr amplification VP2 fragment 660bp, and product is reclaiming after electrophoresis on 1% sepharose; With primer Loop 23, Loop 14 carries out pcr amplification VP2 fragment 1047bp, and product is reclaiming after electrophoresis on 1% sepharose; Reclaim product as template, primer Loop 11, Loop 14PCR amplification VP2Loop 2 deletion fragments take above-mentioned 2 again.Respectively with primer Loop11, Loop 42 and primer Loop 43, Loop 14 amplification VP2 fragment 1227bp and 465bp, and reclaim respectively and make template, then with primer Loop 11, Loop 14 pcr amplification VP2Loop 4 deletion fragments.Deletion fragment that VP2 Loop2 deletion fragment is connected with VP2 Loop is connected with pMD19-T Vector respectively, Transformed E .coli DH5 α, the alkaline lysis method of extracting plasmid, identify with sal I and xho I double digestion respectively, obtain positive plasmid pT Δ VP2-2 and pT Δ VP2-4, and the precious Bioisystech Co., Ltd order-checking through Dalian.

2) recombinant plasmid pShuttle-Δ VP2-2, the Construction and identification of pShuttle-Δ VP2-4

Utilize double enzyme site sal I and xho I that pT Δ VP2-2 and pT Δ VP2-4 are cloned in transfer vector pShuttle-CMV; Identify with PCR, sal I and xho I double digestion and sequencing analysis, respectively called after pShuttle-Δ VP2-2 and pShuttle-Δ VP2-4.

3) restructuring PPV Δ VP2-2, the acquisition of PPV Δ VP2-4 virus-like particle

Cut this recombinant plasmid with Pme I enzyme, make its linearizing, then with skeleton carrier pAdEasy ^TMVector electricity under 1.8kV, 25 μ F and 200 Ω conditions is converted into the E.coli BJ5183 competent cell, make pShuttle-Δ VP2-2 and pShuttle-Δ VP2-4 and pAdEasyTM Vector that homologous recombination occur, be converted into DH5 α Host Strains, pick out positive colony, cut with PCR, PacI enzyme and identify whether restructuring is correct, be built into recombinant plasmid, respectively called after pAd-Δ VP2-2 and pAd-Δ VP2-4.

4) with recombinant plasmid pAd-Δ VP2-2 and pAd-Δ VP2-4 transfection HEK-293A cell, obtain recombinant virus rAd-Δ VP2-2 and rAd-Δ VP2-4; Recombinant adenovirus expressing protein oneself in the HEK-293A cell is assembled into virus-like particle PPV: Δ VLPs is the restructuring PPVVP2 virus-like particle of Loop 2,4 genetically deficients of acquisition.

The molecular designing of above-mentioned pig parvoviral sample particle B cell epitope insertion point reaches with this restructuring PPV Δ VP2 virus-like particle can obtain pig parvoviral sample particulate vector polyvalent recombinant vaccine in the application aspect the preparation vaccine.

The beneficial effect the features and advantages of the invention are as follows:

1, the present invention gland virus expression system of using is the adenovirus hominis Serotype 5 (Ad5) that has lacked E1 and E3 gene, have that no pathogenicity, duplicating efficiency are high, clone space large (the external source fragment that can hold 7.5Kb), can express recombinant protein in most of mammalian cells and tissue, can express simultaneously the characteristics such as a plurality of genes in allogenic cell and tissue, can carry out correct translation and modification to foreign gene, make the albumen of expression have the biological activity identical with native protein.Use the recombinant virus of this system constructing to be used for the pig body, can also avoid affecting immune effect due to the adenovirus antibody that pig body self produces.

2, use bioinformatics software sybyl software to carry out the structural modeling of vp2 albumen, use simultaneously Discovery Studio software and NAMD software to carry out Molecular Dynamics Calculation at " rolling reamer machine " enterprising row, drawn correct vp2 protein three-dimensional structure, analyze its three-dimensional structure, determine VP24 outstanding Loop structure location: Loop 1:86aa-101aa; Loop 2:212aa-245aa; Loop 3:279aa-333aa; Loop 4:413aa-424aa.On the basis that molecular simulation and document are supported, we infer that loop 2nd, 4 district can be used as foreign epitope gene insertion point.

