CN100379454C - Immunologic adjuvant and vaccine contacning said adjuvant - Google Patents
Immunologic adjuvant and vaccine contacning said adjuvant Download PDFInfo
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- CN100379454C CN100379454C CNB2004100338781A CN200410033878A CN100379454C CN 100379454 C CN100379454 C CN 100379454C CN B2004100338781 A CNB2004100338781 A CN B2004100338781A CN 200410033878 A CN200410033878 A CN 200410033878A CN 100379454 C CN100379454 C CN 100379454C
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Images
Abstract
The present invention provides a novel immunologic adjuvant, the main component of which is CpG-DNA extracted from bacill calmette-guerin, and the CpG-DNA is DNA rich in CpG basic sequences. The adjuvant is an immunostimulation type adjuvant having immunological activity action, and Th1 type immune response is induced in vivo; The novel immunologic adjuvant can be used as a vaccine adjuvant for treatment and prevention.
Description
Technical field
The present invention relates to vaccine adjuvant and contain the vaccine of this adjuvant, relate to the n DNA that is rich in the CpG motif specifically, can be used for treating and prevent with the adjuvant in the vaccine from the bacill calmette-guerin extract.
Background technology
Adjuvant is an indispensable composition in the vaccine, aspect immunological adjuvant, though the application of aluminium adjuvant is near 80 years, the check of prolonged application has also been passed through in its safety, but because its pair cell immunization is not obvious, especially the pair cell immunity is main vaccine, as second filial generation recombiant vaccine and the third generation dna vaccination to high purification, have little effect, so people are devoted to the research of novel adjuvant always.
CpG-DNA stimulates the natural immunity, promotes the body fluid and the cellular immunization of antigen-specific, has the record of laboratory research as the adjuvant of vaccine.CpG-DNA vaccine adjuvant activity is mainly reflected in the following aspects: activation APC cell, and the expression of cellular elementss such as rise CD40, CD54, CD80, CD86 and mhc class ii molecule, ability is presented in enhancement antigen; As costimulating signal, the antigen activation signals that collaborative TCR/BCR imports into, specific T cell of active antigen and B cell; Reply by induction of cytokines Th1 para-immunities such as IFN-α/β, IL-12, IL-18; Induce stronger ctl response.The antibody type of replying is based on IgG2a.But present CpG-DNA research all is the oligonucleotide that contains the CpG motif (ODN) at synthetic, and the preparation cost height has hindered the popularization of clinical practice.
Bacterial extract is another source of CpG-DNA, but so far not about from the CpG-DNA of the antibacterial report as adjuvant.
Summary of the invention
One of purpose of the present invention provides a kind of CpG-DNA immunological adjuvant that derives from bacterial extract, and this adjuvant itself has immunocompetence, with antigen in the vaccine in vivo interaction energy to induce the Th1 type be main immunne response.
The present invention provide simultaneously this immunological adjuvant as treatment and prevention with the purposes of vaccine adjuvant with contain the vaccine of this adjuvant.
CpG-DNA adjuvant of the present invention is from the extract antibacterial, especially is rich in the bacill calmette-guerin extract of CpG motif.The present invention as vaccine adjuvant, has confirmed its inductive immunoreation characteristics with this bacill calmette-guerin DNA (BCG-CpG-DNA), develops novel immunological adjuvant with this.
CpG-DNA is meant and contains the not DNA of methylated CpG dinucleotide motif, and the BCG-CpG-DNA that the present invention obtains contains the not natural CpG-DNA of methylated CpG motif, and it is from antibacterial, preferably extracts from bacill calmette-guerin.
BCG-CpG-DNA provided by the invention, an important characteristic is exactly from natural product, and its CpG content can detect by high performance liquid chromatogram and obtain, that is and, active constituent content wherein is known.It obtains by the following method: collect thalline when bacterial classification inoculation is cultured to logarithmic (log) phase in the culture medium that is fit to the mycobacterium growth; Thalline is centrifugal collection supernatant after fragmentation; Supernatant is collected no albumin layer with NaCl solution dissolving back with the organic solvent extracting through the precipitate of CTAB (cetyltrimethylammonium base amine), and this no albumin layer is the supernatant Ethanol Treatment collecting precipitation after the extracting once more, and this precipitation is carried out post processing.The quality percentage composition of CpG is at 15.75%-24.75% among the BCG-CpG-DNA that obtains, and preferably at 21.5-23.5%, the quality percentage composition of CpG can reach about 22.5% among the BCG-CpG-DNA that obtains according to the concrete extracting method of the present invention.
