CN102008735B - Small molecular DNA vaccine and preparation method thereof - Google Patents
Small molecular DNA vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a small molecular DNA vaccine and a preparation method thereof. In the small molecular DNA vaccine, a linear DNA segment is formed by transcription promoters and target genes, the target gene comprises initiation and termination codons. Compared with the existing DNA vaccine, the small molecular DNA vaccine can be rapidly induced to generate immune body, and the cellular immunity level of the generated immune body is high. The small molecular DNA vaccine only carries eucaryon gene transcription promoters and target genes or target gene segments, and besides the target genes, the small molecular DNA vaccine does not carry unrelated genes or nucleotide sequences, such as antibiotics resistance genes, PolyA tails, replication initial points and the like, and the small molecular DNA vaccine has good biological safety. As the whole length of the linear DNA vaccine is short, the amplification is easier; and meanwhile, the amplification accuracy is high and the vaccine titer ratio is high.
Description
Technical field
The present invention relates to a kind of dna vaccination and preparation method thereof, particularly a kind of small molecular DNA vaccine and preparation method thereof.
Background technology
Nineteen ninety, Wolff etc. (Wolff et al, 1990) chance on dna vaccination when white mice being carried out the gene therapy test.1992, the U.S. had simultaneously four development in laboratory to work out dna vaccination and reports simultaneously in the vaccinology new development conference of JIUYUE in the same year.Dna vaccination causes people's great attention since then, is considered to for the third time vaccine revolution after attenuation, inactivated vaccine and subunit vaccine.
Dna vaccination is with the exogenous gene (DNA) of certain antigen protein of coding and after containing the eukaryotic expression plasmid restructuring that is necessary expression regulation element, recombinant plasmid dna is directly injected animal body, and by the transcribing of host cell, translation system synthetic antigen albumen, induce the host to produce immunne response to this antigen protein, to reach the purpose of prevention and treatment disease.
Dna vaccination demonstrates great potential in the control of infectious disease, parasite and tumor, it is that carrier again can be at eukaryotic expression antigen, the expression of antigen is similar to natural infection in vivo, the antigen protein of expression in vivo has native conformation, the antibody response feature of inducing is similar to wild virus, induces simultaneously body to produce cellular immunization and humoral immune reaction.At present carried out the research of the dna vaccination of multiple pathogens, such as influenza, hepatitis B, acquired immune deficiency syndrome (AIDS) etc., these result of the tests show that dna vaccination not only has prophylactic effect, also have simultaneously the effect for the treatment of disease.Therefore will bring hope for preventing or prevent for a long time undesirable infectious disease.The difference of it and traditional vaccine, the antigen protein that mainly is nucleic acid vaccine produces in immune subject, expression product has natural conformation, can be processed according to normal approach, modify, offer again to immune system, finally cause comprehensive immunoreation, whole process is identical with natural infection or the inoculation attenuated live vaccine of virus, so the advantage of nucleic acid vaccine maximum had both had the safety of subunit vaccine and inactivated vaccine, there are again attenuated live vaccine and recombiant vaccine to induce the advantage of comprehensive immunne response ability.
Existing dna vaccination is that genes of interest is cloned on the plasmid vector, places under the control of eukaryotic gene promoter, and with this plasmid DNA injection animal or human, immune response stimulating reaches the purpose of preventing and treating disease.For filtering out the DNA plasmid of efficient loading, carry other exogenous genes on the existing dna vaccine vector, enhancer, immunomodulatory gene, marker gene such as green fluorescent protein (GFP) gene that improves immune effect and the sweet enzyme gene of β galactose etc. of transcribing or expressing such as the various antibiotic resistance genes that are used for colony screening, the replication initiation sequence, the regulator gene that are used for plasmid replication.Because entrained gene or DNA sequence are too much, many disadvantages will occur: the expression of other antigens of the first may work the mischief or uncertainty to body; It two is because the expression of other genes, has formed resource contention with the expression of genes of interest, causes immune effect to reduce; It three also is that the existence that the most important thing is too much other genes or non-the most necessary DNA sequence has consisted of very large bio-safety problem, increase foreign DNA and be incorporated into probability in the host genome, increased the chance of the problems such as induced tumor, the uncertainty of the use existence of dna vaccination is greatly increased; In addition in the situation of the existence that other genes or non-the most necessary DNA sequence are arranged too much, the corresponding reduction of copy number of DNA in the unit mass, this will reduce the Immune efficiency of dna vaccination, also will improve the production cost of vaccine, thereby other exogenous genes that carry on the while carrier may affect the processing of purpose antigen protein, modify the formation native conformation.
