CN101096680A - NDA vaccine eucaryon expression carrier and application in preparation of gene vaccine - Google Patents
NDA vaccine eucaryon expression carrier and application in preparation of gene vaccine Download PDFInfo
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Abstract
The invention discloses a eucaryon expression vector of a DNA vaccine and the application. The eucaryon expression vector of DNA vaccine is a recombination pVAX1 eucaryon expression vector which is got by adding the internal ribosome in pVAX1 vector skeleton and entering the site coded sequence. The eucaryon expression vector may show the wide spectrum curing function for breast cancer, pancreatic cancer, restocarcinoma and multi-type malignancy solid tumor, which has good humoral immunity and cell-mediated immunity effect, and inhibits the growth of the tumor.
Description
Technical field
The present invention relates to carrier and application thereof, particularly relate to a kind of dna vaccination carrier for expression of eukaryon and its application in gene vaccines such as preparation is antitumor.
Background technology
Current, malignant tumour has become one of most important killer who threatens human health, and therefore, the study on prevention of strengthening malignant tumour is significant.At present, excision, chemotherapy and radiation remain the main methods of treatment of treatment malignant tumour.Though traditional therapeutic method of surgery can make patient's survival rate increase, but the recurrence rate in 5 years of postoperative is still more than half, though this is because operation can be excised the knurl piece of finding of naked eye effectively, but can't guarantee to remove all tumour cells, also fail to change tumorigenic interior environment; And chemotherapy and radiation is except having serious toxic side effect, and many tumours such as kidney etc. are insensitive or produce resistance to it.The transfer of tumour and recurrence are the important factors that influences patient's prognosis, especially malignant tumour take place and evolution in the most dangerous stage, at present, there is significant limitation in above-mentioned traditional therapy aspect tumor recurrence and the transfer overcoming, and becomes the bottleneck of oncotherapy.The immunogene treatment means of development progressively because of it has specificity height, with strong points and characteristics that side effect is little, has become the important development direction for the treatment of malignant tumor in recent years.
Dna vaccination also claims gene vaccine or nucleic acid vaccine, is a kind of new generation vaccine that grows up the nineties in 20th century, and it is meant the exogenous antigen gene clone to carrier for expression of eukaryon, and then the plasmid DNA immune animal that will recombinate.The exogenous antigen gene can be at host's expression in vivo, and stimulates it to produce humoral immunization and cellular immunization.Many animal models and human experimentation prove that all dna vaccination is the humoral immunoresponse(HI) and the cellullar immunologic response of excitating organism effectively, is described as vaccine revolution for the third time.
Dna vaccination has many outstanding advantages: 1. the antigen of DNA plasmid expression is near native conformation, and antigenicity is strong; 2. the comprehensive immunne response of excitating organism, the protective immune response of its conservative antigen has the effect of resisting that intersects to the pathogenic agent of different subtype; 3. preparation is simple, with low cost, easily carries out large-scale production, and transports, preserves conveniently; 4. safe in utilization, there is not the danger of pathogenic infection, dna vaccination only is certain antigenic gene fragment of pathogenic agent, rather than the gene of whole pathogenic agent, and utilizes plasmid to make carrier, does not relate to infectious factor; 5. immunity has persistence, and once inoculation can obtain permanent immunity power, has avoided inactivated vaccine, recombinant subunit vaccine etc. to need repeatedly the loaded down with trivial details of booster immunization; 6. can combined immunization, can be with the different antigenic gene constructed multiple plasmid combined utilization that in same plasmid, maybe will contain different antigen genes of coding, preparation Multivalent DNA Vaccine; 7. the existing prophylactic effect of dna vaccination also has therapeutic action; 8. route of inoculation variation can be adopted injection system inoculation (comprising intracutaneous, subcutaneous, intramuscular injection, particle gun injection technique), also can adopt oral or spray inoculation mode.
Survivin belongs to IAP (IAP) family member, studies show that, Survivin is the strongest survivin of finding up to now, also is one minimum among the IAP family member.The people Survivin assignment of genes gene mapping is in karyomit(e) (17q25), comprise 4 exons and 3 introns, a kind of molecular weight of encoding is the cytoplasmic protein of 16.5kD, contains a baculovirus and repeats (BIR) structure function district, and C-terminal lacks zinc fingers and replaces αLuo Xuanjiegou.Its mRNA can produce three kinds of isomer: Survivin, Survivin-2B and Survivin-EX3 through different cut modes in vivo.
Survivin generally expresses in embryonic tissue and malignant tumour, as cancer of the stomach, colorectal cancer, mammary cancer and lung cancer etc.; Do not express but in the tissue of differentiation and maturation and the other healthy tissues of cancer, have.These characteristics make Survivin become the important target spot of neoplasm targeted therapy, have good specificity and security.Studies confirm that target Survivin treatment can promote apoptosis of tumor cells and suppress its propagation, and healthy tissues almost had no adverse effects that this significant advantage makes Survivin become a big focus in the anti-tumor immunotherapy research gradually.
