CN102839186A - Preparation and application of recombinant Mycobacterium smegmatis expressing NY-ESO-1 - Google Patents
Preparation and application of recombinant Mycobacterium smegmatis expressing NY-ESO-1 Download PDFInfo
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Abstract
The invention relates to preparation and application of recombinant Mycobacterium smegmatis expressing NY-ESO-1. Specifically, the invention provides a plasmid for expressing a recombinant cancer-testis antigen NY-ESO-1 protein; and the plasmid comprises a beta-galactosidase signal peptide coding sequence or a beta- galactosidase coding sequence, and an NY-ESO-1 coding sequence. The invention also provides a recombinant Mycobacterium smegmatis containing the plasmid provided by the invention and its composition, and application thereof. The recombinant Mycobacterium smegmatis provided by the invention has significant killing effect on tumor cells expressing NY-ESO-1; therefore the recombinant Mycobacterium smegmatis can be used in effective prevention and / or treatment of the tumors.
Description
Technical field
The invention belongs to biology and genetic engineering field, in particular to M. smegmatics, its preparation and the application of this recombinant Mycobacterium smegmatis in the immunotherapy tumour of a kind of expressing tumor/testis antigen NY-ESO-1.
Background technology
M. smegmatics (M.smegmatis) is a kind of atypical mycobacterium; Compare with typical mycobacterium BCG; The M. smegmatics growth to equal no pathogenicities of animal such as people, mouse, cavys, has wide application prospect as a kind of bacterial vaccine carrier fast.
BCG is as the existing very long history of the immune adjuvant therapy of superficial bladder cancer, but still there is toxicity in the BCG that lives, might cause the infection of tubercule bacillus, and especially there is very big harm in the patient to immunodeficiency type.Preliminary study shows that acellular Mycobacterium smegmatis vaccine has good immunoregulation effect, and the cavy that mycobacterium tuberculosis (MTB) is infected has obvious protection effect, and has higher-security, has got into II phase clinical study [1] at present.In addition, the secreted protein output in the M. smegmatics unit time be BCG 50-100 doubly.Therefore, become the focus in the present vaccine research with M. smegmatics as a kind of low cost and effective vaccine carrier expression exogenous antigen.
[2] such as Jae-Sung Yu have been studied and have been expressed the immunization of the proteic recombinant Mycobacterium smegmatis of HIV-1 to mouse, and the result shows through the recombinant Mycobacterium smegmatis mice immunized and successfully produced HIV antibody and special T lymphocyte reaction and IFN-γ.[3] such as Lin Lu have made up the recombinant Mycobacterium smegmatis of expressing human helicobacter pylori 26K outer membrane protein (OMP6); Mouse after this recombinant Mycobacterium smegmatis immunity has produced tangible immanoprotection action to the infection of helicobacter pylori; The helicobacter pylori bacterium colony sharply reduces in its enteron aisle, and this author thinks that this recombinant Mycobacterium smegmatis can be used as a kind of combined vaccine that cheaply, effectively is directed against helicobacter pylori thus.
NY-ESO-1 antigen is the important member in the tumor-testis antigen family; On kinds of tumor cells, express; Comprise melanoma, mammary cancer, prostate cancer, lung cancer, ovarian cancer and bladder cancer, and be one of immunogenic tumour antigen of tool of finding so far.
NY-ESO-1 antigen can cause spontaneous humoral immune reaction and specific T-cells immunoreation [4] in NY-ESO-1 expresses male tumour patient body; Therefore this antigen is considered to one of at present best tumour candidate vaccine, in immunotherapy of tumors, has much development potentiality.
At present, be based on its different epi-positions (like p156-167, p157-160, p157-170) mostly, these antigen peptide of synthetic or these antigen peptide and chaperone (like HSP) fusion studied as vaccine to the polypeptide vaccine of NY-ESO-1.
Yet, still press for the polypeptide vaccine of developing more effective NY-ESO-1 in this area, to obtain more significant immune effect.
Summary of the invention
One of the object of the invention provides the mycobacterium-bacillus coli shuttle expression plasmid that has the NY-ESO-1 gene, its can with NY-ESO-1 respectively with beta-galactosidase enzymes signal peptide and complete sequence amalgamation and expression.
Another object of the present invention provides and a kind ofly can tumour/testis antigen NY-ESO-1 grappling be expressed in the M. smegmatics engineering strain on cell walls or the cytolemma.
A further object of the present invention has provided the application of engineering bacillus strain in immunotherapy of tumors.Adopt recombinant Mycobacterium smegmatis immunity object of the present invention; Can in subject, improve antigen NY-ESO-1 offer and bring out body produce cell, humoral immune reaction; And then kill and wound the tumour cell of expressing NY-ESO-1, play the effect that prevents and/or treats tumour.
In first aspect of the present invention, a kind of plasmid that is used for recombinant tumor-testis antigen NY-ESO-1 protein expression is provided, said plasmid comprises:
(a) beta-galactosidase enzymes signal coding sequence or beta-galactosidase enzymes encoding sequence; With
(b) NY-ESO-1 encoding sequence.
In an embodiment of the invention, said reorganization NY-ESO-1 albumen is the fusion rotein of beta-galactosidase enzymes signal peptide sequence and NY-ESO-1 or the fusion rotein of beta-galactosidase enzymes full length sequence and NY-ESO-1.
In a preference, said fusion rotein is positioned on the cell walls or cytolemma of recombinant Mycobacterium smegmatis, on the preferred cell wall.
In another preference; Beta-galactosidase enzymes signal peptide sequence in the said fusion rotein or full length sequence directly link to each other with NY-ESO-1 or link to each other through the connection peptides sequence; The length of preferred said connection peptides is 1～30 amino-acid residue; Preferred 5～25 amino-acid residues, more preferably 10～23 amino-acid residues.
In another preference, said fusion rotein comprises the sequence shown in SEQ ID NO:6 or the SEQ ID NO:8, is preferably the sequence shown in SEQ ID NO:6 or the SEQ ID NO:8.
In yet another embodiment of the present invention; Said beta-galactosidase enzymes signal coding sequence is selected from sequence as follows: GenBank number: the polypeptide of sequence encoding or their conservative property variant protein or its active fragments or aminoacid sequence wherein shown in L25634.1 [14], the SEQ ID NO:3 are through 1-20 (preferred 1-10; More preferably 1-5) replacement, disappearance or the interpolation of amino-acid residue form, and instruct beta-galactosidase enzymes to be positioned the signal sequence on cell walls or the cytolemma;
Said beta-galactosidase enzymes encoding sequence is selected: GenBank number: sequence shown in L25634.1, the SEQ ID NO:4, its homologous sequence or under stringent condition can with the sequence of this sequence hybridization; Have at least 75%, 80%, 90%, 95%, 98% or the polynucleotide of higher homogeny with the sequence of said coding nucleic acid;
Said NY-ESO-1 encoding sequence is selected from sequence as follows: the sequence shown in sequence shown in the GenBank U87459.1, the SEQ ID NO:1, its homologous sequence or under stringent condition can with the sequence of this sequence hybridization; Have at least 75%, 80%, 90%, 95%, 98% or the polynucleotide sequence of higher homogeny with the sequence of said coding nucleic acid.
