CN1872346A - Vaccine for diabetes in type I, and constructing method - Google Patents
Vaccine for diabetes in type I, and constructing method Download PDFInfo
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Abstract
A DNA vaccine able to express the antisense peptide of diabetes A for preventing diabetes is disclosed and prepared. Its configuration method features that the coding genes at the diabetes A's early-phase antigen (insulin, preinsulin, IA-2 beta, IGRG and GAD 65) specific epitopes T, B and CTL are inserted in the eukaryon expression plasmid or the terminal 3' of T7 phage DNA capsid protein B10 coding gene.
Description
Technical field
The present invention relates to a kind of construction method of plasmid DNA, especially express the construction method of the plasmid DNA of type i diabetes early self antigen molecule specificity T epi-position, B epi-position and CTL epi-position antisense peptide, and further relate to the structure and the preparation method of the phage vaccine of presenting antisense peptide.
Background technology
Diabetes have become one of principal disease of harm public health in the global range.Diabetes mainly are divided into I type and II type.The I type makes the β cell impaired with autoimmune system attack beta Cell of islet, causes insulin secretion definitely to be reduced to feature; The II type is a feature with insulin resistant and islet cell function defective then.At present, mainly be to use non-obese diabetes (NOD) mice as animal model to the research of the type i diabetes cause of disease and treatment thereof, the NOD mice can the organic growth one-tenth diabetic symptom very similar to the human I type diabetes.
The main hereditary material of organism is a double-stranded DNA, and the DNA macromole is with two chain formation double-spiral structures of reverse complemental.Therefore, in a cistron, the information of a chain encoding polypeptide structure, the sense strand of meaning, and complementary with it, not the coded polypeptide structure, do not have transcriptional capability yet and only play a chain of template action, the meaningless chain of meaning.According to generally being called the custom of antisense RNA with the complementary RNA of mRNA, the polypeptide that is called for short by the antisense RNA translation is an antisense peptide.
Experimental results demonstrate that the characteristic of mutual identification is arranged between antisense peptide and the native peptides.Discover that the symbol of encode complementary amino acid whose " hydration index " is normally opposite, promptly one is hydrophilic, and another then is hydrophobic.Therefore, Bost (Proc Natl Acad Sci USA., 1985,82 (5): 1372-1375) think, under water environment, complementary two chains of encoding are because corresponding amino acid whose hydration index difference may be passed through their hydrophilic or hydrophobic surfaces, mutually combine along the whole piece peptide chain, this may be exactly the mechanism of action of antisense peptide and the identification of native peptides selectivity.
(Biochem Biophys Res Commun, 1984,121 (1): 203-207) propose " molecular recognition theory ", think that interacting proteins and polypeptide encoded by complementary dna fragmentation such as Blalock.The new not necessarily strictly complementary of two interactional proteic DNA of high-affinity of coding that studies have shown that, but, its aminoacid sequence has significantly hydrophobic-water-wet behavior, promptly a hydrophobic amino acid correspondence a hydrophilic amino acid, amino acid whose hydrophobic-the aqueous favoring mutual effect makes two highly affine albumen present complementary form, promptly " lock-key " recognition mode produces specific affinity interaction.
At present, the treatment of type i diabetes mainly is an insulin injection, does not still have proper prophylactic methods.In recent years, utilize the Th1 class to be converted to the generation of Th2 para-immunity therapeutic strategy prevention type i diabetes in the world.Yet many researchs have proposed query to Th1-Th2 immunne response switching strategy.Not necessarily valid approach of generation that the mode of utilizing Th1-Th2 immunne response conversion suppresses type i diabetes is described, means to have more complicated immunoregulation mechanism.Therefore, be necessary to study new technical scheme, more effectively prevent type i diabetes.
Summary of the invention
Purpose of the present invention has proposed type i diabetes antisense peptide vaccine and construction method thereof for solving the prior art above shortcomings just, and the antisense DNA vaccine of structure and the phage vaccine of presenting antisense peptide immunity in early days can suppress the NOD mice diabetes take place.
The present invention finishes by following technical proposal: a kind of type i diabetes vaccine, it is characterized in that antisense dna sequence catcaagttcaagttttcgatcaagtc by insulin C TL epi-position, the antisense dna sequence gctccttccgctgtccatcaagtccaaggtttccatcaagtcgct of proinsulin T epi-position, the antisense dna sequence gtccacaagctcctgctgctgctggtcatgctcctgtcgtccctgctcaaaaatga aacttggtcatggtcctggtttcgtcggtcttcaagtcttccaagtcaaagctcct catcaaaaagtcttcgctcctgatgtcttcgctgatcaagtccatttcgtccatgt cgtccaagtcgctgctttcgtcggtcctgtcgtcgtcgtccttcctcatgctgtct tcggtcaaggtggtcaatcatgatcatcatgctgctgctttccctcatcatctt of the antisense dna sequence caattccttgtcggtggtggtggtgtcggtgctggtgtcgctgct of two T epi-positions of receptor type Protein-tyrosine-phosphatase sample protein I A-2 β and catcctgctgtcgtcgtcggtcaacatggtcaagccctgtcttc and B epi-position, the antisense dna sequence caaaaacaagtcggtttccaagtcggt of the associated protein IGRP T of islets of langerhans G-6-Pase catalytic subunit epi-position, the antisense dna sequence gtctctgtccaacaaggtgctcaattcgtcgctgatgctgctggtaaattccttgg tcatcttg ctcctggtcctggtggtgctggtgtcttcgctgtcgctgct of the antisense dna sequence ggtggtggtggtgtcgctgatcatcatcctaaaaaagtc of glutamate decarboxylase GAD65T epi-position and B epi-position and the antisense dna sequence catcatgtccttcaacaagatgtccatcat of CTL epi-position, and be connected in introns encoding gene and expression vector between each antisense dna sequence.
Described introns encoding gene is the encoding gene of GPGPG or AAY.
Described expression vector is eucaryon pCDNA3.1 or T7 phage, is commercial product.
