CN1468957A - Plasmid used as human therapeutic vaccine adjuvant - Google Patents

Abstract

The present invention provides one kind of high-copied and stable plasmid, which is prepared through combining oligonucleotide sequence ODN with immunostimulation to specific vector. The ODN inside the plasmid can stimulate immune cell without being modified with thiophosphate. The plasmid may be used as immune adjuvant and especially as adjuvant of therapeutic vaccine for treating hepatitis B and other chronic contagious diseases and tumors.

Description

A kind of plasmid as human therapeutic vaccine adjuvant

Invention field

The invention belongs to genetic engineering, relate to vaccine and nucleic acid molecule.The oligonucleotide sequence (ODN) that the present invention relates to have immunostimulation combines with specific support, the high copy of preparation, plasmid that can stable existence, this plasmid can be as vaccine adjuvant, especially can be as the adjuvant of treatment chronic infectious disease and tumor treatment vaccine.

Background of invention

Recent discovers, what derive from bacterial genomes DNA is that the oligonucleotide sequence (ODN) of core has very strong immunostimulation with non-methylated cytosine(Cyt) and guanine (CpG), can effectively activate mammiferous B cell, immunocytes such as NK cell and dendritic cell, these oligonucleotide sequences are except the CpG core, the nucleotide sequence of CpG both sides is also extremely important for playing immunostimulation, and determine the species specificity of its effective stimulus, such as, this class oligonucleotide that can activate the mouse immune cell does not have activity for people's immunocyte, and the oligonucleotide that can effectively activate people's immunocyte to the hormesis of mouse immune cell not as good as hormesis to people's immunocyte.The nucleotide sequence of CpG both sides also determines the immunostimulatory activity that they are different, finding two kinds at present is the ODN sequence of core with CpG, its 26S Proteasome Structure and Function feature is respectively: K type ODN, the TCGTT sequence that contains one or several copies, its 5 ' and 3 ' end is thymus pyrimidine T, and whole nucleotide chain must be modified with thiophosphoric acid, thiophosphoric acid is modified not only can improve the resistance of ODN to the DNA enzyme greatly, and can improve its immunostimulation greatly, representational K type ODN that people's immunocyte is worked is as 5 '-TCGTCGTTTTGTCGTTTTGTCGTT-3 '.D type ODN, the oligonucleotide sequence that can form palindrome that contains single copy, wherein contain CpG, link to each other with phosphodiester bond, immunostimulatory activity significantly reduces if modify then with thiophosphoric acid, this type ODN 3 ' end mostly is poly guanine G, and representational D type ODN that people's immunocyte is worked is as 5 '-GGTGCATCGATGCAGGGGGG-3 '.This ODN of two types has different immunostimulation function: but K type ODN polyclone activates the B cell, promotes monocyte propagation, secretion IL-6 and is divided into scavenger cell; And D type ODN can stimulate NK emiocytosis IFN-γ, strengthens the killing activity of NK cell, promotes monocyte to be divided into and has the active dendritic cell of efficient angtigen presentation.

Because its energy effective stimulus and activating immune cell, above-mentioned ODN has application promise in clinical practice for the new human immunological adjuvant of exploitation.With K type or D type ODN is that the experimentation on animals of the adjuvant mouse, rhesus monkey and the chimpanzee that carry out shows; ODN is enhancement antigen inductive body fluid and cellullar immunologic response significantly; and security is good, and D type ODN is better than K type ODN as the obtained immune protective effect of vaccine adjuvant.