3, Sadeyen etc. [2003] inserts immunodominant epitopes's polypeptide DPASRE of HbcAg respectively 6 ring districts of HPV16 L1-VLPs outside surface, these 6 kinds of chimeric VLPs all can induce the HBc specific antibody, but after BC ring district's insertion exogenous peptide, the neutrality antibody titre of the anti-HPV16 L1 that body produces obviously descends.Prompting BC ring district is the key position that forms HPV16 L1 conformational epitope, inserts herein and often can disturb correct VLPs to form.The present invention lacks respectively PPV VP2 loop 2, corresponding gene in 4, use again the gland virus expression system expression, loop 2 as a result, 4 deletion mutantion strain all can give expression to virus-like particle, preliminary identification loop 2 (212aa-245aa), Loop 4:(413aa-424aa) can hold the exogenous antigen determinant.

4, virus-like particle (VLPs) as the vaccine delivery instrument, has two outstanding characteristics and advantage: the first, can further modify transformation to its surface with chemically crosslinked or engineered method; The second, VLPs can wrap up molecular weight and suitable nucleic acid or the non-nucleic acid molecule of electric charge.These two characteristics are for providing desirable platform according to different purposes ground versatile and flexible Design and optimization vaccine.In brief, different vaccine forms such as polypeptide, nucleic acid, even adjuvant can carry or load on VLPs alone or in combination, and it is not enough separately thereby overcome.Saini[2003] with an epitope polypeptide of japanese encephalitis virus (JEV) E albumen, after flexibly connecting son (Gly4-Ser) 3 and the C end disappearance capsid protein of Johnson showy flowers of herbaceous plants pinta poison (JGMV) is connected by one, form a kind of chimeric VLPs; After this chimeric VLPs did not need the adjuvant immunity mouse, the neutralizing antibody of generation can effectively be protected mouse to avoid the lethality japanese encephalitis virus and attack.The present invention builds restructuring PPV Δ VP2 virus-like particle and carries out the development of polyvalent recombinant vaccine as B cell epitope carrier, and is not only safe, inexpensive, and the chimeric PPV VLps vaccine of preparation multiple pathogens and/or a plurality of hypotype pathogenic agent.

Four, description of drawings

Fig. 1: PPV VP2 goal gene amplification

Fig. 2: the VP2 protein three-dimensional structure is analyzed

Fig. 3: Δ VP2 ( Loop 2,4 disappearances) goal gene amplification Δ VP2-2 (a), Δ VP2-4 (b)

Fig. 4: recombinant plasmid pShuttle-Δ VP2-2 (a), pShuttle-Δ VP2-4 (b) sal I/xhoI double digestion enzyme is cut evaluation

Fig. 5: recombinant plasmid pAd-Δ VP2-2 (a) and pAd-Δ VP2-4 (b) PacI enzyme are cut the evaluation collection of illustrative plates

Fig. 6: recombinant adenovirus rAd-Δ VP2-2 and rAd-Δ VP2-4 indirect immunofluorescence figure: rAd-Δ VP2-2 (a), rAd-Δ VP2-4 (b), and cell contrast (c)

Fig. 7: immunoblotting is identified

Fig. 8: PPV Δ VP2 virus-like particle Electronic Speculum figure rAd-Δ VP2-2 (a), rAd-Δ VP2-4 (b)

Five, embodiment

1) PPVVP2 gene cloning

Be template according to NADL-2 in GenBank (NC:001718) gene order, with software PrimerPremier 5.0 design primer P1 and P2, add respectively restriction enzyme site, primer sequence is as follows:

P1 5-acc-gga-tcc-atg-ggg-ggg-gtt-ggt-gtg-tct-ac-3(BamH I)

P2 5-atc-ctc-gag-cta-gta-taa-ttt-tct-tgg-3(xho I)

With the PPVNADL-2 low virulent strain (document sees reference: Pan Qunxing, etc., detect the multiple PCR method of PCV2, PPV, PRV vaccine strain and street strain, Chinese virusology, 2005,20 (6) 603～606; Available from China Veterinary Drugs Supervisory Inst.; platform resource number: 1511C0006H00000087, country's veterinary microorganism bacterial classification is preserved center bacterial strain deposit number: HVRIPPV0003) be template, carry out pcr amplification with primer P1 and P2, product is after electrophoresis on 1% sepharose, reclaim and it is connected with pMD19-T Vector (precious biotechnology (Dalian) company limited), Transformed E .coli DH5 α (precious biotechnology (Dalian) company limited), the alkaline lysis method of extracting plasmid, identify with BamH I and xhoI double digestion respectively, obtain positive plasmid pT VP2, and the precious Bioisystech Co., Ltd order-checking through Dalian.

