CN102008735A - Small molecular DNA vaccine and preparation method thereof - Google Patents

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

A kind of small molecule DNA vaccine and preparation method thereof

Technical field

The present invention relates to a kind of dna vaccination and preparation method thereof, particularly a kind of small molecule 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 four development in laboratory to work out dna vaccination simultaneously 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 the revolution of vaccine for the third time 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 reorganization 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 reach the purpose of prevention and treatment disease to this antigen protein.

Dna vaccination demonstrates great potential in the control of infectious disease, parasite and tumor, it be carrier again can be in eukaryotic cell antigen expressed, antigenic in vivo expression is similar to natural infection, the antigen protein of expression in vivo has native conformation, inductive antibody response feature is similar to wild virus, induces body to produce cellular immunization and humoral immune reaction simultaneously.At present carried out the research of the dna vaccination of multiple pathogen, 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 the effect of treatment disease simultaneously.Therefore will bring hope for preventing or prevent unfavorable infectious disease for a long time.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 the natural infection or the inoculation attenuated live vaccine of virus, so the nucleic acid vaccine biggest advantage had both had the safety of subunit vaccine and inactivated vaccine, there are attenuated live vaccine and recombiant vaccine to induce the advantage of comprehensive immunne response ability again.

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, as be used for colony screening various antibiotic resistance genes, be used for 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. that replication initiation sequence, the regulator gene of plasmid replication are transcribed or expressed.Because genes carried or DNA sequence are too much, many disadvantages will occur: other antigenic expression of the first may work the mischief or uncertainty to body; It two is because other expression of gene, 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 constituted very big bio-safety problem, increase foreign DNA and be incorporated into probability in the host genome, increased the chance of problems such as induced tumor, the uncertainty of the use existence of dna vaccination is greatly increased; In addition under 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 efficient of dna vaccination, also will improve the production cost of vaccine, thereby other exogenous genes that carry on the while carrier may influence the processing of purpose antigen protein, modify the formation native conformation.

Traditional in theory dna vaccination only uses superhelix or cyclic plasmid DNA as carrier, 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, though the conventional plasmid DNA vaccine of its length has substantial degradation, its overall length still is more considerable, and existing P CR technology is difficult to guarantee the accuracy of its amplification.

Summary of the invention

A kind of small molecule DNA vaccine constitutes 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 gene transcripting promoter.

The preparation method of above-mentioned small molecule 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 transcripting promoter in the universal support, according to the downstream sequence of genes of interest, the design downstream primer is a template with the recombinant vector, 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 molecule 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 immunity of inductor fluidity and cellular immunity, effective protection is provided.Compare with existing dna vaccination, small molecule DNA vaccine of the present invention can be induced generation antibody quickly, and the cellular immune level of generation is higher.

Small molecule DNA vaccine of the present invention, only carry eukaryotic gene transcripting promoter and genes of interest or target gene fragment, do not carry beyond any genes of interest independent basis because of or nucleotide sequence, 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 molecule 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 the small molecule DNA vaccine, whole process can be finished in 24 hours, and with respect to the vaccine production technology of routine, its production cycle shortens greatly.Simultaneously, small molecule DNA vaccine of the present invention can adopt the preparation of sophisticated round pcr, 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 molecule DNA vaccine universal support of the present invention.

Fig. 2 is the preparation process sketch map of small molecule DNA vaccine of the present invention.

The specific embodiment

Below in conjunction with embodiment, further specify the present invention.

Small molecule DNA vaccine production process of the present invention as shown in Figure 1 and Figure 2, comparatively concrete preparation technology is as follows:

At 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 segmental template of clone CMV, CMV promoter gene with primer P1 and P2 amplification CMV, reaction condition is: 94 ℃ of pre-degeneration 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, 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 then;

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);

With pCMV-T-Target is template, and P1, P4 are that primer amplification obtains linear DNA segment pMD-CMV-Target, obtain small molecule DNA vaccine of the present invention behind the purification.

The structure of the ORF2 gene small molecule DNA vaccine of embodiment 1 pig annulus 2 type 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 pre-degeneration 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;

With pCMV-T-ORF2 is template, amplifies the linear DNA segment that only contains CMV promoter and ORF2 gene, and the purify DNA segment obtains pMD-CMV-ORF2 small molecule DNA vaccine.