But present ODN invention all has some problems.(people CpG oligodeoxynucleotide is to the influence of plasmid dna immunization stimulating activity such as the invention that has, 2002 the 82nd the 7th phases of volume of Chinese Medical Journal) make up the plasmid pMini that contains kalamycin resistance gene and puc plasmid replication starting point earlier, in this plasmid, insert 9 and 16 copies more respectively and can stimulate the K type oligodeoxynucleotide CpG 2006 of people's immunocyte, discovery is along with the increase of inserting CpG 2006 copy numbers, plasmid strengthens gradually in the ability of external activation mouse boosting cell secretory antibody, but the ability that induces IFN-γ reduces gradually.With the adjuvant immunity mouse of plasmid as hepatitis B surface antigen HBsAg, can obviously improve the antibody horizontal of mouse, its function is for humoral immunoresponse(HI) promoter action is arranged, and it is not obvious for the promoter action of cellullar immunologic response, in addition, K type oligodeoxynucleotide has only the modification through thiophosphoric acid that immunostimulation is well just arranged, and plasmid duplicates in the intestinal bacteria bacillus, can not carry out thiophosphoric acid and modify, this also greatly reduces its immunostimulation.Use kalamycin resistance gene as the protokaryon selection markers, though meet the protokaryon selection markers requirement of U.S. FDA about the human plasmid, but the molecular weight of kalamycin resistance gene itself is bigger, and inserting the plasmid molecule of 16 copy oligodeoxynucleotides, to reach 2150 deoxynucleotides right.

The oligodeoxynucleotide that contains CpG with synthetic adds the business-like hepatitis B vaccine inoculation orangutan extremely low to this vaccine reaction, the result adds seroconversion rate of serum antibody and the antibody titers that the CpG oligodeoxynucleotide can significantly improve orangutan showing in vaccine, shows that the CpG oligodeoxynucleotide may have important use to be worth as treating hepatitis B as vaccine adjuvant.

Contain the oligodeoxynucleotide of CpG as leishmania vaccine adjuvant immunity rhesus monkey with K type and D type respectively, detect of the reaction of rhesus monkey peripheral blood lymphocytes to LA, K type CpG is the peripheral blood lymphocytes energy secreting high levels IL-6 of adjuvant immunity monkey as a result, and D type CpG is the peripheral blood lymphocytes energy secreting high levels IFN-γ of adjuvant immunity monkey.Attack the rhesus monkey of immunity with the leishmania that lives, D type CpG is that to be better than K type CpG be the immune protective effect of adjuvant for the immune protective effect of adjuvant as a result.More than show with the oligodeoxynucleotide that contains CpG and can improve the immune efficient of vaccine greatly with the vaccine of conventional adjuvant immunity weak effect or as the immunological adjuvant of therapeutic vaccine good development prospect is arranged for some as vaccine adjuvant in the experiment of primate.

Relevant document is with plasmid as dna vaccination, but not as simple immunological adjuvant, perhaps use artificial synthetic oligodeoxynucleotide as vaccine adjuvant, find to make up the plasmid that contains multiple copied immunostimulation oligodeoxynucleotide, with report or patent as vaccine adjuvant.

Goal of the invention

Purpose of the present invention at first is to provide a kind of oligodeoxynucleotide that can be used as vaccine adjuvant; Next provides a kind of simple, stable oligodeoxynucleotide for preparing, and a kind of exist with the plasmid form, plasmid that can use in tumour and chronic infectious disease therapeutic vaccine especially is provided; Purpose of the present invention comprises that also providing non-just modifies effectively immune cell activated through thiophosphoric acid, and has reached multiple copied in plasmid, the therapeutic vaccine adjuvant of more effective activating cells immunne response.Technical scheme

The present invention has used 3 kinds of D type ODN that can stimulate the human body cell immunne response as the adjuvant unit, has been built into a kind of plasmid that contains multiple copied D type ODN.Select D type ODN and do not select K type ODN as immunostimulation unit, be based on following reason: 1, D type ODN can and K type ODN can not be divided into dendritic cell by the effective stimulus monocyte, and dendritic cell is the strongest antigen presenting cell, is even more important for the activated cell immunne response.2, D type ODN can and K type ODN can not activate the NK cell and promote its secretion of gamma-IFN, FN-γ can induce static property CD4+T lymphocyte to differentiation of Thl type and the lymphocytic cytotoxic activity of enhancing CD8+T, also can directly suppress the propagation of virus and the division of tumour cell.And chronic viral infection person and tumour patient all show as the cellullar immunologic response incapability, and strengthening its cellullar immunologic response is to generally acknowledge effective immunotherapy method.3, K type ODN needs thio-modification just effective, and this plasmid for self-replication in bacterium can not be realized.4, D type ODN also can effectively strengthen humoral immunoresponse(HI), improves the antibody response of body.