Traditional dna vaccination only uses the plasmid DNA of superhelix or ring-type as carrier in theory, the linear DNA that does not use linear DNA or amplify with round pcr, therefore existing dna vaccination can be called plasmid DNA vaccine (Plasmid DNA Vaccine, pDNA Vaccine).
Linear DNA is easy to degraded in vivo, and it is generally acknowledged in vivo can not effectively expressing, not can be used as vaccine or other gene formulations and uses.(number of patent application: 200610033861.5) in the patent application before us, break through conventional, the eukaryotic gene transcripting promoter is linked to each other with genes of interest with transcription terminator, obtain a kind of short chain linear DNA, and find that this kind DNA in animal body can effectively expressing, can be used as dna vaccination or other gene formulations and use.Simultaneously, compare with the plasmid DNA vaccine, the linear DNA vaccine of this application has better safety, the more advantages of higher of tiring.
In this application in the disclosed dna molecular, must contain complete promoter, genes of interest and terminator, although the conventional plasmid DNA vaccine of its length has substantial degradation, its overall length still is more considerable, and existing round pcr is difficult to guarantee the accuracy of its amplification.
Summary of the invention
A kind of small molecular DNA vaccine consists of the linear DNA segment by transcripting promoter and genes of interest, and genes of interest comprises its initial sum termination codon.
Preferably, transcripting promoter is the eukaryotic transcription promoter.
The preparation method of above-mentioned small molecular DNA vaccine may further comprise the steps:
1) transcripting promoter is cloned in the carrier that contains cloning site, obtains universal support;
2) genes of interest is cloned in the universal support, obtains containing the recombinant vector of transcripting promoter and genes of interest;
3) according to the upstream sequence design universal primer of universal support transcription promoter, according to the downstream sequence of genes of interest, design downstream primer, take recombinant vector as template, the linear DNA segment that amplification is made of transcripting promoter and genes of interest.
Preferably, the carrier that contains cloning site is the polyclone carrier.
Preferably, the polyclone carrier is the T carrier that contains multiple clone site.
Small molecular DNA vaccine of the present invention is used the human or animal by modes such as injection, spray nose, suctions, can the human or animal in the immunity of inductor fluidity and cellular immunity, effective protection is provided.Compare with existing dna vaccination, small molecular DNA vaccine of the present invention can be induced generation antibody quickly, and the cellular immune level of generation is higher.
Small molecular DNA vaccine of the present invention, only carry eukaryotic gene transcripting promoter and genes of interest or genes of interest fragment, do not carry beyond any genes of interest independent basis because of or nucleotide sequence, such as antibiotics resistance gene, PolyA tail, replication origin etc., has better biological safety.Because the entire length of linear DNA vaccine is shorter, be easier to amplification, simultaneously, the accuracy of amplification is higher, and the potency ratio of vaccine is also higher.
Small molecular DNA vaccine of the present invention, preparation method is simple, and preparation time is short, from genes of interest being cloned into universal support to preparing small molecular DNA vaccine, whole process can be finished in 24 hours, and with respect to the vaccine technology of preparing of routine, its production cycle shortens greatly.Simultaneously, small molecular DNA vaccine of the present invention can adopt ripe round pcr preparation, but large-scale industrial production is specially adapted to the accidents such as outburst of infectious disease.
Description of drawings
Fig. 1 is the structure sketch map of small molecular DNA vaccine universal support of the present invention.
Fig. 2 is the preparation process sketch map of small molecular DNA vaccine of the present invention.
The specific embodiment
Below in conjunction with embodiment, further specify the present invention.