Detecting CpG content can be by anti-phase-high-efficient liquid phase technique (RP-HPLC): adopting the cytosine (dC) of special methylase SssI modification CpG dinucleotide is 5-methylcytosine (m
5-dC), utilize nuclease P 1 and bacterial alkaline phosphatase (BAP) that DNA is hydrolyzed to single deoxynucleoside, utilize anti-phase-high-efficient liquid phase technique (RP-HPLC) to m in the DNA hydrolyzation sample of modification and unmodified
5The difference of-dC detected level and CpG is carried out quantitatively.
According to another aspect of the present invention, be exactly to explore by a large amount of tests to confirm, this the novel CpG of being rich in motif adjuvant itself has immunocompetence, as the inductive Th1 of the being type of adjuvant is main immunoreation, body produces the antibody type based on IgG2a, so this is rich in CpG motif adjuvant and can gives vaccine and can induce humoral immunization, also can the inducing cell immunity, inducing the Th1 type in vivo is main immunoreactive function, thus the preventive effect and the therapeutical effect of performance vaccine.
The present invention more provides a kind of BCG-CpG-DNA of containing vaccine as immunological adjuvant.This vaccine can be the aqueous vaccine that BCG-CpG-DNA and antigen are mixed with, and also can be BCG-CpG-DNA mixes the back preparation with antigen freeze dried vaccine.That is, this vaccine of the present invention can be that BCG-CpG-DNA adjuvant and antigen mix, and also can be that BCG-CpG-DNA and present aluminium adjuvant vaccine mix.
In vaccine provided by the invention, the content of this immunological adjuvant BCG-CpG-DNA is 10-250 μ g/ person-portion in the per unit dosage vaccine, and this consumption is relevant with used antigen or vaccine.
Description of drawings
Fig. 1 is that the result of different immunity times of BCG-CpG-DNA adjuvant effect compares, among the figure:
1 group--2 groups of 3 μ gHBsAg--3 μ gHBsAg+50 μ gBCG-CpG-DNA
3 groups of--4 groups of 3 μ gHBsAg+100 μ gBCG-CpG-DNA--1 μ gHBsAg+10 μ gBCG-CpG-DNA
5 groups--6 groups of 3 μ gHBsAg-Al--3 μ gHBsAg-Al+50 μ gBCG-CpG-DNA
7 groups--3 μ gHBsAg-Al+100 μ gBCG-CpG-DNA
Fig. 2 resists-HbsIgG subclass analysis result for BCG-CpG-DNA;
Fig. 3 is the influence of BCG-CpG-DNA to epidemic encephalitis polysaccharide vaccine immune result;
Fig. 4 is for adding the epidemic encephalitis polysaccharide antibody IgG2a analysis result of BCG-CpG-DNA;
Fig. 5 is for adding the epidemic encephalitis polysaccharide antibody IgG1 analysis result of BCG-CpG-DNA.
The specific embodiment
Below introduce innovation of the present invention and application value place in detail by specific embodiment and result of the test, understand spirit of the present invention and essence better to help the reader, but do not constitute qualification the scope of the present invention.
One, the preparation of BCG-CpG-DNA
Wherein,
The preparation method of the logical culture medium of Rhizoma Solani tuber osi Soviet Union can be:
Get and clean fresh Rhizoma Solani tuber osi (1), wear into cylinder, be cut into 4 centimeters long inclined-planes again by knife with perforator;
Washed potato slope 1 hour with mobile drinking water;
With purified water flushing potato slope piece;
With Soviet Union's logical culture medium flushing ramp blocks;
Get the logical culture medium 20ml of Soviet Union, add in the 100ml sterilization bassoon;
Potato slope after the flushing is put into the sterilization bassoon that the logical culture medium of Soviet Union is housed;
10 pounds of sterilizations in 20 minutes.