The D type ODN that we select comprises following 3 kinds, but equal activation of human immunocyte.5’-GGTGCATCGATGCAGGGGGG-3’，5’-GGGGGACGATCGTCGGGGGG-3，5’-ACCGATAACGTTGCCGGTGACGGCACCACG-3，

D type ODN of the present invention can promote NK emiocytosis IFN-γ and promote that monocyte is divided into dendritic cell that therefore can strengthen cellullar immunologic response, the adjuvant of more suitable being used for the treatment of property vaccine has using value for chronic viral infection and tumour.As the protokaryon selection markers, molecular weight is little with the supF gene, insert 30 the copy oligodeoxynucleotides plasmid molecule by 1796 deoxynucleotides to forming.Technique effect

The plasmid pCG30 that makes up contains 3 kinds verifiedly has the D type oligodeoxynucleotide of efficient activation to people's immunocyte, and totally 30 copies have overcome with synthetic oligodeoxynucleotide unfavorable as adjuvant as adjuvant with it.The synthetic oligodeoxynucleotide is a single stranded DNA, even terminal deoxynucleoside is modified through thiophosphoric acid, also easily be degraded in vivo, modify the immuno-stimulating effect that greatly reduces D type oligodeoxynucleotide and whole strand is carried out thiophosphoric acid, and plasmid is a double-stranded DNA, stability in vivo improves greatly, in addition, as adjuvant, make preparation process very simple with this plasmid, greatly reduce cost.

The oligodeoxynucleotide of multiple copied is concentrated on energy self-replicating and purifying plasmid easily in intestinal bacteria, immunostimulatory activity is not only arranged, and good stability, with low cost, very big using value is arranged.Contain the nucleotide sequence that multiple copied has immunostimulatory activity, can be used as the vaccine for man adjuvant, especially can be used as chronic viral infection and tumor treatment vaccine adjuvant; Such plasmid does not contain any viral associated nucleic acid sequences, and security is good; Can screen with tsiklomitsin, meet the requirement of U.S. FDA about the relevant screening sign of human plasmid; The molecular weight of this plasmid is little, adjuvant component content height.

The embodiment material

Plasmid pcDM8, pcDNA3, intestinal bacteria MC1061/P3 are American I nvitrogen company product; T cloning vector pMD18T is that the product Taqase of Takara company archaeal dna polymerase, restriction endonuclease, T4 dna ligase are Sangon biotechnology company limited product; Bacillus coli DH 5 alpha is the conventional preservation in this chamber; Single stranded DNA comprises that the DNA cloning primer is synthetic by Sangon biotechnology company limited.Method

The replication initiation subsequence of pcr amplification puc plasmid

Primer 1:5 '-GAATTCAAGCTTCTGCAGGCGGCGAGCGGTATCAG-3 '

Primer 2: 5 '-CTCGAGCTAGCGTCGACTGCTGCTTGCAAAC-3 '

Set up following reaction system at 0.5 milliliter of centrifuge tube:

pcDNA3 10ng

10 * Taqase damping fluid, 5 μ l

Primer 1 (5mM) 2 μ l

Primer 2 (5mM) 2 μ l

dNTP(10mM) 2μl

MgCl ₂(25mM) 2μl

Taqase(2U/μl) 0.5μl

Replenish and twoly to boil off ionized water, react at the PE2400PCR thermal cycler to total reaction volume 50 μ l: 94 ℃ of sex change earlier 2 minutes, 94 ℃ of sex change are 40 seconds again, annealed 40 seconds for 60 ℃, 72 ℃ were extended 50 seconds, and carried out 30 circulations altogether, 72 ℃ were extended 5 minutes then, and temperature is reduced to 4 ℃ and finished reaction.The PCR reaction product is carried out agarose gel electrophoresis, and the purpose band is reclaimed.

