CN101289658A - Recombination virus particles for expressing 2-typed porcine circovirus nucleocapsid protein Cap gene - Google Patents
Recombination virus particles for expressing 2-typed porcine circovirus nucleocapsid protein Cap gene Download PDFInfo
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Abstract
The invention relates to construction and application of recombinant nucleocapsids of Cap genes of porcine circovirus expression type 2 nucleocapsid protein, belonging to the genetic engineering bacterin field. C-terminal gene fragments of porcine parvovirus (PPV) VP2 genes are cloned into a type 5 adenovirus shuttle vector of the human beings, and recombinant adenovirus rAd-deltaVP2 is obtained; deltaVP2 proteins are expressed successfully and highly efficiently and can be self-assembled into the nucleocapsids [PPV:VLPs]; the PPV VP2 nucleocapsids are used as antigen transport vectors and 165 to 200 sites of amino acid (deltaCap) genes of the porcine circovirus type 2 (PCV2) nucleocapsid proteins (Cap) are embedded into an N-terminal (deltaVP2) of the PPV VP2, and then recombinant adenovirus rAd-deltaCap-deltaVP2 is obtained; embedded VP2 (deltaCap-deltaVP2) proteins are expressed successfully and highly efficiently and can be self-assembled into nucleocapsids [PPV:VLP(PCV2)]. The invention also relates to application of the recombinant virus and recombinant PPV VP2 nucleocapsids of the expression Cap genes of the recombinant virus in the aspects of bacterin immunity and so on.
Description
One, technical field
The present invention relates in the gland virus expression system, express the structure and the application of the recombinant virus like-particles of 2 type porcine circovirus nucleocapsid protein Cap genes, belong to the recombinant vaccine field.
Two, background technology
PPV (Porcine Parvovirus) is not only one of main pathogens that causes the sow breeding difficulty, and can cause multiple swine disease.VP2 is the virus particle main component, accounts for 80%, and the coat protein of parvovirus can independently be assembled under state of nature.VP2 has good immunogenicity, and numerous scientists utilizes the good immunological characteristic of VP2 to make up recombiant vaccine and prevents this disease, has obtained good effect, still has the material of hemagglutination activity simultaneously.Martinez etc. with PPV VP2 gene clone in baculovirus expression system, and successfully in insect cell, efficiently express, the VP2 polypeptide energy oneself who expresses is assembled into virus like particle, and (Virus-Like Particles VLPs), can induce the generation immunne response with its immune sow.Although do not see about this kind vaccine further research and the report used, the VP2 gene can the oneself be packaged into the characteristic of virus like particle at the albumen of vivoexpression, for the research of reorganization multivalent subunit vaccine lays the foundation.Sedlik etc. link to each other PPV VP2 with the antigenic determinant district of the 118-132 amino acids that comprises lymphocyte choroid plexus encephalitis (LCMV), clone in rhabdovirus expression vector PACYM transfection expression PPV:VP2-LCMV albumen then.Utilize this recombinant protein immunized mice can induce the intensive ctl response, the time length reaches 9 months in vivo, and can resist the LCMV attack of lethal quantity.Afterwards, Sedlik is covalently bonded in the many skins of CD8+T cell antigen determinant of LCMV on the lipid microsphere of 1 μ m again, carried out the comparison of immune mouse with the PPV:VP2-LCMV albumen of above-mentioned expression, found that the two can both induce the very strong CD8+T cell response of generation.When but the antigen amount that PPV:VP2-LCMV carries was lacked 100 times than the liposome microballoon, inductive CTL activity was still active high 6 times than liposome microballoon inductive CTL, and has only the PPV:VP2-LCMV mice immunized can resist the attack of the LCMV of lethal quantity.The antigenic determinant district polypeptide of PPV VP2 covalent attachment hepatitis B virus HBSAg such as Richard, immune mouse is induced and has been produced the very strong t cell responses at the HBSAg determinant of inserting the people then, and can resist the lethal hit of hepatitis B virus.These results illustrate that all PPV VP2 virus like particle has very big potential value as a kind of antigenic transport vehicle, and are that good condition has been created in the research of carrying out the polyvalent recombinant vaccine.
Three, summary of the invention
Technical problem
The object of the invention is 1) utilize the method for the reorganization PPV VP2 virus like particle of gland virus expression system constructing expression alien gene, and carry out the development of polyvalent recombinant vaccine as a kind of antigen transport vehicle with this reorganization PPV VP2 virus like particle; 2) the recombinant virus like-particles of expression 2 type porcine circovirus nucleocapsid protein Cap genes in the gland virus expression system and the application on vaccine immunity thereof have been made up.
