CN103191442A - Anti-HIV-1-virus tuberculosis gene vaccine, and preparation method and application thereof - Google Patents
Anti-HIV-1-virus tuberculosis gene vaccine, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an anti-HIV-1-virus tuberculosis gene vaccine composed of a vector. An exogenous target gene is inserted into the vector. The exogenous target gene comprises a gene of a capsid protein p24 sourced from a HIV-1NL4-3 strain. T cell epitope polypeptide genes MPT6476-84, Ag85A242-250, Ag85B184-192 and TB10.474-82 from mycobacterium tuberculosis antigen are embedded in the capsid protein p24 gene. The invention also provides a preparation method and an application of the anti-HIV-1-virus tuberculosis gene vaccine. When the gene vaccine is used in intramuscular injection for immunizing mice, Th1-type immune response and specific killing response secreting high-level IFNgamma aiming at tuberculosis antigens can be induced, and specific serum IgG and T-cell immune response aiming at HIV-1 antigen p24 can be induced. Therefore, the vaccine can be used for preventing tuberculosis and inhibiting HIV.
Description
Technical field:
The invention belongs to the biological gene engineering field, relate to a kind of gene vaccine, relate in particular to and a kind ofly can prevent difunctional vaccine of tuberculosis and acquired immune deficiency syndrome (AIDS) and its preparation method and application simultaneously.
Background technology:
Tuberculosis remains and causes one of primary infectious disease that the mankind die of illness, and is the great health problem in the global range.There are 8,000,000 active tuberculosis patients in the whole world and cause 300 to die ten thousand deaths and die every year; China has 5.5 hundred million populations once to infect tuberculosis approximately, and annual 130000 people die from tuberculosis, and 120,000 New Development patients were arranged in every month approximately.In recent years the patient of concurrent infection tuberculosis and HIV grows with each passing day, tuberculosis that the whole world has had 1/3rd HIV patient infection at least, and they develop into tuberculosis than the HIV patient Geng Yi that does not infect tuberculosis and case fatality rate increases greatly.The ill rapid increase of tuberculosis often appears in the area of the high prevalence rate of HIV.Existing BCG vaccine only produces effect to the outer tuberculosis of child and adult's lung, lacks the protection effect for tuberculosis in adult's the lung.Also there are not effective HIV prevention or treatment vaccine at present.Therefore developing the difunctional Vaccinum Calmette-Guerini that has anti-HIV potential novel the time has great importance.
Former studies shows, induces Th1 type cellullar immunologic response most important for infecting the color mycobacterium tuberculosis in the scavenger cell, induces CD4
+High-caliber IFN γ and the CD8 of Th1 and secretion thereof
+The generation of CTL, the opposing tubercule bacillus that can effectively watch for animals is attacked.For the protective immune response of HIV-1, think that at present serum neutralizing antibody and Th1 type cellullar immunologic response all play a significant role.
Gene vaccine claims dna vaccination again, it is the third generation vaccine that the external source genes of interest is implemented in eukaryon expression plasmid, direct injection animal, owing to can express purpose antigen in vivo, offer pathway activation DC cell by exogenous, endogenous and cross-reacting antigen and offer antigen, not only can cause humoral immunoresponse(HI), Th1 type cellullar immunologic response such as CD4+Th1 and CD8+T cell response be can activate effectively especially, pathogen infection such as mycobacterium tuberculosis and HIV in the scavenger cell therefore are conducive to.
Because ineffectivity and the appearance of tuberculosis Resistant strain and the tuberculosis infection that the AIDS disease causes of conventional BCG vaccine, make the poor effect of vaccine prevention of the prior art or treatment tuberculosis and acquired immune deficiency syndrome (AIDS).
Summary of the invention:
The object of the present invention is to provide a kind of tuberculosis gene vaccine with anti-HIV-1 virus and its preparation method and application, described this tuberculosis gene vaccine with anti-HIV-1 virus will solve the technical problem of the poor effect of vaccine prevention of the prior art or treatment tuberculosis and acquired immune deficiency syndrome (AIDS).
The invention provides a kind of tuberculosis gene vaccine with anti-HIV-1 virus, constituted by a carrier, in described carrier, be inserted with one section external source genes of interest, it is characterized in that: described external source genes of interest comprises the full-length gene of the capsid protein p24 in a HIV (human immunodeficiency virus) HIV-1NL4-3 strain source, chimeric 4 t cell epitope polypeptide genes that have from negre antigen in the full-length gene of the capsid protein p24 that described HIV (human immunodeficiency virus) HIV-1NL4-3 strain is originated, described 4 t cell epitopes are respectively 76～84 genes of the MPT64 albumen in mycobacterium tuberculosis H37Rv strain source, 242～250 genes of the Ag85A albumen in mycobacterium tuberculosis H37Rv strain source, 184～192 genes of the Ag85B albumen in mycobacterium tuberculosis H37Rv strain source, 74～82 genes of the TB10.4 albumen in mycobacterium tuberculosis H37Rv strain source, from 5 ' of the capsid protein p24 full-length gene in HIV (human immunodeficiency virus) HIV-1NL4-3 strain source, before the 1st bit base, insert 76～84 genes of the MPT64 albumen in mycobacterium tuberculosis H37Rv strain source, behind the 273rd bit base, insert 242～250 genes of the Ag85A albumen in mycobacterium tuberculosis H37Rv strain source, behind the 303rd bit base, insert 184～192 genes of the Ag85B albumen in mycobacterium tuberculosis H37Rv strain source, behind the 441st bit base, insert 74～82 genes of the TB10.4 albumen in mycobacterium tuberculosis H37Rv strain source, the DNA sequence of the capsid protein p24 full-length gene in described HIV (human immunodeficiency virus) HIV-1NL4-3 strain source is shown in SEQ ID NO:2, the DNA sequence of 76～84 genes of the MPT64 albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:4, the DNA sequence of 242～250 genes of the Ag85A albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:6, the DNA sequence of 184～192 genes of the Ag85B albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:8, and the DNA sequence of 74～82 genes of the TB10.4 albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:10.
The DNA sequence of the external source genes of interest that the full-length gene of the capsid protein albumen p24 in the HIV-1NL4-3 strain source of 4 t cell epitope genes in further, chimeric mycobacterium tuberculosis H37Rv strain source constitutes is shown in SEQ ID NO:12.
Further, described carrier is eukaryon expression plasmid, and described eukaryon expression plasmid is selected from pcDNA3.1 plasmid or pVAX1 plasmid.
Further, the aminoacid sequence of the capsid protein albumen p24 in described HIV (human immunodeficiency virus) HIV-1NL4-3 strain source is shown in SEQ ID NO:1.
Further, the aminoacid sequence of 76～84 genes of described mycobacterium tuberculosis H37Rv strain source tubercule bacillus MPT64 albumen is shown in SEQ ID NO:3.
Further, the aminoacid sequence of 242～250 genes of described mycobacterium tuberculosis H37Rv strain source tubercule bacillus Ag85A albumen is shown in SEQ ID NO:5.
Further, the aminoacid sequence of 184～192 genes of described mycobacterium tuberculosis H37Rv strain source tubercule bacillus Ag85B albumen is shown in SEQ ID NO:7.
Further, the aminoacid sequence of 74～82 genes of described mycobacterium tuberculosis H37Rv strain source tubercule bacillus TB10.4 albumen is shown in SEQ ID NO:9.
The aminoacid sequence of the external source genes of interest that the full-length gene of the capsid protein albumen p24 in the HIV (human immunodeficiency virus) HIV-1NL4-3 strain source of 4 t cell epitope genes in further, chimeric mycobacterium tuberculosis H37Rv strain source constitutes is shown in SEQ ID NO:11.