Rong Xiang etc. are connected to the gene of mouse total length Survivin and secretor type chemokine CCL21 on the pBudCE4.1 carrier for expression of eukaryon, be prepared into dna vaccination, intravital immunization experiment result shows the C57BL/6J mouse, the carry out the coffin upon burial CD8+CTL reaction of this dna vaccination, lung carcinoma cell had good fragmentation effect [Rong X, Noriko M, Yunping L, et al.A DNA vaccine targeting survivin combines apoptosiswith suppression of angiogenesis in lung tumor eradication.Cancer Research.2005,65 (2): 553-561].What people such as Marc Schmitz confirmed Apoptosis Inhibitor Protein Survivin can be at external specific C D8+CTL killing tumor cell (the Marc S that excites effectively from aminoterminal 5-14 amino acids (TLPPAWQPFL) and 95-104 amino acids (ELTLGEFLKL), Petra D, Bernd W, et al.Generationof Survivin-specific CD8+T Effector Cells by Dendritic Cells Pulsed withProtein or Selected Peptides.Cancer research, 2000,60 (17): 4845-4849).People such as Kerstin Otto have carried out functional experiment with this section small peptide again in IV phase melanoma patients body, the ELISPOT detected result shows, this small peptide can excite intensive t cell immune response (Kerstin O, Mads HA, AndreasE, et al.Lack of toxicity of therapy-induced T cell responses against theuniversal tumour antigen surviving.Vaccine, 2005,23 (7): 884-889).The tumor protein p53 vaccine of developments such as YoshihikoHirohashi is to be target spot with people Survivin-2B from aminoterminal 80-88 amino acids (AYACNTSTL), this section small peptide is the restricted T lymphocyte epitopes of HLA-A24, can be by CD8+ cytotoxic T cell specific recognition, experimental result proves, with this section small peptide is that the vaccine of target antigen preparation has colon cancer cell and well kills and wounds restraining effect, present this vaccine (Yoshihiko H that entered the II clinical trial phase stage, Toshihiko T, Akiko M, et al.An HLA-A24-restricted Cytotoxic T LymphocyteEpitope of a Tumor-associated Protein, Survivin.Clinical Cancer Research, 2002,8 (6): 1731-1739).
Human chorionic is urged sexual hormoue, and (human chorionic gonadotropin is by the secreted glycoprotein hormones of embryo's plamoditrophoblast cell hCG), and normal hCG connects and composes dimer by α chain and β chain subunit with non covalent bond.Nearly all malignant cell all can be expressed dystopy hCG.HCG β is the characteristic albumen of kinds of tumors, the core fragment of equal selectivity secretion single chain hCG β of the tumour cell of multiple tissue or hCG β, studies show that, this albumen and malignant tumour transfer characteristics, the formation of malignization degree and tumor microenvironment and immunological tolerance etc. all has certain relation, be embodied in: the glycosyl sequence of hCG concentrates on shuttle base terminal polypeptide (hCGp-CTP) zone of hCG β mostly, the hCG β chain of tumor cell surface expression is many more, degree of glycosylation is high more, tumor cell surface with negative charge just many more, and the electronegative adhesion that promotes tumour cell and vascular endothelial cell of tumor cell surface, stimulate the generation of relevant adhesion molecule and chemokine, thereby make the easy more transfer of tumour cell.Simultaneously, the existence owing to strong negative charge can suppress the activation of immunocytes such as NK, T cell, scavenger cell, causes activity of immune cells to reduce, even incompetent, causes the immunological tolerance of tumour.
Because hCG β subunit and metakentrin (LH) have higher homology, be that the antibody that immunogen produces can produce cross reaction with LH therefore with hCG β subunit.(hCG β-CTP37) is that the hCG molecule is peculiar, and has the specific antigen epi-position of hCG, and it induces the antibody of generation just to have better antigen-specific to have only 37 peptides that hCG β carboxyl terminal forms from 109-145 amino acids residue.
With dystopy hCG β subunit is that body fluid and the cell immune response that target antigen excites can be attacked tumour cell effectively, has become the new research focus of tumor biotherapy.The pancreatic tumour vaccine Avicine of this target spot design of overseas utilization has finished the II clinical trial phase; the colorectal cancer vaccine has entered the critical period of III clinical trial phase; show that this target spot to multiple malignant tumour postoperative recurrence and transfer tool immunity protection function, has a good application prospect.
Core fragment-the CTP37 of hCG β chain is the characteristic albumen of kinds of tumors, and shift with malignant tumour, the formation of malignization degree and tumor microenvironment and immunological tolerance etc. all has certain relation.Studies show that, be body fluid and the cell immune response that target antigen excites with CTP37, can attack tumour cell effectively, now become another research focus of tumor biotherapy.
Li-Zhen He etc. are connected the full gene of hCG β 3 ' end formation fusion gene of anti-DC cell antibody B11 heavy chain, be inserted into again among the carrier for expression of eukaryon pMMV4 and constitute dna vaccination, experiment in vitro proves that this vaccine can excite generation intensive t cell immune response kill tumor cell (He LZ, Ramakrishna V, Connolly JE, etal.A novel human cancer vaccine elicits cellular responses to thetumor-associated antigen, human chorionic gonadotropin beta[J] .Clin CancerRes, 2004,10 (6): 1920-7.).Moulton etc. will contain the small peptide and the mutually coupled protein vaccine that is prepared into of diphtheria toxoid of terminal 37 amino-acid residues of COOH-(CTP37) of hCG β, but excitating organism produces the antibody inhibiting tumor growth of anti-CTP37, the experiment of this vaccine therapy colorectal cancer has now entered critical period (the Moulton HM of III clinical trial phase, Yoshihars PH, Mason DH, et al.Active specificimmunotherapy with a beta-human chorionic gonadotropin peptide vaccine inpatients with metastatic colorectal cancer:antibody response is associatedwith improved survival[J] .Clin Cancer Res, 2002,8 (7): 2044-51).Having introduced a kind of novel of their development in one piece of paper that He Yi etc. delivers in the near future is the nucleic acid vaccine of target spot with CTP37, be that CTP37 is linked to each other with mycobacterium heat shock protein HSP65, and, in the mouse body, carried out the experiment of tumor killing effect with this section fusion gene insertion carrier for expression of eukaryon formation nucleic acid vaccine.Found that this nucleic acid vaccine can bring out the affinity antibody that body produces high titre, show all that with the experiment in vitro result this vaccine can effectively suppress the growth of breast cancer cell in the body, for the immunotherapy of tumour provides a new approaches (Yi H, Rong Y, Yankai Z, et al.Improved efficacy of DNA vaccination against breast cancer by boosting withthe repeat beta-hCG C-terminal peptide carried by mycobacterial heat-shockprotein HSP65[J] .Vaccine, 2006,24 (14): 2575-84.).