In a preference, said tumor-testis antigen NY-ESO-1 derives from testis tissue or the tumor tissues of people, rat, mouse, preferably derives from people's testis tissue.
In yet another embodiment of the present invention, said plasmid comprises the sequence shown in SEQ ID NO:5 or the SEQ ID NO:7.
In yet another embodiment of the present invention, said plasmid is a shuttle plasmid, and it can duplicate amplification in intestinal bacteria, M. smegmatics or mycobacterium tuberculosis.
In a preference, said recombinant plasmid is mycobacterium-bacillus coli shuttle expression plasmid pLA73NY or pLA71NY, preferred pLA71NY.
In yet another embodiment of the present invention, the plasmid of the said NY-ESO-1 protein expression that is used to recombinate is based on the plasmid construction that is selected from down group: pLA71, pLA73, pJH152 or pJEM17
In a preference, the plasmid of the said NY-ESO-1 protein expression that is used to recombinate is based on the pLA71 plasmid construction.
In second aspect of the present invention, a kind of recombinant Mycobacterium smegmatis is provided, it comprises plasmid of the present invention.
In a preference, said recombinant Mycobacterium smegmatis is based on that the mycobacterium that is selected from down group makes up: M. smegmatics mc
2155 bacterial strains, mycobacterium tuberculosis, preferred M. smegmatics mc
2155 bacterial strains.
In an embodiment of the invention, the NY-ESO-1 albumen of its express recombinant, and the albumen that will recombinate is positioned cell walls.
In a preference, said reorganization NY-ESO-1 albumen is the fusion rotein of beta-galactosidase enzymes signal peptide sequence or full length sequence and NY-ESO-1.
In the third aspect of the invention, a kind of immune composition is provided, said compsn comprises:
(i) plasmid of the present invention, recombinant Mycobacterium smegmatis of the present invention or its lysate or cell walls part; With
(ii) acceptable carrier and/or adjuvant on the immunology.
In a preference, said immune composition also comprises NY-ESO-1 antigen.
In another preference; Said immune composition is used to prevent and/or treat the NY-ESO-1 positive tumor; Said tumour includes but not limited to: melanoma, mammary cancer, prostate cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, preferred bladder cancer or prostate cancer.
In another preference, said recombinant Mycobacterium smegmatis is viable bacteria body, deactivation thalline or deactivation thalline.
In fourth aspect of the present invention, provide plasmid of the present invention, recombinant Mycobacterium smegmatis of the present invention or its lysate or cell walls part to be used for preventing and/or treating the purposes of the pharmaceutical composition or the vaccine of NY-ESO-1 positive tumor in preparation.
In a preference, said NY-ESO-1 positive tumor is selected from: melanoma, mammary cancer, prostate cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, preferred bladder cancer or prostate cancer.
In aspect the of the present invention the 5th, a kind of method for preparing recombinant Mycobacterium smegmatis of the present invention is provided, said method comprises the steps:
(1) plasmid of the present invention is provided;
(2) transform M. smegmatics to obtain described recombinant Mycobacterium smegmatis with said plasmid.
In a preference, said method also comprises the step of cultivating amplification and/or separating the recombinant Mycobacterium smegmatis that is obtained.
In another aspect of the present invention; A kind of test kit is provided; Said test kit comprises: just exempt from the recombinant Mycobacterium smegmatis of significant quantity and booster immunization significant quantity, perhaps just exempt from the recombinant Mycobacterium smegmatis of significant quantity and the NY-ESO-1 antigen of booster immunization significant quantity; And optional: thinner, solvent, adjuvant, carrier etc.
In another aspect of the present invention, the present invention is provided also the purposes of plasmid and/or recombinant Mycobacterium smegmatis as stated, it is used to prepare immune composition of the present invention.Said immune composition is used to prevent and/or treat the NY-ESO-1 positive tumor, and said tumour includes but not limited to: melanoma, mammary cancer, prostate cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, preferred bladder cancer or prostate cancer.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and specifically described each technical characterictic can mutual combination in (like embodiment) hereinafter, thus constitute new or optimized technical scheme.As space is limited, this tired no longer one by one stating.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Shown in Figure 1 is the design of graphics of plasmid pLA71NY (Figure 1A), pLA73NY (Figure 1B).
Shown in Figure 2 is the bacterium colony PCR checking of recombinant Mycobacterium smegmatis.
The detection of in recombinant Mycobacterium smegmatis, expressing for NY-ESO-1 shown in Figure 3.
Shown in Figure 4 is the location of NY-ESO-1 in recombinant Mycobacterium smegmatis.
Shown in Figure 5 is through rMS-pLA71NY immune serum antibody and the inferior really analysis of type of antibody.
Shown in Figure 6 is propagation through rMS-pLA71NY immune mouse SPL.
Shown in Figure 7 is secretion through rMS-pLA71NY immune mouse SPL cytokine after antigenic stimulation.
Shown in Figure 8 is detection through the external kill capability of rMS-pLA71NY immune mouse SPL.
Embodiment
The inventor is through long-term and deep research; Find to pass through with NY-ESO-1 and beta-galactosidase enzymes signal peptide sequence or complete sequence fusion; Can the NY-ESO-1 grappling be expressed on the cell walls or cytolemma (preferred cell wall) of M. smegmatics; The difficulty of NY-ESO-1 vivoexpression and the restriction of epi-position thereof have been avoided on the one hand; On the other hand can be by the superior cell adjuvant effect of M. smegmatics, offering after enhancing NY-ESO-1 antigen gets in the body brought out body and produced significant cell and humoral immunization.On this basis, the inventor has accomplished the present invention.
Particularly; The M. smegmatics cell wall polysaccharides has good immunostimulation, tropina has better immunogenicity, bacterial nucleic acid because of being rich in the immunoloregulation function that the CpG segment has excellence; Therefore mycobacterium Smegmatis preparation can strengthen the immunologic function of normal body, promotes the recovery [6] of immunocompromised body's immunity.At present also obtained progress certain as the vector construction recombiant vaccine of foreign gene with M. smegmatics; Behind GLS and IL-12 coupling recombinant Mycobacterium smegmatis intranasal mucosa-immune mouse, the body specific immunity is cellular immunization and mucosa-immune enhancing [7] particularly; [8] such as Sarah L propose also to have shown good antitumor action with the recombinant Mycobacterium smegmatis that cytokine IFN-γ, TNF α modify, and can be used as effective new tool in the immunotherapy of tumors.
