The application be that January 23, application number in 2008 are 200810004736.0 the applying date, denomination of invention divides an application for the application of " sulpho-oligodeoxynucleotidewith and application thereof with immunostimulatory activity ".
Summary of the invention
The object of the present invention is to provide sulpho-oligodeoxynucleotidewith a kind of non-species specificity, that have immunostimulatory activity.More particularly, the invention provides a kind of sulpho-oligodeoxynucleotidewith that people and mouse is had the cross immunity stimulating activity.
The present invention also aims to provide this sulpho-oligodeoxynucleotidewith in preparation vaccine adjuvant and the application in preparation prevention or treatment tumour, infection and medicine hypersensitive with immunostimulatory activity.
The inventor studies on the basis of existing technology, finds: the CpG primitive of (1) activation people immunocyte is 5 '-NTCGTPy-3 ' (wherein N represents A, G, C or T, and Py represents pyrimidine C or T); (2) the CpG primitive of activation mouse immune cell is 5 '-NA/TCGTT-3 ' (wherein N represents A, G, C or T).On this research basis, find out that the CpG-ODN that contains 5 '-NTCGTT-3 ' primitive (comprising 5 '-ATCGTT-3 ', 5 '-GTCGTT-3 ', 5 '-CTCGTT-3 ' and 5 '-TTCGTT-3 ') has the cross immunity stimulating activity to people and mouse.The contriver studies show that further having 5 ' of two or more copies-NTCGTT-3 ' primitive (N does not represent A or G) in the nucleotide sequence is sulpho-oligodeoxynucleotidewith of the present invention has the cross immunity stimulating activity to people and mouse key.
Given this, the invention provides a kind of sulpho-oligodeoxynucleotidewith with immunostimulatory activity, have 5 ' of two or more copies-NTCGTT-3 ' primitive in its sequence, described N does not represent A or G, is preferably T or C.The length of described sulpho-oligodeoxynucleotidewith is 15~35 Nucleotide, is preferably 20~30 Nucleotide.Described CpG right and wrong are methylated.
When N was T, the sequence preference of described sulpho-oligodeoxynucleotidewith was:
5’-TCGTTCGTTCGTTCGTTCGTT-3’、
5’-TCGTTCGTTCGTTCGTTCGTTCGTT-3’、
5’-TCGTTCGTTCGTTCGTTCGTTCGTTCGTT-3’、
5’-TCGTTTCGTTTCGTTTCGTT-3’、
5’-TCGTTTCGTTTCGTTTCGTTTCGTT-3’、
5’-TCGTTTCGTTTCGTTTCGTTTCGTTTCGTT-3’、
5’-TTCGTTTTTCGTTTTTCGTT-3’、
5’-TTCGTTATTCGTTATTCGTT-3’、
5’-TTCGTTGTTCGTTGTTCGTT-3’、
5’-TTCGTTCTTCGTTCTTCGTT-3’、
5’-TCTTCGTTTTTCGTTTTTCGTT-3’、
5’-TCTTCGTTATTCGTTATTCGTT-3’、
5’-TCTTCGTTGTTCGTTGTTCGTT-3’、
5’-TCTTCGTTCTTCGTTCTTCGTT-3’、
5’-TTCGTTTTCGTTTTCGTTTTCGTT-3’、
5’-TCTTCGTTTTCGTTTTCGTT-3’、
5’-TTCGTTTCGTTTCGTTTCGTT-3’、
5’-TTCGTTTCGTTTCGTTTCGTTTCGTT-3’、
5’-TTCGTTCGTTCGTTCGTT-3’、
5’-TCTTCGTTCGTTCGTTCGTT-3’、
5 '-TTCGTTCGTTCGTTCGTTCGTT-3 ' or
5’-TTCGTTCGTTCGTTCGTTCGTTCGTT-3’。
When described N was C, the sequence preference of described sulpho-oligodeoxynucleotidewith was:
5 '-TCGTCTCGTTTCGTCTCGTT-3 ' or
5’-TCGTCTCGTTTCGTCTCGTTTCGTCTCGTT-3’。
The preparation method of sulpho-oligodeoxynucleotidewith of the present invention is well-known to those skilled in the art, for example can adopt the chemosynthesis of solid phase phosphoramidite triester method, this method has efficient and advantage such as quick coupling, has been widely used in DNA the field of chemical synthesis.Entrust gene Synesis Company of specialty to handle for the oligodeoxynucleotide synthetic at present, for example domestic Shanghai living worker biotech company that entrusts is synthetic more.