The present invention also provides the preparation method of the above-mentioned tuberculosis gene vaccine with anti-HIV-1 virus, may further comprise the steps:
1) extracts HIV-1NL4-3 strain DNA as template, by PCR method amplification HIV-1 capsid protein p24 encoding gene;
2) with HIV-1 capsid protein p24 gene as template, by the pcr amplification band 18 or 24 double chain DNA fragment 1, fragment 2, fragments 3 that base is overlapping are arranged each other respectively, the sequence of the double-stranded DNA of described fragment 1 is:
atgaagttcc?tcagcgcggc?aacatcgtcc?cctatagtgc?agaacctcca?ggggcaaatg
tacttcaagg?agtcgcgccg?ttgtagcagg?ggatatcacg?tcttggaggt?ccccgtttac
gtacatcagg?ccatatcacc?tagaacttta?aatgcatggg?taaaagtagt?agaagagaag
catgtagtcc?ggtatagtgg?atcttgaaat?ttacgtaccc?attttcatca?tcttctcttc
gctttcagcc?cagaagtaat?acccatgttt?tcagcattat?cagaaggagc?caccccacaa
cgaaagtcgg?gtcttcatta?tgggtacaaa?agtcgtaata?gtcttcctcg?gtggggtgtt
gatttaaata?ccatgctaaa?cacagtgggg?ggacatcaag?cagccatgca?aatgttaaaa
ctaaatttat?ggtacgattt?gtgtcacccc?cctgtagttc?gtcggtacgt?ttacaatttt
gagaccatca?atgaggaagc?tgcagaatgg?gatagattgc?atccagtgca?tgcagggcct
ctctggtagt?tactccttcg?acgtcttacc?ctatctaacg?taggtcacgt?acgtcccgga
attaagctga?tcgccaacaa?cacccgcgtc?gcaccaggcc?agatgagaga?acca
taattcgact?agcggttgtt?gtgggcgcag?cgtggtccgg?tctactctct?tggt
The sequence of the double-stranded DNA of described fragment 2 is
ggccagatga?gagaaccaag?gggaatctac?gccggctcgc?tgtcggccct?gagtgacata
ccggtctact?ctcttggttc?cccttagatg?cggccgagcg?acagccggga?ctcactgtat
gcaggaacta?ctagtaccct?tcaggaacaa?ataggatgga?tgacacataa?tccacctatc
cgtccttgat?gatcatggga?agtccttgtt?tatcctacct?actgtgtatt?aggtggatag
ccagtaggag?aaatctataa?aagatggata?atcctgggat?taaataaaat?agtaagaatg
ggtcatcctc?tttagatatt?ttctacctat?taggacccta?atttatttta?tcattcttac
tatagcccta?gcacccatga?agccaacacc?atggcgacca?gcattctgga?cata
atatcgggat?cgtgggtact?tcggttgtgg?taccgctggt?cgtaagacct?gtat
The sequence of the double-stranded DNA of described fragment 3 is
atggcgacca?gcattctgga?cataagacaa?ggaccaaagg?aaccctttag?agactatgta
taccgctggt?cgtaagacct?gtattctgtt?cctggtttcc?ttgggaaatc?tctgatacat
gaccgattct?ataaaactct?aagagccgag?caagcttcac?aagaggtaaa?aaattggatg
ctggctaaga?tattttgaga?ttctcggctc?gttcgaagtg?ttctccattt?tttaacctac
acagaaacct?tgttggtcca?aaatgcgaac?ccagattgta?agactatttt?aaaagcactg
tgtctttgga?acaaccaggt?tttacgcttg?ggtctaacat?tctgataaaa?ttttcgtgac
ggaccaggag?cgacactaga?agaaatgatg?acagcatgtc?agggagtggg?gggacccggc
cctggtcctc?gctgtgatct?tctttactac?tgtcgtacag?tccctcaccc?ccctgggccg
cataaagcaa?gagttttgta?a
gtatttcgtt?ctcaaaacat?t
3) 3 above-mentioned fragments are mixed, degeneration becomes strand, by 18 or 24 base complementrities connections that overlap each other, as template, obtained the HIV (human immunodeficiency virus) capsid protein p24 external source genes of interest of 4 t cell epitopes chimeric by PCR method amplification, genes of interest is connected with the carrier of the same double digestion of warp behind double digestion, obtains the described tuberculosis gene vaccine with anti-HIV-1 virus of claim 1.
Concrete, be that above-mentioned fragment 1 and fragment 2 are mixed, thermal denaturation makes becomes single stranded DNA separately, because fragment 1,2 has the overlapping DNA sequence of 18 bases, therefore can complementation become two strands, can increase by the PCR method again and obtain the fragment of fragment (1+2), mix with fragment 3 again, thermal denaturation becomes strand, because fragment 2 and fragment 3 have the overlapping DNA sequence of 24 bases, therefore can complementation become two strands, can increase through PCR obtains fragment (1+2+3), the namely chimeric HIV-1 viral capsid proteins p24 external source genes of interest of 4 t cell epitopes.
Further, pass through pcr amplified fragment 1 with the p24F2 primer sequence shown in the p24F1 primer sequence shown in the SEQ ID NO:13 and the SEQ ID NO:14.
Further, pass through pcr amplified fragment 2 with the p24T2 primer sequence shown in the p24T1 primer sequence shown in the SEQ ID NO:15 and the SEQ ID NO:16.
Further, pass through pcr amplified fragment 3 with the p24R2 primer sequence shown in the p24R1 primer sequence shown in the SEQ ID NO:17 and the SEQ ID NO:18.
Further, be template with the p24F1 primer sequence shown in the SEQ ID NO:13 and the p24R2 primer sequence shown in the SEQ ID NO:18 with fragment 1,2,3 mixing, degeneration, gene after complimentary to one another the connection, by the PCR method amplification chimeric the HIV-1 capsid protein p24 external source genes of interest of 4 t cell epitopes;
The p24 external source genes of interest of further, chimeric 4 t cell epitopes and the restriction enzyme site that inserts carrier are respectively NheI and EcoRI site.
Further, its antigen is from mycobacterium tuberculosis H37Rv strain and HIV (human immunodeficiency virus) HIV-1NL4-3 strain.
Further, described carrier is selected from pcDNA3.1 plasmid or pVAX1 plasmid.
The present invention also provides a kind of pharmaceutical composition, contains the above-mentioned tuberculosis gene vaccine with anti-HIV-1 virus of effective dose.
Further, also contain pharmaceutically acceptable carrier or excipient.
The present invention also provides the above-mentioned application of the tuberculosis gene vaccine with anti-HIV-1 virus in the medicine of preparation treatment or prevention tuberculosis and acquired immune deficiency syndrome (AIDS).
The present invention is by the DNA forecasting software, from known 4 kinds of crucial antigen MPT64, Ag85A, Ag85B and TB10.4 of mycobacterium tuberculosis H37Rv strain each self-sizing a t cell epitope, totally 4 t cell epitopes intend making up the tuberculosis gene vaccine that contains 4 t cell epitopes.Because the aminoacid sequence of epi-position only has 9, structure is too little and simple, and immunogenicity is very weak, can not induce very strong immunne response usually.Therefore intend selecting a kind of vector gene, 4 t cell epitopes are inserted in the vector gene, constitute tuberculosis external source genes of interest jointly.
The present invention selects the capsid protein p24 in HIV (human immunodeficiency virus) HIV-1NL4-3 strain source as the genophore that embeds t cell epitope.Is p24 that (capsid protein p24 is that the people of this area knows for the capsid protein of HIV-1?).Total length 693bp, 231 aminoacid of encoding, molecular weight 24kD.P24 albumen plays an important role in processes such as virion maturation, has suppressed the effect that the function of p24 can produce anti-HIV-1.P24 is a good immunogenic protein in addition, contains a plurality of T cells and B cell epitope, effectively the specific CD4 of activation antigen
+/ CD8
+The T cellullar immunologic response; The tuberculosis t cell epitope is inserted into the non-key structure position of p24, thereby thereby not only can make the tuberculosis epi-position supported and obtain native conformation and be identified better to produce and reply, can also produce replying at HIV-1 antigen p24.
4 t cell epitopes that the present invention selects are from following 4 kinds of tuberculosis antigens:
1) MPT64,205 aminoacid of protein total length, molecular weight 24KD claims MPB64, Rv1980c again, is a kind of secretory protein in the tulase culturing filtrate.But MPT64 activated t cell immunne response produces the T cell proliferative response, and induces CD8
+CTL produces the specific killing reaction, and tuberculosis is produced protective response.
2) Ag85A, 295 aminoacid of protein total length.Secretory protein main component is Ag85 complex (antigen 85 complex) in the culturing filtrate of tulase, and the relative molecular mass of being made up of Ag85A, Ag85B, Ag85C is 38000 protein families.Ag85A can stimulate body to produce humoral immunization, also can excite Th1 type cellular immunization, induces CD8
+Risings such as T cell proliferation and IL-2 and IFN-γ.
3) Ag85B, 285 aminoacid of protein total length, molecular weight 34.6KD claims MPT59, Rv1886 again, is a kind of mycobacteria transferring enzyme, and is synthetic relevant with bacteria cell wall, has a plurality of t cell epitopes, can induce the generation of Th1 reaction, IFN-γ.