M. smegmatics is as another advantage of vaccine carrier, in reorganization gets into body, can be engulfed degraded by phagosome rapidly; Fast and effeciently offer to the effector cell; And BCG can the interference body phagosome maturation, escape and catch degraded, the cell wall polysaccharide, tropina and the CpG segment that discharge of the M. smegmatics of degraded can be protected exotic antigen on the one hand in addition; Simultaneously also can effectively promote antigenic intersection to offer, stimulate the immunoreation [9] of body.M. smegmatics is as the cell adjuvant, and main inductive is Th1 type immunne response, i.e. CD4
+The release of a large amount of differentiation of T cell and the cytokine of Th1 type.
The inventor's result of study shows, behind the recombinant Mycobacterium smegmatis immune mouse that makes up among the present invention, has produced tangible CD4
+The secretion of t cell response and IFN-γ, IL-2 also is accompanied by CD8 simultaneously
+The increase of T cell; And the rising of mice serum antibody behind the booster immunization, in-vitro multiplication and CTL killing experiments also fully prove through recombinant Mycobacterium smegmatis mice immunized of the present invention and have produced the special booster immunization reaction of NY-ESO-1 in vivo, and produced the T cellullar immunologic response of memory.
This area generally believes the CD8 of memory
+The T cell has important effect in mediation T lymphocyte kills and wounds, research in recent years shows CD4
+The Th1 cytokines IFN-γ of T emiocytosis can activate the expression of the MHC II molecule on APC surface, promotes directly offering of tumour antigen, in addition CD4
+The T cell can activate effect cells such as tumor-infiltrated property BMDC, eosinophil, scavenger cell, promotes anti tumor immune response, and can produce cytolysis molecule pore-forming protein, directly acts on tumour cell, and does not receive the restriction [10,11] of MHC molecule.That is to say the CD4 that recombinant Mycobacterium smegmatis of the present invention brought out
+T cell, IFN-γ and CD8
+T cell synergy has produced significant lethal effect to the NY-ESO-1-B16 cell.
Therefore, with recombinant Mycobacterium smegmatis vector expression tsa of the present invention bigger potentiality are arranged in immunotherapy of tumors.
What deserves to be mentioned is; NY-ESO-1 has expression in various degree in most of bladder cancer patients; [12] such as Padmanee S are with NY-ESO-1 and BCG, GM-CSF combined immunization bladder cancer patients; Discovery has produced significantly NY-ESO-1 antibody horizontal and specific CD4 in the patient body under the effect of adjuvant
+The T cell levels, research shows that also the recombinant Mycobacterium smegmatis of expressing IL-2 is inhibited to the mouse bladder cancer, and can prolong the lifetime [13] of bladder cancer mice with tumor.Therefore, the recombinant Mycobacterium smegmatis of the expression NY-ESO-1 that makes up among the present invention especially has great application potential to prevention of recurrence at the bladder cancer patients immune adjuvant therapy.
As used herein, have comprised " containing ", " having " or " comprising " " comprising ", " mainly by ... constitute ", " basically by ... constitute " and " by ... constitute "; " mainly by ... constitute ", " basically by ... constitute " belong to the subordinate concept of " containing ", " having " or " comprising " with " by ... formation ".
As used herein, " isolating " or " separation and purification " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification like polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
NY-ESO-1 albumen and NY-ESO-1 encoding sequence
As used herein; Term " NY-ESO-1 albumen " or " NY-ESO-1 " interchangeable use; Be meant a kind of important member in the tumor-testis antigen family; It is expressed on kinds of tumor cells, and can in NY-ESO-1 expresses male tumour subject, cause spontaneous humoral immune reaction and specific T-cells immunoreation.
The proteic aminoacid sequence of NY-ESO-1 that can be used among the present invention can include, but is not limited to: GenBank number: sequence shown in U87459.1, the SEQ ID NO:2, their homologous sequence, conservative property variant protein or its active fragments or aminoacid sequence wherein are through 1-20 (preferred 1-10; More preferably 1-5) replacement, disappearance or the interpolation of amino-acid residue form; And can induce body to produce the antibody of anti-NY-ESO-1, to prevent and/or treat the antigen of tumour.
Term " NY-ESO-1 albumen " or " NY-ESO-1 " also comprise the variant form that has with the albumen identical function of GenBank U87459.1 or SEQ ID NO:2.These variant forms comprise (but being not limited to): several (are generally 1-50; Preferably 1-30; 1-20 more preferably, 1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20; Preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of NY-ESO-1 and reactive derivative.
Invention also provides the analogue of NY-ESO-1 albumen or polypeptide.The difference of these analogues and NY-ESO-1 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain through various technology, as through radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(like D-amino acid), and has non-natural analogue that exist or synthetic amino acid (like β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to above-mentioned representational polypeptide of giving an example.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, in the synthetic and processing of polypeptide or further, carries out glycosylation modified and polypeptide that produce in the procedure of processing like those.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).
Should understand through conservative substituted sequence mentioned above and also can be used for the present invention; Preferred reactive derivative refers to compare with the original acid sequence, has 5 at the most, preferably at the most 3; More preferably at the most 2,1 amino acid is replaced by similar performance or close amino acid and is formed polypeptide best.
As used herein, term " NY-ESO-1 encoding sequence ", " NY-ESO-1 gene " interchangeable use all refer to the NY-ESO-1 albumen of encoding, and the nucleotide sequence that can in recombinant Mycobacterium smegmatis of the present invention, express.The NY-ESO-1 encoding sequence that can be used among the present invention can include, but is not limited to: the sequence shown in sequence shown in the GenBank U87459.1, the SEQ ID NO:1, its homologous sequence or under stringent condition can with the sequence of this sequence hybridization; Have at least 75%, 80%, 90%, 95%, 98% or the polynucleotide sequence of higher homogeny with the sequence of said coding nucleic acid.
Among the present invention, " stringent condition " is meant: (1) than hybridization under LIS and the comparatively high temps and wash-out, like 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, like 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with polypeptide of the present invention.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (like PCR) of nucleic acid to confirm and/or the proteic polynucleotide of separation coding NY-ESO-1.
In an embodiment of the invention; Can obtain described encoding sequence through conventional molecular biology method; Said method can be according to people such as normal condition such as Sambrook; Condition described in " molecular cloning: lab guide " (New York:Cold Spring Harbor Laboratory Press, 1989) is carried out.