The structure of the ORF1 gene small molecule DNA vaccine of embodiment 2 pig annulus 2 type viruses (PCV2)

Structure similar to Example 1 obtains the small molecule DNA vaccine of the ORF1 gene of pig annulus 2 type 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 pre-degeneration 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 molecule 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 pMD-CMV-ORF2 of chitosan bag quilt, 5. the pMD-CMV-ORF2 of liposome bag quilt, the 6. pMD-CMV-ORF1 of chitosan bag quilt, the 7. pMD-CMV-ORF1 of liposome bag quilt, 8. the pMD-CMV-ORF1+2 of chitosan bag quilt, the 9. pMD-CMV-ORF1+2 of liposome bag quilt.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 molecule DNA vaccine of chitosan bag quilt: the 40mg chitosan, add 280 μ L acetic acid, add tri-distilled water then 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 the small molecule 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 50～55 ℃ respectively, vibration mixes 30s rapidly, forms milky suspension, i.e. the small molecule DNA vaccine of chitosan bag quilt.After the small molecule 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 molecule DNA vaccine of liposome bag quilt: the small molecule DNA vaccine 0.3mL and the liposome Lipofectamine that get 2 μ g/ μ L ^TM2000 0.3mL mix the back room temperature and place 15min, promptly form the small molecule DNA vaccine of liposome bag quilt.PcrDNA content is 1 μ g/ μ L, each every injected in mice 100 μ L.

Chitosan (the do not contain small molecule DNA vaccine) immune mouse of chitosan matched group with the small molecule DNA vaccine immunity group equivalent of chitosan bag quilt.

Liposome (the do not contain small molecule DNA vaccine) immune mouse of liposome matched group with the small molecule DNA vaccine immunity group equivalent of liposome bag quilt.

The every fortnight immunity of all mices once is total to immunity three times.

Experimental result:

2,4,6,8,10 weeks took 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 molecule DNA vaccine, pMD-CMV-ORF1 small molecule DNA vaccine and pMD-CMV-ORF1+2 small molecule DNA vaccine all can bring out the mice specific antibody and produce, and promptly induce humoral immunization to take place.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 molecule DNA vaccine immune mouse trigger cell

1, lymphocyte transformation test

1) get the mouse spleen isolated lymphocytes, 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 the lymphocyte, 50 of 100 μ L separator wells respectively μThe concanavalin A, Con A (ConA) of L 40 μ g/mL and the RPMI-1640 of 10% calf serum; Matched group adds the lymphocyte of 100 μ L separator wells, the RPMI-1640 of 100 μ L10% calf serums respectively; 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 every then 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 molecule DNA vaccine immunity Mus 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) separate the mice 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 group (Mixture), target cell nature release group (Spon), maximum release groups (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, imitating the 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 a target cell nature release group (Spon); 100 μ L target cells and 100 μ L, 10% TritonX-100 are maximum release group, and application of sample finishes, mixing, and the centrifugal 2min of 500r/min is hatched 2h for 37 ℃, respectively manages cold saline 50 μ L on the rocks then and stops imitating cytotoxicity between 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 on enzyme-linked immunosorbent assay instrument ₆₃₀Value.

Calculate the NKT rate (%) of natural killer cell as follows.The killing activity of this fraction values and natural killer cell is proportionate.

The NKT rate=(Mixture manages A ₆₃₀Value-Spon manages A ₆₃₀Value)/(Max manages A ₆₃₀Value-Spon manages A ₆₃₀Value)

The result shows that 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 anti-CD4+, the CD8a+ of FITC labelling, the CD3+ of PE-Cy5 labelling of PE labelling respectively, rearmounted 4 ℃ of lucifuge 30min are mixed;

3) take out, wash 2 times, add 300 μ L fluorescence washing liquids, with flow cytometry analysis T cell subsets with the fluorescence washing liquid.

The result shows, pMD-CMV-ORF2, pMD-CMV-ORF1 and pMD-CMV-ORF1+2 pcrDNA vaccine immunity all can make the Th subgroup in the mouse boosting cell and the percentage composition of Tc subgroup be higher than normal saline group, chitosan matched group, liposome matched group respectively.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

The healthy piglet of 7 ages in days is divided into 9 groups, every group 3, be respectively: 1. normal saline group, 2. chitosan matched group, 3. liposome matched group, 4. the pMD-CMV-ORF2 of chitosan bag quilt organizes, 5. the pMD-CMV-ORF2 of liposome bag quilt organizes, 6. the pMD-CMV-ORF1 of chitosan bag quilt group, the 7. pMD-CMV-ORF1 of liposome bag quilt group, 8. the pMD-CMV-ORF1+2 of chitosan bag quilt organizes, 9. the pMD-CMV-ORF1+2 of liposome bag quilt group.Every pig isolated rearing respectively, intramuscular injection is 3 times altogether, each 2 weeks at interval.Inject the small molecule DNA vaccine of 1mL through chitosan or liposome bag quilt in left and right sides neck sidepiece muscle for every group, immunizing dose is 1000 μ L.

The preparation of the small molecule DNA vaccine of chitosan bag quilt: the 40mg chitosan, add 280 μ L acetic acid, add tri-distilled water then 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 the small molecule 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 50～55 ℃ respectively, vibration mixes 30s rapidly, forms milky suspension, i.e. the small molecule DNA vaccine of chitosan bag quilt.After the small molecule DNA vaccine suspension room temperature of the chitosan bag quilt for preparing 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 small molecule DNA vaccine of liposome bag quilt is equipped with in following ratio preparation: lecithin 30mg, cholesterol 15 mg and 18-amine. 2mg are dissolved in fully and form solution 1 in the 15m1 ether; 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 small molecule DNA vaccine of liposome bag quilt.4 ℃ of preservations are standby, and small molecule DNA content is 1 μ g/ μ L, and each every pig is injected 1000 μ L.