4) TB10.4,96 aminoacid of protein total length, molecular weight 10.4KD claims Rv0288 again, is the virulence factor of the early stage secretion of tulase, belongs to ESAT-6 family.TB10.4 is the albumen with high immunogenicity, can induce very strong t cell response and discharge a large amount of IFN-γ, can play a protective role in anti-tubercle bacillus infects.
The present invention is by BLAST network data base comparison, the analysis of DNAstar biosoftware and hydrophilic and hydrophobic, flexibility, antigenic index, the isoparametric analysis of surperficial accessibility, select all kinds of parameters all preferably structural region as candidate vaccine epi-position section; Then by network data base (http://www.syfpeithi.com/scripts/MHCServer.dll/home.htm) but the T cell antigen epitope that exists in these sections of analyses and prediction, the epi-position of being combined with HLA I, II quasi-molecule high-affinity simultaneously.Through last 4 t cell epitopes that derive from mycobacterium tuberculosis H37Rv strain that obtain of computer molecule prediction be respectively: 76～84 gene (MPT64 of MPT64 albumen
76-84); 242～250 gene (Ag85A of Ag85A albumen
242-250); 184～192 gene (Ag85B of Ag85B albumen
184-192) 74～82 gene (TB10.4 of TB10.4 albumen
74-82).Before being inserted into the 1st bit base of p24 gene respectively, behind the 273rd bit base, the 303rd bit base and the 441st bit base, make up the difunctional tuberculosis gene vaccine of pP24-Mtb.
The present invention is in pcDNA3.1 or pVAX1 plasmid, insert one section HIV-1p24 external source genes of interest, and 4 non-key locations of structures in this external source genes of interest are chimeric goes into 4 tuberculosis t cell epitopes, by analysis, therefore the embedding of 4 epi-positions can not influence normal three grades, the level Four space structure of p24 itself, chimericly has the tuberculosis gene vaccine of the p24 gene of 4 t cell epitopes to be called the pP24-Mtb tuberculosis gene vaccine this.
Among the present invention, described eukaryon expression plasmid comprises any highly effective eukaryon expression plasmid vector that can transfection mammalian cell, includes but not limited to: pcDNA3.1, pVAX and common and commercially available eucaryon plasmid carrier.
Among the present invention, the acquisition of p24 protein coding gene and 4 t cell epitopes can obtain in several ways, includes but not limited to: 1) chemical synthesising DNA; 2) obtain DNA by suitable primer with the round pcr amplification.
Among the present invention, the method employing muscle injection mode with pP24-Mtb gene vaccine injection mice carries out with routine techniques well known to those skilled in the art: draw 50 μ l plasmid (PBS) direct injection mice tibialis anterior meat with syringe.Except syringe, also can select for use other effectively by means of the mode of the genetic immunization of commercially available instrument, include but not limited to: gene gun technology, electroporation ancillary technique etc.
Among the present invention, " pharmaceutically acceptable " composition is to be applicable to humans and animals and not have excessive bad side reaction (as toxicity, stimulation, allergy), the material of reasonable benefit/risk ratio is arranged.
Among the present invention, " pharmaceutically acceptable carrier " composition is pharmaceutically acceptable solvent, suspending agent and the excipient that sends the human or animal for the effective ingredient that will have activity to.Described carrier can be liquid, solid or semisolid.Carrier includes but not limited to: water, PBS buffer, normal saline, glucose, glycerol, sodium azide and combination thereof.
Pharmaceutical composition of the present invention also contains above-mentioned effective ingredient and pharmaceutically acceptable carrier.Usually it is formulated in nontoxic, neutral, inertia and the pharmaceutically acceptable aqueous carrier medium, pH value is about 6-8 usually.
The present invention and contrast with prior art, its effect is actively and significantly.Tuberculosis gene vaccine with anti-HIV-1 virus of the present invention is a kind of plasmid DNA vaccine, wherein the external source genes of interest of Cha Ruing is the chimeric HIV-1 source p24 gene that 4 tuberculosis t cell epitopes are arranged, but they are equal inducing specific t cell response separately, and chimeric epi-position can not influence the space structure of p24 albumen.Therefore this gene vaccine can be induced tuberculosis and HIV-1 specific antigen simultaneously in theory, can activate the specific T-cells immunne response at tuberculosis and HIV-1 by force effectively.And p24 is a big antigen, except containing T northwest epi-position, also contains B northwest epi-position, therefore also can induce HIV-1p24 specific serum IgG antibody response, and antibody have in and the potential of HIV virus.By with difunctional gene vaccine intramuscular injection immune mouse of the present invention, confirmation can be induced at the specific antibody of HIV-1p24 antigen and be replied and t cell response, can also induce simultaneously stronger specificity T h1 type immunne response at the tuberculosis t cell epitope and CTL to kill and wound replys, therefore secreting high levels IFN γ is a kind of potential difunctional vaccine that tuberculosis has certain anti-HIV function simultaneously that prevents and treat.
Description of drawings
Fig. 1 has shown that the external source genes of interest with tuberculosis gene vaccine of anti-HIV-1 virus of the present invention (chimeric the HIV-1p24 protein gene of 4 tuberculosis t cell epitopes) inserts the structure ideograph of pcDNA3.1 vector plasmid.
Fig. 2 has shown the construction method of this plasmid: by F1/F2, T1/T2, three pairs of primers of R1/R2, be template with HIV-1p24DNA, amplification obtains double chain DNA fragment 1 (1-351bp), fragment 2 (333-567bp), fragment 3 (543-801bp) respectively, 18 by overlapping each other or 24 base complementrities p24 full-length gene of chimeric 4 t cell epitopes of obtaining that links to each other then, wherein, the sequence of bold Italic is 4 t cell epitope sequences inserting.
Shown Fig. 3 the plasmid PCR (Fig. 3 A) with bifunctional polyepitope tuberculosis gene vaccine of anti-HIV-1 potential of the present invention and the part qualification result (Fig. 3 B) that checks order.
Fig. 4 has shown the specific IgG antibodies that the tuberculosis gene vaccine immune mouse with anti-HIV-1 virus of the present invention is induced, and Fig. 4 A shows the level of p24 antigenic specificity IgG; Fig. 4 B shows that p24 antigenic specificity IgG tires; Fig. 4 C shows p24 antigen-specific antibodies subclass.
Fig. 5 has shown tuberculosis antigen specificity IFN γ+T cell (Fig. 5 A) and the HIV-1 antigenic specificity IFN γ+t cell response (Fig. 5 B) that the tuberculosis gene vaccine immune mouse with anti-HIV-1 virus of the present invention is induced.
Fig. 6 has shown tuberculosis antigen specificity (Fig. 6 A) and the HIV-1p24 antigenic specificity cytokine secretion level (Fig. 6 B) that the tuberculosis gene vaccine immune mouse with anti-HIV-1 virus of the present invention is induced.
Fig. 7 has shown tuberculosis and HIV-1 antigenic specificity lymphopoiesis (Fig. 7 A) and the killing activity (Fig. 7 B) that the tuberculosis gene vaccine immune mouse with anti-HIV-1 virus of the present invention is induced.
Fig. 8 has shown the protective effect of the anti-tubercle bacillus attack that the tuberculosis gene vaccine immune mouse with anti-HIV-1 virus of the present invention produces.Figure A has shown the HE of pulmonary dyeing pathologic condition; Figure B has shown the colony counting of lungs tubercule bacillus.
The specific embodiment
Below be specific embodiments of the invention, described embodiment is for description use of the present invention and purposes, rather than restriction the present invention.
The plasmid that adopts among the embodiment, strain, cell, animal and reagent are as follows:
Plasmid, strain, cell, animal.Plasmid pcDNA3.1 (-), pET32a, host bacteria DH5 α, BL21 (purchase of Invitrogen company).Mycobacterium tuberculosis H37Rv strain provides HIV (human immunodeficiency virus) HIV-1NL4-3 strain by the anti-section of Shanghai Disease Prevention and Control Centre's knot
From where?,Synthetic match Parkson, the Shanghai company that entrusts of gene.Age in 4-6 week female BALB/c (H-2K
d) mice is available from Chinese Academy of Sciences's animal center.
Molecular biology reagent: restriction endonuclease Nhe I, EcoR I (TaKaRa company), T4DNA ligase (MBI company); Taq archaeal dna polymerase (Promega company); RNase A (Ameresco company); DNTP (Promega and magnificent biotech firm); LB culture medium (Britain OXOID company); agar powder, agarose, SDS, EB (Shanghai chemical reagent purchasing and supply station); Tris (USB company), agarose gel reclaim test kit (Shanghai China Shun biological product company), a large amount of plasmid extraction test kits (Qiagen company).