Beta-galactosidase enzymes signal peptide sequence or full length sequence and encoding sequence thereof
As used herein, term " beta-galactosidase enzymes signal peptide sequence " is meant to can be used in the beta-galactosidase enzymes full length sequence expressing and instructs this enzyme to be positioned the signal sequence on cell walls or the cytolemma (preferred cell wall).
The aminoacid sequence that can be used for the beta-galactosidase enzymes signal peptide sequence among the present invention can include, but is not limited to: GenBank number: the polypeptide of sequence encoding or their conservative property variant protein or its active fragments or aminoacid sequence wherein shown in L25634.1, the SEQ ID NO:3 are through 1-20 (preferred 1-10; More preferably 1-5) replacement, disappearance or the interpolation of amino-acid residue form, and can instruct beta-galactosidase enzymes to be positioned the signal sequence on cell walls or the cytolemma (preferred cell wall).
The aminoacid sequence that can be used for the beta-galactosidase enzymes full length sequence among the present invention can include, but is not limited to: GenBank number: the polypeptide of sequence encoding or their conservative property variant protein or its active fragments or aminoacid sequence wherein shown in L25634.1, the SEQ ID NO:4 are through 1-20 (preferred 1-10; More preferably 1-5) replacement, disappearance or the interpolation of amino-acid residue form, and can instruct beta-galactosidase enzymes to be positioned the signal sequence on cell walls or the cytolemma (preferred cell wall).
Should understand through conservative substituted sequence mentioned above and also can be used for the present invention; Preferred reactive derivative refers to compare with the original acid sequence, has 5 at the most, preferably at the most 3; More preferably at the most 2,1 amino acid is replaced by similar performance or close amino acid and is formed polypeptide best.
As used herein, term " beta-galactosidase enzymes signal coding sequence " is meant the coding beta-galactosidase signal peptide, and the nucleotide sequence that can in recombinant Mycobacterium smegmatis of the present invention, express.The beta-galactosidase enzymes signal coding sequence that can be used among the present invention can include, but is not limited to: GenBank number: the sequence shown in L25634.1, the SEQ ID NO:3, its homologous sequence or under stringent condition can with the sequence of this sequence hybridization; Have at least 75%, 80%, 90%, 95%, 98% or the polynucleotide of higher homogeny with the sequence of said coding nucleic acid.
As used herein, term " beta-galactosidase enzymes encoding sequence " is meant coding beta-galactosidase (comprising the beta-galactosidase enzymes signal peptide), and the nucleotide sequence that can in recombinant Mycobacterium smegmatis of the present invention, express.The beta-galactosidase enzymes encoding sequence that can be used among the present invention can include, but is not limited to: GenBank number: sequence shown in L25634.1, the SEQ ID NO:4, its homologous sequence or under stringent condition can with the sequence of this sequence hybridization; Have at least 75%, 80%, 90%, 95%, 98% or the polynucleotide of higher homogeny with the sequence of said coding nucleic acid.
Should be understood that the derived sequences such as hybridization sequences that also comprised in aforesaid term " beta-galactosidase enzymes signal peptide sequence ", " beta-galactosidase enzymes signal coding sequence ", " the beta-galactosidase enzymes encoding sequence " under its homologous sequence, conservative property series of variation, its active fragments, the stringent condition.The definition of said sequence with "
NY-ESO-1 albumen and NY-ESO-1 encoding sequence" part in description similar, repeat no more at this.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (like PCR) of nucleic acid to confirm and/or the proteic polynucleotide of separation coding NY-ESO-1.
In an embodiment of the invention; Can obtain described encoding sequence through conventional molecular biology method; Said method can be according to people such as normal condition such as Sambrook; Condition described in " molecular cloning: lab guide " (New York:Cold Spring Harbor Laboratory Press, 1989) is carried out.
Reorganization NY-ESO-1 albumen and be used for the proteic plasmid of express recombinant NY-ESO-1
As used herein; Term " reorganization NY-ESO-1 albumen ", " NY-ESO-1 recombinant protein " or " recombinant protein of the present invention " interchangeable use; All be meant and obtain through genetic engineering means; By the recombinant protein that beta-galactosidase enzymes signal peptide sequence that is blended in the NY-ESO-1 albumen n end or full length sequence are formed jointly, the beta-galactosidase enzymes signal peptide sequence of this fusion rotein or full length sequence can directly be connected with NY-ESO-1 albumen or link to each other through optional connection peptides.Recombinant protein of the present invention is compared with nonrecombinant NY-ESO-1 albumen has the NY-ESO-1 positive tumor cell lethal effect that significantly improves.
As used herein, term " connection peptides " or " amino acid connecting arm " interchangeable use refer to small peptide between beta-galactosidase enzymes signal peptide sequence or full length sequence and NY-ESO-1 Argine Monohydrochloride sequence, that play ligation.Connection peptides can exist or not exist, and its length is generally 1～30 amino acid, preferably is 5～25 amino acid, is 10～23 amino acid best.The technician can be according to this area ordinary method (as referring to PNAS 1998; 95:5929-5934; Protein Eng, 2000; 13 (5): design connection peptides documents such as 309-312).Usually, connection peptides does not influence or the remarkably influenced recombinant protein forms correct folding and space conformation.
In preferred implementation of the present invention, the sequence of said recombinant protein is shown in SEQ ID NO:6 or SEQID NO:8.
A kind of plasmid that can be used for expressing recombinant protein of the present invention is provided among the present invention.This plasmid comprises beta-galactosidase enzymes signal coding sequence or the beta-galactosidase enzymes encoding sequence that is connected in series; The NY-ESO-1 encoding sequence; And the connection peptides encoding sequence of optional (but nonessential).Plasmid of the present invention is preferably shuttle plasmid, and it can duplicate amplification in intestinal bacteria and M. smegmatics, mycobacterium tuberculosis.
In a preference, said recombinant plasmid is mycobacterium-bacillus coli shuttle expression plasmid pLA73NY or pLA71NY, preferred pLA71NY.
In preferred implementation of the present invention, can be based on following plasmid construction plasmid of the present invention, for example: pLA71, pLA73, pJH152 or pJEM17, preferred pLA71.The fusion of gene fragment can adopt usual manner as known in the art to carry out.For example, can the encoding sequence of recombinant protein be fixed a point to insert the appropriate location of plasmid, for example when used carrier is pLA71, preferably the recombinant protein encoding sequence inserted between Kpn I restriction enzyme site and the NotI restriction enzyme site.Can adopt ordinary method such as the PCR method expression cassette that from the expression plasmid that has made up, increases, and change the restriction enzyme site at two ends.
Recombinant Mycobacterium smegmatis and preparation thereof
As used herein, term " recombinant Mycobacterium smegmatis " or " recombinant Mycobacterium smegmatis of the present invention " interchangeable use all refer to express recombinant protein of the present invention, and this recombinant protein are positioned the M. smegmatics of cell walls.