Chitosan (the do not contain small molecule DNA vaccine) immune swine of chitosan matched group with the small molecule DNA vaccine immunity group equivalent of chitosan bag quilt.

Liposome (the do not contain small molecule DNA vaccine) immune swine of liposome matched group with the small molecule DNA vaccine immunity group equivalent of liposome bag quilt.

The counteracting toxic substances of pig annulus 2 types virus small molecule DNA vaccine immunity pig

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 handling, get toxic tissue suspension and be inoculated in the PK15 cell, (Immunoperoxidase monolayer assay IPMA) measures TCID with the test of immunoperoxidase cell monolayer ₅₀After, packing, frozen standby in-80 ℃ of refrigerator-freezers.

IPMA measures TCID ₅₀Test method reference literature (Ladekjaer-Mikkelsen et al, 2002).

In last 1 immunity 4 week of back, 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 molecule DNA vaccine immunity pig detects

Respectively at before the immunity, back 14 days, the 3rd time immunity of back 14 days, the 2nd time immunity of the 1st immunity back 14 days and 28 days, and behind the counteracting toxic substances 1,2,3,4,5 weeks were 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.The 3rd immunity 4 weeks of back are infected PCV2 tissue poison (GD strain) by oral cavity and nasal.

The result shows, each small molecule DNA vaccine immunity 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 molecule DNA vaccine immunity pig

1, lymphocyte proliferation assay

1) gets pig vena cava anterior blood, place the anticoagulant heparin pipe.Isolated lymphocytes, 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 the lymphocyte, 50 of 100 μ L separator wells respectively μThe ConA of L 40 μ g/mL and the RPMI-1640 of 10% calf serum; Test control group adds the lymphocyte of 100 μ L separator wells, the RPMI-1640 of 100 μ L10% calf serums respectively; 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, every then 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) isolated lymphocytes, 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 group (Mixture), target cell nature release group (Spon), maximum release groups (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, imitating the 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 a target cell nature release group (Spon); 100 μ L target cells and 100 μ L, 10% TritonX-100 are maximum release group.Application of sample finishes, mixing, and the centrifugal 2min of 500 r/m in is hatched 2h for 37 ℃, respectively manages cold saline 50 μ L on the rocks then and stops imitating cytotoxicity between 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 on enzyme-linked immunosorbent assay instrument ₆₃₀Value.

Calculate the NKT rate (%) of natural killer cell as follows.The killing activity of this fraction values and natural killer cell is proportionate.

The NKT rate=(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) isolated lymphocytes, 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 anti-CD4+, the CD8a+ of FITC labelling, the CD3+ of PE-Cy5 labelling of PE labelling respectively, after being mixed, put 4 ℃ of lucifuge 30min;

4) take out, wash 2 times, add 300 μ L fluorescence washing liquids, with flow cytometry analysis T cell subsets with the fluorescence washing liquid.

The result shows, pMD-CMV-ORF2, pMD-CMV-ORF1 and pMD-CMV-ORF1+2 pcrDNA vaccine immunity all can make the Th subgroup in the pig peripheral blood lymphocyte and the percentage composition of Tc subgroup be higher than normal saline group, chitosan matched group, liposome matched group respectively.The percentage composition of the Th subgroup in 4～9 groups of pig peripheral blood lymphocyte rises to nearly 30% by 20% of normal saline group, the percentage composition of Tc subgroup also slightly raises.

Small molecule DNA vaccine of the present invention, only carry eukaryotic gene transcripting promoter and genes of interest or target gene fragment, do not carry beyond any genes of interest independent basis because of or nucleotide sequence, 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.

Claims (5)

1. a small molecule DNA vaccine constitutes linear DNA fragment by transcripting promoter and genes of interest, and genes of interest comprises its initial sum termination codon.

2. a kind of small molecule DNA vaccine according to claim 1 is characterized in that: transcripting promoter is the eukaryotic gene transcripting promoter.

3. the preparation method of claim 1 or 2 described small molecule DNA vaccines 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 transcripting promoter in the universal support, according to the downstream sequence of genes of interest, the design downstream primer is a template with the recombinant vector, the linear DNA segment that amplification is made of transcripting promoter and genes of interest.

4. the preparation method of small molecule DNA vaccine according to claim 3 is characterized in that: the carrier that contains cloning site is the polyclone carrier.

5. the preparation method of small molecule DNA vaccine according to claim 4 is characterized in that: the polyclone carrier is the T carrier that contains multiple clone site.

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