Immunology reagent and material: HRP labelling goat anti-mouse igg polyclonal antibody (Sant Clous company); FITC-CD4 antibody, FITC-CD8 antibody, PE-IFN gamma antibodies, Brefeldin A (Gorky's blocker) are available from BD company; 2ml and 1ml asepsis injector (Mi Shawa medical courses in general Industrial Co., Ltd).
The prediction of the purpose antigen gene of embodiment 1:pcDNA3-P24-Mtb gene vaccine
Analyze by BLAST network data base, DNAstar biosoftware, network data base (http://www.syfpeithi.com/scripts/MHCServer.dll/home.htm), comprehensive hydrophilic and hydrophobic, flexibility, antigenic index, surperficial accessibility, be combined with HLAI, II quasi-molecule, etc. parameter, predict 4 t cell epitopes that derive from mycobacterium tuberculosis H37Rv strain of acquisition: 76～84 gene (MPT64 of MPT64 albumen
76-84); 242～250 gene (Ag85A of Ag85A albumen
242-250); 184～192 gene (Ag85B of Ag85B albumen
184-192); 74～82 gene (TB10.4 of TB10.4 albumen
74-82).
As shown in Figure 1, be plasmid vector with pcDNA3.1 or pVAX, with the genophore of HIV (human immunodeficiency virus) HIV-1p24 gene as chimeric epi-position, on the basis of its secondary structure of computer forecast, do not influence the preceding MPT64 of insertion of position the 1st bit base of key structure therein
76-84Insert Ag85A behind epitope gene, the 273rd bit base
242-250Insert Ag85B behind epitope gene, the 303rd bit base
184-192Insert TB10.4 behind epitope gene, the 441st bit base
74-82Epitope gene.
The structure of embodiment 2 pcDNA3-P24-Mtb tuberculosis gene vaccines
In order between p24 gene and tuberculosis t cell epitope gene, not introduce restriction enzyme site, the present invention utilizes the directly method of synthetic and PCR of dna primer, increase successively three sections 18 or 24 genetic fragments that comprise part p24 gene and tuberculosis t cell epitope that base is overlapping are arranged each other from 5 ' and 3 ' end, connect by the complementary series that overlaps each other after the degeneration, use the upper and lower primer PCR of 5 ' and 3 ' two ends p24 to amplify the chimeric HIV-1p24 full-length gene that 4 tuberculosis t cell epitopes are arranged at last.Simultaneously be with Nhe I and EcoR I restriction enzyme site respectively at the gene two ends, behind double digestion, can connect into carrier for expression of eukaryon pcDNA3.1 (-) or prokaryotic expression carrier pET32a through same double digestion.
Be template with HIV-1 capsid protein p24 gene (SEQ ID NO:2) at first, (F1 is relative with F2 as shown in Figure 2 by pcr amplified fragment 1 with F2 primer (SEQ ID NO:14) with primers F 1 (SEQ ID NO:13) respectively, the 1-351bp double chain DNA fragment that obtains through pcr amplification), the sequence of the double-stranded DNA of described fragment 1 is:
atgaagttcc?tcagcgcggc?aacatcgtcc?cctatagtgc?agaacctcca?ggggcaaatg
tacttcaagg?agtcgcgccg?ttgtagcagg?ggatatcacg?tcttggaggt?ccccgtttac
gtacatcagg?ccatatcacc?tagaacttta?aatgcatggg?taaaagtagt?agaagagaag
catgtagtcc?ggtatagtgg?atcttgaaat?ttacgtaccc?attttcatca?tcttctcttc
gctttcagcc?cagaagtaat?acccatgttt?tcagcattat?cagaaggagc?caccccacaa
cgaaagtcgg?gtcttcatta?tgggtacaaa?agtcgtaata?gtcttcctcg?gtggggtgtt
gatttaaata?ccatgctaaa?cacagtgggg?ggacatcaag?cagccatgca?aatgttaaaa
ctaaatttat?ggtacgattt?gtgtcacccc?cctgtagttc?gtcggtacgt?ttacaatttt
gagaccatca?atgaggaagc?tgcagaatgg?gatagattgc?atccagtgca?tgcagggcct
ctctggtagt?tactccttcg?acgtcttacc?ctatctaacg?taggtcacgt?acgtcccgga
attaagctga?tcgccaacaa?cacccgcgtc?gcaccaggcc?agatgagaga?acca
taattcgact?agcggttgtt?gtgggcgcag?cgtggtccgg?tctactctct?tggt
With p24 gene (SEQ ID NO:2) as template, (the T1 primer is relative with T2 as shown in Figure 2 by pcr amplified fragment 2 with T2 primer (SEQ ID NO:16) with T1 primer (SEQ ID NO:15), the 333-567bp double chain DNA fragment that obtains through pcr amplification), the sequence of the double-stranded DNA of described fragment 2 is:
ggccagatga?gagaaccaag?gggaatctac?gccggctcgc?tgtcggccct?gagtgacata
ccggtctact?ctcttggttc?cccttagatg?cggccgagcg?acagccggga?ctcactgtat
gcaggaacta?ctagtaccct?tcaggaacaa?ataggatgga?tgacacataa?tccacctatc
cgtccttgat?gatcatggga?agtccttgtt?tatcctacct?actgtgtatt?aggtggatag
ccagtaggag?aaatctataa?aagatggata?atcctgggat?taaataaaat?agtaagaatg
ggtcatcctc?tttagatatt?ttctacctat?taggacccta?atttatttta?tcattcttac
tatagcccta?gcacccatga?agccaacacc?atggcgacca?gcattctgga?cata
atatcgggat?cgtgggtact?tcggttgtgg?taccgctggt?cgtaagacct?gtat
With p24 gene (SEQ ID NO:2) as template, (the R1 primer is relative with the R2 primer as shown in Figure 2 by pcr amplified fragment 3 with R2 primer (SEQ ID NO:18) with R1 primer (SEQ ID NO:17), the 543-801bp double chain DNA fragment that obtains through pcr amplification), the sequence of the double-stranded DNA of described fragment 3 is:
atggcgacca?gcattctgga?cataagacaa?ggaccaaagg?aaccctttag?agactatgta
taccgctggt?cgtaagacct?gtattctgtt?cctggtttcc?ttgggaaatc?tctgatacat
gaccgattct?ataaaactct?aagagccgag?caagcttcac?aagaggtaaa?aaattggatg
ctggctaaga?tattttgaga?ttctcggctc?gttcgaagtg?ttctccattt?tttaacctac
acagaaacct?tgttggtcca?aaatgcgaac?ccagattgta?agactatttt?aaaagcactg
tgtctttgga?acaaccaggt?tttacgcttg?ggtctaacat?tctgataaaa?ttttcgtgac
ggaccaggag?cgacactaga?agaaatgatg?acagcatgtc?agggagtggg?gggacccggc
cctggtcctc?gctgtgatct?tctttactac?tgtcgtacag?tccctcaccc?ccctgggccg
cataaagcaa?gagttttgta?a
gtatttcgtt?ctcaaaacat?t
Above-mentioned fragment 1 and fragment 2 are mixed, degeneration makes becomes single stranded DNA separately, because fragment 1,2 have the overlapping DNA sequence of 18 bases, therefore can complementation become two strands, can increase by the PCR method with F1 (SEQ ID NO:13) and T2 (SEQ ID NO:16) primer again and obtain the fragment of fragment (1+2), mix with fragment 3 again, degeneration becomes strand, because fragment 2 and fragment 3 also have the overlapping DNA sequence of 24 bases, therefore can complementation become two strands, with F1 (SEQ ID NO:13) and R2 primer (SEQ ID NO:18) as primer, obtain fragment (1+2+3) by the PCR method amplification, the HIV-1p24 genes of interest of 4 tuberculosis t cell epitopes that has been chimeric, i.e. P24-Mtb encoding gene, length is 801bp (as shown in Figure 2).This P24-Mtb encoding gene turns out to be 801bp through electrophoresis, as shown in Figure 3A.Again with amplified production through Nhe I and EcoR I double digestion, reclaim endonuclease bamhi, is connected with carrier pcDNA3.1 (-) through corresponding enzyme action, will connect product transformed into escherichia coli DH5 α, ampicillin screens the conversion bacterium colony.Identify sequence correct (Fig. 3 B) through order-checking, point out and successfully made up the pcDNA3-P24-Mtb eukaryon expression plasmid.Such 4 tuberculosis epitope genes have just inserted the p24 gene in the mode that does not have restriction enzyme site.