The solid phase phosphoramidite triester method is by 3 ' end beginning, and concrete reactions steps is as follows:
1. deprotection base: slough the blocking group DMT (dimethoxytrityl) that is connected the Nucleotide on the CPG (Controlled Pore Glass) with trichoroacetic acid(TCA), obtain free 5 ' hydroxyl, use for next step condensation reaction;
2. activation: with the nucleotide monomer and the tetrazole activator mix of phosphoramidite protection and enter synthetic post, (its 3 ' end is activated to form phosphoramidite tetrazolium active intermediate, but 5 ' end is protected by DMT still), the Nucleotide generation condensation reaction of deprotection base on this intermediate and the CPG;
3. connect: when phosphoramidite tetrazolium active intermediate runs on the CPG the Nucleotide of deprotection base, will with its 5 ' hydroxyl generation affinity reaction, condensation is also sloughed tetrazolium, this moment, oligonucleotide chain prolonged a base forward;
4. oxidation: nucleotide monomer is to be connected with oligonucleotide on being connected in CPG by inferior phosphide key during condensation reaction, and inferior phosphide key instability, easily, use this moment sulfo-reagent that phosphoramidite is oxidized to the phosphotriester of the two keys of sulphur phosphorus, thereby obtain stable oligonucleotide by acid or basic hydrolysis;
5. sealing: for the 5 ' hydroxyl that has neither part nor lot in reaction that prevents to be connected on the CPG is extended, often seal this terminal hydroxy group after the condensation reaction in circulating reaction subsequently by acetylize.
Through after above five steps, a deoxynucleotide is just linked on the Nucleotide of CPG.Repeat above deprotection base, activation, connection, oxidation, closed process and can obtain a dna fragmentation crude product.At last to it cuts, deprotection base, purifying (commonly used have methods such as PAGE method, HPLC method, C18 method and OPC method), synthetic aftertreatment such as quantitative can obtain meeting requirement of the present invention sulpho-oligodeoxynucleotidewith.
The present invention also provides the application of sulpho-oligodeoxynucleotidewith in the preparation vaccine adjuvant.
The present invention also provides the application of sulpho-oligodeoxynucleotidewith in the medicine of preparation prevention or treatment tumour.
The present invention also provides the application of sulpho-oligodeoxynucleotidewith in the medicine of preparation prevention or treatment infection.
The present invention also provides sulpho-oligodeoxynucleotidewith in the preparation prevention or treat application in the medicine hypersensitive.
Described sulpho-oligodeoxynucleotidewith all has good immunostimulatory activity external to people and mouse immune cell, can be from the immunostimulatory activity result the mouse is estimated its immunostimulatory activity in human body.As vaccine adjuvant, it can significantly strengthen the immunne response of mouse to recombinant hepatitis b vaccine, and can make immunne response deflection TH
1Direction.Because sulpho-oligodeoxynucleotidewith of the present invention can induce out very strong TH
1Para-immunity is replied, and shows that it also can be used for prevention and treatment irritated and that infect.Simultaneously, sulpho-oligodeoxynucleotidewith of the present invention also has the activity of good inhibition tumor growth in the mouse body.
Embodiment
The invention will be further described below in conjunction with the description of accompanying drawing by embodiment, but this is not to be limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modifications or improvement, but only otherwise break away from basic thought of the present invention, all within the scope of the present invention.
Embodiment 1. has people's immunostimulatory activity of the oligodeoxynucleotide of 5 '-NTCGTPy-3 ' (wherein N represents A, G, C or T, and Py represents pyrimidine C or T) primitive
In order to illustrate the CpG primitive of activation people immunocyte, the inventor has carried out mutation research to four Nucleotide of all the other except that core Nucleotide CpG in the CpG primitive.For this reason, the contriver has designed one group of CpG-ODN, and is as shown in table 1, and every sequence length is 12 Nucleotide, all contains the CpG primitive of two identical copies.With the human peripheral blood single nucleus cell (PBMC) of these CpG-ODN stimulation vitro culture,, can estimate them to people's activity of immune cells by measuring the IgM content in the culture supernatant.