2) structural modeling of vp2 albumen and analysis

After relatively confirming by the database multisequencing, we use bioinformatics software sybyl software to carry out the structural modeling of vp2 albumen, use simultaneously Discovery Studio software and NAMD software to carry out Molecular Dynamics Calculation at " rolling reamer machine " enterprising row, drawn correct vp2 protein three-dimensional structure, analyze its three-dimensional structure, determine VP24 outstanding Loop structure location (Loop 1:86aa-101aa; Loop2:212aa-245aa; Loop3:279aa-333aa; Loop4:413aa-424aa).

3) clone of Δ VP2 ( Loop 2,4 disappearances) goal gene

Be template according to NADL-2 in GenBank (NC:001718) gene order, with software PrimerPremier 5.0 design primer Loop 11, Loop 22, Loop 23, Loop 14, Loop 42 and Loop 43, wherein Loop 11 and Loop 14 add respectively restriction enzyme site, and primer sequence is as follows:

Loop 11：5-gtc-gac-atg-agt-gaa-aat-gtg-gaa-caa-c-3(sal I)

Loop 22：5-tgg-att-tag-gtt-tct-gat-g-3

Loop 23：5-cat-cag-aaa-cct-aaa-tcc-aga-ctc-aat-aca-aac-agg-act-3

Loop 14：5-gcc-ctc-gag-cta-gta-taa-ttt-tct-tgg-3(xhoI)

Loop 42：5-ttg-ttg-ctt-tgg-agc-tct-tcc-3

Loop 43：5-gga-aga-gct-cca-aag-caa-caa-ctt-tta-cct-tca-gat-cc-3

By SOE method gene splicing: take plasmid pT VP2 as template, with primer Loop 11, Loop 22 carries out pcr amplification VP2 fragment 660bp, and product is reclaiming after electrophoresis on 1% sepharose; With primer Loop 23, Loop 14 carries out pcr amplification VP2 fragment 1047bp, and product is reclaiming after electrophoresis on 1% sepharose; Reclaim product as template, primer Loop 11, Loop 14 pcr amplification VP2 Loop 2 deletion fragments take above-mentioned 2 again.Respectively with primer Loop 11, Loop 42 and primer Loop 43, Loop 14 amplification VP2 fragment 1227bp and 465bp, and reclaim respectively and make template, then with primer Loop 11, Loop 14 pcr amplification VP2 Loop 4 deletion fragments.VP2 Loop2 deletion fragment is connected deletion fragment ((precious biotechnology (Dalian) company limited) Vector is connected with pMD19-T respectively with VP2 Loop, Transformed E .coli DH5 α, the alkaline lysis method of extracting plasmid, identify with sal I and xhoI double digestion respectively, obtain positive plasmid pT Δ VP2-2 and pT Δ VP2-4, and the precious Bioisystech Co., Ltd order-checking through Dalian.

4) recombinant plasmid pShuttle-Δ VP2-2, the Construction and identification of pShuttle-Δ VP2-4

(document sees reference: Construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus 2 (PCV2) in mice.Vaccine.2006,12 to utilize double enzyme site sal I and xho I that pT Δ VP2-2 and pT Δ VP2-4 are cloned into transfer vector pShuttle-CMV; 24 (16): 337～480); Identify with PCR, sal I and xho I double digestion and sequencing analysis, respectively called after pShuttle-Δ VP2-2 and pShuttle-Δ VP2-4.