The preparation method of recombinant Mycobacterium smegmatis of the present invention can comprise step: (1) provides the plasmid that can be used for expressing recombinant protein of the present invention; (2) transform M. smegmatics to obtain recombinant Mycobacterium smegmatis of the present invention with said plasmid; And the recombinant Mycobacterium smegmatis that (3) optional cultivation is increased and/or separation is obtained.
Conversion can adopt the conventional method for transformation of the present invention such as electricity conversion, protoplast transformation to carry out.The fusion rotein that is produced by recombinant Mycobacterium smegmatis of the present invention can stimulate immune system to produce the antibody of anti-NY-ESO-1.
In preferred implementation of the present invention, can adopt the M. smegmatics bacterial strain that is selected from down group to make up recombinant bacterial strain of the present invention: M. smegmatics mc
2155 bacterial strains, mycobacterium tuberculosis is like BCG.
Vaccine composition
The invention provides a kind of vaccine composition, it contains significant quantity (like 0.000001-50wt%; Preferable 0.00001-20wt%; Better, recombinant Mycobacterium smegmatis of the present invention 0.0001-10wt%) or its lysate or cell walls part (preferably purified), and acceptable carrier on the immunology.
Vaccine composition of the present invention can be used for treating and/or preventing tumour or the NY-ESO-1 male tumour of expressing NY-ESO-1; Said tumour includes but not limited to: melanoma, mammary cancer, prostate cancer, lung cancer, ovarian cancer, bladder cancer or liver cancer, preferred bladder cancer or prostate cancer.
As used herein, the composition of " acceptable on the immunology " is applicable to people and/or Mammals and does not have excessive bad side reaction (like toxicity, stimulation and transformation reactions), promptly has the material of rational benefit/risk ratio.Term " acceptable carrier on the immunology " refers to be used for the carrier of immune-active agent administration, comprises various vehicle and thinner.
This type carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Usually vaccine preparation should be complementary with administering mode, and vaccine composition of the present invention can be made into the injection form, for example with saline water or contain glucose and the aqueous solution of other assistant agent prepares through ordinary method.Described vaccine composition should be made under aseptic condition.The dosage of activeconstituents is treatment or prevention significant quantity.Preparation of the present invention also can be made into sustained release preparation.
The significant quantity of immune-active agent can change with the severity of the pattern of administration and disease to be treated etc. in the present composition.The selection of preferred significant quantity can be confirmed (for example through clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio of immune-active agent, metabolism, transformation period etc.; The patient the severity, patient's body weight, patient's immune state, the approach of administration etc. of the disease that will treat.For example, by an urgent demand of treatment or prevention situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
PH in the present composition or carrier etc. can be as indicated above, and can be selected with general knowledge as required by those of ordinary skills.
The administering mode of vaccine composition of the present invention has no particular limits, can be whole body or partial.Preferably; Vaccine composition of the present invention can give object through the mode of intramuscular injection; Usually, when the dosage of immune-active agent of the present invention with about 0.00001-50mg/kg the weight of animals (preferable 0.0001-10mg/kg the weight of animals) gives, can obtain gratifying effect.Also can adopt the means of gene therapy to carry out administration, such as can be directly with immune-active agent through delivering medicine to the experimenter such as methods such as injections; Perhaps can be delivered on the target spot, and make it to express recombinant protein of the present invention through the ceneme (such as expression vector etc.) that certain approach will carry immune-active agent.
Can directly give Mammals (such as the people) with recombinant Mycobacterium smegmatis of the present invention, or it is carried out giving object after the processing such as deactivation or deactivation, broken bacterium collecting cell wall fraction, purifying.
Can when just exempting from, distinguish the recombinant Mycobacterium smegmatis of the present invention of empoly effective amount with booster immunization, also can be when just exempting from the recombinant Mycobacterium smegmatis of the present invention of empoly effective amount, other NY-ESO-1 antigen of empoly effective amount when booster immunization.
Test kit
A kind of test kit that prevents and/or treats of the tumour that can be used for expressing NY-ESO-1 also is provided among the present invention.Test kit of the present invention comprises: just exempt from the recombinant Mycobacterium smegmatis of significant quantity and booster immunization significant quantity, perhaps just exempt from the recombinant Mycobacterium smegmatis of significant quantity and the NY-ESO-1 antigen of booster immunization significant quantity; And optional: thinner, solvent, adjuvant, carrier, container, specification sheets etc.
Those of ordinary skills can select the reagent in the test kit or material and consumption thereof according to the needs of concrete application.
Advantage of the present invention
Obtained a kind of recombinant Mycobacterium smegmatis of novelty among the present invention through genetic engineering technique, but its expressing tumor antigen NY-ESO-1 and it is anchored on the cell walls of this recombinant Mycobacterium smegmatis.This recombinant Mycobacterium smegmatis can bring out CD4
+T cell, IFN-γ and CD8
+T cell synergy can produce remarkable lethal effect to the tumour cell of expressing NY-ESO-1, can be used for effectively preventing and/or treating of these tumours thus.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook " molecular cloning: lab guide " (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Experiment material
1. bacterial strain and carrier
M. smegmatics mc
2155, e. coli jm109 is available from Shanghai Vaccine and Serum Institute;
M. smegmatics expression plasmid pLA71, pLA73 prepare according to reference [15];
The NY-ESO-1-B16 cell strain is that the strain of NY-ESO-1 male mouse melanin tumor cell is available from Shanghai Vaccine and Serum Institute.
2. gene and reagent
People's testis tissue mRNA purchases the ability lottery industry biotech firm in the Shen, Shanghai;
Each restriction enzyme, reversed transcriptive enzyme, Taq archaeal dna polymerase, T4 ligase enzyme and DNA operation test kit are all available from Takara biotech firm;
NY-ESO-1 monoclonal antibody E978 is available from Millipore company;
CD4
+, CD8
+Traget antibody is available from crystalline substance U.S. biotech firm;
The female BALB/c mouse in ages in SPF level 4～6 week is bought the Experimental Animal Center in Shanghai City;
The mouse boosting cell parting liquid is biological scientific & technical corporation available from reaching section;
WST-8 is available from the green skies, Hangzhou biotech firm;
IFN-γ and IL-2ELISA test kit are biotech firm available from reaching section;
The CTL detection kit is available from Promega company;
RPMI1640 is available from GIBICO company.