Technical scheme
1) obtains Δ Cap-Δ VP2 goal gene and be cloned into the pMD19-T carrier
With PCV2-js2002 gene accession number among the GenBank is that the gene order of AY691679 and PPV VP2 recombinant plasmid is a template, and with software Primer 5.0 design primer: P3, P4, P5, P6 and P7 add restriction enzyme site respectively:
P3 5′-ctggtaccatggtccaccgcccaggag-3′ KpnI
P4 5′-cagctagccgttaccgctggagaagg-3′ NheI
P5 5′-gagctagcgggggggttggtgtgt-3′ NheI
P6 5′-cggctgcagctagtataattttcttgg-3′ PstI
P7 5′-cgggcggccgcctagtataattttcttgg-3′ NotI
With P3, the fragment Δ Cap of antigenic determinant district polypeptide gene 108 bp of the proteic 165-200 amino acids of P4 primer amplification PCV2 Cap, with P5, the fragment Δ VP2 of P6 primer amplification PPV VP2 PROTEIN C end 1640 bp, amplification PCR products is behind electrophoresis on 1% sepharose, reclaim and it is connected Transformed E .coli DH with pMD19-T Vector
5α, the alkaline lysis method of extracting plasmid is identified with KpnI/NheI and NheI/PstI double digestion respectively, obtains positive plasmid pT Δ Cap and pT Δ VP2.
Utilize restriction endonuclease NheI on the primer and the PstI on the pMD19-T Vector with positive plasmid pT Δ Cap double digestion, reclaim the fragment of 2.8Kb, utilize restriction endonuclease NheI on the primer and PstI with positive plasmid pT Δ VP2 double digestion simultaneously, reclaim the small segment of 1.64Kb, two fragments that will reclaim then connect Transformed E .coli DH with T4 DNA Ligase
5α, the alkaline lysis method of extracting plasmid, the KpnI/PstI double digestion is identified, positive plasmid called after pT Δ Cap-Δ VP2.
2) structure and the evaluation of recombinant plasmid pAd-Δ Cap-Δ VP2
With P3, P7 is a primer, and positive plasmid pT Δ Cap-Δ VP2 is a template, pcr amplification Δ Cap-Δ VP2 fragment, and introduce restriction endonuclease Kpn I/NotI, according to pAdEasy
TMThe carrier system operation instructions utilizes double enzyme site KpnI/NotI that external source fragment Δ Cap-Δ VP2 is cloned among the transfer vector pShuttle-CMV; Identify called after rShuttle-Δ Cap-Δ VP2 with PCR, KpnI/NotI double digestion and sequencing analysis;
3) acquisition of the recombinant virus like-particles of expression 2 type porcine circovirus nucleocapsid protein Cap genes
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 intestinal bacteria BJ5183 competent cell, makes rShuttle-Δ Cap-Δ VP2 and pAdEasy
TMHomologous recombination takes place in Vector, is converted into DH
5α host bacterium is picked out positive colony, cuts with PCR, PacI enzyme and identifies whether reorganization is correct, is built into recombinant plasmid, called after pAd-Δ Cap-Δ VP2;
With recombinant plasmid pAd-Δ Cap-Δ VP2 transfection HEK-293A cell, obtain recombinant virus rAd-Δ Cap-Δ VP2; Recombinant adenovirus rAd-Δ Cap-Δ VP2 expresses Δ Cap-Δ VP2 chimeric protein oneself in the 293A cell that this recombinant adenovirus infects and is assembled into virus like particle PPV:VLP (PCV2), is the recombinant virus like-particles of the expression 2 type porcine circovirus nucleocapsid protein Cap genes of acquisition.The recombinant virus like-particles of above-mentioned expression 2 type porcine circovirus nucleocapsid protein Cap genes can obtain a kind of protein carrier polyvalent recombinant vaccine in the application aspect the preparation vaccine.
Beneficial effect characteristics of the present invention and advantage are as follows:
1, the gland virus expression system of the present invention's application is adenovirus hominis serum 5 types (Ad5) that lacked E1 and E3 gene, have no pathogenicity, duplicating efficiency height, clone space big (the external source fragment that can hold 7.5Kb), can be in most of mammalian cells and tissue express recombinant protein, in allogenic cell and tissue, can express characteristics such as a plurality of genes simultaneously, can carry out correct translation and modification to foreign gene, make expressed proteins 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 because the adenovirus antibody that pig body self produces influences immune effect.
2, utilize the reorganization PPV VP2 virus like particle of gland virus expression system constructing expression alien gene: PPV VP2 C end group is cloned in the gland virus expression system because of fragment Δ VP, transfection HEK-293 A cell, RT-PCR, indirect immunofluorescence and Western-blotting experimental result show that Δ VP2 albumen efficiently expresses in HEK-293 A cell; The crude extract of expression product after transmission electron microscope observing can see that under Electronic Speculum a large amount of virus-like particles exist, is round with 3% phospho-wolframic acid negative staining, and diameter is about 30nm, and its form size is all similar to the totivirus particle.
3, the reorganization PPV VP2 virus like particle of expression alien gene that utilized the gland virus expression system constructing, with PPV VP2 virus like particle as a kind of antigen transport vehicle, for the development of reorganization multivalent subunit vaccine lays the foundation.