With recombiant plasmid pP24-Mtb transformed into escherichia coli DH5 α, screening positive clone through LB (Amp100 μ g/ml) fluid medium shaken cultivation 15h, is collected thalline, remove foreign protein, bacterial endotoxin according to QIAGEN Plasmid Mega Kit, obtain plasmid purification.
For obtaining a large amount of p24 or P24-Mtb proteantigen, the P24-Mtb encoding gene that p24 and amplification are obtained is with NheI and EcoRI double digestion, be connected with the prokaryotic expression carrier pET32a through corresponding enzyme action, made up pET32a-p24 and pET32a-P24-Mtb prokaryotic expression plasmid.
PET32a-P24-Mtb or pET32a-p24 are transformed into e. coli bl21 (DE3) competent cell, 37 ℃ of overnight incubation, screening positive clone respectively.Reach about 0.75 to A600 through LB (Amp100 μ g/ml) fluid medium shaken cultivation, adding isopropylthio half glucosides (IPTG) to final concentration is 0.1mM.Continued shaken cultivation 6 hours, the centrifugal 20min of 4000r/min collects thalline, the resuspended back ultrasonication of 1 * PBS, and 12000r/min is in 4 ℃ of centrifugal 20min, and results go up cleer and peaceful precipitation respectively.Supernatant is crossed affinity column, with variable concentrations eluent eluting, collects every 1ml eluent, preserves A280 greater than 1.0 eluent, identifies expressed proteins finally by the 12%SDS-PAGE electrophoresis.Obtain purified fusion protein p24 or P24-Mtb at last.
Eukaryon expression plasmid pcDNA3.1-P24-Mtb is dissolved among aseptic, the apyrogenic PBS, and concentration is adjusted to 1 μ g/ μ l.Age in 6-8 week female BALB/c (H-2
d) 18 of healthy mices, be divided into 3 groups: the contrast of (1) pcDNA3.1 empty plasmid; (2) pcDNA3.1-p24 plasmid group; (3) pcDNA3.1-P24-Mtb plasmid.The immunity of employing intramuscular injection: at BALB/c mouse tibialis anterior meat center injection 50 μ g plasmids, every each 50 μ g/ μ l of mice.In 0 week, 2 weeks, 4 weeks, 6 all intramuscular injection immune mouses four times.Per two weeks take a blood sample through eye socket, and blood sample spends the night for 4 ℃, and the centrifugal 15min of 6000 commentaries on classics/min obtains serum, and-20 ℃ frozen.
The specific antibody that embodiment 5 pcDNA3.1-P24-Mtb gene vaccines are induced is replied
Indirect elisa method detects HIV-1p24 specific IgG in the immune serum.The p24 protein 10 μ g/ml bag of the purification that obtains with embodiment 3 is spent the night 100 μ l/ holes for 4 ℃ by polystyrene micropore plate.With confining liquid (5% defatted milk powder, 0.05%Tween-20, PBS) closure plate, 200 μ l/ holes, 37 ℃ of 2h.Antiserum after dilution in 1: 100,100 μ l/ holes, 37 ℃ of 1h, washing back adds the HRP-sheep anti-mouse igg of dilution in 1: 5000,37 ℃ of 45min wash behind the plate with TMB colour developing 20min, survey A after the cessation reaction
450Value.
Show as Fig. 4 A: the pcDNA3.1-P24-Mtb dna gene vaccine is induced in the mice body later for four times and has been produced the p24 specific IgG, the serum IgG titre reaches the highest when the 10th week, reach 1: 4000 (as Fig. 4 B), and empty plasmid injection group there is not the specific antibody generation.And the pcDNA3.1-P24-Mtb gene vaccine can produce higher IgG2a (as Fig. 4 C), prompting Th1 type cellullar immunologic response.In a word, pcDNA3.1-P24-Mtb vaccine intramuscular injection immune mouse has induced stronger HIV-1p24 antigen-specific antibodies to reply.
IFN γ+t cell response that embodiment 6 pcDNA3.1-P24-Mtb gene vaccines are induced
The 8th week of mouse immune puts to death, extracting spleen cell, and washing is 2 times behind the removal erythrocyte, with 3 * 10
6Cells/well adds 24 orifice plates, adds the p24 albumen (20 μ g/ml) of 4 hybrid junctions nuclear antigen polypeptide (20 μ g/ml) or purification respectively, 37 ℃, 5%CO
2Cultivate.After 4 hours, add Gorky and block body BFA, continue to cultivate 2h, collecting cell carries out dyeing in surface and the born of the same parents with FITC-CD4 antibody, FITC-CD8 antibody and PE-IFN gamma antibodies.The T cell of secretion IFN γ is detected in the washing back with flow cytometer.As shown in Figure 5, compare with matched group, if stimulate through the HIV-1p24 proteantigen, pcDNA3.1-P24-Mtb and pcDNA3.1-P24 genetic immunization have all significantly been induced CD4+T and the CD8+T cell (as Fig. 5 B) of p24 specific secretion IFN γ; If with 4 mixed polypeptide irritation cells, should then only having the pcDNA3.1-P24-Mtb immune group to induce CD4+T (2.3%) and the CD8+T cell (2.57%) of secretion IFN γ (be 5.57%?) reply (as Fig. 5 A), be significantly higher than pcDNA3.1-P24 and pcDNA3.1 group (p＜0.01).Prove that this difunctional gene vaccine can induce the stronger Th1 type cellullar immunologic response at tuberculosis and HIV-1p24 antigen simultaneously.
Embodiment 7 pcDNA3.1-P24-Mtb gene vaccines are induced high-level IFN γ secretion
The 8th week of mouse immune puts to death, extracting spleen cell, and washing is 2 times behind the removal erythrocyte, with 5 * 10
6Cells/well adds 12 orifice plates, adds the p24 albumen (10 μ g/ml) of 4 hybrid junctions nuclear antigen polypeptide (10 μ g/ml) or purification respectively, 37 ℃, 5%CO
2Cultivate.After 72 hours, collect supernatant, detect the level of IFN γ, TNF α, IL-4, IL-10 cytokine with the ELISA method.As shown in Figure 6, pcDNA3.1-P24-Mtb genetic immunization group can significantly strengthen the secretion of IFN γ and TNF α, is significantly higher than matched group, points out this gene vaccine can effectively activate tuberculosis t cell epitope and HIV-1p24 T cells with antigenic specificity secretion IFN γ.
Proliferated specifically and lethal effect that embodiment 8 pcDNA3.1-P24-Mtb gene vaccines are induced
The 8th week of mouse immune puts to death, extracting spleen cell, and washing is 2 times behind the removal erythrocyte, with 5 * 10
5Cells/well adds 96 orifice plates, adds the p24 albumen (20 μ g/ml) of 4 hybrid junctions nuclear antigen polypeptide (20 μ g/ml) or purification respectively, 37 ℃, 5%CO
2Cultivate.After 72 hours, add CCK-8 reagent, 10 μ l/ holes, 37 ℃, 5%CO
2Cultivate after 6 hours, read the OD450 value with microplate reader.With each numerical value divided by blank, the stimulation index that obtains breeding, as Fig. 7 A, pcDNA3.1-P24-Mtb immune group lymphopoiesis ability is significantly higher than matched group, takes place that specificity activates and propagation.
In the lymphocyte fragmentation test, with 2 * 10
7Cells/well adds in 6 orifice plates, stimulate with tuberculosis antigen mixtures of polypeptides or p24 albumen, and add the IL-2 of 50U/ml, cultivate action effect cell after 6 days, with the SP2/0 cell of tuberculosis mixed polypeptide or the stimulation of p24 albumen as target cell, add 96 orifice plates with 25: 1 effect targets than mixing, 37 ℃ of effects collecting cell after 4 hours adds CCK-8 reagent, cultivates 3h for 37 ℃, read the OD450 value with microplate reader, calculate and kill and wound percentage ratio.The lymphocytic specific killing situation of different plasmid immune mouses is shown in Fig. 7 B, after the tuberculosis antigen peptide stimulates, pcDNA3.1-P24-Mtb genetic immunization group kill rate all reaches more than 45%, be significantly higher than pcDNA3.1-P24 (5%) and pcDNA3.1 (3%) matched group, prove that this gene vaccine can induce stronger tuberculosis antigen specific killing to reply; And after stimulating with p24 albumen, pcDNA3.1-P24 is vaccine-induced p24 specific killing (32%) necessarily, but the specific killing that the pcDNA3.1-P24-Mtb genetic immunization is induced (51%) still significantly (p＜0.01) be higher than the pcDNA3.1-P24 vaccine.Point out this difunctional gene vaccine to have the function of opposing mycobacterium tuberculosis and HIV-1 infection.