Small molecular DNA vaccine preparation process of the present invention as shown in Figure 1 and Figure 2, comparatively concrete preparation technology is as follows:
For people's cytomegalovirus CMV promoter design primer, the upstream and downstream primer of design is as follows:
CMV forward primer P1:5 '-gcggccgccttatatattctttccca-3 ' (SEQ ID NO.1)
CMV downstream primer P2:5 '-ctgacggttcactaaaccagctctgct-3 ' (SEQ ID NO.2)
The plasmid of pAdTrack-CMV is as the template of clone CMV fragment, CMV promoter gene with primer P1 and P2 amplification CMV, reaction condition is: 94 ℃ of denaturation 5min, then with 94 ℃ of degeneration 1min, 48 ℃ of annealing 1min, 72 ℃ are extended 1.5min, carry out 30 circulations, and last 72 ℃ are extended 10min;
Reclaim the PCR product that purification obtains, the clone enters between the EcoR ⅴ cloning site of pMD18-T carrier, positive single bacterium colony of picking Amp resistance is cultivated, after extracting plasmid wherein, double digestion is identified recombiant plasmid, then order-checking confirmation CMV promoter has been cloned and has been entered in the carrier recombiant plasmid called after pCMV-T that contains the CMV promoter of acquisition;
The purpose group is cloned among the pCMV-T, and the recombiant plasmid of acquisition is called pCMV-T-Target;
According to the upstream sequence of CMV promoter among the pCMV-T-Target, design forward primer P1, according to the downstream sequence of genes of interest, design downstream primer P4, its middle and upper reaches universal primer is:
Forward primer P1:5 '-gcggccgccttatatattctttccca-3 ' (SEQ ID NO.1);
Take pCMV-T-Target as template, P1, P4 are that primer amplification obtains linear DNA segment pMD-CMV-Target, obtain small molecular DNA vaccine of the present invention behind the purification.
The structure of the ORF2 gene small molecular DNA vaccine of embodiment 1 pig annulus 2 types viruses (PCV2)
Use forward primer A1:5 '-ttaatgacgtatccaaggag-3 ' (SEQ ID NO.3) and
Downstream primer A2:5 '-tacaggggttaagtgggg-3 ' (SEQ ID NO.4) amplification PCV2 GD strain (AY613854) ORF2 gene, amplification condition is: 94 ℃ of denaturation 5min, then with 94 ℃ of degeneration 1min, 48 ℃ of annealing 1min, 72 ℃ are extended 1.5min, carry out 30 circulations, last 72 ℃ are extended 10min;
The PCV2 GD strain ORF2 gene clone that the amplification of recovery purification obtains enters among the pCMV-T, obtain recombiant plasmid called after pCMV-T-ORF2, use pCMV-T-ORF2 transformed competence colibacillus cell bacillus coli DH 5 alpha, recombiant plasmid is identified, confirmed to be cloned into PCV2 GD strain ORF2 gene in the plasmid;
Take pCMV-T-ORF2 as template, amplify the linear DNA segment that only contains CMV promoter and ORF2 gene, the purify DNA segment obtains the pMD-CMV-ORF2 small molecular DNA vaccine.
The structure of the ORF1 gene small molecular DNA vaccine of embodiment 2 pig annulus 2 types viruses (PCV2)
Structure similar to Example 1 obtains the small molecular DNA vaccine of the ORF1 gene of pig annulus 2 types viruses (PCV2), and wherein, the primer that uses during amplification ORF1 gene is:
Forward primer D1:5 '-taaggtaccaatgcccagcaagaagaatg-3 ' (SEQ ID NO.5)
Downstream primer D2:5 '-taatctagaatttcatatggaaattcagg-3 ' (SEQ ID NO.6)
The condition of amplification is 94 ℃ of denaturation 5min, then with 94 ℃ of degeneration 1min, and 53 ℃ of annealing 1min, 72 ℃ are extended 1.5min, carry out 30 circulations, and last 72 ℃ are extended 10min.
The immunity test of small molecular DNA vaccine
Mouse immuning test
Choose 6～8 all 54 healthy of female BALB/C mice and be divided into 9 groups, 6 every group, be respectively:
1. normal saline group, 2. chitosan matched group, 3. liposome matched group, 4. the coated pMD-CMV-ORF2 of chitosan, 5. the coated pMD-CMV-ORF2 of liposome, the 6. coated pMD-CMV-ORF1 of chitosan, the 7. coated pMD-CMV-ORF1 of liposome, 8. the coated pMD-CMV-ORF1+2 of chitosan, the 9. coated pMD-CMV-ORF1+2 of liposome.Before every mouse immune with injecting 0.2% lignocaine in 24 hours in the back leg quadriceps femoris, every 100 μ L, the injection site of test reagent is identical.