Employed CpG-ODN is designed by the inventor in the present embodiment, entrust Shanghai to give birth to worker biotech company and carry out synthetic, and carry out full chain thio-modification, polyacrylamide gel electrophoresis (PAGE) purifying, be dissolved in physiological saline, preserve standby in-20 ℃ of refrigerators.
Table 1
Sequence number | Numbering |
Sequence | |
1 |
1444 |
5’-ATCGTT?ATCGTT-3’ |
2 |
2444 |
5’-GTCGTT?GTCGTT-3’ |
3 |
3444 |
5’-CTCGTT?CTCGTT-3’ |
4 |
4444 |
5’-TTCGTT?TTCGTT-3’ |
5 |
2144 |
5’-GACGTT?GACGTT-3’ |
6 |
2244 |
5’-GGCGTT?GGCGTT-3’ |
7 |
2344 |
5’-GCCGTT?GCCGTT-3’ |
8 |
2444 |
5’-GTCGTT?GTCGTT-3’ |
9 |
2414 |
5’-GTCGAT?GTCGAT-3’ |
10 |
2424 |
5’-GTCGGT?GTCGGT-3’ |
11 |
2434 |
5’-GTCGCT?GTCGCT-3’ |
12 |
2444 |
5’-GTCGTT?GTCGTT-3’ |
13 |
2441 |
5’-GTCGTA?GTCGTA-3’ |
14 |
2442 |
5’-GTCGTG?GTCGTG-3’ |
15 |
2443 |
5’-GTCGTC?GTCGTC-3’ |
16 |
2444 |
5’-GTCGTT?GTCGTT-3’ |
Get healthy people's venous blood under the aseptic condition, the lymphocyte separation medium of choosing behind the anticoagulant heparin (available from the together positive biotech company in the north) separates PBMC; The counting back is adjusted to 1 * 10 with RPMI 1640 substratum (available from U.S. Gibco company) with cell concn
6/ ml is seeded in the 96 porocyte plates, every hole 200 μ l; Adding CpG-ODN is 2 μ M to final concentration, 5%CO
2Condition was cultivated 8 days for following 37 ℃.Wrap by 96 hole enzyme plates with goat-anti people IgM (available from U.S. SIGMA company), every hole 200ng, 4 ℃ are spent the night; Add above-mentioned PBMC culture supernatant after washing plate 3 times, every hole 100 μ l, 37 ℃ of effects 1 hour; Wash the goat-anti people IgM (available from U.S. SIGMA company) that adds the horseradish peroxidase-labeled of dilution in 1: 10000 behind the plate 3 times, every hole 100 μ l, 37 ℃ of effects 1 hour; Wash behind the plate 3 times with O-Phenylene Diamine (OPD) colour developing, 2M sulfuric acid termination reaction is with the spectrophotometric determination 490nm absorbance OD490 of place.Measured OD490 value is big more, shows that this CpG-ODN is strong more to people's activity of immune cells.
The result sees Fig. 1 for details, and wherein " contrast " be not for adding the cell contrast of CpG-ODN.Sequence number is four sequences of 1~4, and only in first Nucleotide difference of CpG primitive, the result shows that they are suitable to people's activity of immune cells, shows that first Nucleotide of the CpG primitive of activation people immunocyte is N (wherein N represents A, G, C or T); Sequence number is four sequences of 5~8, and only in second Nucleotide difference of CpG primitive, the result shows that this position is necessary for T, otherwise very weak to people's activity of immune cells; Sequence number is four sequences of 9~12, and only in the 5th Nucleotide difference of CpG primitive, the result shows that this position also is necessary for T, otherwise very weak to people's activity of immune cells; Sequence number is four sequences of 13~16, and only in the 6th Nucleotide difference of CpG primitive, the result shows that this position has immunostimulatory activity preferably when being C or T.In sum, the CpG primitive of activation people immunocyte is 5 '-NTCGTPy-3 ' (wherein N represents A, G, C or T, and Py represents pyrimidine C or T).