Described type i diabetes vaccine makes up by following method: at the antisense dna sequence catcaagttcaagttttcgatcaagtc of insulin C TL epi-position, the antisense dna sequence gctccttccgctgtccatcaagtccaaggtttccatcaagtcgct of proinsulin T epi-position, the antisense dna sequence gtccaacaagctcctgctgctgctggtcatgctcctgtcgtccctgctcaaaaatg aaacttggtcatggtcctggtttcgtcggtcttcaagtcttccaagtcaaagctcc tcatcaaaaagtcttcgctcctgatgtcttcgctgatcaagtccatttcgtccatg tcgtccaagtcgctgctttcgtcggtcctgtcgtcgtcgtccttcctcatgctgtc ttcggtcaaggtggtcaatcatgatcatcatgctgctgctttccctcatcatctt of the antisense dna sequence caattccttgtcggtggtggtggtgtcggtgctggtgtcgctgct of two T epi-positions of receptor type Protein-tyrosine-phosphatase sample protein I A-2 β and catcctgctgtcgtcgtcggtcaacatggtcaagccctgtcttc and B epi-position, the antisense dna sequence caaaaacaagtcg gtttccaagtcggt of the associated protein IGRP T of islets of langerhans G-6-Pase catalytic subunit epi-position, between the antisense dna sequence catcatg tccttcaacaagatgtccatcat of the antisense dna sequence ggtggtg gtggtgtcgctgatcatcatcctaaaaaagtc of glutamate decarboxylase GAD65 T epi-position and the antisense dna sequence gtctctgtc caacaaggtgctcaattcgtcgctgatgctgctggtaaattccttggtcatcttgc tcctggtc ctggtggtgctggtgtcttcgctgtcgctgct of B epi-position and CTL epi-position, link to each other with the introns encoding gene, after synthesizing, insert carrier for expression of eukaryon, obtain the type i diabetes antisense DNA, perhaps insert 3 ' end of T7 phage DNA capsid protein B10 encoding gene, the reorganization T7 phage that obtains presenting antisense peptide.
The aminoacid sequence of described antisense peptide is:
HQVQGFDQVGPGPGAPFAAHQVQGFHQVAGPGPGQFLVGGGGVGAGVAAGPGPGHPAVVVGQHGQAPVFGPGPGVQQAPAAAGHAPVVPAQKMKLGHGPGFVGLQVFQVKAPHQKVFAPDVFADQVHFVHVVQVAAFVGPVVVALPHAVFGQGGQHHDHHAAAFPHHLGPGPGQKQVGFQVGGPGPGGGGGVADHHPKKVGPGPGVSVQQGAQFVADAAGKFLGHLAPGPGGAGVFAVAAGPGPGHHVLQQDVHH。
Described insulin C TL epi-position (Gabrielle GM Pinkse, Proc.Natl.Acad.Sci.USA, 2005,102 (51): 18425-18430); Described proinsulin T epi-position (Kent.Nature, 2005,435:224); Described receptor type Protein-tyrosine-phosphatase sample protein I A-2 β (receptor-type protein tyrosine phosphatase-like protein) T epi-position (KatalinKelemen,
J Immunol,2004,172 (6): 3955-3962.), and B epi-position (Hoppu S, JAutoimmun.2004,23 (4): 361-370.); The described islets of langerhans G-6-Pase associated protein IGRP of catalytic subunit (islet glucose-6-phosphatase catalytic subunit-related protein) T epi-position (Scott M.Lieberman, Proc Natl Acad Sci U S A.2003,100 (14): 8384-8388); Described glutamate decarboxylase GAD65 (glutamic acid decarboxylase) T epi-position, B epi-position (HelenaReijonen,
Diabetes.2004,53 (8): 1987-1994.), and the CTL epi-position (ConstadinaPanagiotopoulos, Current Diabetes Reports, 2004,4:87-94.).
Described introns encoding gene GPGPG (Livingston BD.J Immunol, 2002,168 (11): 5499-506), introns encoding gene AAY (Velders MP.J Immunol, 2001,166 (9): 5366-5373).
The preparation method of described type i diabetes vaccine, after getting the type i diabetes antisense DNA and it being increased in escherichia coli with existing processes, through existing alkaline lysis method of extracting, purification, press desired concn with aseptic dissolved in distilled water DNA, obtain type i diabetes antisense DNA vaccine.
The preparation method of described type i diabetes vaccine, after the reorganization T7 phage that it is characterized in that presenting antisense peptide increases in coli strain BL5403 (available from Novagen company), gather in the crops phage according to a conventional method, measure the titre of phage, titre is pressed the pfu/ml counting, equals " plaque number * extension rate * 10 " on the numerical value.Adjusting phage concentration is 10
14Pfu/ml adds formaldehyde by 1: 4000 volume ratio, and the conventional method deactivation gets type i diabetes antisense peptide vaccine.
765 nucleotide of described antisense DNA total length.255 amino acid residues of described antisense peptide total length.
Described antisense peptide is illustrated in the surface of T7 phage.
The following test of the present invention's process:
1, antisense DNA vaccine immunity NOD mice
4 ages in week, female NOD mice was divided into 3 groups at random, i.e. antisense DNA vaccine group, empty plasmid group and positive controls, 20 every group.In the 0th week, 1 week, 3 weeks and the immunity of 8 weeks, vaccine group intramuscular injection antisense DNA vaccine, immunizing dose is 100ug/; Empty plasmid group intramuscular injection empty plasmid PcDNA3.1,100ug/ are only; Positive controls is injecting normal saline only, and 0.1ml/ only.
2, antisense peptide vaccine immunity NOD mice
4 ages in week, female NOD mice was divided into 3 groups at random, i.e. antisense peptide vaccine group, empty phage group and positive controls, 20 every group.In the 0th week, 1 week, 3 weeks and the immunity of 8 weeks, vaccine group subcutaneous injection antisense peptide vaccine, immunizing dose is 10
12Pfu/ only; Empty phage group subcutaneous injection T7 phage, 10
12Pfu/ only; Positive controls is injecting normal saline only, and 0.1ml/ only.
3, the ELISA method detects antisense DNA vaccine, the vaccine-induced antibody of antisense peptide
Different time behind antisense DNA vaccine, antisense peptide vaccine immunity is gathered the immune mouse tail vein, separation of serum, and indirect elisa method detects the special serum IgG level of antisense peptide.Its method is to wrap by 96 hole ELISA Plate with the about 1 μ g of synthetic antisense peptide, and the test serum behind the adding serial dilution adds the peroxidase sheep anti-mouse antibody, washes plate after the reaction 3 times, adds tmb substrate colour developing liquid subsequently, cessation reaction, 450nm wavelength readings.