The preparation of the small molecular DNA vaccine that chitosan is coated: the 40mg chitosan, add 280 μ L acetic acid, then add tri-distilled water to 2mL, 37 ℃ of 180～200r/min sway to spend the night to chitosan and dissolve fully, and solution is transparence.Add water to 190mL, regulating PH with NaOH is 5.5, last standardize solution 200mL, and 0.22 μ m membrane filtration degerming gets solution a.The NaSO that small molecular DNA vaccine is added 50mmol/L
4Obtain solution b to 100 μ g/mL in the solution.Respectively get isopyknic solution a, b, be preheated to respectively 50～55 ℃, vibration mixes 30s rapidly, forms milky suspension, i.e. the coated small molecular DNA vaccine of chitosan.After the small molecular DNA vaccine suspension room temperature for preparing placed 1 hour, the centrifugal 30min of 4000r/min removed the part supernatant, adds sterilized water again and is settled to pcrDNA content 1 μ g/ μ L.Each every injected in mice 100 μ L.
The preparation of the small molecular DNA vaccine that liposome is coated: the small molecular DNA vaccine 0.3mL and the liposome Lipofectamine that get 2 μ g/ μ L
TMRoom temperature was placed 15min after 2000 0.3mL mixed, and namely formed the coated small molecular DNA vaccine of liposome.PcrDNA content is 1 μ g/ μ L, each every injected in mice 100 μ L.
Chitosan matched group chitosan (the not containing small molecular DNA vaccine) immune mouse of the small molecular DNA vaccine immune group equivalent that is coated with chitosan.
Liposome matched group liposome (the not containing small molecular DNA vaccine) immune mouse of the small molecular DNA vaccine immune group equivalent that is coated with liposome.
The every fortnight immunity of all mices once is total to immunity three times.
Experimental result:
2,4,6,8,10 weeks taken a blood sample separation of serum by mouse tail before the immunity and behind the initial immunity.Show with ELISA detection kit testing result, pMD-CMV-ORF2 small molecular DNA vaccine, pMD-CMV-ORF1 small molecular DNA vaccine and pMD-CMV-ORF1+2 small molecular DNA vaccine all can bring out the mice specific antibody and produce, and namely induce humoral immunization to occur.Above-mentioned vaccine test group is compared with normal saline group, chitosan matched group, liposome matched group has significant difference.4～9 groups is respectively 149.6%, 169.7%, 142.4%, 164.1%, 149.6%, 189.9% of normal saline group antibody OD value; Chitosan matched group, liposome matched group and normal saline group do not have significant difference.
The immunoreation of small molecular DNA vaccine immune mouse trigger cell
1, lymphocyte transformation test
1) get mouse spleen and separate lymphocyte, adjusting cell concentration is 1 * 10
7/ mL; Test group, test control group and blank group are established in test.
2) specifically add quadrat method: in 96 porocyte culture plates, every part of lymphocyte specimen is divided two groups (stimulating group and matched groups), every group of 5 repeating holes, and stimulating group adds respectively the lymphocyte, 50 of 100 μ L separator wells
μThe concanavalin A, Con A (ConA) of L 40 μ g/mL and the RPMI-1640 of 10% calf serum; Matched group adds respectively the lymphocyte of 100 μ L separator wells, the RPMI-1640 of 100 μ L10% calf serums; The every hole of blank group adds the RPMI-1640 of 200 μ L10% calf serums;
3) 37 ℃ of 5% CO
2, incubation 24 hours adds 10 μ L MTT (5 μ g/mL), continue to cultivate 4 hours, and then every hole adds 100 μ L, 10% SDS-0.04mol/L stop buffer cessation reaction, after 37 ℃ of black purple crystals after 4 hours dissolve fully, measures OD with enzyme-linked immunosorbent assay instrument
630Value.
Calculate stimulation index (SI), SI=(stimulating group-blank group)/(test is according to group-blank group).
The result shows that post-stimulatory breeder reaction all is significantly higher than normal saline group, chitosan matched group, liposome matched group to ConA for pMD-CMV-ORF2, pMD-CMV-ORF1 and pMD-CMV-ORF1+2 small molecular DNA vaccine immunized mice spleen lymphocyte.4～9 groups of immunostimulation indexes (SI) are 149.3,133.7%, 125.7%, 129.2%, 127.1%, 150.6% of normal saline groups; Chitosan matched group, liposome matched group and normal saline group do not have significant difference.
Natural killer cell activity (NK) detects
Adopt the 2h short distance to discharge improved method.