The key link of gene therapy is the most special except correctly choosing, efficiently, widely the target antigen, also comprise the carrier for expression of eukaryon that choose reasonable is suitable, this also is the precondition of nucleic acid vaccine development.The carrier for expression of eukaryon that is used for the clinical use of vaccine that the pVAX1 carrier is admitted by united states drug and food evaluation committee, it has kalamycin resistance, is very suitable for human body and uses.
Current, a critical problem of dna vaccination research field is the immune protective efficiency that how to improve vaccine, and can this problem is related to dna vaccination finally be converted into product.Past people is in order to make up the immunogenicity of Multivalent DNA Vaccine and enhancing dna vaccination, the double promoter expression vector that make up more, recombinant expressed fusion rotein or plasmid immunity jointly by carrying different antigen genes and adjuvant gene, though the double-promoter carrier can be used for the coordinate expression of two genes, but causing, the phase mutual interference meeting between the promotor transcribes the inconsistent of effect, weaken even lose some expression of gene (Tahara H, Zitvogel L, Storkus WJ, et al.Effective eradication of establishedmurine tumors with IL-12 gene therapy using a polycistronic retroviralvector[J] .J Immunol, 1995,154 (12): 6466-74.); In addition, the coimmune efficient of multichip carrier is also lower, usually can not obtain gratifying effect.Adopt the IRES element to make up two-cistron expression vector, can allow two goal gene effective expression (Wagstaff M simultaneously, Lilley C, Smith J, et al.Gene transferusing a disabled herpes virus vector containing the EMCV IRES allows multiplegene expression in vitro and in vivo.Gene Ther.1998,5 (11): 1566-70.), be the important method of multi-gene expression in recent years.
Internal ribosome entry site (internal ribosome entry site, IRES) be the section of DNA sequence that is found in animal virus genome 5 ' non-coding region the earliest, it has the translation Initiation Function that does not rely on 5 ' cap sequence, 2 open reading frames that derive from same mRNA are correctly expressed, and are a kind of bicistronic mRNA Expression elements.Between two albumen coded sequences, insert IRES, under the control of upstream promoter, transcribe the back and be same mRNA; During translation, IRES upstream gene translation initiation is followed the translation initiation rule (being that cap structure relies on mode) of general eukaryotic gene, the IRES downstream gene then enters by IRES guiding rrna, open primordium because of translation, thereby express when realizing two genes.Because two gene translations are from same mRNA, the albumen that gives expression to is approaching on time and space, especially be applicable to interactional two protein of research or follow the trail of and to be detectedly proteicly transcribe and express, also can be used as the expression vector of Polyepitope DNA vaccine by reporter gene.
Under the normal circumstances, body has tolerance to autoantigen, does not produce immunne response.Utilize the xenogenesis homologous gene as antigen; homology by gene between biological species and the kind; manage to induce body to produce cross-immune reaction; help breaking the autoimmunization tolerance of body; can make vaccine induce body fluid and cellular immunization protective reaction effectively, efficiently bring into play the effect of anti metastasis and recurrence.
The xenogenesis isogeneic is an available strategy of breaking the tumour immunity tolerance.Discover, biological by rudimentary species in senior spore evolution process, the same gene of different genera shows as certain homology.Can utilize the autoimmune reaction of xenogenesis homologous cell or molecule inducing tumor cell to reach antineoplastic purpose.By utilizing the homology of gene between biological species and the kind, manage to induce body to produce cross-immune reaction (cross-reaction), may break autoimmunization tolerance (Ciesielski MJ, Apfel L, Barone TA, et al.Antitumor effects ofa xenogeneic surviyin bone marrow derived dendritic cell vaccine againstmurine GL261 gliomas.Cancer Immunol Immunother, 2006,55 (12), 1491-503).Studies show that, use and tumour antigen (HER-2/neu) height homologous protein, can induce than using the much bigger organism immune response of tumour antigen itself.Domestic Wei in full academician wait to find with the heterogenous blood vessel endothelial cell growth factor (ECGF) extremely acceptor as vaccine immune mouse, can induce at tumor neovasculature autoimmune response, thereby can suppress tumor growth, obtained tumor killing effect (Wei YQ preferably in animal body, Huang MJ, Yang L, et al.Immunogene therapy of tumors with vaccine based on Xenopus homologous vascularendothelial growth factor as a model antigen.[J] Proc Natl Acad Sci USA.2001 Sep 25; 98 (20): 11545-50).At present, become important layout strategy as immunogen development anti-tumor vaccine, on multiple vaccine design, all used this strategy both at home and abroad, and obtained gratifying immune protective effect with heterologous antigen.
The main purpose of tumor immune gene therapy is body to be produced have the T lymphocyte of antineoplastic specificity and cytotoxic activity, thereby reaches the effect that specificity is attacked tumour.The activation of T cell needs dual signal to stimulate: first signal is combined and provides with antigen peptide-MHC molecular complex on the antigen presenting cell (APC) by T cell antigen receptor (TCR), and second signal is combined with respective ligand on the T cell by the costimulatory molecules on the APC and provides.Have only dual signal to stimulate jointly and could effectively activate specific T cell, lack second signal and cause T cell clone apoptosis, make tumour cell produce immune evasion.Costimulatory molecules on the APC mainly is some adhesion molecules, and wherein, the B7 molecule is current research focus.