4, the present invention with PPV VP2 virus like particle as the antigenic transport vehicle of PCV2: the N end (Δ VP2) that 165-200 amino acids (Δ Cap) gene of PCV2 nucleocapsid protein (Cap) is entrenched in PPV VP2, be cloned into then in the gland virus expression system, transfection HEK-293A cell, RT-PCR, indirect immunofluorescence and Western-blotting experimental result show that Δ VP2 albumen efficiently expresses in HEK-293 A cell; The crude extract of expression product after transmission electron microscope observing can see that under Electronic Speculum a large amount of virus-like particles exist, is round with 3% phospho-wolframic acid negative staining, and diameter is about 30nm, and its form size is all similar to the totivirus particle.
5, the embedded virus like-particles of the recombinant adenovirus expression of the present invention's structure, the immune mouse experimental result shows: exempt from the 14th day experimental group in back at head and can detect the PCV2 specific antibody, the titre of antibody increases along with the prolongation of time, and Hangzhoupro body level reaches and is up to 1: 10000 when two exempt from back 35 days; Two exempted from back 35 days, and experimental group PPV NAT is 20, PCV2 NAT position 16; Two exempt from 28 to the 42 days experimental group in back and control group Con A/LPS stimulation index difference extremely remarkable.The embedded virus like-particles that reorganization is described has the immunogenicity of good PPVVP2 and PCV2 Cap, can induce the generation protective immunological reaction behind the immune animal.
6, the embedded virus like-particles [PPV:VLP (PCV2)] of the recombinant adenovirus expression of the present invention's structure is not only safe, inexpensive as a kind of protein carrier polyvalent recombinant vaccine, and can effectively prevent PCV2 to infect and the generation of the PMWS that causes by PCV2 and PPV polyinfection, be a kind of novel candidate vaccine safely and effectively.
Four, description of drawings
Fig. 1: PPV Δ VP2 goal gene amplified fragments
Fig. 2: recombinant plasmid rShuttle-Δ VP2 Kpn I/Not I double digestion enzyme is cut the evaluation collection of illustrative plates
Fig. 3: recombinant plasmid pAd-Δ VP2 PacI enzyme is cut the evaluation collection of illustrative plates
Fig. 4: recombinant adenovirus rAd-Δ VP2 indirect immunofluorescence figure: recombinant adenovirus (a) and cell contrast (b)
Fig. 5: express the proteic immunoblotting of Δ VP2 and identify
Fig. 6: PPVVP2 virus like particle Electronic Speculum figure
Fig. 7: PCV2 Δ Cap goal gene amplified fragments
Fig. 8: recombinant plasmid rShuttle-Δ Cap-Δ VP2 Kpn I/Not I double digestion enzyme is cut the evaluation collection of illustrative plates
Fig. 9: recombinant plasmid pAd-Δ Cap-Δ VP2 PacI enzyme is cut the evaluation collection of illustrative plates
The corresponding aperture of Figure 10: recombinant virus indirect immunofluorescence figure: anti-PPV of pig (a) and PCV2 (b) serum, cell control well (c)
Figure 11: the immunoblotting of expressing chimeric protein is identified: anti-PPV of pig (1) and PCV2 (2) serum, cell control well (3)
Figure 12: embedded virus like-particles Electronic Speculum figure
Figure 13: PCV2 specific antibody figure
Figure 14: lymph transformation experiment figure (ConA stimulation)
Figure 15: lymph transformation experiment figure (LPS stimulation)
Figure 16: embedded virus like-particles [PPV:VLP (PCV2)] makes up synoptic diagram
Five, embodiment
1, the structure and the application of the PPV VP2 virus like particle of expression alien gene
1) obtains Δ VP2 goal gene and be cloned into the pMD19-T carrier
With NADL-2 gene accession number among the GenBank is that the NC:001718 gene order is a template, designs primers with software Primer 5.0: add KpnI and Not I restriction enzyme site respectively.PPV low virulent strain (NADL-2) is available from China Veterinary Drugs Supervisory Inst..
P1 5′-gaggtaccgggggggttggtgtgt-3′
P2 5′-cgggcggccgcctagtataattttcttgg-3′
With P1, the P2 primer amplification contains the fragment (Fig. 1) of VP2 (1740bp) gene C end 1640bp.Amplification PCR products reclaims and it is connected Transformed E .coli DH with pMD19-T Vector behind electrophoresis on 1% sepharose
5α, alkaline lysis method of extracting plasmid, KpnI and Not I double digestion are identified, obtain positive plasmid (pT Δ VP2).
2) structure and the evaluation of recombinant plasmid pAd-Δ VP2
Utilize double enzyme site (KpnI/NotI) with the purpose fragment cloning to shuttle vectors pShuttle-CMV; Identify called after rShuttle-Δ VP2 with KpnI/NotI double digestion (Fig. 2) and sequencing analysis; Cut this recombinant plasmid with Pmne I enzyme, make its linearizing, then with adenovirus skeleton carrier (pAdEasy
TMVector) electricity is converted into intestinal bacteria BJ5183 competent cell under 1.8kV, 25 μ F and 200 Ω conditions, makes rShuttle-Δ VP2 and pAdEasy
TMHomologous recombination takes place in Vector, is converted into DH
5α host bacterium is picked out positive colony, cuts (Fig. 3) with PCR, PacI enzyme and identifies whether reorganization is correct, is built into recombinant plasmid, called after pAd-Δ VP2.