Embodiment 9 pcDNA3.1-P24-Mtb gene vaccines produce tuberculosis protectiveness effect
2 weeks behind the last immune mouse are with 1 * 10
7BCG (Beijing institute of Biological Products) gives the mice counteracting toxic substances by the collunarium mode.4 weeks were put to death mice behind the counteracting toxic substances, got spleen and lungs homogenate, were uniformly coated on the mycobacterium tuberculosis solid medium 7H11.After 4 weeks, the bacterium colony of growing on the flat board is counted.As shown in the figure, its lungs clump count (10 of pcDNA3.1-P24-Mtb genetic immunization group
45) all obviously be less than matched group (10
6) (Fig. 8 B), prove that this difunctional gene vaccine can protect the infection of mice opposing mycobacterium tuberculosis better.And the HE of pulmonary dyeing pathological examination confirms, pcDNA3.1-P24 and pcDNA3.1 matched group can not be protected mice, and tuberculose focus appears in its pulmonary, and remarkable inflammatory infiltration and alveolar cell wall thickening are arranged simultaneously.And pcDNA3.1-P24-Mtb genetic immunization group lungs pathology degree is very light, near normal lungs cell, points out immanoprotection action (Fig. 8 A) preferably.
Sequence table
＜110〉Fudan University
＜120〉a kind of tuberculosis gene vaccine with anti-HIV-1 virus and its preparation method and application
<160> 18
<170> PatentIn?version?3.1
<210> 1
<211> 231
<212> PRT
＜213〉HIV (human immunodeficiency virus) HIV-1 NL4-3 strain capsid protein p24
<400> 1
Pro?Ile?Val?Gln?Asn?Leu?Gln?Gly?Gln?Met?Val?His?Gln?Ala?Ile?Ser
1 5 10 15
Pro?Arg?Thr?Leu Asn?Ala?Trp?Val?Lys?Val?Val?Glu?Glu?Lys?Ala?Phe
20 25 30
Ser?Pro?Glu?Val?Ile?Pro?Met?Phe?Ser?Ala?Leu?Ser?Glu?Gly?Ala?Thr
35 40 45
Pro?Gln?Asp?Leu?Asn?Thr?Met?Leu?Asn?Thr?Val?Gly?Gly?His?Gln?Ala
50 55 60
Ala?Met?Gln?Met?Leu?Lys?Glu?Thr?Ile?Asn?Glu?Glu?Ala?Ala?Glu?Trp
65 70 75 80
Asp?Arg?Leu?His?Pro?Val?His?Ala?Gly?Pro?Ile?Ala?Pro?Gly?Gln?Met
85 90 95
Arg?Glu?Pro?Arg?Gly?Ser?Asp?Ile?Ala?Gly?Thr?Thr?Ser?Thr?Leu?Gln
100 105 110
Glu?Gln?Ile?Gly?Trp?Met?Thr?His?Asn?Pro?Pro?Ile?Pro?Val?Gly?Glu
115 120 125
Ile?Tyr?Lys?Arg?Trp?Ile?Ile?Leu?Gly?Leu?Asn?Lys?Ile?Val?Arg?Met
130 135 140
Tyr?Ser?Pro?Thr?Ser?Ile?Leu?Asp?Ile?Arg?Gln?Gly?Pro?Lys?Glu?Pro
145 150 155 160
Phe?Arg?Asp?Tyr?Val?Asp?Arg?Phe?Tyr?Lys?Thr?Leu?Arg?Ala?Glu?Gln
165 170 175
Ala?Ser?Gln?Glu?Val?Lys?Asn?Trp?Met?Thr?Glu?Thr?Leu?Leu?Val?Gln
180 185 190
Asn?Ala?Asn?Pro?Asp?Cys?Lys?Thr?Ile?Leu?Lys?Ala?Leu?Gly?Pro?Gl?y
195 200 205
Ala?Thr?Leu?Glu?Glu?Met?Met?Thr?Ala?Cys?Gln?Gly?Val?Gly?Gly?Pro
210 215 220
Gly?His?Lys?Ala?Arg?Val?Leu
225 230
<210> 2
<211> 693
<212> DNA
＜213〉HIV (human immunodeficiency virus) HIV-1 NL4-3 strain capsid protein p24
<400> 2
cctatagtgc?agaacctcca?ggggcaaatg?gtacatcagg?ccatatcacc?tagaacttta 60
aatgcatggg?taaaagtagt?agaagagaag?gctttcagcc?cagaagtaat?acccatgttt 120
tcagcattat?cagaaggagc?caccccacaa?gatttaaata?ccatgctaaa?cacagtgggg 180
ggacatcaag?cagccatgca?aatgttaaaa?gagaccatca?atgaggaagc?tgcagaatgg 240
gatagattgc?atccagtgca?tgcagggcct?attgcaccag?gccagatgag?agaaccaagg 300
ggaagtgaca?tagcaggaac?tactagtacc?cttcaggaac?aaataggatg?gatgacacat 360
aatccaccta?tcccagtagg?agaaatctat?aaaagatgga?taatcctggg?attaaataaa 420
atagtaagaa?tgtatagccc?taccagcatt?ctggacataa?gacaaggacc?aaaggaaccc 480
tttagagact?atgtagaccg?attctataaa?actctaagag?ccgagcaagc?ttcacaagag 540
gtaaaaaatt?ggatgacaga?aaccttgttg?gtccaaaatg?cgaacccaga?ttgtaagact 600
attttaaaag?cactgggacc?aggagcgaca?ctagaagaaa?tgatgacagc?atgtcaggga 660
gtggggggac?ccggccataa?agcaagagtt?ttg 693
<210> 3
<211> 9
<212> PRT
＜213〉mycobacterium tuberculosis H37Rv strain MPT64
76-84Antigen
<400> 3
Lys?Phe?Leu?Ser?Ala?Ala?Thr?Ser?Ser
1 5
<210> 4
<211> 27
<212> DNA
＜213〉mycobacterium tuberculosis H37Rv strain MPT64
76-84Antigen
<400> 4
aagttcctca?gcgcggcaac?atcgtcc 27
<210> 5
<211> 9
<212> PRT
＜213〉mycobacterium tuberculosis H37Rv strain Ag85A
242-250Antigen
<400> 5
Lys?Leu?Ile?Ala?Asn?Asn?Thr?Arg?Val
1 5
<210> 6
<211> 27
<212> DNA
＜213〉mycobacterium tuberculosis H37Rv strain Ag85A
242-250Antigen
<400> 6
aagctgatcg?ccaacaacac?ccgcgtc 27
<210> 7
<211> 9
<212> PRT
＜213〉mycobacterium tuberculosis H37Rv strain Ag85B
184-192Antigen
<400> 7
Ile?Tyr?Ala?Gly?Ser?Leu?Ser?Ala?Leu
1 5
<210> 8
<211> 27
<212> DNA
＜213〉mycobacterium tuberculosis H37Rv strain Ag85B
184-192Antigen
<400> 8
atctacgccg?gctcgctgtc?ggccctg 27
<210> 9
<211> 9
<212> PRT
＜213〉mycobacterium tuberculosis H37Rv strain TB10.4
74-82Antigen
<400> 9
Ser?Thr?His?Glu?Ala?Asn?Thr?Met?Ala
1 5
<210> 10
<211> 27
<212> DNA
＜213〉mycobacterium tuberculosis H37Rv strain TB10.4
74-82Antigen
<400> 10
agcacccatg?aagccaacac?catggcg 27
<210> 11
<211> 267
<212> PRT
＜213〉the chimeric people HIV-1 capsid protein p24 external source genes of interest albumen of 4 tuberculosis t cell epitopes
<400> 11
Lys?Phe?Leu?Ser?Ala?Ala?Thr?Ser?Ser?Pro?Ile?Val?Gln?Asn?Leu?Gln
1 5 10 15
Gly?Gln?Met?Val?His?Gln?Ala?Ile?Ser?Pro?Arg?Thr?Leu?Asn?Ala?Trp
20 25 30
Val?Lys?Val?Val?Glu?Glu?Lys?Ala?Phe?Ser?Pro?Glu?Val?Ile?Pro?Met
35 40 45
Phe?Ser?Ala?Leu?Ser?Cys?Gly?Ala?Thr?Pro?Gln?Asp?Leu?Asn?Thr?Met
50 55 60
Leu?Asn?Thr?Val?Gly?Gly?His?Gln?Ala?Ala?Met?Gln?Met?Leu?Lys?Glu
65 70 75 80
Thr?Ile?Asn?Glu?Glu?Ala?Ala?Glu?Trp?Asp?Arg?Leu?His?Pro?Val?His
85 90 95
Ala?Gly?Pro?Ile?Lys?Leu?Ile?Ala?Asn?Asn?Thr?Arg?Val?Ala?Pro?Gly
100 105 110
Gln?Met?Arg?Glu?Pro?Arg?Gly?Ile?Tyr?Ala?Gly?Ser?Leu?Ser?Ala?Leu
115 120 125
Ser?Asp?Ile?Ala?Gly?Thr?Thr?Ser?Thr?Leu?Gln?Glu?Gln?Ile?Gly?Trp?Met
130 135 140
Thr?His?Asn?Pro?Pro?Ile?Pro?Val?Gly?Glu?Ile?Tyr?Lys?Arg?Trp
145 150 155 160
Ile?Ile?Leu?Gly?Leu?Asn?Lys?Ile?Val?Arg?Met?Tyr?Ser?Pro?Ser?Thr
165 170 175
His?Glu?Ala?Asn?Thr?Met?Ala?Thr?Ser?Ile?Leu?Asp?Ile?Arg?Gln?Gly?Pro