3. primer
The required primer of gene clone is given birth to worker bio-engineering corporation by Shanghai and is synthesized, and primer sequence is:
5’-ATGGTACCTCAGGCCGAAGGCCGG-3’(Kpn?I，SEQ?ID?NO：9)
5’-TAGCGGCCGCTTAGCGCCTCTGCCCTG-3’(Not?I，SEQ?ID?NO：10)。
Is cDNA by ordinary method with people's testis tissue mRNA rt; With people's testis tissue cDNA (GenBank number: AJ003149.1; Available from Shen, Shanghai ability lottery industry biotech firm) be template; Pcr amplification goes out the NY-ESO-1 gene, and the sequence of used forward primer and reverse primer is respectively shown in SEQ ID NO:9 and SEQ ID NO:10.The PCR reaction system is (the μ l of unit) as follows:
| Template | Forward | Reverse primer | 10 * Taq damping fluid | 10mMdNTP | ddH 2O | |
|
| 1 | 1 | 1 | ?5 | 1 | 40 | 1 |
The PCR reaction conditions is: 30 → 72 ℃ of 5min of circulation of 96 ℃ of 5min → (94 ℃ 30 seconds → 60 ℃ 30 seconds → 72 ℃ 50 seconds)
Reclaim the purpose fragment; NY-ESO-1 is passed through BamH I, Not I double digestion with carrier mycobacterium expression shuttle plasmid pLA71 or pLA73 respectively; Reclaim endonuclease bamhi; And connect, make NY-ESO-1 merge be cascaded (as shown in Figure 1) with beta-galactosidase enzymes signal peptide sequence or complete sequence respectively.
With gained recombinant plasmid transformed e. coli jm109, on the LB solid plate that contains 50 μ g/ml Km, screen positive transformant, and through enzyme cut, sequence verification.The checking result shows that structure has obtained and beta-galactosidase enzymes signal peptide sequence or the correct recombinant clone that merges of full length sequence.
The structure and the checking of embodiment 2. recombinant Mycobacterium smegmatis
1. M. smegmatics mc
2155 competent cell preparation
A. with the M. smegmatics inoculation in 3ml M7H 9-Tween80 substratum, 37 ℃ of 250rpm overnight cultures;
B. get 150 μ l bacterium liquid and be inoculated in the 150ml M7H 9-Tween80 substratum 37 ℃ of 250rpm overnight cultures;
C. survey OD
600, when reaching 1.2-1.6,4 ℃ of centrifugal 5min of 2500rpm;
D. abandon supernatant, the 150ml frozen water is resuspended, 4 ℃ of centrifugal 5min of 2500rpm;
E. abandon supernatant, the 75ml frozen water is resuspended, 4 ℃ of centrifugal 5min of 2500rpm;
F. abandon supernatant, 10ml 10% glycerine is resuspended, 4 ℃ of centrifugal 5min of 2500rpm;
G. abandon supernatant, 1.5ml 10% glycerine is resuspended, is competent cell.
2. electricity transforms
Get 10 μ l pLA71NY, pLA73NY and mix with 100 μ l competent cells respectively, adopt GenePulser (Bio-Rad) to carry out electricity and change, condition is 2.5kV, 25 μ F, 1000 Ω.
3. the checking of recombinant Mycobacterium smegmatis
Electric shock back adds 1ml M7H9 liquid nutrient medium, and mixing is cultivated 2h for 37 ℃ gently, gets 0.1ml and is applied on the M7H9 culture medium flat plate that contains 50 μ g/ml kantlex 37 ℃ and hatches.After 2-3 days, choose transformant and do bacterium colony PCR checking (PCR system and reaction conditions such as embodiment 1 are said), primer is NY-ESO-1 amplimer (shown in SEQ ID NO:9 and 10).
The result is as shown in Figure 2 in bacterium colony PCR checking; The result shows: recombinant Mycobacterium smegmatis rMS-pLA71NY (swimming lane 2), rMS-pLA73NY (swimming lane 3) have specific band at the 540bp place; Consistent with the NY-ESO-1 size, and M. smegmatics contrast (swimming lane 1) does not have specific band.
This result verification recombinant plasmid pLA71NY, pLA73NY be transferred in the M. smegmatics, form recombinant Mycobacterium smegmatis rMS-pLA71NY and rMS-pLA73NY respectively.
Expression and the location of NY-ESO-1 in embodiment 3. recombinant Mycobacterium smegmatis
The single transformant of picking recombinant Mycobacterium smegmatis rMS-pLA71NY is gone in the 4ml M7H 9-Tween80 substratum, and shaken overnight is cultivated, switching next day 2%.Treat that cell grows to 10
6～10
8CFU, centrifugal collection thalline; After PBS+0.02%Tween 20 damping fluid washed twice, resuspended with 1ml PBS+0.02%Tween20 damping fluid, carrying out ultrasonic bacteria breaking obtains the whole cell lysate; With the centrifugal 15min of 27000g, deposition is the cell walls enriching section with lysate; In supernatant, add 20 μ l Triton X-114 dissolving film albumen [5].Ultrafiltration and concentration nutrient solution supernatant obtains secretory protein.
With reference to " molecular cloning: lab guide "; Whole cell albumen to M. smegmatics and rMS-pLA71NY carries out western blotting (Western-blot) detection respectively: the SDS-PAGE gel electrophoresis of getting each several part protein sample warp 12%; Change film, analyze expression and the location of NY-ESO-1 with NY-ESO-1 monoclonal antibody E978 (available from Millipore company).
The result that western blotting detects is as shown in Figure 3, and wherein swimming lane 1-2 is respectively: wild-type M. smegmatics (MS), rMS-pLA73NY whole cell lysate, swimming lane 3 is the EY-ESO-1 of purifying.This result shows: rMS-pLA71NY has the purpose band at 30kDa (NY-ESO-1 and beta-galactosidase enzymes signal peptide merge) and 26kDa (albumen has the part degraded).
As shown in Figure 4 with the NY-ESO-1 monoclonal antibody to the result that rMS-pLA71NY cytolemma, cell walls, kytoplasm and last white protein detect; Wherein swimming lane 1 and 2 is respectively the whole cell lysate of wild-type M. smegmatics (MS) and rMS-pLA71NY, and swimming lane 3-6 is respectively rMS-pLA71NY cytolemma, cell walls, kytoplasm and goes up white protein.The result shows: NY-ESO-1 is expressed on the cell walls mostly, and some is not transported fully and rests on the cytolemma yet.
The preparation and the mouse immune of embodiment 4. recombinant Mycobacterium smegmatis (rMS-pLA71NY) vaccine
The single transformant of picking recombinant Mycobacterium smegmatis rMS-pLA71NY is gone in the 4ml M7H 9-Tween80 substratum, and shaken overnight is cultivated, switching next day 2%; Treat that cell grows to 10
7～10
10CFU, centrifugal collection thalline; After PBS+0.05%Tween 80 washed twice, resuspended, cell concn is adjusted to 10
9CFU promptly gets recombinant Mycobacterium smegmatis (rMS-pLA71NY) vaccine.