3) acquisition of recombinant virus
According to the lipofectamine working method, will use the linearizing recombinant plasmid pAd-of PacI Δ VP2 transfection HEK-293A cell, 5%CO in advance
2, 37 ℃ leave standstill more than the cultivation 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) is 10
11.2TCID
50/ mL.
4) biological property of virus like particle research
1. indirect immunofluorescence
To be inoculated in the HEK-293A cell that covers with individual layer, 5%CO after the recombinant adenovirus dilution
2, 37 ℃ of cultivations, about 24h, supernatant discarded, with the PBS washing once, 37 ℃ of dry 45min ,-20 ℃ of freezing 45min are again with the fixing 45min of 4 ℃ of cold dehydrated alcohols, PBST washing 3 times; The serum that adds 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: 100 FITC-SPA, 37 ℃ of effect 1h wash 3 times; Microscopic examination.Present stronger fluorescence in the recombinant adenovirus rAd-Δ VP2 corresponding aperture (a), and cell control well (b) there is not fluorescence (Fig. 4) to occur, illustrates that recombinant adenovirus rAd-Δ VP2 has expressed PPV in cell: Δ VP2 albumen.
2. protein imprinted
Concentrate recombinant adenovirus HEK-293A cell culture with 8%PEG-6000, set normal HEK-293A cell culture simultaneously and be contrast.Above-mentioned protein concentrate is carried out the SDS-PAGE electrophoresis, be transferred to the fine film of nitre after, the sealing of 10% skimming milk 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; Behind the thorough washing, add chemoluminescence method colour developing liquid (DAB), observe the differential protein band.Spissated recombinant adenovirus poisons electrophoresis transfer printing colour developing back shows at the 66KD place a tangible band is arranged all, does not have (Fig. 5) and contrast normal HEK-293A cell, illustrates that recombinant adenovirus rAd-Δ VP2 has expressed PPV in cell: Δ VP2 albumen.
3. the thick purifying and the electron microscopic observation of virus like particle
Collect sick cell, wash twice with PBS, 1000r/min 15min is centrifugal, is resuspended among the 50mmol/l NaHCO3, and behind the ultrasonic treatment, the centrifugal 15min of 24000r/min contains the chimeric protein of expression in the supernatant liquor.Adding ammonium sulfate spends the night to 20%, 4 ℃ of stirring of final concentration and precipitates in the supernatant of collecting, and then from the centrifugal 10min of 20000r/min, during precipitation was resuspended in, the dialysis desalination was the crude extract of embedded virus like-particles.After doing same processing, the wild poison of adenovirus does contrast.Add behind 37 ℃ of anti-PPV serum of the pig effect 1h of dilution in 1: 10 4 ℃ again and spend the night, with the centrifugal 90min of 12000r/min, precipitation is resuspended in the less water, and the phospho-wolframic acid negative staining with 3% is after transmission electron microscope observing.Can see that under Electronic Speculum a large amount of virus like particle exist, be round, diameter is about 30nm, and its form size is similar to the totivirus particle (Fig. 6) all, and the PPV that recombinant adenovirus rAd-Δ VP2 expresses in cell is described: Δ VP2 albumen can the oneself be assembled into virus like particle.
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 hemagglutinative titer is 1: 64, and the wild poison of empty adenovirus does not have hemagglutination activity, illustrates that the Δ VP2 albumen that rAd-Δ VP2 expresses keeps the original biologic activity of PPV.
2, with PPV VP2 virus like particle as a kind of antigen transport vehicle, the recombinant virus like-particles (Figure 16) of construction expression 2 type porcine circovirus nucleocapsid protein Cap genes
1) obtains Δ Cap-Δ VP2 goal gene and be cloned into the pMD19-T carrier
With PCV2-js2002 gene accession number among the GenBank is that the gene order of AY691679 and PPV VP2 recombinant plasmid is a template, with software Primer 5.0 design primer: P3, P4, P5, P6 and P7, add restriction enzyme site respectively, (public, PCV2 js2002 sees reference document: Pan Qunxing etc. by this laboratory isolation identification, detect the multiple PCR method of PCV2, PPV, PRV vaccine strain and street strain, China's virusology, 2005,20 (6) 603～60).