The logical culture medium prescription of liquid Soviet Union and the preparation of improvement:
Asparagine (AR): 4g
Citric acid (AR): 2g
K
2HPO
4.3H
2O(AR)： 0.5g
MgSO
4.7H
2O(AR)： 0.5g
Ferric ammonium citrate (CP): 0.05g
Glycerol (medicinal): 60ml
Distilled water: 940ml
With NH
3.H
2O regulates between the pH7.2-7.4,
Add 1% (W/V) zinc sulfate 1ml then, 10 pounds of sterilizations in 20 minutes.
Step 4, the preparation of BCG-CpG-DNA: the concentration that the thalline suspension of fragmentation is diluted to 200mg/ml with ultra-pure water, 4 ℃ of High speed refrigerated centrifuges, 12000rpm/min is centrifugal, 15min * 2 time, collect supernatant, in supernatant, add 5MNaCl, 5% (w/v) CTAB/0.3MNaCl, 2000rpm/min, the centrifugal precipitation of staying of 10min, precipitation is dissolved in the NaCl solution of 1M, the phenol that adds equal volume: chloroform: isoamyl alcohol (25: 24: 1) extracting is to there not being albumin layer, chloroform: isoamyl alcohol (24: 1) extracting, the dehydrated alcohol that in the supernatant of collecting, adds 2 times of volumes, centrifugal collecting precipitation, again with 70% washing with alcohol 3 times, in the air air-dry after, be dissolved in the TE buffer (Tris-Cl pH8.0), deionized water, the dialysis of TE buffer, 0.45 μ m sterile filters is filtered, and is the BCG-CpG-DNA adjuvant, in-20 ℃ of preservations.
Two, the detection of CpG effective ingredient in the extract
1, the modification reaction that methylates
The modification reaction that methylates carries out in strict accordance with the method that supplier provides, be that DNA handles in suitable NEBuffer with the SssI methylase, 37 ℃ add S-ademetionine (SAM), per 4 hours additional SAM, phenol after 24 hours: chloroform: isoamyl alcohol (25: 24: 1) extracting 2 times, chloroform: isoamyl alcohol (24: 1) extracting 2 times, use ether respectively, ethanol precipitation, 70% washing with alcohol, air-dry in the air, be dissolved in the TE buffer.
2, the methylate detection of degree of modification
The DNA that DNA stock solution and methylase were handled handled 1 hour with restricted enzyme HpaII, 1% agarose gel electrophoresis 60 minutes, EB dyeing.
3, enzymatic hydrolysis reaction
Utilize (Sigma) hydrolysis DNA of nuclease P 1 (Nuclease P1), bacterial alkaline phosphatase (BAP).
Get 50 μ l dna solutions (500 μ g/ml are dissolved in the TE buffer, pH8.0) to the 1.5ml centrifuge tube, the deactivation in 5 minutes of the rearmounted frozen water of boiling water degeneration in 10 minutes, sample adds 100 μ l 30mM sodium acetates, pH5.3.
5 μ l 20mM zinc sulfate and 10 μ l nuclease Pl (1mg/ml 200units per mg in30mM sodium acetate, pH5.3) in 37 ℃ of water-baths 2 hours, add 20 μ l 0.5M Tris-cl and transfer pH8.5, add 37 ℃ of water-baths of 10 μ l bacterial alkaline phosphatases (BAP) (10.8mg prot./ml 43.7units/mgprot.) 2 hours again ,-20 ℃ of preservations are standby.
4、RP-HPLC
HPLC system: waters 600 high performance liquid chromatographs, 717plus automatic sampler, 2487 pairs of session UV-detector, Millennium
32Chromatographic work station;
The HPLC condition:
Chromatographic column: 25-cm Supelcosil LC-18-DB C
18Reversed-phase column (Supelco, U.S.A);
Mobile phase: buffer A (2.5%v/v methanol, 0.05M KH
2PO
4, pH4.0) 22.5 minutes;
Buffer B (8.0%v/v methanol, 0.05M KH
2PO
4KH
2PO
4, pH4.0) 30 minutes;
Eluent: 70% methanol-water 10 minutes;
Flow velocity: 1ml/min;
Column temperature: 35 ℃;
Detect wavelength: 254nm, the 280nm dual wavelength detects simultaneously.