4, the detection of blood glucose, glucose in urine, body weight
Different time behind antisense DNA vaccine, antisense peptide vaccine immunity, blood glucose, glucose in urine, the body weight level of detection NOD mice.
The present invention compared with prior art has following advantage and effect: process evidence 1) the antisense DNA vaccine has played significant protective effect to the NOD mice; the blood glucose of immune mouse, glucose in urine and body weight all are in normal range; and the NOD mice of positive controls, empty plasmid group all shows blood glucose, glucose in urine raises, the symptom that loses weight.2) the antisense peptide vaccine has played significant protective effect to the NOD mice, and the blood glucose of immune mouse, glucose in urine and body weight all are in normal range, and the NOD mice of positive controls, empty phage group all shows blood glucose, glucose in urine raises, the symptom that loses weight.
The invention solves following technological difficulties:
Type i diabetes is a kind of autoimmune disease, and immune t-cell is a target spot with the β islet cells, causes β islet cells apoptosis.The immunization therapy of antigenic specificity is that a kind of selectivity suppresses and self t cell response that resists of exempting from disease association certainly, is characterized in the regulation and control Th cell of inducing antigen-specific, suppresses from the generation of exempting from disease.Yet the mode of Th1-Th2 immunne response conversion might not suppress the generation of type i diabetes, there are some researches show this method even can quicken the generation of type i diabetes.So; the inventor of this patent novelty on the basis of " molecular recognition theory " and antisense peptide notion takes the lead in proposing " type i diabetes antisense DNA vaccine "; the research thinking of " type i diabetes antisense peptide vaccine "; through concentrating on studies and testing for many years; and paid performing creative labour; successfully made up type i diabetes antisense DNA vaccine; and further make up the antisense peptide vaccine; behind the immunity NOD mice; immune animal has been played significant protective effect; the blood glucose of immune animal; glucose in urine and body weight all are in normal range; and the positive control treated animal shows blood glucose; glucose in urine raises, the symptom that loses weight.Its result is as follows: (one) type i diabetes antisense DNA vaccine is to the protective effect of immune NOD mice
(1) NOD mouse anti antisense peptide antibody test
Respectively at 0,4,8,12,16,20,24,28 weeks gathering mouse tail vein blood, separation of serum as antigen coated plank, detects anti-antisense peptide antibody with indirect elisa method with synthetic antisense peptide.
The anti-antisense peptide antibody titer of table 1 (X ± S)
| 0 week | 4 weeks | 8 weeks | 12 weeks | 16 weeks | 20 weeks | 24 weeks | 28 weeks | |
| Vaccine group empty plasmid group matched group | 1∶1.2±1 1∶1.4±1 1∶1.2±1 | 1∶64±5 1∶2.2±1 1∶1.4±2 | 1∶120±8 1∶3.2±1 1∶2.4±2 | 1∶160±13 1∶2.4±2 1∶2.1±2 | 1∶201±23 1∶3.2±1 1∶1.5±1 | 1∶180±15 1∶2.4±2 1∶1.4±1 | 1∶142±13 1∶2.1±1 1∶3.6±3 | 1∶110±9 1∶2.4±2 1∶21±1 |
Survey is the result show, behind the antisense DNA vaccine immunity NOD mice, induced anti-antisense peptide production of antibodies, and antibody horizontal rises to the highest when 16 weeks, reaches 201 ± 23, compares with other group, significant difference (P＜0.01), begin afterwards to descend gradually, and the empty plasmid group is compared with the antibody horizontal of positive controls, difference does not have significance (P＞0.05).
(2) NOD mouse blood sugar, glucose in urine, body weight detect
Table 2 NOD mouse blood sugar level changes (X ± S mmol/L)
| 0 week | 4 weeks | 8 weeks | 12 weeks | 16 weeks | 20 weeks | 24 weeks | 28 weeks | |
| Vaccine group empty plasmid group positive controls | 3.2±1.2 3.5±1.2 3.0±1.0 | 4.1±1.5 4.8±1.5 4.5±1.2 | 4.5±1.2 5.6±1.2 5.3±1.4 | 5.0±1.4 7.0±1.6 7.8±1.3 | 5.8±1.6 9.8±1.5 10.4±2.4 | 6.2±1.3 12.4±2.1 14.1±5.6 | 3 12.1 ± 5.8, all the other 4.3 ± 1.2 25.0 ± 7.1 28.5 ± 8.4 | 3 14.3 ± 6.5, all the other 4.5 ± 1.5 18 death, 17.8 17 death of all the other 2 averages, all the other are 18.5 ± 6.3 years old |
As can be seen from Table 2, the blood sugar level of positive controls and empty plasmid group surpasses normal value in 16 whens week, and rises to the highlyest when 24 weeks, is respectively 28.5 ± 8.4,25.0 ± 7.1, when 28 weeks, 17,18 NOD dead mouses is arranged respectively.And the vaccine group blood sugar level is in range of normal value substantially, except have 3 the 28th when week blood glucose rise to 14.3 ± 6.5.Illustrate that the antisense DNA vaccine protected immune mouse effectively, avoided the rising of NOD mouse blood sugar level, the effective percentage of antisense DNA vaccine protection NOD mice prevent diabetes reaches 85%.
Table 3 NOD mouse retention sugar level changes
| 0 week | 4 weeks | 8 weeks | 12 weeks | 16 weeks | 20 weeks | 24 weeks | 28 weeks | |
| Vaccine group empty plasmid group positive controls | Negative negative | Negative negative | Negative++ | Negative ++ ++ | Negative ++ +++ | Negative ++++++ | Negative ++ ++ ++ ++ | 3 ++, all the other negative 18 dead 17 death |
As can be seen from Table 3, the level of sugar of positive controls and empty plasmid group raise when 8 weeks, continues afterwards to rise, and reach the highest when 24 weeks ++ ++, when 28 weeks, 17,18 NOD dead mouses are arranged respectively.And the level of sugar of vaccine group is in feminine gender substantially, except have 3 28 when week glucose in urine ++.Illustrate that the antisense DNA vaccine protected immune mouse effectively, avoided the rising of NOD mouse retention sugar level.