P3 5′-ctggtaccatggtccaccgcccaggag-3′ KpnI
P4 5′-cagctagccgttaccgctggagaagg-3′ NheI
P5 5′-gagctagcgggggggttggtgtgt-3′ NheI
P6 5′-cggctgcagctagtataattttcttgg-3′ PstI
P7 5′-cgggcggccgcctagtataattttcttgg-3′ NotI
With P3, the fragment Δ Cap of P4 primer amplification PCV2 Cap proteic 165-200 amino acids gene 108bp (Fig. 7), with P5, the fragment Δ VP2 of P6 primer amplification PPV VP2 PROTEIN C end 1640bp (Fig. 1), amplification PCR products is behind electrophoresis on 1% sepharose, reclaim and it is connected Transformed E .coli DH with pMD19-T Vector
5α, the alkaline lysis method of extracting plasmid is identified with KpnI/NheI and NheI/PstI double digestion respectively, obtains positive plasmid (pT Δ Cap, pT Δ VP2).
Utilize restriction endonuclease NheI on the primer and the PstI on the pMD19-T Vector with positive plasmid pT Δ Cap double digestion, reclaim the fragment of 2.8Kb, utilize restriction endonuclease NheI on the primer and PstI with positive plasmid pT Δ VP2 double digestion simultaneously, reclaim the small segment of 1.64Kb, two fragments that will reclaim then connect Transformed E .coli DH with T4 DNA Ligase
5α, the alkaline lysis method of extracting plasmid, the KpnI/PstI double digestion is identified, positive plasmid called after pT Δ Cap-Δ VP2.
2) structure and the evaluation of recombinant plasmid pAd-Δ Cap-Δ VP2
With P3, P7 is a primer, and positive plasmid pT Δ Cap-Δ VP2 is a template, pcr amplification Δ Cap-Δ VP2 fragment, and introduce restriction endonuclease KpnI/NotI.According to pAdEasy
TMVector carrier system operation instructions utilizes double enzyme site (KpnI/NotI) that external source fragment (Δ Cap-Δ VP2) is cloned among the transfer vector pShuttle-CMV; Identify (Fig. 8) with PCR, KpnI/NotI double digestion and sequencing analysis, called after rShuttle-Δ Cap-Δ VP2.
Cut this recombinant plasmid with the PmeI enzyme, make its linearizing, then with pAdEasy
TMVector electricity under 1.8kV, 25 μ F and 200 Ω conditions is converted into intestinal bacteria BJ5183 competent cell, makes rShuttle-Δ Cap-Δ VP2 and skeleton carrier (pAdEasy
TMVector) homologous recombination taking place, is converted into DH
5Whether correctly α host bacterium is picked out positive colony, cut with PCR, PacI enzyme and identify reorganization (Fig. 9), is built into recombinant plasmid, called after pAd-Δ Cap-Δ VP2.
3) acquisition of recombinant virus and purifying
According to the lipofectamine working method, will use the linearizing recombinant plasmid pAd-of PacI Δ Cap-Δ VP2 transfection HEK-293A cell, 5%CO in advance
2, 37 ℃ leave standstill more than the cultivation 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) is 10
11.8TCID
50/ mL.
4) biological property of embedded virus like-particles research
1. indirect immunofluorescence
To be inoculated in the HEK-293A cell that covers with individual layer, 5%CO2,37 ℃ of cultivations, about 24h after the recombinant adenovirus dilution, supernatant discarded, with the PBS washing once, 37 ℃ of dry 45min,-20 ℃ of freezing 45min are again with the fixing 45min of 4 ℃ of cold dehydrated alcohols, PBST washing 3 times; Add 50 μ L1 respectively: the anti-PCV2 serum of pig (a) of 40 dilutions and the serum (b) of the anti-PPV of pig, 37 ℃ of effect 1h are with PBST washing 3 times; Add 1: 100 FITC-SPA, 37 ℃ of effect 1h wash 3 times; Microscopic examination.Recombinant adenovirus rAd-Δ Cap-Δ VP2 presents stronger fluorescence, and cell control well (c) does not have fluorescence (Figure 10) to occur.Illustrate that recombinant adenovirus rAd-Δ Cap-Δ VP2 has expressed PPV in cell: Δ VP2 and PCV2: Δ Cap albumen.
2. protein imprinted
Concentrate recombinant adenovirus HEK-293A cell culture with 8%PEG-6000, set normal HEK-293A cell culture simultaneously and be contrast.Above-mentioned protein concentrate is carried out the SDS-PAGE electrophoresis, be transferred to the fine film of nitre after, the sealing of 10% skimming milk is spent the night; The serum room temperature effect 2h that adds 1: 40 anti-PCV2 serum of pig and the 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 colour developing liquid (DAB), observe the differential protein band.Spissated recombinant adenovirus poisons electrophoresis transfer printing colour developing back shows, one is anti-during for the anti-PCV2 serum of the anti-PPV serum of pig or pig, at the 66KD place a tangible band is arranged all, do not have (Figure 11), illustrate that recombinant adenovirus expressed Δ Cap-Δ VP2 albumen in cell and contrast normal HEK-293A cell.