Goal gene is connected with T cloning vector pMD18T

Set up following reaction system at 0.5 milliliter of centrifuge tube:

The PCR reaction product 6 μ l that reclaim

5 * T4DNA ligase enzyme damping fluid, 2 μ l

T cloning vector pMD18T 1 μ l

T4 dna ligase (2U/ μ l) 1 μ l

Mixing, place 16 ℃ of water-baths 16 hours, get 5 μ l transformed competence colibacillus bacillus coli DH 5 alphas, the amicillin resistance colony inoculation that obtains is in the LB liquid nutrient medium that contains penbritin 100 μ g/ml, extracting recombinant plasmid again carries out enzyme and cuts evaluation, called after pMD18T-puc ori.Pcr amplification tRNA suppresses sub-supF gene

Primer 3:5 '-GAATTCGGATCCGATTACCGCGGTCTTTCTC-3 '

Primer 4:5 '-CTCGAGTCTAGAAAGCAGGGAGCAGATTC-3 '

Suppress sub-supF gene with plasmid pcDM8 for template amplification tRNA; Amplified production reclaims the back and is connected with T cloning vector pMD18T; Obtains recombinant plasmid pMD18T-supF, the synthetic ODN4:5 ' of the synthetic ODN3:5 ' of synthetic ODN2:5 ' the GCTAGCTCGAGCTGCAGAATTCCCCCCGACGATCGTCCCCCCATCCCCCCCTGCAT CGATGCACCCATCGTGGTGCCGTCACCGGCAACGT-3 ' of synthetic ODN1:5 ' the GTCGACTCTAGAAGCTTGGATCCGGTGCATCGATGCAGGGGGGGATGGGGGGACGA TCGTCGGGGGGACCGAIAACGTTGCCGGTGACGGCAC-3 ' of the splicing of and method is the same. immunostimulation ODN-GTCGACTCTAGAAGCTTG-3 '-GCTAGCTCGAGCTGCAG-3 ' sets up following reaction system at 0.5 milliliter of centrifuge tube:

ODN1(1mM) 1μl

ODN2(1mM) 1μl

10 * Taqase damping fluid, 5 μ l

dNTP(10mM) 2μl

MgCl ₂(25mM) 2μl

Taqase(2U/μl) 0.5μl

Replenish and twoly to boil off ionized water, react at the PE2400PCR thermal cycler to total reaction volume 50 μ l: 94 ℃ of sex change earlier 2 minutes, 94 ℃ of sex change are 40 seconds again, annealed 40 seconds for 60 ℃, 72 ℃ were extended 20 seconds, and carried out 10 circulations altogether, 72 ℃ were extended 5 minutes then, and temperature is reduced to 4 ℃ and finished reaction.Get above-mentioned reaction product 2 μ l, place another 0.5 milliliter of centrifuge tube, add again:

10 * Taqase damping fluid, 5 μ l

ODN3(5mM) 2μl

ODN4(5mM) 2μl

dNTP(10mM) 2μl

MgCl ₂(25mM) 2μl

Taqase(2U/μl) 0.5μl

Replenish and twoly to boil off ionized water, react at the PE2400PCR thermal cycler to total reaction volume 50 μ l: 94 ℃ of sex change earlier 2 minutes, 94 ℃ of sex change are 40 seconds again, annealed 40 seconds for 6 ℃, 72 ℃ were extended 30 seconds, and carried out 35 circulations altogether, 72 ℃ were extended 5 minutes then, and temperature is reduced to 4 ℃ and finished reaction.The PCR reaction product is carried out agarose gel electrophoresis, and the purpose band is reclaimed, and contains 5 D type immunostimulation ODN sequences, and pMD18T is connected with the T cloning vector, obtains recombinant plasmid pMD18T-CpG5.