Table 4 NOD mice body weight change (X ± S g)
| 0 week | 4 weeks | 8 weeks | 12 weeks | 16 weeks | 20 weeks | 24 weeks | 28 weeks | |
| Vaccine group empty plasmid group positive controls | 22.5±2.3 22.8±2.6 22.2±1.5 | 23.4±2.5 23.7±2.1 23.5±2.0 | 24.6±2.8 24.7±2.3 24.4±2.4 | 25.7±2.4 21.4±3.0 23.3±2.6 | 27.4±3.1 20.4±2.6 21.6±2.4 | 28.0±3.4 16.0±3.0 14.2±2.7 | 28.3±4.1 14.5±4.3 13.3±3.6 | 27.6±3.1 11.2±3.5 10.7±4.3 |
As can be seen from Table 4, before 12 weeks, each organizes the mice body weight all increases gradually, after 12 weeks, positive controls and empty plasmid group mice body weight begin to descend, and when 28 weeks, reduce to minimumly, be respectively 10.7 ± 4.3,11.2 ± 3.5, and the body weight of vaccine group does not obviously descend.Illustrate that the antisense DNA vaccine protected immune mouse effectively, avoided the remarkable decline of NOD mice body weight.(3) detection of NOD mice Th1, Th2 type cytokines
Respectively at 6,12,18,24 weeks collection tail vein, separation of serum, with cytokine IL-1, the IFN-γ of test kit detection Th1 emiocytosis, and the cytokine IL-4 of Th2 emiocytosis, IL-10.
Table 5.IL-1 level changes (X ± S pg/ml)
| 6 weeks | 12 weeks | 18 weeks | 24 weeks | |
| Vaccine group empty plasmid group positive controls | 10±3 23±5 28±5 | 13±7 27±9 31±8 | 8±5 33±4 36±10 | 12±8 31±6 33±7 |
Table 6 IFN-γ level changes (X ± S pg/ml)
| 6 weeks | 12 weeks | 18 weeks | 24 weeks | |
| Vaccine group empty plasmid group positive controls | 8±2 25±8 30±6 | 10±5 29±10 33±8 | 13±3 35±9 42±12 | 9±4 33±7 40±10 |
As can be seen from Table 6, positive controls and the excretory IFN-γ of empty plasmid group level reach the highest when 18 weeks, and compare significant difference, P＜0.05 with vaccine group; And the secretion level of positive controls and empty plasmid group does not have significant difference, P＞0.05.
Table 7 IL-4 level changes (X ± S pg/ml)
| 6 weeks | 12 weeks | 18 weeks | 24 weeks | |
| Vaccine group empty plasmid group positive controls | 34±7 23±5 20±10 | 42±10 19±7 15±7 | 50±15 22±4 23±4 | 47±6 20±4 22±8 |
As can be seen from Table 7, the IL-4 level of vaccine group secretion reaches the highest when 18 weeks, and is significantly higher than empty plasmid group and positive controls, P＜0.05; And the secretion level of positive controls and empty plasmid group does not have significant difference, P＞0.05.
Table 8 IL-10 level changes (X ± S pg/ml)
| 6 weeks | 12 weeks | 18 weeks | 24 weeks | |
| Vaccine group empty plasmid group positive controls | 30±8 23±6 18±5 | 37±12 20±9 22±7 | 45±9 22±5 19±4 | 48±10 18±7 16±2 |
As can be seen from Table 8, the IL-10 level of vaccine group secretion reaches the highest when 18 weeks, and is significantly higher than empty plasmid group and positive controls, P＜0.05; And the secretion level of positive controls and empty plasmid group does not have significant difference, P＞0.05.
From the testing result of above cytokine as can be seen, the Th2 cytokines level of vaccine group secretion is significantly higher than positive controls, and excretory Th1 cytokines level significantly is lower than positive controls, shows that the effect of vaccine makes immunoreation transform to the Th2 type from the Th1 type.
(4) sensitized T cell proliferation inhibition test
1. the preparation of sensitized T cell
Positive controls mice when getting for 26 weeks, disconnected neck is put to death, and aseptic condition separates lymph node down.Cross stainless steel mesh and collect lymphocyte.By centrifugal fragment of tissue and the dead cell removed of Ficoll-Hypaque, lymphocyte is resuspended in complete culture solution, and (RPMI 1640,10%FCS, 100U/ml penicillin, 100mg/ml streptomycin, 1%NaHCO3, the 10mM non essential amino acid, the 20mM L-glutaminate).Remove macrophage by the adherent method of plastic plate.
2. the preparation of radiation splenocyte
Put to death orthoglycemic NOD mice, aseptic condition takes out spleen down.By the centrifugal collection splenocyte of Ficoll-Hypaque, splenocyte is suspended from above-mentioned culture fluid.Splenocyte 3000rad radiation.With the splenocyte behind the synthetic just peptide adding irradiation of 10ug/ml, at 5%CO
2Incubation is 1 day in the incubator.These irradiation cells are as APC.
3. the propagation of sensitized T cell suppresses
T cell (2 * 10 with sensitization
5/ hole), be combined with the APC (5 * 10 of just peptide
5/ hole) hatched altogether 4 days at 37 ℃, 5%CO2 with the anti-antisense peptide serum of variable concentrations or normal NOD mice serum, each sample is done 3 multiple holes.Cultivate to finish preceding 16 hours with 0.5 μ Ci [
3H] the TdR labelling, cultivate end back collecting cell and measure the cpm value with liquid scintillation instrument.
The anti-antisense peptide antibody of table 9 suppresses (cpm) to the propagation of sensitized T cell
| Antibody concentration (%) | ||||
| 0.01 | 0.1 | 1 | 10 | |
| The normal NOD mice of vaccine group | 1287±56 1342±61 | 954±49 1254±45 | 812±53 1316±37 | 357±41 1297±72 |
As can be seen from Table 9, anti-antisense peptide antibody can suppress the propagation of sensitized T cell, and the concentration of inhibitory action and anti-antisense peptide serum is dose dependent, and concentration is high more, and inhibitory action is obvious more; The serum of normal NOD mice does not then have inhibitory action to the propagation of sensitization T.