5) restructuring PPV Δ VP2-2, the acquisition of PPV Δ VP2-4 virus-like particle

Cut this recombinant plasmid with Pme I enzyme, make its linearizing, then with skeleton carrier pAdEasy ^TM(Vector sees reference document: Construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus 2 (PCV2) in mice.Vaccine.2006,12; 24 (16): 337～480) electricity is converted into E.coli BJ5183 (document sees reference: Construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus 2 (PCV2) in mice.Vaccine.2006,12 under 1.8kV, 25 μ F and 200 Ω conditions; 24 (16): 337～480) competent cell, make pShuttle-Δ VP2-2 and pShuttle-Δ VP2-4 and pAdEasyTM Vector that homologous recombination occur, be converted into DH ₅The α Host Strains is picked out positive colony, cuts to identify whether restructuring is correct, is built into recombinant plasmid with PCR, PacI enzyme, respectively called after pAd-Δ VP2-2 and pAd-Δ VP2-4.

3) acquisition of recombinant virus and purifying

According to the lipofectamine working method, will be in advance (document sees reference: Construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus 2 (PCV2) in mice.Vaccine.2006,12 with the linearizing recombinant plasmid pAd-of PacI Δ VP2-2 and pAd-Δ VP2-4 transfection HEK-293A cell; 24 (16): 337～480), 5%CO ₂, 37 ℃ of standing cultivations are more than 10d, and the microscope observing cell pathology when cytopathy reaches 70%, is received poison.Through 3 plaque purification experiments, the titre that obtains recombinant virus rAd-Δ VP2-2 and rAd-Δ VP2-4 is respectively 10 ^11.8TCID ₅₀/ mL and 10 ^11.5TCID ₅₀/ mL.

4) PPV Δ VP2 virus-like particle biological property research

1. indirect immunofluorescence

To be inoculated in the HEK-293A cell that covers with individual layer, 5%CO2,37 ℃ of cultivations, approximately 24h after the recombinant adenovirus dilution, supernatant discarded, with the PBS washing once, 37 ℃ of dry 45min,-20 ℃ of freezing 45min, then with the fixing 45min of 4 ℃ of cold dehydrated alcohols, PBST washing 3 times; The serum (b) that adds respectively the anti-PPV of pig of 50 μ L dilution in 1: 40,37 ℃ of effect 1h are with PBST washing 3 times; Add 1: 100FITC-SPA, 37 ℃ of effect 1h wash 3 times; Microscopic examination.Recombinant adenovirus (rAd-Δ VP2-2a) and rAd-Δ VP2-4 (b) present stronger fluorescence, and cell control well (c) does not have fluorescence (Fig. 6) to occur.Illustrate recombinant adenovirus at cells PPV: Δ VP2 albumen.

2. protein imprinted

With the concentrated recombinant adenovirus HEK-293A cell culture of 8%PEG-6000, set simultaneously normal HEK-293A cell culture and be contrast.Above-mentioned protein concentrate is carried out the SDS-PAGE electrophoresis, and after being transferred to the fine film of nitre, 10% skimming milk sealing is spent the night; The serum room temperature effect 2h that adds 1: 40 anti-PPV of pig; Wash 3 times, add the goat-anti pig IgG of the horseradish peroxidase-labeled of dilution in 1: 20000, room temperature effect 1.5h; Wash 4 times, add chemoluminescence method nitrite ion (DAB), observe the differential protein band.Show at the 66KD place, an obvious band is arranged all after concentrated recombinant adenovirus poisons electrophoresis transfer printing colour developing, there is no (Fig. 7) and contrast normal HEK-293A cell, illustrate recombinant adenovirus at cells Δ VP2 albumen.

3. thick purifying and the electron microscopic observation of embedded virus like-particles

Collect sick cell, wash the centrifugal 15min of twice, 1000r/min with PBS, be resuspended in 50mmol/l NaHCO3, after ultrasonic treatment, the centrifugal 15min of 24000r/min contains expressing protein in supernatant liquor.After doing same processing, the wild poison of adenovirus does contrast.Add after 37 ℃ of anti-PPV serum of the pig effect 1h of dilution in 1: 10 4 ℃ to spend the night, with the centrifugal 90min of 12000r/min, precipitation is resuspended in a small amount of water again, and the phospho-wolframic acid negative staining with 3% is by transmission electron microscope observing.Can see that under Electronic Speculum a large amount of virus like particle exist, be round, diameter is slightly larger than totivirus particle about 30～40nm, its form is similar to the totivirus particle (Fig. 8) all, illustrates that recombinant adenovirus rAd-Δ VP2-2 can become virus-like particle by self assembly with rAd-Δ VP2-4 at the Δ VP2 of cells albumen.