5, date processing
Because the special base complementrity pair principle of double-stranded DNA, dC/dG=1dT/dA=1 in theory, therefore to the detection of the mol ratio of dC/dG, dT/dA can reaction detection accuracy.Various deoxynucleosides with known quantity are standard, with 8Br-Guo is interior mark, various deoxynucleosides among the BCG-CpG-DNA of institute's hydrolysis are carried out the mol ratio that mole calculates dC/dG, dT/dA after quantitatively respectively, and the molar content of GC, the accumulation of the molecular weight of the mole of various deoxynucleosides and its corresponding deoxymononucleotide is added the quality that calculates nucleic acid; According to m
5The mole of-dC and CpG molecular weight product calculate the quality of CpG, and the two is compared, and can obtain CpG quality percentage composition.
Example as a result:
The table 1 BCG-CpG-DNA testing result that do not methylate
The table 2 BCG-CpG-DNA testing result that methylates
Three, the pharmaceutical research of BCG-CpG-DNA
By testing in experiment in vitro and the body, pharmacotoxicological effect mechanism to BCG-CpG-DNA is studied, and the result shows that the external B cell that can stimulate of BCG-CpG-DNA produces IgM, promote antigen-presenting cell (APC) to produce IL-12, the enhancing ConA of energy significance is to the ability that induces of IFN-γ; Can strengthen the proliferation function of T, bone-marrow-derived lymphocyte in the body, improve the content of each subgroup of T cell in the animal body, and the CD4+/CD8+ ratio still keeps normal, can strengthen the kill capability of NK cells in mice, strengthen the generation of Th1 cytokines IL-2, IFN-γ in the body, the immunologic hypofunction mouse model had promote the immunologic function restitution, the mouse allelgic model of porcine blood serum sensitization is had tangible desensitization.
Therefore immunologic adjuvant BCG-CpG-DNA provided by the invention itself has the immunocompetence effect, is main immunoreation as the inductive Th1 of the being type of adjuvant, and the antibody type that body produces is based on IgG2a.Illustrate that this novel CpG of being rich in motif adjuvant gives vaccine and can induce humoral immunization, also can the inducing cell immunity, the preventive effect and the therapeutical effect of performance vaccine.
Four, pharmacodynamic study
Meaning of the present invention especially is the BCG-CpG-DNA from the bacill calmette-guerin extract is used for vaccine as adjuvant, and this adjuvant can be separately and antigenic action, also can with the aluminium adjuvant synergy.
1, hepatitis B BCG-CpG-DNA vaccine:
1.1 by certain proportioning mixing, obtaining with BCG-CpG-DNA is the Hepatitis B virus vaccine of adjuvant with BCG-CpG-DNA, viral surface antigen or Hepatitis B virus vaccine (aluminium adjuvant) solution.
1.2 the Hepatitis B virus vaccine ice bath 30min of above-mentioned gained, subcutaneous immune animal.
1.3BCG-CpG-DNA be used for the pharmacodynamic experiment result of adjuvant effect:
(1) different adjuvants and antigen dose immune effect are relatively
The BCG-CpG-DNA that in the HBs Ag of various dose (1 μ g, 3 μ g) and HBs aluminum vaccine (HBsAg-Al), mixes various dose (10 μ g, 100 μ g, 500 μ g), cumulative volume is 200 μ l, the subcutaneous immune BALB/c mouse in back, quantitative I is used in the back blood sampling of 4 week of immunity
125-HBsAg is put the method for exempting from and is detected anti--total antibody horizontal of HBs, the results are shown in Table 3,4.
By table 3 as seen, after the common immunity of BCG-CpG-DNA of 1 μ g HbsAg and various dose, antibody horizontal all improves, in it and 10 μ g dna immunizations as a result antibody horizontal near 2 times of 1 μ g HBsAg-Al; The antibody horizontal of the common immunity of 3 μ g HBsAg and 100 μ g DNA is 2 times of 3 μ g HBsAg-Al, and and the immune effect of 3 μ g HbsAg+10 μ g DNA significant difference (* P＜0.05) is arranged.