Get 200 μ l rMS-pLA71NY, the BALB/C mice in ages in abdominal injection 4 week, 12 every group, other establishes PBS+0.05%Tween 80 (200 μ l), M. smegmatics mc
2155 (200 μ l) and NY-ESO-1 (50 μ g/200 μ l) control group.Whenever once at a distance from 3 all tail vein bloods; And in the 10th week with the vaccine inoculation immunity of same dosage once; The 12nd week, each group was killed 6 mouse, won spleen under the aseptic condition, isolated lymphocytes; With NY-ESO-1 antigen (50 μ g) the residue mouse is carried out booster immunization simultaneously, after the week its serum antibody is analyzed.
The serum N Y-ESO-1 of embodiment 5. recombinant Mycobacterium smegmatis (rMS-pLA71NY) vaccine immune mouse is anti-
The detection of body and the analysis of antibody subtype
Each immune group mice serum of learning from else's experience adds the 96 hole enzyme plates that encapsulate through NY-ESO-1 albumen behind 10 times of gradient dilutions, and each diluted sample degree is done 3 multiple holes; Place 1h for 37 ℃, it is inferior to give a baby a bath on the third day after its birth with PBS-0.1%Tween20 (PBST), adds the sheep anti mouse ELIAS secondary antibody through the PBST of 4% skim-milk dilution; Place 1h for 37 ℃; It is inferior to give a baby a bath on the third day after its birth with PBS-0.1%Tween20 (PBST), presses the ELISA operation steps and adds developer, terminator; Detect absorbancy down in the 490nm wavelength, analyze antibody horizontal.Resist analyzing with sheep anti mouse enzyme mark IgG, IgG1, IgG2a, IgG3 two through the mice serum antibody subtype of NY-ESO-1 booster immunization after one week.
The antibody horizontal detected result is shown in Fig. 5 A; This result shows: through the rMS-pLA71NY mice immunized; When the 3rd, 6,9,12 weeks, all do not detect the antibody of NY-ESO-1, analysis possibly be because the NY-ESO-1 antigen quantity not sufficient of expressing is to let body produce high titer antibody.To the mouse booster immunization, after the week, rMS-pLA71NY immune group mouse produced antibody mediated immunity and replied with NY-ESO-1 antigen the 12nd week, and antibody horizontal is consistent with the protein immunization group.
The antibody subtype classification results is shown in Fig. 5 B, and this result shows: through the initial mice immunized of rMS-pLA71NY, IgG2a wants high than the initial immune group of albumen NY-ESO-1, promptly is partial to the Th1 immunoreation, but still is the mixed type immunoreation of Th1 and Th2 mediation.
CD4 in embodiment 6. recombinant Mycobacterium smegmatis (rMS-pLA71NY) vaccine immune mouse
+
, CD8
+
The T cell
The detection that distributes
Separate each experimental mice (the 12nd week) splenocyte, and adjust to consistent cell concn (2 * 10
6Individual cell/m1), respectively with CD4
+, CD8
+Antibody labeling detects through flow cytometer (BD FACS Calibur, U.S. company BD), and calculates CD4
+, CD8
+The per-cent of T cell.
The fluidic cell detected result is as shown in table 1 (with CD4
+, CD8
+Per-cent compare), this result shows no matter rMS-pLA71NY immune group mouse is CD4
+Or CD8
+The T cell all has increase clearly, and promptly recombinant Mycobacterium smegmatis has promoted antigenic offering, and has activated the mouse T lymphocyte crowd and has been divided into CD4
+, CD8
+Effector T cell has stimulated the immunne response of body.
The different immune group mouse of table 1. CD4
+, CD8
+The comparison of T cell per-cent:
Splenocyte propagation and thin in embodiment 7. recombinant Mycobacterium smegmatis (rMS-pLA71NY) vaccine immune mouse
Intracellular cytokine (IFN-γ, IL-2) excretory detects
Separate each experimental mice splenocyte, and the adjustment cell concn is 2 * 10
4Individual cell/ml adds 96 porocyte plates with every hole 100 μ l, and adding 5 μ l NY-ESO-1 antigens (final concentration is 5 μ g/ml) stimulates, and does the blank that does not add antigenic stimulation simultaneously.After placing incubator to cultivate 3d the cell, every hole adds 10 μ lWST-8, and 0.5-4h surveys the absorbancy under the 450nm wavelength respectively.
Calculate the splenocyte proliferation rate according to following formula:
Proliferation rate %=(the A in antigenic stimulation hole
450-do not add the A in antigen hole
450)/do not add the antigen hole A
450* 100%
Amount with every hole 1ml adds 24 porocyte plates with splenocyte in addition, and the NY-ESO-1 antigen that adds 5 μ g/ml stimulates, and every 24h gets culture supernatant, detects the secretion level of splenocyte cytokine after antigenic stimulation according to the indication of test kit specification sheets.
Test of splenocyte proliferation rate and calculation result are as shown in Figure 6; This result shows: the mouse boosting cell that stimulates through rMS-pLA71NY has proliferative response clearly; Proliferation rate is about 40%, and the proliferation rate of NY-ESO-1 protein immunization group is 22%, and PBS and MS immune group then are merely 3% and 13%.This result shows, possessed persistent, special to NY-ESO-1 immunological memory effect through rMS-pLA71NY mice immunized splenocyte.
Splenocyte secretion level detected result of cytokine after antigenic stimulation is as shown in Figure 7; This result shows: after the NY-ESO-1 antigenic stimulation; The splenocyte of rMS and NY-ESO-1 immune mouse; The secretion level of its cytokine IFN-γ and IL-2 significantly improves than PBS and MS immune group, and rMS immune group splenocyte excretory cytokine levels will be significantly higher than the NY-ESO-1 immune group.These cytokines can further be regulated CD4
+The differentiation of T cell assists the T cell to carry out cellular immunization simultaneously.
The CTL killing experiments of embodiment 8. recombinant Mycobacterium smegmatis (rMS-pLA71NY) vaccine immune mouse
Separate each experimental mice splenocyte, the adjustment cell concn is 3.2 * 10
6Individual cell/ml adds 50 μ l NY-ESO-1-B16 cells (about 4000 cells) and 50 μ l splenocytes in every hole of 96 porocyte plates, make that the ratio (be splenocyte with the quantity of NY-ESO-1-B 16 cells than) of effector cell's number and target cell number is 40: 1.Place cell culture incubator to cultivate 4h cell plate, detect the lethal effect of splenocyte NY-ESO-1-B16 according to the indication of test kit specification sheets.
The result of Fig. 8 shows: NY-ESO-1 immune group mouse boosting cell is merely 19% to the average kill rate of NY-ESO-1-B 16 cells; And the High Fragmentation rate of rMS-pLA71NY immune group can reach 53%; Average kill rate also reaches 42%, is significantly higher than the NY-ESO-1 immune group.