B7.1 is the main costimulatory molecules on the APC, and the interaction of it and part CD28 molecule is the second signal of t cell activation.B7.1 is with after its part CD28 combines, can promote T cell proliferation, stop t cell proliferation, produce T cell chemotactic factor (Herold K, Lu J, Rulifson T, et al.Regulation of C-Cchemokineproduction by murine T cells by CD28/B7 costimulation[J] .J Immunol, 1997,159 (9): 4150-53.); Raise IL-2R α, β and γ, increase transcribing of IL-2 mRNA, strengthen cytokine secretion.Raise the expression of CD40L and the mRNA level of CTLA 24; Also can strengthen the susceptibility of T cell to superantigen, mediation CTL is to the effector function of target cell; The intercellular adhesion of mediation T-B promotes humoral immunoresponse(HI) etc.B7.1 mainly stimulate altogether the CD8+T cell to CTL differentiation CD4+T cell to the Th1 cytodifferentiation, this plays a key effect strengthening and keep in effective immunne response.Simultaneously, B7.1 also has effect (the Boussiotis V that stronger common stimulation GM-CSF produces, Freeman G, Gribben J, et al.The role of B7.1/B7.2:CD28/CTLA-4 pathways in the prevention of anergy, induction of productiveimmunity and down-regulation of the immune response[J] .Immunol Rev, 1996,153:5-26.).
RHuGM-CSF (granulocyte macrophage colony stimulatingfactor, GM-CSF) mainly produce by T cell and scavenger cell, it is a kind of multiple bioactive cytokine that has, can promote APC differentiation such as DC, the expression of maturation and activation and rise CD86 is to excite immunne response (the Kass E to tumour, Parker J, Schlom, et al.Comparat ive studies of the effects ofrecombiant GM-CSF and GM-CSF administered via a poxvirus to enhance theconcentration of antigen presenting cells in regional lymphnodes[J] .Cytokine, 2000,12 (7): 960-71.); External have promote marrow sample progenitor cell proliferation, strengthen neutrophil leucocyte, mononuclear macrophage, eosinophilic granulocyte phagolysis and ADCC effect isoreactivity (Yu J to tumour cell, Burwick J, Dranoff G, et al.Gene therapy for metastatic brain tumors byvaccination with granulocyte-macrophage colony-stimulating factor transducedtumors cells[J] .Human gene therpy, 1997,8 (9): 1065-72.); Promote Th, Tc, NK to soak at tumor locus, thereby killing tumor cells (Nakazaki Y, Tani K, Lin ZT, et al.Vaccine effectof granulocyte-macrophage colony-stimulating factor or CD80 gene-transducedmurine hematopoietic tumor cells and their cooperative enhancement ofanti-tumor immunity[J] .Gene Ther, 1998,5 (10): 1355-62.).Lot of documents shows, GM-CSF has good synergistic antitumor effect (Armstrong C, Botella R, Galloway T, et al.Anti-tumor effects of granulocyte macrophage colony-stimulating factorproduction by melanoma cells[J] .Cancer Res, 1996,56 (9): 2191-98; WakimotoH, Abe J, Tsunoda R, et al.Intensified anti-tumor immunity by a cancer vaccinethat produces granulocyte-macrophage colony stimulating factor plusinterleukin 4[J] .Cancer Res, 1996,56 (8): 1828-33.).
Only depend on tumour specific antigen to be difficult to effective excitating organism antineoplastic immune, still need B7.1 costimulatory molecules and its part etc. interact and participate in activated T cell.Many researchs have confirmed that also the most tumors cell do not express the B7.1 costimulatory molecules, thereby escaped the immunity of organism supervision, so that tumour is escaped immunosurveillance and the (Yang Xuwei that unrestrictedly grows in vivo, Chen Yuanzhong, the woods development. the clone of people B7.1 gene cDNA and retrovirus expression vector thereof make up [J]. Medical University Of Fujian's journal, 2001,35 (2): 109-111.).And GM-CSF has full-time antigen presenting cell effects such as the dendritic cell of activation, makes the helper T lymphocyte and the anti-tumour effect hyperplasia of antigen-specific, can activate stronger anti tumor immune response in vivo.If The combined is used, can start immunity at tumour antigen, can promote helper T lymphocyte and antineoplastic immune effector cell hyperplasia again, produce collaborative anti-tumour effect.Existing at present studies confirm that can be improved the lethal effect of vaccine to tumour cell with GM-CSF and B7.1 molecule combined utilization.People's such as Sumimoto result of study shows, the knurl seedling (LLC/GM+B7) of the lung cancer cell line LLC preparation of B7.1 gene and GM-CSF gene co-transfection, the CTL cell killing activity that brings out is apparently higher than LLC cell (the Sumimoto H of independent transfection B7.1 or GM-CSF gene, Tani K, Nakazaki Y, et al.GM-CSF and B7-1 (CD80) co-stimulatory signals co-operate in the induction of effective anti-tumorimmunity in syngeneic mice.Int J Cancer.1997,73 (4): 556-61.).People's such as Nakazaki result of study shows, thymic lymphoma oncocyte/GM-CSF and thymic lymphoma oncocyte/B7.1 knurl seedling is united use and is had the synergistic antitumor immune effect, and its mechanism may be that the knurl seedling of GM-CSF gene transfer mainly is to influence the Th cell and then activate CTL by the indirect approach that APC mediates; The knurl seedling of B7.1 genetic transcription is to influence CTL by direct way, and the The combined use can be brought out systemic anti tumor immune response, cause tumor section to eradicate (Nakazaki Y, Tani K, L in Z, et al.Vaccine effect of granulocyte macrophagecolony stimulating factor or CD80 gene transduced murine hematopoietic tumorcells and their cooperative enhancement of anti-tumor immunity[J] .Gene Ther, 1998,5 (10): 1355-62.).