Above presentation of results; the antisense DNA vaccine has played significant protective effect to immune mouse; the blood glucose of immune mouse, glucose in urine and body weight all are in normal range, and the NOD mice of positive controls and empty plasmid group all shows blood glucose, glucose in urine raises, the symptom that loses weight.After the mechanism of antisense DNA vaccine protective effect is vaccine immunity NOD mice, induced anti-antisense peptide production of antibodies, and suppressed the propagation of pathologic T cell, thereby reduced the damage of beta Cell of islet by antibody.
(2) the antisense peptide vaccine is to the protective effect of immune NOD mice
(1) NOD mouse anti antisense peptide antibody test
Respectively at 0,4,8,12,16,20,24,28 weeks gathering tail vein, separation of serum as antigen coated plank, detects anti-antisense peptide antibody with indirect elisa method with synthetic antisense peptide.
The anti-antisense peptide antibody horizontal variation of table 10 (X ± S)
| 0 week | 4 weeks | 8 weeks | 12 weeks | 16 weeks | 20 weeks | 24 weeks | 28 weeks | |
| The empty phage group of vaccine group positive controls | 1∶1.6±2 1∶1.2±1 1∶1.4±2 | 1∶80±7 1∶2.4±2 1∶1.2±1 | 1∶190±15 1∶3.4±1 1∶2.4±2 | 1∶220±13 1∶2.1±2 1∶2.1±2 | 1∶300±34 1∶2.4±2 1∶3.2±2 | 1∶240±27 1∶3.2±4 1∶2.6±2 | 1∶210±13 1∶3.6±3 1∶2.8±3 | 1∶160±9 1∶2.6±1 1∶2.4±1 |
Testing result shows, has induced anti-antisense peptide production of antibodies behind the antisense peptide vaccine immunity NOD mice, and when the 16th week, the antibody horizontal of vaccine group rises to the highest, 300 ± 34, compare with other group, and significant difference, P＜0.01 descends afterwards to some extent; And the difference of empty phage group and positive controls does not have significance (P＞0.05).
(2) NOD mouse blood sugar, glucose in urine and body weight detect
Table 11 NOD mouse blood sugar level changes (X ± S mmol/L)
| 0 week | 4 weeks | 8 weeks | 12 weeks | 16 weeks | 20 weeks | 24 weeks | 28 weeks | |
| The empty bacteriophage group of vaccine group positive controls | 3.2±1.2 3.1±1.4 2.8±1.0 | 4.3±1.6 4.5±1.5 4.0±1.2 | 4.8±1.4 5.3±1.2 5.6±1.3 | 5.4±1.3 7.8±1.4 8.1±1.3 | 6.0±1.4 10.2±1.3 10.7±2.1 | 6.5±1.3 14.6±2.8 15.2±5.4 | 3 13.1 ± 4.5, all the other 6.2 ± 1.3 24.7 ± 6.1 25.5 ± 7.4 | 3 17.6 ± 5.8, all the other 4.6 ± 1.8 16 dead 17 death |
As can be seen from Table 11, the blood sugar level of positive controls and empty phage group surpasses normal value in 16 whens week, rises to the highlyest when 24 weeks, is respectively 25.5 ± 7.4,24.7 ± 6.1, when 28 weeks, 17,16 NOD dead mouses is arranged respectively.And the vaccine group blood sugar level is in range of normal value substantially, except have 3 the 24th when week blood glucose rise to 13.1 ± 4.5, rise to 17.6 ± 5.8 during in the 28th week.Illustrate that the antisense peptide vaccine protected immune mouse effectively, avoided the rising of NOD mouse blood sugar level, protection efficient reaches 85%.
Table 12 NOD mouse retention sugar level changes
| 0 week | 4 weeks | 8 weeks | 12 weeks | 16 weeks | 20 weeks | 24 weeks | 28 weeks | |
| The empty phage group of vaccine group positive controls | Negative negative | Negative negative | Negative++ | Negative ++ ++ | Negative ++++++ | Negative ++++++ | Negative ++ ++ ++ ++ | 3 ++, all the other negative 16 dead 17 death |
As can be seen from Table 12, the level of sugar of positive controls and empty phage group raise when 8 weeks, and continue afterwards to rise, and when 24 weeks, reach the highest, ++ ++, when 28 weeks, 17,16 NOD dead mouses are arranged respectively.And the level of sugar of vaccine group is in feminine gender substantially, except have 3 NOD mices 28 when week glucose in urine ++.Illustrate that the antisense peptide vaccine protected immune mouse effectively, avoided the rising of NOD mouse retention sugar level.
Table 13 NOD mice body weight change (X ± S g)
| 0 week | 4 weeks | 8 weeks | 12 weeks | 16 weeks | 20 weeks | 24 weeks | 28 weeks | |
| The empty phage group of vaccine group positive controls | 21.2±1.2 21.5±2.2 21.0±1.3 | 22.1±2.0 22.4±2.3 22.1±2.2 | 23.5±2.4 23.2±2.1 23.3±2.0 | 24.6±2.2 23.0±2.4 23.2±2.1 | 25.4±3.1 22.7±2.6 22.5±2.4 | 26.0±3.4 21.9±2.4 21.2±2.8 | 26.3±4.1 20.5±4.3 19.3±3.6 | 26.1±3.6 16.5±4.1 15.2±3.5 |
As can be seen from Table 13, before 12 weeks, each organizes the mice body weight all increases gradually, after 12 weeks, positive controls and empty phage group mice body weight begin to descend, and when 28 weeks, reduce to minimumly, be respectively 15.2 ± 3.5,16.5 ± 4.1, and the body weight of vaccine group does not obviously descend.Illustrate that the antisense peptide vaccine protected immune mouse effectively, avoided the remarkable decline of NOD mice body weight.
(3) detection of NOD mice Th1, Th2 type cytokines
Respectively at 6,12,18,24 weeks collection tail vein, separation of serum, with cytokine IL-1, the IFN-γ of test kit detection Th1 emiocytosis, and the cytokine IL-4 of Th2 emiocytosis, IL-10.