Embodiment 2. has the immunostimulatory activity of the oligodeoxynucleotide of 5 '-NA/TCGTT-3 ' (wherein N represents A, G, C or T) primitive to mouse
In order to illustrate the CpG primitive of activation mouse immune cell, the inventor has carried out mutation research to four Nucleotide of all the other except that core Nucleotide CpG in the CpG primitive.The CpG-ODN sequence of using in the present embodiment sees Table 1.Stimulate mouse spleen mononuclearcell (SMMC) with these CpG-ODN,, can estimate them the mouse immune cell activity by measuring the IgM content in the culture supernatant.Employed is the Balb/c mouse, female, and 6~8 weeks are available from Chinese Academy of Medical Sciences's Experimental Animal Center.
Get the Balb/c mouse spleen under the aseptic condition, with RPMI 1640 medium preparation SMMC suspensions; The counting back is adjusted to 1 * 10 with RPMI 1640 substratum with cell concn
6/ ml is seeded in the 96 porocyte plates, every hole 200 μ l; Adding CpG-ODN is 2 μ M to final concentration, 5%CO
2Condition was cultivated 3 days for following 37 ℃.Wrap by 96 hole enzyme plates with goat-anti mouse IgM (available from U.S. SIGMA company), every hole 200ng, 4 ℃ are spent the night; Add above-mentioned SMMC culture supernatant after washing plate 3 times, every hole 100 μ l, 37 ℃ of effects 1 hour; Wash the goat-anti mouse IgM (available from U.S. SIGMA company) that adds the horseradish peroxidase-labeled of dilution in 1: 10000 behind the plate 3 times, 1,37 ℃ of effect of every hole 100 μ 1 hour; Wash behind the plate 3 times with the OPD colour developing, 2M sulfuric acid termination reaction is with the spectrophotometric determination 490nm absorbance OD490 of place.Measured OD490 value is big more, shows that this CpG-ODN is strong more to the mouse immune cell activity.
The result sees Fig. 2 for details, and wherein " contrast " be not for adding the cell contrast of CpG-ODN.Sequence number is four sequences of 1~4, and only in first Nucleotide difference of CpG primitive, the result shows that they are suitable to the mouse immune cell activity, shows that first Nucleotide of the CpG primitive of activation mouse immune cell is N (wherein N represents A, G, C or T); Sequence number is four sequences of 5~8, and only in second Nucleotide difference of CpG primitive, the result shows that this position is necessary for A or T, otherwise very weak to the mouse immune cell activity; Sequence number is four sequences of 9~12, and only in the 5th Nucleotide difference of CpG primitive, the result shows that this position is necessary for T, otherwise very weak to the mouse immune cell activity; Sequence number is four sequences of 13~16, and only in the 6th Nucleotide difference of CpG primitive, the result shows that this position also is necessary for T, otherwise very weak to the mouse immune cell activity.In sum, the CpG primitive of activation mouse immune cell is 5 '-NA/TCGTT-3 ' (wherein N represents A, G, C or T).
By the result of study of embodiment 1 and 2 as can be known, the CpG primitive of activation people immunocyte is 5 '-NTCGTPy-3 ' (wherein N represents A, G, C or T, and Py represents pyrimidine C or T); The CpG primitive of activation mouse immune cell is 5 '-NA/TCGTT-3 ' (wherein N represents A, G, C or T).Comprehensive above-mentioned result of study is 5 '-NTCGTT-3 ' (wherein N represents A, G, C or T) to the CpG primitive that people and mouse have a cross immunity stimulating activity as can be seen.
Embodiment 3. oligodeoxynucleotide of the present invention are to the immunostimulatory activity of people's immunocyte
According to what find in embodiment 1 and 2 people and mouse had the CpG primitive of cross immunity stimulating activity, the contriver designs and has synthesized one group of CpG-ODN, as shown in table 2, length is 15~35 Nucleotide, contains two or (be numbered T1~T22) and 5 '-CTCGTT-3 ' primitive (being numbered C1 and C2) more than 5 '-TTCGTT-3 ' primitive of two copies respectively.