3. the thick purifying and the electron microscopic observation of embedded virus like-particles
Collect sick cell, wash twice with PBS, the centrifugal 15min of 1000r/min is resuspended among the 50mmol/l NaHCO3, and behind the ultrasonic treatment, the centrifugal 15min of 24000r/min contains the chimeric protein of expression in the supernatant liquor.Adding ammonium sulfate spends the night to 20%, 4 ℃ of stirring of final concentration and precipitates in the supernatant of collecting, and then from the centrifugal 10min of 20000r/min, during precipitation was resuspended in, the dialysis desalination was the crude extract of embedded virus like-particles.After doing same processing, the wild poison of adenovirus does contrast.Add behind 37 ℃ of anti-PPV serum of the pig effect 1h of dilution in 1: 10 4 ℃ again and spend the night, with the centrifugal 90min of 12000r/min, precipitation is resuspended in the less water, and the phospho-wolframic acid negative staining with 3% is after transmission electron microscope observing.Can see that under Electronic Speculum a large amount of virus like particle exist, be round, diameter is about 30nm, and its form size is similar to the totivirus particle (Figure 12) all, illustrates that the Δ Cap-Δ VP2 albumen that recombinant adenovirus rAd-Δ Cap-Δ VP2 expresses in cell can the oneself be assembled into virus like particle.
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 Cap-Δ VP2 hemagglutinative titer is 1: 64, and the wild poison of empty adenovirus does not have hemagglutination activity, illustrates that rAd-Δ Cap-Δ VP2 expresses Δ Cap-Δ VP2 albumen and keeps the original biologic activity of PPV.
5) mouse immune experiment
1. immune programme for children
60 of the male mices in age in 6-8 week are divided into 2 groups, 30 every group at random.First group of subcutaneous multi-point injection 10 in back
10TCID
50Recombinant adenovirus rAd-Δ Cap-Δ VP2; Second group of every mouse subcutaneous injection 10
10TCID
50Recombinant adenovirus rAd-Cap; The 3rd group of every mouse subcutaneous injection 10
10TCID
50The adenovirus of sky that does not have foreign gene is as negative control; The 4th group every an amount of PBS of mouse subcutaneous injection compares; Carry out identical immunity after two weeks.Exempt from head respectively, head exempts from back 14,28,35,42,49 and 56 days, gathers determination of serum PCV2 antibody and PPV/PCV neutralizing antibody.Head exempted from the back 28,42 and 56 days, slaughtered 3 mouse for every group, got its spleen, and isolated lymphocytes is measured the lymphocytic hyperplasia ability.
2. ELISA detection of antibodies
PCV2 ORF2 prokaryotic expression product (this prepared in laboratory) is through His-band purification kit purifying, and measuring proteic mean concns is 1.10mg/mL.Can draw by ELISA square formation test, when antigen coated concentration is 1.8 μ g/mL, 100 times of antibody dilutions, one anti-37 ℃ hatches 1.5h respectively, ELIAS secondary antibody is hatched 45min for 37 ℃, adds TMB-H
2O
2The colour developing of (tetramethyl benzidine-hydrogen peroxide) solution, 37 ℃ of reaction 10-15min.Add 2M H
2SO4 solution, termination reaction, 50 μ L/ holes.Reaction conditions is proper, and this moment, the P/N value was bigger, and the OD value of negative serum is less, and is promptly non-specific smaller.The result judges: P
x-N
xExperiment in＞0.15 o'clock effectively.(sample OD value-N
x)/(P
x-N
x) 〉=0.45 positive; (sample OD value-N
x)/(P
x-N
x)＜0.45 feminine gender.P wherein
xExpression: positive control mean value; N
xExpression: negative control mean value.
Indirect ELISA antibody test result: rAd-Δ Cap-Δ VP2 immune group mouse is exempted from the back at head can detect the PCV2 specific antibody on the 14th day, the titre of antibody increases along with the prolongation of time, ELISA Hangzhoupro body level reaches and is up to 1: 10000 when two exempt from back 35 days, apparently higher than the rAd-Cap immune group; And empty adenovirus and PBS immune group mouse do not produce PCV2 specific antibody (Figure 13), illustrate that the recombinant adenovirus of expressing the embedded virus like-particles has good immunogenicity, but inducing producing specificity immunoprotection reaction behind the immune animal.
3. the detection of neutralizing antibody
Detect the neutralizing antibody of PCV2 with IFA.Concrete grammar is as follows: 56 ℃ of deactivation 30min of mice serum, and the centrifugal 10min of 12000r/min gets supernatant, and by 1: 4,1: 8,1: 16,1: 32 and 1: 64 doubling dilution: viral PCV2 was diluted as 2000 TCID5 with 1640 substratum that contain 2%FCS
0/ mL; The diluent of above-mentioned serum and virus is respectively got 50 μ L, mixes 37 ℃ of water-bath 1h; Take out the back and add the PK-15 cell that has covered with cell monolayer, every hole 100 μ L, each serum dilution is inoculated four holes, sets positive control hole and the normal cell negative control hole of a virus inoculation PCV2 simultaneously, and 37 ℃ leave standstill cultivation 72h.Carry out IFA experiment according to a conventional method, with reduce 70% or the inverse of the high dilution of the serum of more fluorescence be NAT.In IFA, tangible fluorescence all appears in all positive control cells, and negative control does not have fluorescence, and the hole of adding immune serum does not have or only have faint fluorescence.Same group in different time NAT difference, and head exempts from back 14 days all mouse NATs all＜4, is 16 to the two neutralizing antibody average titers of exempting from back 35 days rAd-Δ Cap-Δ VP2 immune group, is higher than rAd-Cap immune group (12); Empty adenovirus and PBS immune group NAT are almost 0.