Table 14 1L-1 level changes (X ± S pg/ml)
| 6 weeks | 12 weeks | 18 weeks | 24 weeks | |
| The empty phage group of vaccine group positive controls | 8±2 23±5 20±3 | 11±4 28±7 31±6 | 9±2 37±6 40±10 | 13±4 30±5 35±8 |
As can be seen from Table 14, positive controls and the excretory IL-1 level of empty phage group reach the highest when 18 weeks, and compare significant difference, P＜0.05 with vaccine group; And the secretion level of positive controls and empty phage group does not have significant difference, P＞0.05.
Table 15 IFN-γ level changes (X ± S pg/ml)
| 6 weeks | 12 weeks | 18 weeks | 24 weeks | |
| The empty phage group of vaccine group positive controls | 4±1 27±6 30±8 | 6±1 29±8 33±10 | 5±2 37±7 42±14 | 7±2 33±5 36±8 |
As can be seen from Table 15, positive controls and the excretory IFN-γ of empty phage group level reach the highest when 18 weeks, and compare significant difference, P＜0.05 with vaccine group; And the secretion level of positive controls and empty phage group does not have significant difference, P＞0.05.
Table 16 IL-4 level changes (X ± S pg/ml)
| 6 weeks | 12 weeks | 18 weeks | 24 weeks | |
| The empty phage group of vaccine group positive controls | 35±7 10±3 12±4 | 42±9 13±5 11±3 | 57±12 16±7 15±4 | 43±7 12±4 10±3 |
As can be seen from Table 16, the IL-4 level of vaccine group secretion reaches the highest when 18 weeks, and is significantly higher than sky phage group and positive controls, P＜0.05; And the secretion level of positive controls and empty phage group does not have significant difference, P＞0.05.
Table 17 IL-10 level changes (X ± S pg/ml)
| 6 weeks | 12 weeks | 18 weeks | 24 weeks | |
| The empty phage group of vaccine group positive controls | 30±6 8±2 10±3 | 37±12 12±4 13±5 | 45±9 11±3 9±4 | 48±10 7±2 11±3 |
As can be seen from Table 17, the IL-10 level of vaccine group secretion reaches the highest when 18 weeks, and is significantly higher than sky phage group and positive controls, P＜0.05; And the secretion level of positive controls and empty phage group does not have significant difference, P＞0.05.
Above cytokines measurement result shows, the excretory Th2 cytokines of antisense peptide vaccine immunity group level is significantly higher than positive controls, and excretory Th1 cytokines level significantly is lower than positive controls, shows that the effect of antisense peptide vaccine makes immunoreation transform to the Th2 type from the Th1 type.
(4) sensitized T cell proliferation inhibition test
1. the preparation of sensitized T cell
Positive controls mice when getting for 26 weeks, disconnected neck is put to death, and aseptic condition separates lymph node down.Cross stainless steel mesh and collect lymphocyte.By centrifugal fragment of tissue and the dead cell removed of Ficoll-Hypaque, lymphocyte is resuspended in complete culture solution, and (RPMI 1640,10%FCS, 100U/ml penicillin, 100mg/ml streptomycin, 1%NaHCO3, the 10mM non essential amino acid, the 20mM L-glutaminate).Remove macrophage by the adherent method of plastic plate.
2. the preparation of radiation splenocyte
Put to death orthoglycemic NOD mice, aseptic condition takes out spleen down.By the centrifugal collection splenocyte of Ficoll-Hypaque, splenocyte is suspended from above-mentioned culture fluid.Splenocyte 3000rad radiation.With the splenocyte behind the synthetic just peptide adding irradiation of 10ug/ml, incubation is 1 day in the 5%CO2 incubator.These irradiation cells are as APC.
3. the propagation of sensitized T cell suppresses
T cell (2 * 10 with sensitization
5/ hole), the serum of the anti-antisense peptide serum of the APC (5 * 105/ hole) that is combined with just peptide and variable concentrations or normal not immune NOD mice hatched 4 days altogether at 37 ℃, 5%CO2, each sample is done 3 multiple holes.Cultivate to finish preceding 16 hours with 0.5 μ Ci [
3H] the TdR labelling, cultivate to finish the back harvesting with liquid scintillation instrument measure [
3H] the incorporation efficiency cpm of TdR.
The anti-antisense peptide antibody of table 18 suppresses (cpm) to the propagation of sensitized T cell
| Antibody concentration (%) | ||||
| 0.01 | 0.1 | 1 | 10 | |
| The normal NOD mice of vaccine group | 1120±64 1211±58 | 896±43 1109±47 | 772±52 1206±35 | 292±48 1157±42 |
As can be seen from Table 18, anti-antisense peptide serum can suppress the propagation of sensitized T cell, and the concentration of inhibitory action and anti-antisense peptide serum is dose dependent, and concentration is high more, and inhibitory action is obvious more; The serum of normal NOD mice does not then have inhibitory action to the propagation of sensitization T.
Above presentation of results; the antisense peptide vaccine has played significant protective effect to immune mouse; the blood glucose of immune mouse, glucose in urine and body weight substantially all are in normal range, and the NOD mice of positive controls and empty phage group all shows blood glucose, glucose in urine raises, the symptom that loses weight.After the mechanism of antisense peptide vaccine protective effect is vaccine immunity NOD mice, induced anti-antisense peptide production of antibodies, and suppressed the propagation of pathologic T cell, thereby reduced the damage of beta Cell of islet by antibody.
The specific embodiment
Below in conjunction with implementing the present invention is described further, is not limited thereto but hold within the present invention.