4. hemagglutination test (HA test)

With reference to " animal virology ", use the physiological saline suspension of 0.7% guinea-pig red blood cell.Recombinant adenovirus rAd-Δ VP2-2 and rAd-Δ VP2-4 hemagglutinative titer are 1: 64, and the wild poison of empty adenovirus does not have hemagglutination activity, illustrate that recombinant adenovirus expression Δ VP2 albumen keeps the original biologic activity of PPV.

5, the PPVVLps insertion point is analyzed

Virus-like particle (VLPs) is as the vaccine delivery instrument, two outstanding characteristics and advantage: the first, can further modify transformation to its surface with chemically crosslinked or engineered method; The second, VLPs can wrap up molecular weight and suitable nucleic acid or the non-nucleic acid molecule of electric charge.These two characteristics are for providing desirable platform according to different purposes ground versatile and flexible Design and optimization vaccine.In brief, different vaccine forms such as polypeptide, nucleic acid, even adjuvant can carry or load on VLPs alone or in combination, thereby it is not enough separately to overcome it: Saini[2003] with an epitope polypeptide of japanese encephalitis virus (JEV) E albumen, after flexibly connecting son (Gly4-Ser) 3 and the C end disappearance capsid protein of Johnson showy flowers of herbaceous plants pinta poison (JGMV) is connected by one, form a kind of chimeric VLPs; After this chimeric VLPs did not need the adjuvant immunity mouse, the neutralizing antibody of generation can effectively be protected mouse to avoid the lethality japanese encephalitis virus and attack.Vietheer[2007] hepatitis C virus envelope protein E2 hypervariable region (HVR1) is inserted the chimeric VLPs of hyperimmunity that HBsAg-S major antigen site (a determinant) forms, still can keep inserting the antigenicity of HVR1 sequence.Kanda[2008] with human papillomavirus (HPV) HPV16 L1 and the chimeric VLPs of L2 type common epitope, successfully the L2 peptide is presented to the VLPs surface, immunizing rabbit has been induced L1 and the L2 neutralizing antibody that is enough to bring into play provide protection.

Be to seek the effectively external source B cell epitope insertion point of assembling of PPV VLps, analyze by PPV VLPs three-dimensional structure and epitope distribution plan, we infer that loop 2nd, 4 district can be used as foreign epitope gene insertion point; And express checking in the gland virus expression system, and the deletion mutantion strain of loop 2,4 as a result all can give expression to virus-like particle, preliminary identification loop2, and 4th district can hold the exogenous antigen determinant.

This paper PPV Δ VP2 virus-like particle of recombinating can be used as B cell epitope carrier and carries out the development of polyvalent recombinant vaccine, structure is the PPV VLPs technology platform of submission different sources B cell epitope effectively, has opened up new thinking for development resists the vaccine of one or more cause of diseases simultaneously.

SEQUENCE LISTING

＜110〉Jiangsu Province Agriculture Science Institute

＜120〉molecular designing of pig parvoviral sample particle B cell epitope display carrier

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<170> PatentIn version 3.1

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Claims (1)

1. the recombination porcine parvovirus PPV VP2 virus-like particle of Loop 2,4 genetically deficients, its construction process is as follows:

1) PPV VP2 gene cloning

The NADL-2 gene order that is NC:001718 according to accession number in GenBank is template, with software PrimerPremier 5.0 design primer P1 and P2, adds respectively restriction enzyme site, and primer sequence is as follows:

P1 5- acc-gga-tcc- atg-ggg-ggg-gtt-ggt-gtg-tct-ac-3 BamH I

P2 5- atc- ctc-gag-cta-gta-taa-ttt-tct-tgg-3 xho I

Take PPV NADL-2 low virulent strain as template, carry out pcr amplification with primer P1 and P2, product reclaims and it is connected with pMD19-T Vector after electrophoresis on 1% sepharose, transforms E.coliDH5 α, the alkaline lysis method of extracting plasmid is used respectively BamH I and xhoThe I double digestion is identified, obtains positive plasmid pT VP2,