1) separating mouse spleen mononuclear cell, 1640 culture medium of 10% calf serum are adjusted to 1 * 10 with cell concentration
6/ mL, as the effector lymphocyte who detects the NK cytoactive, as target cell, culture tube is managed with aseptic 1.5mL Eppondof with the SP2/0 oncocyte.Totally 3 groups of NK NKT groups (Mixture), target cell Spontaneous release group (Spon), maximum release group (Max) are established in test, and respectively establish 3 multiple pipes;
2) specifically add quadrat method: 100 μ L effector lymphocytes and 100 μ L target cells, effect target cell concentration ratio is 50:1, as NK NKT group (Mixture); The RPMI-1640 of 100 μ L target cells and 2% calf serum is target cell Spontaneous release group (Spon); 100 μ L target cells and 100 μ L, 10% TritonX-100 are maximum release group, and application of sample is complete, mixing, and the centrifugal 2min of 500r/min is hatched 2h for 37 ℃, then respectively manages cold saline 50 μ L on the rocks and stops cytotoxicity between the effect target cell;
3) with developmental tube with the centrifugal 5min of 3 000r/min, getting supernatant is added in the 40 hole polystyrene micro-reaction plates and carries out enzymatic reaction, 100 μ L/ holes, put 37 ℃ of pre-temperature 20min, each hole adds LDH substrate solution 100 μ L, is put in 37 ℃ of calorstats and reacts 10min, takes out, each hole adds 0.1mol/L citric acid 30 μ L to stop enzymatic reaction, reads OD at enzyme-linked immunosorbent assay instrument
630Value.
Calculate as follows the nature killing efficiency (%) of natural killer cell.The killing activity of this fraction values and natural killer cell is proportionate.
Nature killing efficiency=(Mixture manages A
630Value-Spon manages A
630Value)/(Max manages A
630Value-Spon manages A
630Value)
The result shows that the vaccine immunities such as pMD-CMV-ORF2, pMD-CMV-ORF1 and pMD-CMV-ORF1+2 all can make the killing activity of NK cell in the mouse spleen obviously strengthen; The killing activity of NK cell reaches 37.2%, 30.4%, 31.6%, 34.8%, 39.1%, 34.8% in the vaccine immunity group mouse spleen such as 4～9 groups.
The mensuration of T cell subsets
1) preparation mice spleen individual cells suspension 6 * 10
6/ L 100 μ L, the centrifugal 5min of 1500r/min after 200 order steel sieve filters abandons supernatant;
2) with fluorescence washing liquid (0.15mol/L PBS pH7.4,2% glucose, 0.1% BSA, 0.05%NaN
3) wash twice.With fluorescence washing liquid standardize solution 100 μ L, add respectively anti-CD4+, the CD8a+ of FITC labelling, the CD3+ of PE-Cy5 labelling of PE labelling, rearmounted 4 ℃ of lucifuge 30min are mixed;
3) take out, wash 2 times with the fluorescence washing liquid, add 300 μ L fluorescence washing liquids, with flow cytometry analysis T cell subsets.
The result shows, pMD-CMV-ORF2, pMD-CMV-ORF1 and pMD-CMV-ORF1+2 pcrDNA vaccine immunity all can make Th subgroup in the mouse boosting cell and the percentage composition of Tc subgroup be higher than respectively normal saline group, chitosan matched group, liposome matched group.4～9 groups of vaccine immunities all can make the Th subgroup percentage composition in the mouse boosting cell rise to 26%, 27.7%, 26.1%, 27.1%, 27.8%, 28.5% by 19.3% of normal saline group; The percentage composition of Tc subgroup has risen to 7%, 6.7%, 6.5%, 6.4%, 6.5%, 7.1% by 6.1% of normal saline group.
The pig immunization experiment
7 age in days sodium selenites are divided into 9 groups, every group 3, be respectively: 1. normal saline group, 2. chitosan matched group, 3. liposome matched group, 4. the coated pMD-CMV-ORF2 of chitosan organizes, 5. the coated pMD-CMV-ORF2 of liposome organizes, 6. the coated pMD-CMV-ORF1 group of chitosan, the 7. coated pMD-CMV-ORF1 group of liposome, 8. the coated pMD-CMV-ORF1+2 of chitosan organizes, 9. the coated pMD-CMV-ORF1+2 group of liposome.Every respectively isolated rearing of pig, intramuscular injection is 3 times altogether, every 2 weeks of minor tick.Inject 1mL through the coated small molecular DNA vaccine of chitosan or liposome in left and right sides neck sidepiece muscle for every group, immunizing dose is 1000 μ L.