CpG-ODN in the table 2 draws respectively from document " Chinese microbiology and IMMUNOLOGY KEY WORDS INDEX 2001 except control sequence T7C and 1826,21 (5), 471-475 page or leaf, " The Journal of Immunology " 1998, the 160 volumes, the 870-876 page or leaf, all the other design by the inventor.The sequence that used control sequence and contriver design voluntarily in the present embodiment all entrusts Shanghai living worker biotech company to synthesize, full chain thio-modification, and the PAGE purifying is dissolved in physiological saline, preserves standby in-20 ℃ of refrigerators.
Table 2
Numbering |
Sequence |
T1 |
5’-TCGTTCGTTCGTTCGTTCGTT-3’ |
T2 |
5’-TCGTTCGTTCGTTCGTTCGTTCGTT-3’ |
T3 |
5’-TCGTTCGTTCGTTCGTTCGTTCGTTCGTT-3’ |
T4 |
5’-TCGTTTCGTTTCGTTTCGTT-3’ |
T5 |
5’-TCGTTTCGTTTCGTTTCGTTTCGTT-3’ |
T6 |
5’-TCGTTTCGTTTCGTTTCGTTTCGTTTCGTT-3’ |
T7 |
5’-TTCGTTTTTCGTTTTTCGTT-3’ |
T8 |
5’-TTCGTTATTCGTTATTCGTT-3’ |
T9 |
5’-TTCGTTGTTCGTTGTTCGTT-3’ |
T10 |
5’-TTCGTTCTTCGTTCTTCGTT-3’ |
T11 |
5’-TCTTCGTTTTTCGTTTTTCGTT-3’ |
T12 |
5’-TCTTCGTTATTCGTTATTCGTT-3’ |
T13 |
5’-TCTTCGTTGTTCGTTGTTCGTT-3’ |
T14 |
5’-TCTTCGTTCTTCGTTCTTCGTT-3’ |
T15 |
5’-TTCGTTTTCGTTTTCGTTTTCGTT-3’ |
T16 |
5’-TCTTCGTTTTCGTTTTCGTT-3’ |
T17 |
5’-TTCGTTTCGTTTCGTTTCGTT-3’ |
T18 |
5’-TTCGTTTCGTTTCGTTTCGTTTCGTT-3’ |
T19 |
5’-TTCGTTCGTTCGTTCGTT-3’ |
T20 |
5’-TCTTCGTTCGTTCGTTCGTT-3’ |
T21 |
5’-TTCGTTCGTTCGTTCGTTCGTT-3’ |
T22 |
5’-TTCGTTCGTTCGTTCGTTCGTTCGTT-3’ |
C1 |
5’-TCGTCTCGTTTCGTCTCGTT-3’ |
C2 |
5’-TCGTCTCGTTTCGTCTCGTTTCGTCTCGTT-3’ |
T7C |
5’-TCGTCGTCGTCGTCGTCGTCG-3’ |
1826 |
5’-TCCATGACGTTCCTGACGTT-3’ |
Measure the immunostimulatory activity of oligodeoxynucleotide of the present invention to people's immunocyte according to embodiment 1 described method, difference is that adding oligodeoxynucleotide of the present invention to final concentration in 96 porocyte plates is 4nM, 8nM, 16nM and 32nM.The results are shown in Table 3, Fig. 3 a, 3b and 3c.
Table 3
By table 3, Fig. 3 a, 3b and 3c as seen, oligodeoxynucleotide of the present invention has immunostimulatory activity highly to people's immunocyte, and very strong activity is just arranged in 4~32nM concentration range.Control sequence 1826 contains two copies 5 '-GACGTT-3 ' primitive, is mouse specific C pG-ODN, therefore to people's activity of immune cells a little less than.
Embodiment 4. oligodeoxynucleotide of the present invention are to the immunostimulatory activity of mouse immune cell
According to embodiment 2 described methods, measure the immunostimulatory activity of oligodeoxynucleotide of the present invention to the mouse immune cell, difference is that adding oligodeoxynucleotide of the present invention to final concentration in 96 porocyte plates is 12.5nM, 25nM, 50nM, 100nM and 200nM.The results are shown in Table 4, Fig. 4 a, 4b and 4c.
Table 4
By table 4, Fig. 4 a, 4b and 4c as seen, oligodeoxynucleotide of the present invention has immunostimulatory activity highly to the mouse immune cell, and very strong activity is just arranged in 12.5~200nM concentration range.Control sequence 1826 contains two copies 5 '-GACGTT-3 ' primitive, is mouse specific C pG-ODN, therefore the mouse immune cell is had the strongest activity.Control sequence T7C contains multiple copied 5 '-GTCGTC-3 ' primitive, is people's specific C pG-ODN, and is therefore very weak to the mouse immune cell activity.