Table 3HBsAg and the common immune effect of various dose BCG-CpG-DNA (X ± SD) (mIU/ml)
| Ag dosage | HBsAg | HBsAg-Al | Ag+10μgDNA | Ag+100μgDNA | Ag+500μgDNA |
| 1μg 3μg | 41.62±11.6 47.42±46.7 | 53.74±54.6 76.09±46.2 | 102.16±63.9 57.42±55.6 | 37.82±47.45 152.03±75.52* | 84.85±46.77 126.01±127.61 |
Annotate: " Ag " in the table is meant " HBsAg ".
By table 4 as seen, after the common immunity of BCG-CpG-DNA of 1 μ g HBsAg-Al and various dose, antibody horizontal all improves, and is the highest with the coefficient effect of 100 μ gDNA in it; After the common immunity of BCG-CpG-DNA of 3 μ g HBsAg-Al and various dose, antibody horizontal also all improves, and the effect with 100 coefficient effects of μ g DNA and 3 μ g HBsAg-Al in it has significant difference (* P＜0.05)
Table 4HBsAg-Al and the common immune effect of various dose BCG-CpG-DNA (X ± SD) (mIU/ml)
| HBsAg-Al dosage | HBsAg-Al | HBsAg-Al+10μgDNA | HBsAg-Al+100μgDNA | HBsAg-Al+500μgDNA |
| 1μg 3μg | 53.74±54.61 76.09±46.22 | 76.82±71.32 140.25±102.21 | 93.96±101.55 171.02±44.28* | 60.65±32.24 126.76±64.83 |
2, render a service result the long term of BCG-CpG-DNA adjuvant effect:
1) in the HBs antigen of various dose (1 μ g, 3 μ g) and HBs vaccine, mixes the BCG-CpG-DNA of various dose (10 μ g, 50 μ g, 100 μ g), cumulative volume is 200 μ l, and the subcutaneous immune BALB/c mouse in a back was strengthened once during 3 weeks, quantitative I is used in blood sampling when 4 weeks, 10 weeks respectively
125The method of exempting from of putting-HBsAg detects anti--total antibody horizontal of HBs in the serum.The results are shown in Figure 1.
As seen from Figure 1, during 4 weeks, the antibody horizontal (2,3,4 groups) that adds the HBs antigen group of BCG-CpG-DNA all is higher than simple antigen group (1 group), and all reaches the level (2,3 groups and 5 groups are compared) of HBs vaccine (HBsAg-Al) group of same dose; 10 whens week, each group antibody horizontal all improve, wherein add the antibody horizontal (2,3, group) of the HBsAg group of BCG-CpG-DNA and merely antigen group (1 group) significant difference (P＜0.05) is arranged; Aluminium adjuvant vaccine group (5 groups) than same antigen dosage is slightly high, there was no significant difference between the two (P＞0.05).When BCG-CpG-DNA adds in the HBs-Al vaccine common immune animal (6,7 groups) to, in 4,10 weeks, its antibody titer all is higher than simple HBs-Al vaccine group (5 groups).In addition, no matter result's demonstration still adds BCG-CpG-DNA at HBs antigen in the HBs-Al vaccine, and the effect of 50 μ g and 100 μ g does not almost have difference (seeing 2,3 groups and 6,7 groups).
2) anti--HBsIgG subclass is analyzed
Get immunity 10 all mice serums, certain dilution factor (generally can be 1: 500), the ELISA method is measured the IgG subclass, the results are shown in Figure 2.As shown in Figure 1, when the HBs antigen immunity jointly of the BCG-CpG-DNA of 50 μ g and 100 μ g and 3 μ g, immune effect reaches and is higher than the effect of the HBs-AL vaccine of 3 μ g, but the two there was no significant difference, in the HBs-AL vaccine, add BCG-CpG-DNA equally, but also enhance immunity effect.And as seen from Figure 2, the level of the IgG2a that the HBs antigen group of interpolation BCG-CpG-DNA produces all has significance difference (* * * P＜0.01 with simple antigen group and HBs-AL vaccine group, * P＜＜0.01,), and the level of 100 μ gBCG-CpG-DNA group IgG2a is higher than 50 μ gBCG-CpG-DNA group, and the level of adding the HBs-AL vaccine group IgG2a of 100 μ gBCG-CpG-DNA also is higher than the HBs-AL vaccine group, the horizontal indifference of IgG1.But the IgG1 level of adding the HBs antigen group of BCG-CpG-DNA is starkly lower than simple antigen group and HBs-AL vaccine group, and IgG1 level and these two matched groups of wherein adding the HBs antigen group of 100 μ gBCG-CpG-DNA have significant difference (* * P＜0.05).