Use TCID
50Reduce the neutralizing antibody of test determination PPV.Concrete grammar is as follows: 56 ℃ of deactivation 30min of mice serum, and the centrifugal 10min of 12000r/min gets supernatant, uses 1640 substratum that contain 2%FCS by 1: 4, and 1: 8,1: 16,1: 32 and 1: 64 doubling dilution; Virus PPV is diluted as 2000 TCID
50/ mL; The diluent equal-volume of dilution serum of above-mentioned difference and virus mixes, 37 ℃ of water-bath 1h; Take out the PK-15 cell that inoculation 40～50% is merged, every hole 100 μ L, each serum dilution is inoculated 4 holes, sets the PK-15 positive control hole and the normal cell negative control hole of a virus inoculation simultaneously.37 ℃ leave standstill cultivation 4-5 days; Microscopic examination.The inverse that can 100% suppresses the high dilution of serum of pathology is a NAT.Tangible cytopathy all appears in all positive control cells, it is 20 that cytopathy rAd-Δ Cap-Δ VP2 immune group PPV NAT does not all appear in negative control cell, the rAd-Cap immune group, empty adenovirus group and PBS control group NAT are almost 0.
4. lymphocyte transformation test
Slaughter mouse, aseptic its spleen of getting is peeled off reticular tissue, places aseptic plate: add a spot of aseptic PBS, sting spleen with syringe needle, with the extruding of looper head, extrude cell wherein then; Grind evenly with the looper head, discard unnecessary reticular tissue and big tissue block; The lymphocyte separation medium that adds 6mL in the centrifuge tube of 10mL adds lightly and grinds the ground tissue homogenate; The centrifugal 15min of 2000r/min: nebulous white cellular layer in the middle of getting, place new centrifuge tube, add an amount of Hank ' liquid; The centrifugal 10min of 2000r/min; Abandon supernatant, add an amount of 1640 nutritive medium re-suspended cells that contain 10%FCS, carry out cell counting then; According to count results cell concn is adjusted into 5 * 10
6/ mL adds in the 96 porocyte plates, every hole 100 μ L cell suspensions, and every mouse is spread 12 holes; Wherein adding final concentration in 4 holes is the ConA of 20 μ g/ μ L, and adding final concentration in 4 holes is the LPS of 15 μ g/ μ L in addition, and all the other 4 holes are as negative control; 37 ℃, 5%C0
2Leave standstill and cultivate 44h; Every hole adds 20 μ L MTT (concentration 5mg/mL), cultivates 4h; Supernatant discarded, every hole add 100 μ L DMSO, make the crystallization dissolving, vibration; Measure OD
570Nm, calculating ConA stimulates the average OD in ground, hole
570Nm/ does not stimulate the average OD in hole
570Nm (SI value).Whether the SI value difference with t method of inspection check immune group and control group is different remarkable.Two exempted from back 14 days, (SI) is approaching for four groups stimulation index, from the SI value rising rapidly of back 28 days to 42 days rAd-Δ Cap-Δ VP2 immune group of immunity for the second time, and the SI value of Wild Ad and control group changes not quite, significantly ((P＜0.01) illustrates that recombinant adenovirus rAd-Δ Cap-Δ VP2 can strengthen mouse cell immunity (Figure 14) to the SI value difference heteropole of 28 to 42 days immune group and control group after t check analysis two is exempted from.
The embedded virus like-particles [PPV:VLP (PCV2)] that the recombinant adenovirus that the present invention makes up is expressed has good immunogenicity, can induce the generation protective immunological reaction behind the immune animal.Embedded virus like-particles [PPV:VLP (PCV2)] is not only safe, inexpensive as a kind of protein carrier polyvalent recombinant vaccine, and can effectively prevent PCV2 to infect and the generation of the PMWS that causes by PCV2 and PPV polyinfection, be a kind of novel candidate vaccine safely and effectively.