By the experimental result in embodiment 3 and 4 as can be seen, can design the oligodeoxynucleotide that people and mouse is had height cross immunity stimulating activity according to CpG the primitive 5 '-NTCGTT-3 ' (wherein N does not represent A or G) that people and mouse is had the cross immunity stimulating activity, also prompting can be estimated potential human clinical's using value of this class oligodeoxynucleotide with mouse as animal model simultaneously.
Embodiment 5. oligodeoxynucleotide of the present invention strengthen the immunne response of mouse to the genetically engineered hepatitis B surface antigen as vaccine adjuvant
Use CpG-ODN T1~T6 and C1~C2 in the present embodiment.Genetically engineered hepatitis B surface antigen (HBsAg) is provided with epi chamber by Virology Inst., Chinese Academy of Preventive Medical Science's virus heredity, expressing cho cell, and purity is greater than 95%.The Balb/c mouse, female, 6~8 weeks are available from Chinese Academy of Medical Sciences's Experimental Animal Center.
HBsAg is adsorbed in the Al (OH) of 1mg/ml
3, final protein concentration is 10 μ g/ml.Through left hind gastrocnemius muscle immunity Balb/c mouse, cumulative volume is 100 μ l, every group of 6 mouse.Every injected in mice of experimental group 1 μ g is through Al (OH)
3The HBsAg and the 10 μ g oligodeoxynucleotide of the present invention of absorption, every injected in mice of control group 1 μ g is through Al (OH)
3The HBsAg of absorption.The 4 week blood sampling of immunity back is also isolated serum, according to ordinary method this serum is carried out 2 times of serial dilutions, is used to detect the total antibody of antigen-specific IgG.
Wrap by 96 hole enzyme plates with HBsAg, every hole 200ng, 4 ℃ are spent the night; Wash behind the plate 3 times with 37 ℃ of sealings of 1% bovine serum albumin 1 hour; Wash the serum to be checked that adds above-mentioned 2 times of serial dilutions behind the plate 3 times, 37 ℃ of effects 1 hour; Wash the goat anti-mouse igg (available from U.S. SIGMA company) that adds the horseradish peroxidase-labeled of dilution in 1: 10000 behind the plate 3 times, every hole 100 μ l, 37 ℃ of effects 1 hour; Wash behind the plate 3 times with the OPD colour developing, 2M sulfuric acid termination reaction is with the also definite terminal point titre (threshold value is made as 0.10) of the absorbance OD490 of spectrophotometric determination 490nm place.
" contrast " expression Al (OH) among Fig. 5
3As the HBsAg specific IgG antibodies titre that induces behind the adjuvant immunity.Immunity the time adds CpG-ODN of the present invention can significantly strengthen immunne response to HBsAg, and specific antibody titre (logarithmic value) can strengthen more than 10 times, shows that they have fabulous vaccine adjuvant activity.Because these CpG-ODN have the cross immunity stimulating activity to people and mouse, point out them also to can be used as the vaccine for man adjuvant.
Embodiment 6. oligodeoxynucleotide of the present invention and other vaccine adjuvant coupling strengthen the immunne response of mouse to the genetically engineered hepatitis B surface antigen
Use CpG-ODN T1 in the present embodiment.Genetically engineered HBsAg is provided with epi chamber by Virology Inst., Chinese Academy of Preventive Medical Science's virus heredity, expressing cho cell, and purity is greater than 95%.The Balb/c mouse, female, 6~8 weeks are available from Chinese Academy of Medical Sciences's Experimental Animal Center.