Effectiveness result in sum shows that behind the interpolation BCG-CpG-DNA, the anti-HBs antibody horizontal that mice produces all improves, and meets and exceeds the immune effect with the aluminum vaccine of dosage; After in the aluminum vaccine, adding BCG-CpG-DNA, then show as the obvious synergistic effect, and significant difference is arranged.Find that simultaneously BCG-CpG-DNA has good safety, because when the dosage of BCG-CpG-DNA reaches 500 μ g, it is any unusual that animal does not have contrary hair, diarrhoea or other intoxicating phenomenons etc.But the adjuvant effect of BCG-CpG-DNA is not enhanced with the increase of dosage, and this may have certain relation with antigenic proportioning.Learning the inductive antibody intensity of result BCG-CpG-DNA from rendeing a service, at 4W and 10W, is the experimental group of adjuvant with BCG-CpG-DNA, all is higher than simple antigen group, when 10W, significant difference is arranged.All reach simultaneously the antibody horizontal that also surpasses the aluminum vaccine experimental group of same antigen dosage.And the experimental result of antibody subtype also shows, compares with aluminium adjuvant, and BCG-CpG-DNA induces the horizontal significance of the IgG2a of generation to increase, and the horizontal significance of IgG1 is low simultaneously; The two coefficient result of BCG-CpG-DNA and aluminium adjuvant is that the level of IgG2a has increased, the horizontal indifference of IgG1.This explanation BCG-CpG-DNA can induce the Th1 type immunoreation of body generation based on IgG2a, also can work in coordination with the aluminium adjuvant combined effect, and part reverses the inductive Th2 type immunoreation based on IgG1 of aluminium adjuvant.
3, epidemic encephalitis Polysaccharide B CG-CpG-DNA immune vaccine
3.1 different immunity time results' comparison
BCG-CpG DNA the results are shown in Figure 3 to the epidemic encephalitis polysaccharide vaccine different immunity time.As seen from Figure 3, immune 2 pins, the antibody horizontal of the epidemic encephalitis polysaccharide vaccine group of interpolation BCG-CpG DNA is unlike simple vaccine group height; But behind immune 3 pins, the antibody horizontal that adds the epidemic encephalitis polysaccharide vaccine group of BCG-CpG DNA generally is higher than simple vaccine group, illustrate that BCG-CpG DNA can strengthen the antibody horizontal of epidemic encephalitis polysaccharide vaccine, but 10 μ g and 100 μ gBCG-CpG-DNA effects there is no very big-difference.
3.2BCG-CpG DNA is to the influence of epidemic encephalitis polysaccharide antibody hypotype
Get mice serum behind immune 3 pins,, measure the IgG subclass, the results are shown in Figure 4,5 with the ELISA method through certain dilution factor (1: 100).Fig. 4 is an epidemic encephalitis polysaccharide antibody IgG2a analysis result, and Fig. 5 is an epidemic encephalitis polysaccharide antibody IgG1 analysis result.
As seen from Figure 4, the level of the IgG2a of the epidemic encephalitis polysaccharide group of interpolation BCG-CpG DNA all is higher than simple antigen group, the IgG2a level and the simple antigen group of wherein adding two groups of 10,100 μ gBCG-CpG-DNA have significant difference (P＜0.01), and the IgG2a level of 100 μ gBCG-CpG-DNA group is higher than other dosage group.