180 185 190
Lys?Glu?Pro?Phe?Arg?Asp?Tyr?Val?Asp?Arg?Phe?Tyr?Lys?Thr?Leu?Arg
195 200 205
Ala?Glu?Gln?Ala?Ser?Gln?Glu?Val?Lys?Asn?Ala?Ala?Thr?Glu?Thr?Leu
210 215 220
Leu?Val?Gln?Asn?Ala?Asn?Pro?Asp?Cys?Lys?Thr?Ile?Leu?Lys?Ala?Leu
225 230 235 240
Gly?Pro?Gly?Ala?Thr?Leu?Glu?Glu?Met?Met?Thr?Ala?Cys?Gln?Gly?Val
245 250 255
Val?Gly?Gly?Pro?Gly?His?Lys?Ala?Arg?Val?Leu
260 265
<210> 12
<211> 801
<212> DNA
＜213〉the chimeric people HIV-1 capsid protein p24 external source genes of interest of 4 t cell epitopes
<400> 12
aagttcctca?gcgcggcaac?atcgtcccct?atagtgcaga?acctccaggg?gcaaatggta 60
catcaggcca?tatcacctag?aactttaaat?gcatgggtaa?aagtagtaga?agagaaggct 120
ttcagcccag?aagtaatacc?catgttttca?gcattatcag?aaggagccac?cccacaagat 180
ttaaatacca?tgctaaacac?agtgggggga?catcaagcag?ccatgcaaat?gttaaaagag 240
accatcaatg?aggaagctgc?agaatgggat?agattgcatc?cagtgcatgc?agggcctatt 300
aagctgatcg?ccaacaacac?ccgcgtcgca?ccaggccaga?tgagagaacc?aaggggaatc 360
tacgccggct?cgctgtcggc?cctgagtgac?atagcaggaa?ctactagtac?ccttcaggaa 420
caaataggat?ggatgacaca?taatccacct?atcccagtag?gagaaatcta?taaaagatgg 480
ataatcctgg?gattaaataa?aatagtaaga?atgtatagcc?ctagcaccca?tgaagccaac 540
accatggcga?ccagcattct?ggacataaga?caaggaccaa?aggaaccctt?tagagactat 600
gtagaccgat?tctataaaac?tctaagagcc?gagcaagctt?cacaagaggt?aaaaaattgg 660
atgacagaaa?ccttgttggt?ccaaaatgcg?aacccagatt?gtaagactat?tttaaaagca 720
ctgggaccag?gagcgacact?agaagaaatg?atgacagcat?gtcagggagt?ggggggaccc 780
ggccataaag?caagagtttt?g 801
<210> 13
<211> 57
<212> DNA
＜213〉F1 primer
<400> 13
ctagctagca?tgaagttcct?cagcgcggca?acatcgtccc?ctatagtgca?gaacctc 57
<210> 14
<211> 69
<212> DNA
＜213〉F2 downstream primer
<400> 14
tggttctctc?atctggcctg?gtgcgacgcg?ggtgttgttg?gcgatcagct?taataggccc?tgcatgcac 69
<210> 15
<211> 69
<212> DNA
＜213〉T1 primer
<400> 15
ggccagatga?gagaaccaag?gggaatctac?gccggctcgc?tgtcggccct?gagtgacata?gcaggaact 69
<210> 16
<211> 69
<212> DNA
＜213〉T2 primer
<400> 16
tatgtccaga?atgctggtcg?ccatggtgtt?ggcttcatgg?gtgctagggc?tatacattct?tactatttt 69
<210> 17
<211> 39
<212> DNA
＜213〉R1 primer
<400> 17
atggcgacca?gcattctgga?cataagacaa?ggaccaaag 39
<210> 18
<211> 39
<212> DNA
＜213〉R2 primer
<400> 18
ccggaattct?tacaaaactc?ttgctttatg?gccgggtcc 39
Claims (20)
1. tuberculosis gene vaccine with anti-HIV-1 virus, constituted by a carrier, in described carrier, be inserted with one section external source genes of interest, it is characterized in that: described external source genes of interest comprises the full-length gene of the capsid protein p24 in a HIV (human immunodeficiency virus) HIV-1NL4-3 strain source, chimeric 4 t cell epitope polypeptide genes that have from negre antigen in the full-length gene of the capsid protein p24 that described HIV (human immunodeficiency virus) HIV-1NL4-3 strain is originated, described 4 t cell epitopes are respectively 76～84 genes of the MPT64 albumen in mycobacterium tuberculosis H37Rv strain source, 242～250 genes of the Ag85A albumen in mycobacterium tuberculosis H37Rv strain source, 184～192 genes of the Ag85B albumen in mycobacterium tuberculosis H37Rv strain source, 74～82 genes of the TB10.4 albumen in mycobacterium tuberculosis H37Rv strain source, from 5 ' of the capsid protein p24 full-length gene in HIV (human immunodeficiency virus) HIV-1NL4-3 strain source, before the 1st bit base, insert 76～84 genes of the MPT64 albumen in mycobacterium tuberculosis H37Rv strain source, behind the 273rd bit base, insert 242～250 genes of the Ag85A albumen in mycobacterium tuberculosis H37Rv strain source, behind the 303rd bit base, insert 184～192 genes of the Ag85B albumen in mycobacterium tuberculosis H37Rv strain source, behind the 441st bit base, insert 74～82 genes of the TB10.4 albumen in mycobacterium tuberculosis H37Rv strain source, the DNA sequence of the capsid protein p24 full-length gene in described HIV (human immunodeficiency virus) HIV-1NL4-3 strain source is shown in SEQ IDNO:2, the DNA sequence of 76～84 genes of the MPT64 albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:4, the DNA sequence of 242～250 genes of the Ag85A albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:6, the DNA sequence of 184～192 genes of the Ag85B albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:8, and the DNA sequence of 74～82 genes of the TB10.4 albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:10.
2. a kind of tuberculosis gene vaccine with anti-HIV-1 virus according to claim 1 is characterized in that: the DNA sequence of the external source genes of interest that the capsid protein p24 full-length gene in the HIV (human immunodeficiency virus) HIV-1NL4-3 strain source of 4 t cell epitope genes in chimeric mycobacterium tuberculosis H37Rv strain source constitutes is shown in SEQ ID NO:12.
3. the tuberculosis gene vaccine with anti-HIV-1 virus according to claim 1, it is characterized in that: described carrier is eukaryon expression plasmid, described eukaryon expression plasmid is selected from any one in pcDNA3.1 plasmid or the pVAX1 plasmid.
4. the tuberculosis gene vaccine with anti-HIV-1 virus according to claim 1 is characterized in that: the aminoacid sequence SEQ ID of the capsid protein p24 in described HIV (human immunodeficiency virus) HIV-1NL4-3 strain source is shown in NO:1.
5. the tuberculosis gene vaccine with anti-HIV-1 virus according to claim 1 is characterized in that: the aminoacid sequence of 76～84 genes of the MPT64 albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:3.
6. the tuberculosis gene vaccine with anti-HIV-1 virus according to claim 1 is characterized in that: the aminoacid sequence of 242～250 genes of the Ag85A albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:5.