Sequence table
＜110〉Jiangsu Province Agriculture Science Institute
＜120〉the recombinant virus like-particles of expression 2 type porcine circovirus nucleocapsid protein Cap genes
＜130〉specification sheets
<140>00
<141>2008-03-25
<160>7
<170>PatentIn?version?3.1
<210>1
<211>24
<212>DNA
＜213〉synthetic
<220>
＜221〉primer P1
<222>(1)..(24)
<223>
<400>1
gaggtaccgg?gggggttggt?gtgt 24
<210>2
<211>29
<212>DNA
＜213〉synthetic
<220>
＜221〉primer P2
<222>(1)..(29)
<223><400>2
cgggcggccg?cctagtataa?ttttcttgg 29
<210>3
<211>27
<212>DNA
＜213〉synthetic
<220>
＜221〉primer P3
<222>(1)..(27)
<223>
<400>3
ctggtaccat?ggtccaccgc?ccaggag 27
<210>4
<211>26
<212>DNA
＜213〉synthetic
<220>
＜221〉primer P4
<222>(1)..(26)
<223><400>4
cagctagccg?ttaccgctgg?agaagg 26
<210>5
<211>24
<212>DNA
＜213〉synthetic
<220>
＜221〉primer P5
<222>(1)..(24)
<223>
<400>5
gagctagcgg?gggggttggt?gtgt 24
<210>6
<211>27
<212>DNA
＜213〉synthetic
<220>
＜221〉primer P6
<222>(1)..(27)
<223>
<400>6
cggctgcagc?tagtataatt?ttcttgg 27
<210>7
<211>29
<212>DNA
＜213〉synthetic
<220>
＜221〉primer P7
<222>(1)..(29)
<223>
<400>7
cgggcggccg?cctagtataa?ttttcttgg 29
Claims (2)
1, expressing the recombinant virus like-particles of 2 type porcine circovirus nucleocapsid protein Cap genes, it is characterized in that, is to be made up by following method to form:
1) obtains Δ Cap-Δ VP2 goal gene and be cloned into the pMD19-T carrier
With PCV2-js2002 gene accession number among the GenBank is that the gene order of AY691679 and pig parvoviral PPV VP2 recombinant plasmid is a template, and with software Primer 5.0 design primer: P3, P4, P5, P6 and P7 add restriction enzyme site respectively:
P35′-ctggtaccatggtccaccgcccaggag-3′KpnI
P45′-cagctagccgttaccgctggagaagg-3′NheI
P55′-gagctagcgggggggttggtgtgt-3′NheI
P65′-cggctgcagctagtataattttcttgg-3′PstI
P75′-cgggcggccgcctagtataattttcttgg-3′NotI
With P3, the fragment Δ Cap of the proteic 165-200 amino acids of P4 primer amplification PCV2Cap gene 108bp, with P5, the fragment Δ VP2 of P6 primer amplification PPV VP2 PROTEIN C end 1640bp, amplification PCR products is behind electrophoresis on 1% sepharose, reclaim and it is connected Transformed E .coli DH with pMD19-T Vector
5α, the alkaline lysis method of extracting plasmid is identified with KpnI/NheI and NheI/PstI double digestion respectively, obtains positive plasmid pT Δ Cap and pT Δ VP2.
Utilize restriction endonuclease NheI on the primer and the PstI on the pMD19-TVector with positive plasmid pT Δ Cap double digestion, reclaim the fragment of 2.8Kb, utilize restriction endonuclease NheI on the primer and PstI with positive plasmid pT Δ VP2 double digestion simultaneously, reclaim the small segment of 1.64Kb, two fragments that will reclaim then connect with T4DNA Ligase, Transformed E .coli DH
5α, the alkaline lysis method of extracting plasmid, the KpnI/PstI double digestion is identified, positive plasmid called after pT Δ Cap-Δ VP2.
2) structure and the evaluation of recombinant plasmid pAd-Δ Cap-Δ VP2
With P3, P7 is a primer, and positive plasmid pT Δ Cap-Δ VP2 is a template, pcr amplification Δ Cap-Δ VP2 fragment, and introduce restriction endonuclease Kpn I/NotI, according to pAdEasy
TMThe carrier system operation instructions utilizes double enzyme site KpnI/NotI that external source fragment Δ Cap-Δ VP2 is cloned among the transfer vector pShuttle-CMV; Identify called after rShuttle-Δ Cap-Δ VP2 with PCR, KpnI/NotI double digestion and sequencing analysis;
3) acquisition of the recombinant virus like-particles of expression 2 type porcine circovirus nucleocapsid protein Cap genes
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 intestinal bacteria BJ5183 competent cell, makes rShuttle-Δ Cap-Δ VP2 and pAdEasyTM Vector that homologous recombination take place, and is converted into DH
5α host bacterium is picked out positive colony, cuts with PCR, PacI enzyme and identifies whether reorganization is correct, is built into recombinant plasmid, called after pAd-Δ Cap-Δ VP2.
With recombinant plasmid pAd-Δ Cap-Δ VP2 transfection transfection HEK-293A cell, obtain recombinant virus rAd-Δ Cap-Δ VP2; Recombinant adenovirus rAd-Δ Cap-Δ VP2 expresses Δ Cap-Δ VP2 chimeric protein oneself in the 293A cell that this recombinant adenovirus infects and is assembled into virus like particle PPV:VLP (PCV2), is the recombinant virus like-particles of the expression 2 type porcine circovirus nucleocapsid protein Cap genes of acquisition.
2, the application of the recombinant virus like-particles of the described expression 2 type porcine circovirus nucleocapsid protein Cap genes of claim 1 aspect the preparation vaccine.
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