HBsAg is directly diluted or is adsorbed in the Al (OH) of 1mg/ml with physiological saline
3On, final protein concentration is 10 μ g/ml.Through left hind gastrocnemius muscle immunity Balb/c mouse, cumulative volume is 100 μ l, every group of 6 mouse.Every injected in mice 1 μ g of HBsAg group does not contain the HBsAg of any adjuvant, and every injected in mice 1 μ g is through Al (OH) for the HBsAg+Al group
3The HBsAg of absorption, the HBsAg of every injected in mice 1 μ g of HBsAg+CpG group and the CpG-ODN T1 of 10 μ g, every injected in mice 1 μ g is through Al (OH) for the HBsAg+Al+CpG group
3The HBsAg of absorption and the CpG-ODN T1 of 10 μ g.The 4 week blood sampling of immunity back, and according to the total antibody of embodiment 5 described methods detection antigen-specific IgG.
As seen from Figure 6, do not use the HBsAg group immune effect of adjuvant the poorest, the specific antibody titre only is 2.7 logarithmic value, with Al (OH)
3Or CpG-ODN T1 can significantly strengthen the immune effect of HBsAg as adjuvant, about specific antibody titre (logarithmic value) all can increase by 500.Al (OH)
3Have synergy with CpG-ODN T1, unite use and have the strongest adjuvanticity, can make the specific antibody titre increase more than 10 times.The above results shows that oligodeoxynucleotide of the present invention both can use separately as vaccine adjuvant, also can with other vaccine adjuvant such as Al (OH)
3The associating use.
Embodiment 7. oligodeoxynucleotide of the present invention strengthen TH in mouse
1Para-immunity is replied
According to embodiment 5 described methods, measure oligodeoxynucleotide of the present invention to mouse TH
1The influence that para-immunity is replied, employed enzyme labelled antibody was the goat anti-mouse igg 2a (available from U.S. SBA company) of horseradish peroxidase-labeled when difference was to detect.The result is presented among Fig. 7.
Antigen-specific immune response is divided into TH
1And TH
2Two types, TH wherein
1Class is replied corresponding with high-caliber antigen-specific IgG2a antibody titers.Al (OH)
3Be a kind of extremely strong TH
2The class vaccine adjuvant can suppress TH
1Para-immunity is replied, and induces extremely low-level specific IgG 2a antibody after showing as immunity.In the present embodiment, Al (OH)
3The specific IgG 2a antibody titers that induces as the HBsAg adjuvant only is 2.3 logarithmic value, shown in " contrast " among Fig. 7.Even add CpG-ODN immunne response deflection TH of the present invention during immunity
1Direction, showing as specific IgG 2a antibody titers increases by 2~3 logarithmic value, promptly 100~1000 times.The above results shows that oligodeoxynucleotide of the present invention is the extremely strong TH of a class
1The para-immunity stimulant.Immunization Update studies show that allergy is a kind of TH
2Para-immunity is replied relevant disease, is TH if type of immune response can be reversed
1Allergy can be prevented and treat to class.In addition, the prevention of tumour and infection and treatment all need to induce stronger TH
1Para-immunity is replied.Can induce very strong TH based on CpG-ODN of the present invention
1Therefore para-immunity is replied, and CpG-ODN of the present invention is except can be as vaccine adjuvant, also can be applicable to other fields such as the prevention of allergy, tumour and infection and treatment.
Embodiment 8. oligodeoxynucleotide of the present invention can suppress tumor growth in the mouse body
C-26 solid tumor (being provided by Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences) is ground with copper mesh, is that 7.2 phosphate buffered saline buffer is adjusted to 1 * 10 with cell concn with pH
6/ ml, through left hind veutro subcutaneous vaccination Balb/c mouse, every mouse 100 μ l, every group of 8 mouse.From inoculating back second day, per two days to tumor inoculation position injection CpG-ODN T1, every mouse 10 μ g, and volume is 100 μ l, control group mice is only injected 100 μ l physiological saline, injects altogether 6 times.The last injection was put to death mouse after 5 days, and the excision tumour is also weighed.The average knurl of calculating every treated animal is heavy, and calculates tumour inhibiting rate as follows: the average knurl of (it is heavy that average knurl is organized in the average knurl weight-treatment of control group)/control group heavy * 100%.The result is presented among Fig. 8.
As seen from Figure 8, the average knurl of control group mice heavily is 1.43 grams, and after 6 CpG-ODN T1 treatments of injection, the average knurl of treatment group mouse heavily is 0.59 gram, inhibitory rate to 58.7%.The above results shows that oligodeoxynucleotide of the present invention has good anti-tumor effect, can be used for the immunotherapy of tumour clinically.