The principle that the LDH method is measured cell CTL effect is as shown in figure 27: serum lactic dehydrogenase (LDH) is to include one of enzyme in the viable cell endochylema, after effector cell and target cell hatch jointly, the damaged membrane of target cell, the permeability of film change the LDH in the endochylema are discharged into outside the born of the same parents.Get the culture supernatant that contains LDH, make substrate (tetrazolium salt) change into a kind of mauve first the part between the ribs and the hips compounds (formazan) through enzymatic reaction, at the 490nm place very high absorption peak is arranged, measure its A490 value and just can reflect the kill and wound situation of effector cell indirectly target cell.
Summary of the invention
The purpose of this invention is to provide a kind of dna vaccination carrier for expression of eukaryon that can be used for making up gene vaccine and can start two kinds of gene co-expressings simultaneously.
For solving the problems of the technologies described above, the present invention takes following technical scheme: a kind of dna vaccination carrier for expression of eukaryon, be in the pVAX1 carrier framework, to contain internal ribosome entry site (internal ribosome entry site, the IRES) recombinant eukaryon expression vector of encoding sequence.
Because all can express by promotor gene the upstream and downstream of described internal ribosome entry site, therefore for making things convenient for the insertion of foreign gene, one or more restriction enzyme enzyme recognition sites also can be added again in the two ends of the internal ribosome entry site encoding sequence in above-mentioned dna vaccination carrier for expression of eukaryon, the selection of described restriction enzyme is widely, comprises EcoR V, BamH I, Cla I, BstB I, Pvu I and Psi I, BssH II, Swa I, Pac I, Nsi I, Sma I, Xho I, Not I, Bst XI, Nhe I and Sal I etc.
Specifically, 5 ' end at described internal ribosome entry site encoding sequence adds restriction enzyme EcoRV, Cla I, BstB I, Pvu I and Psi I recognition site, and the dna vaccination carrier for expression of eukaryon that adds restriction enzyme BssH II, Swa I, Pac I, Nsi I and Xho I recognition site at its 3 ' end is pVAX1-IRES.
Described dna vaccination carrier for expression of eukaryon can make up according to the ordinary method in genetically engineered field.
The application of described dna vaccination carrier for expression of eukaryon in the preparation gene vaccine also belongs to protection scope of the present invention; described gene vaccine comprises the gene vaccine at multiple disease, as gene vaccines such as antitumor, anti influenza, anti-AIDS, antiviral hepatitis, tuberculosis, rabies or anti-malarials.
Another purpose of the present invention is to provide a kind of application of described dna vaccination carrier for expression of eukaryon, and a kind of high-efficiency broad spectrum treatment of solid tumor gene vaccine promptly is provided.
Wherein, with dna vaccination carrier for expression of eukaryon of the present invention is the Antioncogene vaccine of vector construction of setting out, its activeconstituents is that the fusion gene of Survivin-2B from aminoterminal 5-28 position and 80-121 amino acids residue encoding sequence formation contained in the upstream of ribosome entry site(RES) encoding sequence in the carrier framework of dna vaccination carrier for expression of eukaryon of the present invention, and the recombinant eukaryon expression vector of the fusion gene of the core fragment CTP37 regional code sequence of the chorion gonadotrophic hormone beta chain of people and monkey formation.
Described Survivin-2B can be connected by connecting arm " nG " with encoding sequence from aminoterminal 80-121 amino acids residue from the encoding sequence of aminoterminal 5-28 amino acids residue, wherein, n can be K (Methionin), R (arginine), C (halfcystine), Q (glutamine) G (glycine) or N (l-asparagine).Described Survivin-2B by connecting arm " nG " connection can be shown in sequence in the sequence table 1 from the nucleotide sequence of the fusion gene of aminoterminal 5-28 position and 80-121 amino acids residue encoding sequence.
Described Survivin-2B from aminoterminal 5-28 amino acids residue encoding sequence be positioned at Survivin-2B from aminoterminal 80-121 amino acids residue encoding sequence 3 ' or 5 ' end all can.
The core fragment CTP37 regional code sequence of the chorion gonadotrophic hormone beta chain of described people and monkey also can be connected by connecting arm " nG ", and wherein, n is K, R, C, Q, G or N.The nucleotide sequence of the fusion gene of the core fragment CTP37 regional code sequence of the chorion gonadotrophic hormone beta chain of described people who is connected by connecting arm " NG " and monkey can be shown in sequence in the sequence table 2.
The core fragment CTP37 regional code sequence of described people's chorion gonadotrophic hormone beta chain can be positioned at monkey the chorion gonadotrophic hormone beta chain core fragment CTP37 regional code sequence 3 ' or 5 ' end all can.
In addition, the fusion gene that constitutes from aminoterminal 5-28 position and 80-121 amino acids residue encoding sequence of described Survivin-2B 3 ' or the 5 ' end that is positioned at the fusion gene that the CTP37 regional code sequence of people and monkey chorion gonadotrophic hormone beta chain constitutes all can.
For the guiding goal gene obtains effective expression and secretion in eukaryotic cell, the upstream of internal ribosome entry site encoding sequence also can be connected with people Ig κ chain targeting signal peptide (sig) encoding sequence and/or human IgG-Fc section encoding sequence and/or glycosyl-phosphatidyl inositol (GPI) grappling signal coding sequence in the carrier framework of the carrier for expression of eukaryon of described Antioncogene vaccine.
In addition, for further strengthening the ability that described Antioncogene vaccine activate immunity is replied, the downstream of internal ribosome entry site encoding sequence also can be connected with human GM-CSF and/or B7.1 costimulatory molecules encoding sequence in the carrier framework of the carrier for expression of eukaryon of described Antioncogene vaccine, and the encoding sequence of immuno-stimulating cytokines such as interleukin-, Interferon, rabbit and/or tumour necrosis factor.