Add level and simple antigen group and the indifference (P＞0.05) of each IgG1 that organizes of epidemic encephalitis polysaccharide of various dose BCG-CpG DNA as seen from Figure 5.
So, BCG-CpG DNA is added in the epidemic encephalitis polysaccharide vaccine, the result shows: compare with the immune effect of simple epidemic encephalitis polysaccharide vaccine, BCG-CpG DNA can strengthen the immune effect of epidemic encephalitis polysaccharide, and antibody horizontal increases, but there was no significant difference.Under the situation of immune two pins, reinforced effects is not obvious, but the reinforced effects behind three pins is more obvious.Adjuvant effect and amount that BCG-CpG DNA is described have relation, but are not to increase and enhanced with dosage, but and the antigen amount of proportioning relation is arranged.The result that epidemic encephalitis polysaccharide antibody hypotype is detected shows simultaneously, with BCG-CpG DNA is that the IgG2a level that the epidemic encephalitis polysaccharide group of adjuvant produces all is higher than simple epidemic encephalitis polysaccharide group, and significant difference arranged, and the IgG1 level that produces is between two groups and there was no significant difference.BCG-CpG DNA induces polysaccharide antigen to produce Th1 type protection antibody IgG2a level apparently higher than matched group, total antibody horizontal there was no significant difference.
Five, intramuscular injection BCG-CpG DNA is to the mice safety evaluation
1, experiment material
BCG-CpG DNA：50μg/100μl
Laboratory animal: BALB/c mouse (SPF), 8-10W, female
2, experimental technique
1) mice is divided into 4 groups at random: normal control group, immunity group, immunity in 22 days were organized in 28 days, 40 days groups of immunity, every group of 3-4 mice.
2) medication: experimental group with BCG-CpG DNA from mice back leg intramuscular injection 50 μ g/100 μ l, the next day once.The normal control group will not any processing.
3) respectively behind 22 days, 28 days, 40 days after the immunity, claim the mice body weight after, put to death mice, get liver, spleen is weighed, and calculates heavy index of liver and the heavy index of spleen, and and the normal control group relatively, the results are shown in Table 5.
Table 5 intramuscular injection BCG-CpG DNA is to mice Safety Evaluation Experiment result
Each BCG-CpG DNA experimental group body weight, liver weigh, spleen is heavy, liver refers to, spleen refers to and the normal control group does not more all have difference (P＞0.05) on the statistics word.
Conclusion: BCG-CpG DNA has safety preferably to mice.
It should be noted that at last: above embodiment is the unrestricted technical scheme of the present invention in order to explanation only, although the present invention is had been described in detail with reference to the foregoing description, those of ordinary skill in the art is to be understood that: still can make amendment or be equal to replacement the present invention, and not breaking away from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (8)
1. immunological adjuvant, its main component is the CpG-DNA that extracts from bacill calmette-guerin; Described CpG-DNA obtains by the following method:
When being cultured to logarithmic (log) phase in the culture medium that is fit to the mycobacterium growth, collects bacterial classification inoculation thalline; Thalline is centrifugal collection supernatant after fragmentation;
Supernatant is collected no albumin layer with NaCl solution dissolving back with the organic solvent extracting through the precipitate of CTAB, and this no albumin layer is the supernatant Ethanol Treatment collecting precipitation after the extracting once more, and this precipitation is carried out post processing.
2. the described immunological adjuvant of claim 1, wherein, the quality percentage composition of CpG is 15.75%-24.75% among the described CpG-DNA.
3. the described immunological adjuvant of claim 2, wherein, the quality percentage composition of CpG is 21.5-23.5% among the described CpG-DNA.
4. the described immunological adjuvant of claim 1 is used the purposes of vaccine adjuvant as treatment and prevention.
One kind the treatment and the prevention use vaccine, adjuvant wherein to comprise the described immunological adjuvant of claim 1 at least.
6. the described vaccine of claim 5, the content of this immunological adjuvant is 10-250 μ g/ person-portion in the per unit dosage vaccine.
7. the described vaccine of claim 5, it is mixed by described immunological adjuvant and antigen.
8. the described vaccine of claim 5, it is mixed by described immunological adjuvant and the vaccine that contains aluminium adjuvant.
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