Embodiment 1
The structure of A, type i diabetes antisense DNA:
Select clearly and the type i diabetes relevant epi-position of falling ill in early days, and infer out by complementary characteristic
The DNA sequence of its antisense peptide and encoding antisense peptide, specifically: at the antisense dna sequence catcaagttcaagttttcgatcaagtc of insulin C TL epi-position, the antisense dna sequence gctccttccgctgtccatcaagtccaaggtttccatcaagtcgct of proinsulin T epi-position, the antisense dna sequence gtccaacaagctcctgctgctgctggtcatgctcctgtcgtccctgctcaaaaatg aaacttggtcatggtcctggtttcgtcggtcttcaagtcttccaagtcaaagctcc tcatcaaaaagtcttcgctcctgatgtcttcgctgatcaagtccatttcgtccatg tcgtccaagtcgctgctttcgtcggtcctgtcgtcgtcgtccttcctcatgctgtc ttcggtcaaggtggtcaatcatgatcatcatgctgctgctttccctcatcatctt of the antisense dna sequence caattccttgtcggtggtggtggtgtcggtgctggtgtcgctgct of two T epi-positions of receptor type Protein-tyrosine-phosphatase sample protein I A-2 β and catcctgctgtcgtcgtcggtcaacatggtcaagcccctgtcttc and B epi-position, the antisense dna sequence caaaaacaagtcggt ttccaagtcggt of the associated protein IGRP T of islets of langerhans G-6-Pase catalytic subunit epi-position, between the antisense dna sequence catcatgtccttcaac aagatgtccatcat of the antisense dna sequence ggtggtggtggtgt cgctgatcatcatcctaaaaaagtc of glutamate decarboxylase GAD65T epi-position and the antisense dna sequence gtctctgtccaacaaggtgctcaattcgtcgctgatgctgctggtaaattccttgg tcatcttgctcctggtcctggtggtgctggtgtcttcgctgtcgctgct of B epi-position and CTL epi-position, encoding gene with introns GPGPG links to each other, and add a start codon before the antisense dna sequence of first epi-position, ' end adds a termination codon to the antisense dna sequence 3 of last epi-position, after synthesizing, insert the PcDNA3.1 carrier, finally be built into the type i diabetes antisense DNA.
The preparation of B, type i diabetes antisense DNA vaccine
Get the type i diabetes antisense DNA that the A process makes up, and increase with existing processes, purification, be about to type i diabetes antisense DNA transformed into escherichia coli, 37 ℃ of incubated overnight, picking list bacterium colony, be inoculated in 5ml LB fluid medium, be cultured to exponential phase at 37 ℃ of shaking tables, go to 600ml LB fluid medium, continue to be cultured to bacterium liquid muddiness, centrifugal results bacterial precipitation carries out a large amount of extractions of plasmid DNA by U.S.'s Sa nurse Brooker " molecular cloning experiment guide " (third edition) alkaline lysis, purification, press desired concn with aseptic dissolved in distilled water DNA, obtain type i diabetes antisense DNA vaccine.
Embodiment 2
The structure of A, type i diabetes antisense peptide
Select clearly and the type i diabetes relevant epi-position of falling ill in early days, and infer out the DNA sequence of its antisense peptide and encoding antisense peptide by complementary characteristic, specifically: at the antisense dna sequence catcaagttcaagttttcgatcaagtc of insulin C TL epi-position, the antisense dna sequence gctccttccgctgtccatcaagtccaaggtttccatcaagtcgct of proinsulin T epi-position, the antisense dna sequence gtccaacaagctcctgctgctgctggtcatgctcctgtcgtccctgctcaaaaatg aaacttggtcatggtcctggtttcgtcggtcttcaagtcttccaagtcaaagctcc tcatcaaaaagtcttcgctcctgatgtcttcgctgatcaagtccatttcgtccatg tcgtccaagtcgctgctttcgtcggtcctgtcgtcgtcgtccttcctcatgctgtc ttcggtcaaggtggtcaatcatgatcatcatgctgctgctttccctcatcatctt of the antisense dna sequence caattccttgtcggtggtggtggtgtcggtgctggtgtcgctgct of two T epi-positions of receptor type Protein-tyrosine-phosphatase sample protein I A-2 β and catcctgctgtcgtcgtcggtcaacatggtcaagcccctgtcttc and B epi-position, the antisense dna sequence caaaaacaagtcggt ttccaagtcggt of the associated protein IGRP T of islets of langerhans G-6-Pase catalytic subunit epi-position, between the antisense dna sequence catcatgtccttcaac aagatgtccatcat of the antisense dna sequence ggtggtggtggtgt cgctgatcatcatcctaaaaaagtc of glutamate decarboxylase GAD65T epi-position and the antisense dna sequence gtctctgtccaacaaggtgctcaattcgtcgctgatgctgctggtaaattccttgg tcatcttgctcctggtcctggtggtgctggtgtcttcgctgtcgctgct of B epi-position and CTL epi-position, coded sequence (introns) with three aminoacid AAY links to each other, and add a start codon before the antisense dna sequence of first epi-position, ' end adds a termination codon to the antisense dna sequence 3 of last epi-position, after synthesizing, insert 3 ' ends of T7 phage DNA capsid protein B10 encoding gene, be built into the reorganization T7 phage of presenting antisense peptide, obtain the type i diabetes antisense peptide.
The preparation of B, type i diabetes antisense peptide vaccine
With existing processes to the phage of presenting antisense peptide increase in a large number, purification, that is: with after a large amount of cultivations of coli strain BL5403, add phage, 37 ℃ of shaking tables cultivate about 3 hours limpid to bacterium liquid, as seen filament, centrifugal collection supernatant, add PEG precipitation phage, centrifugal collecting precipitation adds the phage lysate, jolting dissolving phage, the centrifuging and taking supernatant, measure the titre of phage according to a conventional method, titre is pressed the pfu/ml counting, equals " plaque number * extension rate * 10 " on the numerical value.Adjusting phage concentration is 10
14Pfu/ml, by 1: 4000 volume ratio adding formaldehyde, 37 ℃, 100 rev/mins shaking tables were cultivated deactivation in 4 days, measure the titre of phage, and titre is zero, adds in the sodium sulfite and formaldehyde, gets type i diabetes antisense peptide vaccine.