Specifically, the fusion gene of Survivin-2B from aminoterminal 5-28 position and 80-121 amino acids residue encoding sequence formation contained in the upstream of internal ribosome entry site encoding sequence in the carrier framework of dna vaccination carrier for expression of eukaryon of the present invention, and the recombinant eukaryon expression vector of the fusion gene of the core fragment CTP37 regional code sequence of the chorion gonadotrophic hormone beta chain of people and monkey formation can be pVAX1-2PAG.
In the carrier framework of dna vaccination carrier for expression of eukaryon of the present invention, be connected with people Ig κ chain targeting signal peptide-coding sequence in turn in the upstream of described internal ribosome entry site encoding sequence, Survivin-2B is from the fusion gene of aminoterminal 5-28 position and 80-121 amino acids residue encoding sequence formation, the fusion gene that the core fragment CTP37 regional code sequence of the chorion gonadotrophic hormone beta chain of people and monkey constitutes, and the recombinant eukaryon expression vector that human IgG-Fc section encoding sequence and glycosyl-phosphatidyl inositol anchor are decided signal coding sequence can be pVAX1-2PAG-Fc-GPI.
In the carrier framework of dna vaccination carrier for expression of eukaryon of the present invention, be connected with people Ig κ chain targeting signal peptide-coding sequence in turn in the upstream of described internal ribosome entry site encoding sequence, Survivin-2B is from the fusion gene (antigen gene) of aminoterminal 5-28 position and 80-121 amino acids residue encoding sequence formation, the fusion gene (antigen gene) that the core fragment CTP37 regional code sequence of people and monkey chorion gonadotrophic hormone beta chain constitutes, human IgG-Fc section encoding sequence and glycosyl-phosphatidyl inositol anchor are decided signal coding sequence (described people Ig κ chain targeting signal peptide-coding sequence, human IgG-Fc section encoding sequence and glycosyl-phosphatidyl inositol anchor are decided signal coding sequence and described antigen gene amalgamation and expression), the recombinant eukaryon expression vector that is connected with human GM-CSF and B7.1 costimulatory molecules encoding sequence in the downstream of described internal ribosome entry site encoding sequence can be pVAX1-sig-2PAG-FC-GPI-GM/B7 (being called for short pVAX1-2PFcGB).
When needing, in the preparation of above-mentioned vaccine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, absorption enhancer or the tensio-active agent etc. of pharmaceutical field routine.
The consumption of above-mentioned vaccine is generally 0.01-200 μ g/kg body weight/day, can use by one or many, is generally the course of treatment 20-40 days, and the dosage and the course of treatment all can be adjusted according to practical situation.
For improving curative effect, medicine of the present invention can also carry out combined therapy with microbiotic, immunostimulant etc.
The invention provides a kind of dna vaccination carrier for expression of eukaryon.This carrier is the advantage that shows in treating malignant tumor at dna vaccination, on the basis of carrier for expression of eukaryon pVAX1, introduced bicistronic mRNA Expression element-IRES sequence, and inserted multiple rare restriction enzyme site at the two ends of IRES sequence, thereby a kind of Novel DNA vaccine carrier pVAX1-IRES that successfully constructs.And on the basis of this NA vaccine carrier pVAX1-IRES, made up the compound Antioncogene vaccine of a kind of many target spots, people Ig κ chain targeting signal peptide (sig) is contained in the upstream that this Antioncogene vaccine is an IRES encoding sequence in pVAX1-IRES, the main t cell epitope of people Survivin-2B zone, the core fragment CTP37 zone of people and monkey chorion gonadotrophic hormone beta chain, the polygene fusion sequence of human IgG-Fc section encoding sequence and glycosyl-phosphatidyl inositol (GPI) anchoring peptide, the recombinant eukaryon expression vector pVAX1-sig-2PAG-FC-GPI-GM/B7 (being called for short pVAX1-2PFcGB) of human GM-CSF and B7.1 fusion gene is contained in the IRES downstream of IRES encoding sequence in pVAX1-IRES.Transfection has the flow cytometer and the immunofluorescence detected result of the 293T cell of this plasmid to show, pVAX1-2PFcGB obtains well to express in the 293T cell, in addition, Antioncogene vaccine of the present invention also has humoral immunization and cellular immunization effect preferably, simultaneously tumor growth is had significant inhibitory effect.Gene vaccine with carrier for expression of eukaryon preparation of the present invention is expected to the effect of multiple malignant solid tumor performance such as mammary cancer, carcinoma of the pancreas, colorectal cancer and prostate cancer broad-spectrum curing, play a significant role in tumor treatment and drug development field thereof, have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is 1.2% an agarose gel electrophoresis detected result of the IRES encoding sequence of pcr amplification
Fig. 2 is EcoR V and the Xho I double digestion qualification result that carries the recombinant vectors pSimple18-EcoRV/BAP-IRES of IRES encoding sequence
Fig. 3 is 1.2% an agarose gel electrophoresis detected result of EcoR V and the Xho I double digestion product of pSimple18-EcoRV/BAP-IRES and carrier for expression of eukaryon pVAX1
Fig. 4 is 1.2% an agarose gel electrophoresis detected result of EcoR V and the Xho I double digestion product of eukaryotic DNA vaccine carrier pVAX1-IRES
Fig. 5 A is 1.2% an agarose gel electrophoresis detected result of the DsRed1 gene fragment of pcr amplification
Fig. 5 B is 1.2% an agarose gel electrophoresis detected result of the EGFP gene fragment of pcr amplification
Fig. 6 A carries the BamH I of recombinant vectors pGEM-T Easy-DsRed1 of DsRed1 gene and 1.2% agarose gel electrophoresis detected result of Pvu I double digestion product