The preparation of the small molecular DNA vaccine that chitosan is coated: the 40mg chitosan, add 280 μ L acetic acid, then add tri-distilled water to 2mL, 37 ℃ of 180～200r/min sway to spend the night to chitosan and dissolve fully, and solution is transparence.Add water to 190mL, regulating PH with NaOH is 5.5, last standardize solution 200mL, and vacuum filtration (0.22 μ m filter membrane) degerming gets solution a.The Na that small molecular DNA vaccine is added 50mmol/L
2SO
4Obtain solution b to 100 μ g/mL in the solution.Respectively get isopyknic solution a, b, be preheated to respectively 50～55 ℃, vibration mixes 30s rapidly, forms milky suspension, i.e. the coated small molecular DNA vaccine of chitosan.After the small molecular DNA vaccine suspension room temperature that the chitosan for preparing is coated was placed 1 hour, the centrifugal 30min of 4000r/min removed supernatant, adds sterilized water again and is settled to pcrDNA content 1 μ g/ μ L.Each every pig is injected 1000 μ L.
The coated small molecular DNA vaccine of liposome is prepared standby in following ratio: lecithin 30mg, cholesterol 15 mg and 18-amine. 2mg are dissolved in form solution 1 in the 15m1 ether fully; 2000ug is dissolved in and forms solution 2 among phosphate buffer (pH7.4) 3m1; Solution 1 and solution 2 are mixed, and ultrasonic emulsification is to not stratified, and rotary evaporation to ether is removed fully.The standardize solution final volume is 2m1, i.e. the coated small molecular DNA vaccine of liposome.4 ℃ save backup, and small molecule DNA content is 1 μ g/ μ L, and each every pig is injected 1000 μ L.
Chitosan matched group chitosan (the not containing small molecular DNA vaccine) immune swine of the small molecular DNA vaccine immune group equivalent that is coated with chitosan.
Liposome matched group liposome (the not containing small molecular DNA vaccine) immune swine of the small molecular DNA vaccine immune group equivalent that is coated with liposome.
The counteracting toxic substances of pig annulus 2 types virus small molecular DNA vaccine immune swine
Get the 3rd generation PCV2 (GD strain) cell toxicant via intranasal application and oral cavity route and infect 3 all specific pathogen free in age (SPF) pigs, inoculum concentration is 5mL.Behind the counteracting toxic substances 24 days, aseptic lymph node, spleen, kidney, the tonsil of getting pig, homogenate, make the tissue suspension of 1:3 (W/V) with sterilization PBS, with 6000r/min centrifuging and taking supernatant, after adding penicillin, streptomycin and processing, get toxic tissue suspension and be inoculated in the PK15 cell, measure TCID with immunopcroxidase monolayer assay (Immunoperoxidase monolayer assay, IPMA)
50After, packing, frozen for subsequent use in-80 ℃ of refrigerator-freezers.
IPMA measures TCID
50Test method reference literature (Ladekjaer-Mikkelsen et al, 2002).
In 4 weeks after last 1 immunity, via intranasal application and oral cavity route infect 10
6-22TCID
50The PCV2 tissue poison of/mL, every inoculation 5mL.
Experimental result
The antibody horizontal of pig annulus 2 types virus small molecular DNA vaccine immune swine detects
Respectively at before the immunity, rear 14 days, the 3rd time immunity of rear 14 days, the 2nd time immunity of the 1st immunity rear 14 days and 28 days, and behind the counteracting toxic substances 1,2,3,4,5 weeks gathered the vena cava anterior blood of respectively organizing pig, separation of serum is with the PCV2 specific antibody in the ELISA detection kit detection serum.4 weeks were infected PCV2 tissue poison (GD strain) by oral cavity and nasal after the 3rd immunity.
The result shows, each small molecular DNA vaccine immune group pig all produced PCV2 specific ELISA antibody and in and the Hangzhoupro body, NAT reaches more than the 1:20; After 28 days 4～9 groups pig specific antibody OD value all double than normal saline group above, 2,3 groups with the normal saline group do not have significant difference.