This result shows through rMS-pLA71NY mice immunized splenocyte and has set up effective memory immunity system to expressing the antigenic tumour cell of NY-ESO-1 special lethal effect is arranged.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Appendix one, reference
[1] leaf Longchang, Chen Baowen. Chinese new drug magazine, 2007,16 (7): 565-568.
[2]Jae-Sung?Yu，James?WP.Clinical?and?Vaccine?Immunology，2006，13(11)：1204-1211.
[3] Lin Lu, Hong-Dan Cao .Vaccine such as Han-Qing Zeng, 2009:972-978.
[4] Monica R, Roberto L .Cancer Res such as Elisabeth S, 2003,63:6948-6955.
[5]Tanya?p，Paul?RW.Methods?in?Moloecular?Biology，101：77-89.
[6] Xu Miao, Chen Baowen. Chinese microbiology and Journal of Immunology, 200525 (9): 253-257
[7] Yang Chun, Li Na. Chinese Journal of Immunology, 2007,23:112-116.
[8] Sarah LY, Michael M .Int.J.Cancer such as Xing WZ, 2004,112:653-660.
[9] Via LE, Fratti RA .J Cell Sci such as McFalone M, 1998,111:897-905.
[10] Akihiro Hosoi, Yayoi Takeda .Cancer Res such as Yoshihiro Furuichi, 2008,68 (10): 3941-3949.
[11] Dominik M, Paul AM .Proc Natl Acad Sci USA such as Sherry W, 1999,96:8633-8638.
[12] Padmanee S, Dean FB .J Immunother such as Achim AJ, 2008,31 (9): 849-957.
[13] Yao Jianzhong, Liu Qigui. Chinese cancer magazine, 2007,17 (10): 792-795.
[14] Timm J, Perilli MG .Mol Microbiol.1994 such as Duez C, 12 (3): 491-504.
[15] Lim EM, Rauzier J .J Bacteriol.1995 such as Timm J, 177 (1): 59-65.
Appendix two, sequence table sequence implication:
Claims (11)
1. plasmid that is used for recombinant tumor-testis antigen NY-ESO-1 protein expression, said plasmid comprises:
(a) beta-galactosidase enzymes signal coding sequence or beta-galactosidase enzymes encoding sequence; With
(b) NY-ESO-1 encoding sequence.
2. plasmid as claimed in claim 1 is characterized in that, said reorganization NY-ESO-1 albumen is the fusion rotein of beta-galactosidase enzymes signal peptide sequence and NY-ESO-1 or the fusion rotein of beta-galactosidase enzymes full length sequence and NY-ESO-1.
3. plasmid as claimed in claim 1 is characterized in that,
Said beta-galactosidase enzymes signal coding sequence is selected from sequence as follows: GenBank number: the polypeptide of sequence encoding or their conservative property variant protein or its active fragments or aminoacid sequence wherein shown in L25634.1, the SEQ ID NO:3 are through 1-20 (preferred 1-10; More preferably 1-5) replacement, disappearance or the interpolation of amino-acid residue form, and instruct beta-galactosidase enzymes to be positioned the signal sequence on cell walls or the cytolemma;
Said beta-galactosidase enzymes encoding sequence is selected: GenBank number: sequence shown in L25634.1, the SEQ ID NO:4, its homologous sequence or under stringent condition can with the sequence of this sequence hybridization; Have at least 75%, 80%, 90%, 95%, 98% or the polynucleotide of higher homogeny with the sequence of said coding nucleic acid;
Said NY-ESO-1 encoding sequence is selected from sequence as follows: the sequence shown in sequence shown in the GenBank U87459.1, the SEQ ID NO:1, its homologous sequence or under stringent condition can with the sequence of this sequence hybridization; Have at least 75%, 80%, 90%, 95%, 98% or the polynucleotide sequence of higher homogeny with the sequence of said coding nucleic acid.
4. plasmid as claimed in claim 1 is characterized in that, said plasmid comprises the sequence shown in SEQ ID NO:5 or the SEQID NO:7.
5. plasmid as claimed in claim 1 is characterized in that said plasmid is a shuttle plasmid, and it can duplicate amplification in intestinal bacteria, M. smegmatics or mycobacterium tuberculosis.
6. plasmid as claimed in claim 1 is characterized in that, the plasmid of the said NY-ESO-1 protein expression that is used to recombinate is based on the plasmid construction that is selected from down group: pLA71, pLA73, pJH152 or pJEM17.
7. recombinant Mycobacterium smegmatis, it comprises each described plasmid among the claim 1-6.
8. recombinant Mycobacterium smegmatis as claimed in claim 7 is characterized in that, the NY-ESO-1 albumen of its express recombinant, and the albumen that will recombinate is positioned cell walls.
9. immune composition, said compsn comprises:
(i) each described recombinant Mycobacterium smegmatis or its lysate or cell walls part among each described plasmid, the claim 7-8 among the claim 1-6; With
(ii) acceptable carrier and/or adjuvant on the immunology.
Among the claim 1-6 among each described plasmid, the claim 7-8 each described recombinant Mycobacterium smegmatis or its lysate or cell walls part be used for preventing and/or treating the purposes of the pharmaceutical composition or the vaccine of NY-ESO-1 positive tumor in preparation.
11. a method for preparing claim 7 or 8 described recombinant Mycobacterium smegmatis, said method comprises the steps:
(1) each described plasmid among the claim 1-6 is provided;
(2) transform M. smegmatics to obtain claim 7 or 8 described recombinant Mycobacterium smegmatis with said plasmid.
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| CN106420838A (en) * | 2016-11-08 | 2017-02-22 | 中国科学院微生物研究所 | Lung cancer cell growth inhibitor |
| AU2019200884B2 (en) * | 2013-06-28 | 2020-11-05 | Auckland Uniservices Limited | Amino acid and peptide conjugates and conjugation process |
| US11464853B2 (en) | 2016-02-26 | 2022-10-11 | Auckland Uniservices Limited | Amino acid and peptide conjugates and conjugation process |
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2011
- 2011-06-21 CN CN2011101684921A patent/CN102839186A/en active Pending
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| AU2019200884B2 (en) * | 2013-06-28 | 2020-11-05 | Auckland Uniservices Limited | Amino acid and peptide conjugates and conjugation process |
| US11464853B2 (en) | 2016-02-26 | 2022-10-11 | Auckland Uniservices Limited | Amino acid and peptide conjugates and conjugation process |
| CN106420838A (en) * | 2016-11-08 | 2017-02-22 | 中国科学院微生物研究所 | Lung cancer cell growth inhibitor |
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