Fig. 6 B carries the BssH II of recombinant vectors pGEM-T Easy-EGFP of EGFP gene and 1.2% agarose gel electrophoresis detected result of Nsi I double digestion product
Fig. 7 is 1.2% an agarose gel electrophoresis detected result of pGEM-T Easy-DsRed1, pGEM-T Easy-EGFP and pVAX1-IRES double digestion product
Fig. 8 A carries the BamH I of carrier for expression of eukaryon pVAX1-IRES-DsRed1 of DsRed1 gene and 1.2% agarose gel electrophoresis detected result of Pvu I double digestion product
Fig. 8 B carries the Nsi I of carrier for expression of eukaryon pVAX1-IRES-EGFP of EGFP gene and 1.2% agarose gel electrophoresis detected result of BssH II double digestion product
Fig. 8 C carries the BamH I of carrier for expression of eukaryon pVAX1-DsRed1-IRES-EGFP of DsRed1 and EGFP gene and 1.2% agarose gel electrophoresis detected result of Xho I double digestion product
Fig. 9 is the laser scanning co-focusing microscope detected results of various recombinant plasmids in 293T cell inner expression situation
Figure 10 is the flow cytometer detected result of various recombinant plasmids transient expression situation in the 293T cell
Figure 11 is the structural formula intention of antigenic compound 2PAG fusion gene
Figure 12 is 1% an agarose gel electrophoresis detected result of Sur-SC and the SC-hmCTP and the antigenic compound 2PAG fusion gene of pcr amplification
Figure 13 is the human GM-CSF factor of pcr amplification and 1% agarose gel electrophoresis detected result of B7.1 fusion gene
Figure 14 is that the bacterium colony PCR and the enzyme that contain the eukaryon expression plasmid pCI-2PAG-Fc-GPI of antigenic compound 2PAG fusion gene are cut qualification result
Figure 15 cuts qualification result for the enzyme of eukaryon expression plasmid pVAX-sig-2P-FC-GPI-IRES
Figure 16 cuts qualification result for the enzyme of eukaryon expression plasmid pVAX1-IRES-GM/B7
Figure 17 is that the enzyme of pVAX1-sig-2PAG-Fc-GPI-GM/B7 (pVAX1-2PFcGB) is cut qualification result
Figure 18 is the flow cytometer detected result of the transient expression efficient of pVAX1-2PFcGB in the 293T transfectional cell
Figure 19 is the immunofluorescence detected result of the transient expression efficient of pVAX1-2PFcGB in the 293T transfectional cell
Figure 20 is the immunization protocol of Balb/c mouse
Figure 21 is the statistics of the average one-tenth knurl time of each group immune mouse subcutaneous transplantation knurl
Figure 22 is the growth curve of transplanted tumor in each group immune mouse body
Figure 23 is the comparative result of each group immune mouse subcutaneous transplantation knurl attack stripped gross tumor volume after 30 days
Figure 24 respectively organizes the heavy statistics of the intravital average knurl of immune mouse for the attack of subcutaneous transplantation knurl after 35 days
Figure 25 is the statistics of subcutaneous transplantation knurl attack tumor control rate of each immune group after 35 days
Figure 26 is the ELISA detected result of 2D group, E group and F group immune serum antibody titer
Figure 27 measures the principle schematic of cell CTL effect for the LDH method
Figure 28 is the killing activity detected result of each immune group mouse boosting cell
Figure 29 is the structural representation of pVAX1-2PFcGB, pVAX1-2PAG, pVAX1-2PAG-Fc-GPI
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3
RdEdition, 2001, NY, Cold SpringHarbor).The primer and dna sequence dna are synthetic by Beijing three rich polygala root biotechnology limited liability companys if no special instructions.All percentage concentrations are volume/volume (V/V) or mass/volume (W/V) percentage concentration if no special instructions.
The structure of embodiment 1, dna vaccine vector pVAX1-IRES
One, the pcr amplification of RES encoding sequence and purifying
At first design its pcr amplification primer according to the IRES encoding sequence in the pIRES-neo carrier (position of IRES sequence is 1295-1880bp), and added restriction enzyme EcoR V at the upstream primer IRES-F that is used for pcr amplification IRES encoding sequence, Cla I, BstB I, Pvu I and Psi I recognition site, added restriction enzyme BssH II at the downstream primer IRES-R that is used for pcr amplification IRES encoding sequence, Swa I, Pac I, Nsi I and Xho I recognition site, concrete primer sequence is as follows: IRES-F (upstream primer): 5 '-CCGATATCGATTCGAACGATCGTTATAACTAGGGCGGCCAATTCCGCC-3 ' IRES-R (downstream primer): 5 '-GCCTCGAGCGCGCATTTAAATTAATTAATGCATTGGCTGCAGGAATTATCCCG-3 '.After primer is synthetic, be mixed with the use liquid of 20 μ mol/L with sterile purified water, packing ,-20 ℃ of preservations are standby.
Then, with plasmid pIRES-neo (Clontech company) is template, under the guiding of primer I RES-F and IRES-R, the encoding sequence of pcr amplification IRES on the GeneAmp PCR of Perkin Elmer company System2400 type PCR instrument, 50 μ lPCR reaction systems are: 5 * PrimeSTAR
TMDamping fluid (contains Mg
2+) 10 μ l, dNTPs (2.5mmol/L) 4 μ l, upstream primer (20 μ mol/L) 1 μ l, downstream primer (20 μ mol/L) 1 μ l, PrimeSTAR
TMHS DNA Polymerase (2.5U/ μ l, TaKaRa company) 0.5 μ l, template (100 times of pIRES-neo plasmid dilutions) 1 μ l, sterile purified water 32.5 μ l.The PCR reaction conditions is: 95 ℃ of 3min of elder generation; 94 ℃ of 45s then, 60 ℃ of 45s, 72 ℃ of 1min 30s circulate 30 times altogether; Last 72 ℃ are extended 7min, 4 ℃ of preservations.After reaction finishes, get 5 μ l pcr amplification products and carry out the detection of 1.2% agarose gel electrophoresis, (swimming lane M is DL 2000 DNA Marker (available from TaKaRa company) to detected result as shown in Figure 1, swimming lane 1 is: the pcr amplification product of IRES encoding sequence), obtained the DNA band of 682bp through amplification, consistent with the expection size.PCR product glue with vast Imtech reclaims test kit and the recovery of reference reagent box specification sheets and this purpose band of purifying.