Plasmid pMD18T-puc ori is digested reaction product agarose gel electrophoresis, the replication initiation subsequence puc ori gene of recovery puc plasmid with restriction endonuclease EcoRI, XhoI; With restriction endonuclease EcoRI, XhoI digestion, the reaction product agarose gel electrophoresis reclaims tRNA and suppresses sub-supF gene with plasmid pMD18T-supF; The pucori gene is connected with the T4 dna ligase with the supF gene, and transformed into escherichia coli intestinal bacteria MC1061/P3 obtains recombinant plasmid puc-supF again.

Puc-supF is with BamHI, EcoRI digestion, and the reaction product agarose gel electrophoresis reclaims linearizing puc-supF; Plasmid pMD18T-CpG5 is with BamHI, EcoRI digestion, and the reaction product agarose gel electrophoresis reclaims CpG ODN; CpG ODN is connected with the T4 dna ligase with linearization plasmid puc-supF, transformed into escherichia coli intestinal bacteria MC1061/P3 obtains recombinant plasmid pCG5 again.

Plasmid pMD18T-CpG5 is with EcoRI, HindIII digestion, and the CpG ODN that obtains inserts EcoRI, the HindIII site of plasmid pCG5, obtains recombinant plasmid pCG10.

Plasmid pMD18T-CpG5 is with HindIII, PstI digestion, and the CpG ODN that obtains inserts HindIII, the PstI site of plasmid pCG10, obtains recombinant plasmid pCG15.

Plasmid pMD18T-CpG5 is with XbaI, XhoI digestion, and the CpG ODN that obtains inserts XbaI, the XhoI site of plasmid pCG15, obtains recombinant plasmid pCG20.

Plasmid pMD18T-CpG5 is with XhoI, SalI digestion, and the CpG ODN that obtains inserts XhoI, the SalI site of plasmid pCG20, obtains recombinant plasmid pCG25.

Plasmid pMD18T-CpG5 is with SalI, NheI digestion, and the CpG ODN that obtains inserts SalI, the NheI site of plasmid pCG25, obtains recombinant plasmid pCG30.PCG30 is the adjuvant plasmid.

This plasmid is made up of 1796 Nucleotide, and (SEQ1) is as follows for the complete nucleotide sequence:

GGATCCGGTG?CATCGATGCA?GGGGGGGATG?GGGGGACGAT?CGTCGGGGGG?ACCGATAACG61 TTGCCGGTGA?CGGCACCACG?ATGGGTGCAT?CGATGCAGGG?GGGGATGGGG?GGACGATCGT121 CGGGGGGGAA?TTCCCCCCGA?CGATCGTCCC?CCCATCCCCC?CCTGCATCGA?TGCACCCATC181 GTGGTGCCGT?CACCGGCAAC?GTATCGGTCC?CCCCGACGAT?CGTCCCCCCA?TCCCCCCCTG241 CATCGATGCA?CCGGATCCAA?GCTTGGATCC?GGTGCATCGA?TGCAGGGGGG?GATGGGGGGA301 CGATCGTCGG?GGGGACCGAT?AACGTTGCCG?GTGACGGCAC?CACGATGGGT?GCATCGATGC361 AGGGGGGGAT?GGGGGGACGA?TCGTCGGGGG?GGAATTCCTG?CAGGCGGCGA?GCGGTATCAG421 CTCACTCAAA?GGCGGTAATA?CGGTTATCCA?CAGAATCAGG?GGATAACGCA?GGAAAGAACA481 TGTGAGCAAA?AGGCCAGCAA?AAGGCCAGGA?ACCGTAAAAA?GGCCGCGTTG?CTGGCGTTTT541 TCCATAGGCT?CCGCCCCCCT?GACGAGCATC?ACAAAAATCG?ACGCTCAAGT?CAGAGGTGGC601 GAAACCCGAC?AGGACTATAA?AGATACCAGG?CGTTTCCCCC?TGGAAGCTCC?CTCGTGCGCT661 CTCCTGTTCC?GACCCTGCCG?CTTACCGGAT?ACCTGTCCGC?CTTTCTCCCT?TCGGGAAGCG721 TGGCGCTTTC?TCATAGCTCA?CGCTGTAGGT?ATCTCAGTTC?GGTGTAGGTC?GTTCGCTCCA781 AGCTGGGCTG?TGTGCACGAA?CCCCCCGTTC?AGCCCGACCG?CTGCGCCTTA?TCCGGTAACT841 ATCGTCTTGA?GTCCAACCCG?GTAAGACACG?ACTTATCGCC?ACTGGCAGCA?GCCACTGGTA901 ACAGGATTAG?CAGAGCGAGG?TATGTAGGCG?GTGCTACAGA?GTTCTTGAAG?TGGTGGCCTA961 ACTACGGCTA?CACTAGAAGA?ACAGTATTTG?GTATCTGCGC?TCTGCTGAAG?CCAGTTACCT1021 TCGGAAAAAG?AGTTGGTAGC?TCTTGATCCG?GCAAACAAAC?CACCGCTGGT?AGCGGTTTTT1081 TTGTTTGCAA?GCAGCAGCTA?GCTCGAGCTG?CAGAATTCCC?CCCGACGATC?GTCCCCCCAT1141 CCCCCCCTGC?ATCGATGCAC?CCATCGTGGT?GCCGTCACCG?GCAACGTATC?GGTCCCCCCG1201 ACGATCGTCC?CCCCATCCCC?CCCTGCATCG?ATGCACCGGA?TCCAAGCTTC?TAGAGTCGAC1261 TCTAGAAGCT?TGGATCCGGT?GCATCGATGC?AGGGGGGGAT?GGGGGGACGA?TCGTCGGGGG1321 GACCGATAAC?GTTGCCGGTG?ACGGCACCAC?GATGGGTGCA?TCGATGCAGG?GGGGGATGGG1381 GGGACGATCG?TCGGGGGGGA?ATTCCTGCAG?CTCGAGCTGC?AGAATTCCCC?CCGACGATCG1441 TCCCCCCATC?CCCCCCTGCA?TCGATGCACC?CATCGTGGTG?CCGTCACCGG?CAACGTATCG1501 GTCCCCCCGA?CGATCGTCCC?CCCATCCCCC?CCTGCATCGA?TGCACCGGAT?CCAAGCTTCT1561 AGACAGCTGG?ATTACCGCGG?TCTTTCTCAA?CGTAACACTT?TACAGCGGCG?CGTCATTTGA1621 TATGATGCGC?CCCGCTTCCC?GATAAGGGAG?CAGGCCAGTA?AAAGCATTAC?CCGTGGTGGG1681 GTTCCCGAGC?GGCCAAAGGG?AGCAGACTCT?AAATCTGCCG?TCATCGACTT?CGAAGGTTCG1741 AATCCTTCCC?CCACCACCAT?CACTTTCAAA?AGTCCGAAAG?AATCTGCTCC?CTGCTT

Claims (6)

1. a plasmid is characterized in that being made up of the nucleotide sequence with immunostimulatory activity and the carrier sequence of multiple copied.

2. the described immunostimulatory activity nucleotide sequence of claim 1 comprises and contains CpG, and linking to each other with phosphodiester bond to form the D type oligonucleotide sequence ODN of palindrome.

3. the described immunostimulatory activity nucleotide sequence of claim 1 is D type ODN, comprises following 3 kinds,

5’-GGTGCATCGATGCAGGGGGG-3’，

5’-GGGGGACGATCGTCGGGGGG-3，

5’-ACCGATAACGTTGCCGGTGACGGCACCACG-3。

4. the copy number of the described immunostimulatory activity nucleotide sequence of claim 1 is in 20-40 scope.

The described plasmid of claim 1 in chronic viral infection and tumor treatment vaccine as adjuvant.

6. the described plasmid of claim 6 possesses the described structure of SEQ1.