7. the tuberculosis gene vaccine with anti-HIV-1 virus according to claim 1 is characterized in that: the aminoacid sequence of 184～192 genes of the Ag85B albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:7.
8. the tuberculosis gene vaccine with anti-HIV-1 virus according to claim 1 is characterized in that: the aminoacid sequence of 74～82 genes of the TB10.4 albumen in described mycobacterium tuberculosis H37Rv strain source is shown in SEQ ID NO:9.
9. the tuberculosis gene vaccine with anti-HIV-1 virus according to claim 1 is characterized in that: the aminoacid sequence of the external source genes of interest albumen that the full-length gene of the capsid protein p24 in the HIV (human immunodeficiency virus) HIV-1 strain source of 4 t cell epitope genes in chimeric mycobacterium tuberculosis H37Rv strain source constitutes is shown in SEQ ID NO:11.
10. the described preparation method with tuberculosis gene vaccine of anti-HIV-1 virus of claim 1 is characterized in that: may further comprise the steps:
1) extracts HIV-1NL4-3 strain DNA as template, by the encoding gene of PCR method amplifying human immunodeficiency virus HIV-1 strain capsid protein p24;
2) with HIV-1 capsid protein p24 gene as template, the overlapping double chain DNA fragment 1 of 18 bases, fragment 2 are arranged each other and 24 double chain DNA fragments 3 that base is overlapping are arranged each other by pcr amplification respectively, the sequence of the double-stranded DNA of described fragment 1 is
atgaagttcc tcagcgcggc aacatcgtcc cctatagtgc agaacctcca ggggcaaatg
tacttcaagg agtcgcgccg ttgtagcagg ggatatcacg tcttggaggt ccccgtttac
gtacatcagg ccatatcacc tagaacttta aatgcatggg taaaagtagt agaagagaag
catgtagtcc ggtatagtgg atcttgaaat ttacgtaccc attttcatca tcttctcttc
gctttcagcc cagaagtaat acccatgttt tcagcattat cagaaggagc caccccacaa
cgaaagtcgg gtcttcatta tgggtacaaa agtcgtaata gtcttcctcg gtggggtgtt
gatttaaata ccatgctaaa cacagtgggg ggacatcaag cagccatgca aatgttaaaa
ctaaatttat ggtacgattt gtgtcacccc cctgtagttc gtcggtacgt ttacaatttt
gagaccatca atgaggaagc tgcagaatgg gatagattgc atccagtgca tgcagggcct
ctctggtagt tactccttcg acgtcttacc ctatctaacg taggtcacgt acgtcccgga
attaagctga tcgccaacaa cacccgcgtc gcaccaggcc agatgagaga acca
taattcgact agcggttgtt gtgggcgcag cgtggtccgg tctactctct tggt
The sequence of the double-stranded DNA of described fragment 2 is
ggccagatga gagaaccaag gggaatctac gccggctcgc tgtcggccct gagtgacata
ccggtctact ctcttggttc cccttagatg cggccgagcg acagccggga ctcactgtat
gcaggaacta ctagtaccct tcaggaacaa ataggatgga tgacacataa tccacctatc
cgtccttgat gatcatggga agtccttgtt tatcctacct actgtgtatt aggtggatag
ccagtaggag aaatctataa aagatggata atcctgggat taaataaaat agtaagaatg
ggtcatcctc tttagatatt ttctacctat taggacccta atttatttta tcattcttac
tatagcccta gcacccatga agccaacacc atggcgacca gcattctgga cata
atatcgggat cgtgggtact tcggttgtgg taccgctggt cgtaagacct gtat
The sequence of the double-stranded DNA of described fragment 3 is
atggcgacca gcattctgga cataagacaa ggaccaaagg aaccctttag agactatgta
taccgctggt cgtaagacct gtattctgtt cctggtttcc ttgggaaatc tctgatacat
gaccgattct ataaaactct aagagccgag caagcttcac aagaggtaaa aaattggatg
ctggctaaga tattttgaga ttctcggctc gttcgaagtg ttctccattt tttaacctac
acagaaacct tgttggtcca aaatgcgaac ccagattgta agactatttt aaaagcactg
tgtctttgga acaaccaggt tttacgcttg ggtctaacat tctgataaaa ttttcgtgac
ggaccaggag cgacactaga agaaatgatg acagcatgtc agggagtggg gggacccggc
cctggtcctc gctgtgatct tctttactac tgtcgtacag tccctcaccc ccctgggccg
cataaagcaa gagttttgta a
gtatttcgtt ctcaaaacat t
3) 3 sections above-mentioned fragments are mixed, degeneration becomes strand, by 18 or 24 base complementrities connections that overlap each other, with this as template, obtained the HIV (human immunodeficiency virus) capsid protein p24 external source genes of interest of 4 t cell epitopes chimeric by PCR method amplification, genes of interest is connected with the carrier of the same double digestion of warp behind double digestion, obtains the described tuberculosis gene vaccine with anti-HIV-1 virus of claim 1.
11. the preparation method with tuberculosis gene vaccine of anti-HIV-1 virus as claimed in claim 10 is characterized in that: pass through pcr amplified fragment 1 with the p24F2 primer shown in the p24F1 primer shown in the SEQ ID NO:13 and the SEQ ID NO:14.
12. the preparation method with tuberculosis gene vaccine of anti-HIV-1 virus as claimed in claim 10 is characterized in that: pass through pcr amplified fragment 2 with the primer p24T2 sequence shown in the primer p24T1 sequence shown in the SEQ ID NO:15 and the SEQ ID NO:16.
13. the preparation method with tuberculosis gene vaccine of anti-HIV-1 virus as claimed in claim 10 is characterized in that: pass through pcr amplified fragment 3 with the p24R2 primer sequence shown in the primer p24R1 sequence shown in the SEQ ID NO:17 and the SEQ ID NO:18.
14. the preparation method with tuberculosis gene vaccine of anti-HIV-1 virus as claimed in claim 10, it is characterized in that: with the p24F1 primer shown in the SEQ ID NO:13 and the p24R2 primer shown in the SEQ ID NO:18 mix with fragment 1,2,3 and degeneration be strand, gene after complementary the connection is template, increased the HIV-1 capsid protein p24 external source genes of interest of 4 t cell epitopes chimeric by PCR method.
15. the preparation method with tuberculosis gene vaccine of anti-HIV-1 virus as claimed in claim 10 is characterized in that: the p24 external source genes of interest of chimeric 4 t cell epitopes and the restriction enzyme site that inserts carrier are respectively NheI and EcoRI site.
16. the preparation method with tuberculosis gene vaccine of anti-HIV-1 virus as claimed in claim 10 is characterized in that: antigen is from mycobacterium tuberculosis H37Rv strain and HIV (human immunodeficiency virus) HIV-1NL4-3 strain.
17. the preparation method with tuberculosis gene vaccine of anti-HIV-1 virus as claimed in claim 10 is characterized in that: described carrier is selected from pcDNA3.1 plasmid or pVAX1 plasmid.
18. a pharmaceutical composition is characterized in that: the described tuberculosis gene vaccine with anti-HIV-1 virus of claim 1 that contains effective dose.
19. a kind of pharmaceutical composition as claimed in claim 18 is characterized in that: also contain pharmaceutically acceptable carrier or excipient.
20. the described application of tuberculosis gene vaccine in the medicine of preparation treatment or prevention tuberculosis and acquired immune deficiency syndrome (AIDS) with anti-HIV-1 virus of claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005079833A1 (en) * | 2004-02-20 | 2005-09-01 | China Agricultural University | T cell immune response inhibitor |
| CN101451145A (en) * | 2007-11-30 | 2009-06-10 | 复旦大学 | Tuberculosis gene vaccine based on T cell epitope as well as preparation method and use thereof |
| CN101455846A (en) * | 2007-12-11 | 2009-06-17 | 复旦大学 | Tuberculosis gene vaccine assembled by chitosan delivery system and preparation method and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005079833A1 (en) * | 2004-02-20 | 2005-09-01 | China Agricultural University | T cell immune response inhibitor |
| CN101451145A (en) * | 2007-11-30 | 2009-06-10 | 复旦大学 | Tuberculosis gene vaccine based on T cell epitope as well as preparation method and use thereof |
| CN101455846A (en) * | 2007-12-11 | 2009-06-17 | 复旦大学 | Tuberculosis gene vaccine assembled by chitosan delivery system and preparation method and use thereof |
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| Title |
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| 徐薇等: "以抗原表位为基础的基因免疫", 《科技导报》 * |
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