2) clone of the △ VP2 goal gene of Loop 2,4 disappearances

The NADL-2 gene order that is NC:001718 according to accession number in GenBank is template, with software PrimerPremier 5.0 design primer Loop 11, Loop 22, Loop 23, Loop 14, Loop 42 and Loop 43, wherein Loop 11 and Loop 14 add respectively restriction enzyme site, and primer sequence is as follows:

Loop 11: 5 -gtc-gac - atg-agt-gaa-aat-gtg-gaa-caa-c- 3 sal I

Loop 22: 5- tgg-att-tag-gtt-tct-gat-g-3

Loop 23: 5- cat-cag-aaa-cct-aaa-tcc-aga-ctc-aat-aca-aac-agg-act-3

Loop 14: 5- gcc -ctc-gag- cta-gta-taa-ttt-tct-tgg- 3 xhoI

Loop 42: 5- ttg-ttg-ctt-tgg-agc-tct-tcc-3

Loop 43: 5- gga-aga-gct-cca-aag-caa-caa- ctt-tta-cct-tca-gat-cc-3

By SOE method gene splicing: take plasmid pT VP2 as template, with primer Loop 11, Loop 22 carries out pcr amplification VP2 fragment 660bp, and product is reclaiming after electrophoresis on mass ratio 1% sepharose; With primer Loop 23, Loop 14 carries out pcr amplification VP2 fragment 1047 bp, and product is reclaiming after electrophoresis on 1% sepharose; Reclaim product as template, primer Loop 11, Loop 14 pcr amplification VP2 Loop2 deletion fragments take above-mentioned 2 again;

Respectively with primer Loop 11, Loop 42 and primer Loop 43, Loop 14 amplification VP2 fragment 1227bp and 465bp, and reclaim respectively and make template, then with primer Loop 11, Loop 14 pcr amplification VP2 Loop 4 deletion fragments; Deletion fragment that VP2 Loop 2 deletion fragments are connected with VP2 Loop is connected with pMD19-T Vector respectively, transforms E.coliDH5 α, the alkaline lysis method of extracting plasmid is used respectively Sal IWith xhoThe I double digestion is identified, obtains positive plasmid pT △ VP2-2 and pT △ VP2-4, and order-checking;

3) recombinant plasmid pShuttle-△ VP2-2, the Construction and identification of pShuttle-△ VP2-4

Utilize double enzyme site Sal IWith xhoI is cloned into pT △ VP2-2 and pT △ VP2-4 in transfer vector pShuttle-CMV respectively; With PCR, Sal IWith xhoI double digestion and sequencing analysis are identified, difference called after recombinant plasmid pShuttle-△ VP2-2, pShuttle-△ VP2-4;

4) restructuring PPV △ VP2-2, the acquisition of PPV △ VP2-4 virus-like particle

With PmeI enzyme respectively cuts recombinant plasmid pShuttle-△ VP2-2 and pShuttle-△ VP2-4, makes its linearizing, then with skeleton carrier pAdEasy ^TMVector electricity under 1.8 kV, 25 μ F and 200 Ω conditions is converted into intestinal bacteria BJ5183Competent cell makes pShuttle-△ VP2-2 and pShuttle-△ VP2-4rShuttle-△ VP2-2 pAdEasyTM Vector that homologous recombination occur, and is converted into DH5 α Host Strains, picks out positive colony, use PCR, PacThe I enzyme cuts to identify whether restructuring is correct, is built into recombinant plasmid, respectively called after pAd-△ VP2-2 and pAd-△ VP2-4;

5) with recombinant plasmid pAd-△ VP2-2 and pAd-△ VP2-4 transfection HEK-293A cell, obtain recombinant virus rAd-△ VP2-2 and rAd-△ VP2-4; Recombinant adenovirus expressing protein oneself in the HEK-293A cell is assembled into virus-like particle PPV: △ VLPs is the restructuring PPV VP2 virus-like particle of Loop 2,4 genetically deficients of acquisition.

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