The detection of the cellular immune level of pig annulus 2 types virus small molecular DNA vaccine immune swine
1, lymphocyte proliferation assay
1) gets pig vena cava anterior blood, place the anticoagulant heparin pipe.Separate lymphocyte, adjusting cell concentration is 1 * 10
7/ mL, test stimulus group, test control group and blank group are established in test;
2) specifically add quadrat method: in 96 porocyte culture plates, every part of lymphocyte specimen is divided two groups (test stimulus group and test control group), every group of 5 repeating holes, and the test stimulus group adds respectively the lymphocyte, 50 of 100 μ L separator wells
μThe ConA of L 40 μ g/mL and the RPMI-1640 of 10% calf serum; Test control group adds respectively the lymphocyte of 100 μ L separator wells, the RPMI-1640 of 100 μ L10% calf serums; The every hole of blank group adds the RPMI-1640 of 200 μ L10% calf serums, 37 ℃ of 5% CO
2, incubation 24 hours;
3) add 10 μ L MTT (5 μ g/mL), continue to cultivate 4 hours, then every hole adds 100 μ L10%SDS-0.04mol/L stop buffer cessation reactions, after 37 ℃ of black purple crystals after 4 hours dissolve fully, measures OD with enzyme-linked immunosorbent assay instrument
630Value.
Calculate stimulation index (SI), SI=(test stimulus group-blank group)/(test control group-blank group).
The result shows that post-stimulatory breeder reaction all is significantly higher than normal saline group, chitosan matched group, liposome matched group to the peripheral blood lymphocyte of pMD-CMV-ORF2, pMD-CMV-ORF1 and pMD-CMV-ORF1+2 vaccine immunity pig to ConA.The peripheral blood lymphocyte of 4～9 groups of immune swines all reaches more than 2 the post-stimulatory breeder reaction SI of ConA, compares the generation significant difference with normal saline SI value 1.62; 2,3 groups with the normal saline group do not have significant difference.
2, natural killer cell activity (NK) detects
Adopt the 2h short distance to discharge improved method
1) gets pig vena cava anterior blood, place the anticoagulant heparin pipe;
2) separate lymphocyte, 1640 culture medium of 10% calf serum are adjusted to 1 * 10 with cell concentration
6/ mL, as the effector lymphocyte who detects the NK cytoactive, with the SP2/0 oncocyte as target cell, culture tube is managed with aseptic 1.5mL Eppondof, totally 3 groups of NK NKT groups (Mixture), target cell Spontaneous release group (Spon), maximum release group (Max) are established in test, and respectively establish 3 multiple pipes;
3) specifically add quadrat method: 100 μ L effector lymphocytes and 100 μ L target cells, effect target cell concentration ratio is 50:1, as NK NKT group (Mixture); The RPMI-1640 of 100 μ L target cells and 2% calf serum is target cell Spontaneous release group (Spon); 100 μ L target cells and 100 μ L, 10% TritonX-100 are maximum release group.Application of sample is complete, mixing, and the centrifugal 2min of 500 r/m in is hatched 2h for 37 ℃, then respectively manages cold saline 50 μ L on the rocks and stops cytotoxicity between the effect target cell.
4) with developmental tube with the centrifugal 5min of 3 000r/min, getting supernatant is added in the 40 hole polystyrene micro-reaction plates and carries out enzymatic reaction, 100 μ L/ holes, put 37 ℃ of pre-temperature 20min, each hole adds LDH substrate solution 100 μ L, is put in 37 ℃ of calorstats and reacts 10min, takes out, each hole adds 0.1mol/L citric acid 30 μ L to stop enzymatic reaction, reads OD at enzyme-linked immunosorbent assay instrument
630Value.
Calculate as follows the nature killing efficiency (%) of natural killer cell.The killing activity of this fraction values and natural killer cell is proportionate.
Nature killing efficiency=(Mixture manages A
630Value-Spon manages A
630Value)/(Max manages A
630Value-Spon manages A
630Value)
The result shows that pMD-CMV-ORF2, pMD-CMV-ORF1 and pMD-CMV-ORF1+2 pcrDNA vaccine immunity all can make the killing activity of NK cell in the pig peripheral blood lymphocyte obviously strengthen, and all reach about 35%, are significantly higher than matched group.