Claims (8)
1, a kind of type i diabetes vaccine, it is characterized in that antisense dna sequence catcaagttcaagttttcgatcaagtc by insulin C TL epi-position, the antisense dna sequence gctccttccgctgtccatcaagtccaaggtttccatcaagtcgct of proinsulin T epi-position, the antisense dna sequence gtccaacaagctcctgctgctgctggtcatgctcctgtcgtccctgctcaaaaatg aaacttggtcatggtcctggtttcgtcggtcttcaagtcttccaagtcaaagctcc tcatcaaaaagtcttcgctcctgatgtcttcgctgatcaagtccatttcgtccatg tcgtccaagtcgctgctttcgtcggtcctgtcgtcgtcgtccttcctcatgctgtc ttcggtcaaggtggtcaatcatgatcatcatgctgctgctttccctcatcatctt of the antisense dna sequence caattccttgtcggtggtggtggtgtcggtgctggtgtcgctgct of two T epi-positions of receptor type Protein-tyrosine-phosphatase sample protein I A-2 β and catcctgctg tcgtcgtcggtcaacatggtcaagcccctgtcttc and B epi-position, the antisense dna sequence caaaaacaagtcggtttccaagtcggt of the associated protein IGRP T of islets of langerhans G-6-Pase catalytic subunit epi-position, the antisense dna sequence gtctctgtccaacaaggtgct caattcgtcgctgatgctgctggtaaattccttggtcatcttgctcctggtcctgg tggtgctg gtgtcttcgctgtcgctgct of the antisense dna sequence ggtggtggtggtgtcgctg atcatcatcctaaaaaagtc of glutamate decarboxylase GAD65T epi-position and B epi-position and the antisense dna sequence catcatgtccttcaacaagatgtccatcat of CTL epi-position, and be connected in introns encoding gene and expression vector between each antisense dna sequence.
2, type i diabetes vaccine according to claim 1 is characterized in that described introns encoding gene is the encoding gene of GPGPG or AAY.
3, type i diabetes vaccine according to claim 1 is characterized in that described expression vector is eucaryon pCDNA3.1 or T7 phage.
4, a kind of construction method of the vaccine of type i diabetes according to claim 1, it is characterized in that antisense dna sequence catcaagttcaagttttcgatcaagtc in insulin C TL epi-position, the antisense dna sequence gctccttccgctgtccatcaagtccaaggtttccatcaagtcgct of proinsulin T epi-position, the antisense dna sequence gtccaacaagctcctgctgctgctggtcatgctcctgtcgtccctgctcaaaaatg aaacttggtc atggtcctggtttcgtcggtcttcaagtcttccaagtcaaagctcctcatcaaaaa gtcttcgctcctgatgtcttcgctgatcaagtccatttcgtccatgtcgtccaagt cgctgctttcgtcggtcctgtcgtcgtcgtccttcctcatgctgtcttcggtcaag gtggtcaatcatgatcatcatgctgctgctttccctcatcatctt of the antisense dna sequence caattcc ttgtcggtggtggtggtgtcggtgctggtgtcgctgct of two T epi-positions of receptor type Protein-tyrosine-phosphatase sample protein I A-2 β and catcctgctgtcgtcgtcggtc aacatggtcaagcccctgtcttc and B epi-position, the antisense dna sequence caaaaacaagtcggtttccaagtcggt of the associated protein IGRP T of islets of langerhans G-6-Pase catalytic subunit epi-position, between the antisense dna sequence gtctctgtccaacaaggtgctcaattcgtcgctgatgctgctggtaaattccttgg tcatct tgctcctggtcctggtggtgctggtgtcttcgctgtcgctgct of the antisense dna sequence ggtggtggtggtgtcgctgatcatcatcctaaaaaagtc of glutamate decarboxylase GAD65T epi-position and B epi-position and the antisense dna sequence catcatgtccttcaacaagatgtccatcat of CTL epi-position, link to each other with the introns encoding gene, after synthesizing, insert eucaryon pCDNA3.1 plasmid, obtain the type i diabetes antisense DNA, perhaps insert 3 ' end of T7 phage DNA capsid protein B10 encoding gene, the reorganization T7 phage that obtains presenting antisense peptide.
5, the construction method of type i diabetes vaccine according to claim 1 is characterized in that described introns encoding gene is the encoding gene of GPGPG or AAY.
6, the construction method of type i diabetes vaccine according to claim 1 is characterized in that the aminoacid sequence of described antisense peptide is:
HQVQGFDQVGPGPGAPFAAHQVQGFHQVAGPGPGQFLVGGGGVGAGVAAGPGPGHPAVVVGQHGQAPVFGPGPGVQQAPAAAGHAPVVPAQKMKLGHGPGFVGLQVFQVKAPHQKVFAPDVFADQVHFVHVVQVAAFVGPVVVALPHAVFGQGGQHHDHHAAAFPHHLGPGPGQKQVGFQVGGPGPGGGGGVADHHPKKVGPGPGVSVQQGAQFVADAAGKFLGHLAPGPGGAGVFAVAAGPGPGHHVLQQDVHH。
7, a kind of method for preparing the described type i diabetes vaccine of claim 1, after it is characterized in that the type i diabetes antisense DNA increased it with existing processes in escherichia coli, through existing alkaline lysis method of extracting, purification, press desired concn with aseptic dissolved in distilled water DNA, obtain type i diabetes antisense DNA vaccine.
8, a kind of method for preparing the described type i diabetes vaccine of claim 1, after the reorganization T7 phage that it is characterized in that presenting antisense peptide increases in coli strain BL5403, gather in the crops phage according to a conventional method, measure the titre of phage, titre is pressed the pfu/ml counting, equal " plaque number * extension rate * 10 " on the numerical value, adjusting phage concentration is 10
14Pfu/ml adds formaldehyde by 1: 4000 volume ratio, uses the conventional method deactivation, gets type i diabetes antisense peptide vaccine.
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| CN101974071A (en) * | 2010-09-26 | 2011-02-16 | 中国人民解放军第三军医大学 | Insulin B chain HLA-A*0201 restrictive CTL epitope modified peptide ligand and acquisition method and application thereof |
| CN102060910A (en) * | 2010-11-12 | 2011-05-18 | 中国人民解放军第三军医大学 | HLA (Human Leukocyte Antigen)-A*0201 limitation CTL (Cytotoxic T Lymphocyte) epitope of zinc translocator and application thereof |
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| US5674978A (en) * | 1990-09-21 | 1997-10-07 | The Regents Of The University Of California | Peptides derived from glutamic acid decarboxylase |
| US6096314A (en) * | 1994-10-07 | 2000-08-01 | Yeda Research And Development Co. Ltd. | Peptides and pharmaceutical compositions comprising them |
| US6171590B1 (en) * | 1998-09-30 | 2001-01-09 | Corixa Corporation | Chemokine receptor peptide for inducing an immune response |
| CN100352502C (en) * | 2004-12-29 | 2007-12-05 | 中国药科大学 | Recombinant protein and recombinant gene capable of preventing and curing I type diabetes mellitus through immunity |
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