3, the mensuration of T cell subsets
1) gets pig vena cava anterior blood, place the anticoagulant heparin pipe;
2) separate lymphocyte, preparation spleen mononuclear cell suspension 6 * 10
6/ L 100 μ L, the centrifugal 5min of 1500r/min after 200 order steel sieve filters abandons supernatant.With fluorescence washing liquid (0.15mol/L PBS pH 7.4,2% glucose, 0.1% BSA, 0.05%NaN
3) wash twice;
3) with fluorescence washing liquid standardize solution 100 μ L, add respectively anti-CD4+, the CD8a+ of FITC labelling, the CD3+ of PE-Cy5 labelling of PE labelling, after being mixed, put 4 ℃ of lucifuge 30min;
4) take out, wash 2 times with the fluorescence washing liquid, add 300 μ L fluorescence washing liquids, with flow cytometry analysis T cell subsets.
The result shows, pMD-CMV-ORF2, pMD-CMV-ORF1 and pMD-CMV-ORF1+2 pcrDNA vaccine immunity all can make Th subgroup in the pig peripheral blood lymphocyte and the percentage composition of Tc subgroup be higher than respectively normal saline group, chitosan matched group, liposome matched group.The percentage composition of the Th subgroup in 4～9 groups of pig peripheral blood lymphocytes rises to nearly 30% by 20% of normal saline group, the percentage composition of Tc subgroup also slightly raises.
Small molecular DNA vaccine of the present invention, only carry eukaryotic gene transcripting promoter and genes of interest or genes of interest fragment, do not carry beyond any genes of interest independent basis because of or nucleotide sequence, such as antibiotics resistance gene, PolyA tail, replication origin etc., has better biological safety.Because the entire length of linear DNA vaccine is shorter, be easier to amplification, simultaneously, the accuracy of amplification is higher, and the potency ratio of vaccine is also higher.
＜110〉Guangdong Provincial Academy Of Agricultural Sciences Veterinary Institute
＜120〉a kind of small molecular DNA vaccine and preparation method thereof
<130>
<160> 6
<170> PatentIn version 3.5
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<210> 5
<211> 29
<212> DNA
＜213〉artificial primer
<400> 5
Claims (4)
1. small molecular DNA vaccine, described small molecular DNA vaccine is for by ORF1, the ORF2 of CMV promoter and genes of interest pig annulus 2 type viruses or the linear DNA fragment that the ORF1+ORF2 gene consists of.
2. the preparation method of the described small molecular DNA vaccine of claim 1 may further comprise the steps:
1) transcripting promoter is cloned in the carrier that contains cloning site, obtains universal support;
2) genes of interest is cloned in the universal support, obtains containing the recombinant vector of transcripting promoter and genes of interest;
3) according to the upstream sequence design universal primer of universal support transcription promoter, according to the downstream sequence of genes of interest, design downstream primer, take recombinant vector as template, the linear DNA fragment that amplification is made of transcripting promoter and genes of interest.
3. the preparation method of small molecular DNA vaccine according to claim 2, it is characterized in that: the carrier that contains cloning site is the polyclone carrier.
4. the preparation method of small molecular DNA vaccine according to claim 3, it is characterized in that: the polyclone carrier is the T carrier that contains multiple clone site.
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| CN 201010535633 CN102008735B (en) | 2010-11-09 | 2010-11-09 | Small molecular DNA vaccine and preparation method thereof |
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| CN 201010535633 CN102008735B (en) | 2010-11-09 | 2010-11-09 | Small molecular DNA vaccine and preparation method thereof |
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| CN102008735B true CN102008735B (en) | 2013-01-09 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1850279A (en) * | 2006-02-23 | 2006-10-25 | 广东省农业科学院兽医研究所 | PcrDNA vaccine, and its preparing method and use |
| CN101716338A (en) * | 2009-11-26 | 2010-06-02 | 南方医科大学 | DNA vaccine for promoting regeneration and functional rehabilitation of nerves of central system |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1850279A (en) * | 2006-02-23 | 2006-10-25 | 广东省农业科学院兽医研究所 | PcrDNA vaccine, and its preparing method and use |
| CN101716338A (en) * | 2009-11-26 | 2010-06-02 | 南方医科大学 | DNA vaccine for promoting regeneration and functional rehabilitation of nerves of central system |
Non-Patent Citations (1)
| Title |
|---|
| P. Johansson等.PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine.《Vaccine》.2002,第20卷第3379-3388页. * |
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| CN102008735A (en) | 2011-04-13 |
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