Two, carry the structure and the evaluation of the pSimple18-EcoRV/BAP recombinant vectors of IRES encoding sequence
1, the preparation (CaCl of bacillus coli DH 5 alpha competent cell
2Method)
A. get the bacillus coli DH 5 alpha bacterial classification of-70 ℃ of preservations, with its streak inoculation on the LB agar plate, overnight incubation in 37 ℃ of incubators (12-24 hour);
B. picking is moistening smooth, and the single colony inoculation of the bacillus coli DH 5 alpha of neat in edge is to 3mL LB liquid nutrient medium, and shaking culture is spent the night under 37 ℃, 200rpm;
C. the activatory bacterial classification is inoculated in the 50mL LB liquid nutrient medium by 1%, 37 ℃ of shaking culture 2-4h make bacterial growth reach logarithmic phase, i.e. OD
600nm=0.3-0.6;
D. under aseptic condition bacterium liquid is transferred in the polypropylene tube of aseptic, precooling, 4 ℃, the centrifugal 10min of 8000rpm precipitate thalline fully behind the ice bath 10min;
E. abandon supernatant, with the 0.1mol/L CaCl of 10mL precooling
2Resuspended bacterial sediment, behind the ice bath 20min, 4 ℃, the centrifugal 10min of 6000rpm;
F. abandon supernatant, with the 0.1mol/L CaCl of 2mL precooling
2Resuspended bacterial sediment behind the ice bath 3.5h, adds 15% glycerine, packing, and-70 ℃ of preservations are standby.
2,5 ' terminal phosphateization of the IRES encoding sequence of pcr amplification
Efficient phosphorylation agent box of DNA Kination Kit and reference reagent box specification sheets with TaKaRa company carry out 5 ' terminal phosphateization to the IRES encoding sequence that step 1 increases, and concrete steps are as follows:
A. prepare following reaction solution in Eppendorf tube, full dose is 50 μ l;
T
4 Polynucleotide Kinase
2μl
(10U/μl)
ATP(10mM) 5μl
10 * Reaction damping fluid, 5 μ l
PCR purified product 4 μ l
ddH
2O 34μl
B.37 ℃ reaction is 30 minutes;
C.70 ℃ heating is 10 minutes, makes enzyme deactivation;
D. the ddH that adds 50 μ l
2O replenishes volume to 100 μ l;
E. the 3M CH that adds 10 μ l (1/10 amount)
3COONa (pH5.2);
F. the DNAmate solution that adds 4 μ l, uniform mixing;
G. the dehydrated alcohol that adds-20 ℃ of precoolings of 250 μ l, fully mixing;
H.4 ℃, centrifugal 10 minutes of 12000rpm, reclaim precipitation, after 70% cold ethanol washing and precipitating, drying at room temperature 10 minutes;
6. with the ddH of resolution of precipitate in 20 μ l
2Among the O.
3, the IRES encoding sequence after the 5 ' terminal phosphateization and pSimple18-EcoRV/BAP carrier is connected
Ligation Mix efficient DNA ligase enzyme with TaKaRa company is connected the IRES encoding sequence of step 2 after 5 ' terminal phosphateization with carrier pSimple18-EcoRV/BAP (TaKaRa company), linked system is as follows:
PSimple18-EcoRV/BAP carrier 1 μ l
Ligation Mix 5μl
Condition of contact is: 16 ℃ connect 1h.
4, will connect product transformed into escherichia coli DH5 α competent cell
Get the fresh competent escherichia coli cell DH5 α 200 μ l of step 1 preparation, place ice bath, the connection product 10 μ l that add step 3, ice bath 30min behind the mixing gently, transfer to heat-shocked 90s in 42 ℃ of water-baths immediately, ice bath leaves standstill 2min again, adds the not LB liquid nutrient medium of added with antibiotic of 0.5mL, behind 37 ℃ of water-bath 15min, 37 ℃ of shaking table jog 45min; Subsequently, get 200 μ l and transform thalline, evenly coat on the LB plate culture medium that contains penbritin (100 μ g/mL), dry up about 10min, be inverted for 37 ℃ and cultivate 12-16h at Bechtop.Then, 5 of the single bacterium colonies that picking grows from the LB resistant panel are inoculated in (5mL/ pipe) in the LB liquid nutrient medium that contains penbritin (100 μ g/mL) 37 ℃ of shaking table overnight incubation respectively.
5, extract plasmid
The conversion that step 4) is obtained has the recombinant plasmid bacterium liquid incubated overnight of the pSimple18-EcoRV/BAP that carries the IRES encoding sequence, with the alkaline lysis test kit and the reference reagent box specification sheets extraction plasmid of vast Imtech.
6, enzyme is cut and is identified and order-checking
10×NEBuffer3 2μl
100×BSA 0.2μl
Recombinant plasmid plasmid DNA 5 μ l
Xho I 1μl
EcoR V 1μl
Sterilization distilled water 10.8 μ l
The enzyme tangent condition is: 37 ℃ of water-bath enzymes are cut and are spent the night.The double digestion product is carried out 1.2% agarose gel electrophoresis to be identified, (swimming lane M is DL 2000 DNA Marker to qualification result as shown in Figure 2, swimming lane 1 is: EcoR V and Xho I double digestion product), cut the DNA band that has obtained 2692bp and 682bp through enzyme, consistent with expected results, show that the IRES encoding sequence successfully is connected in the pSimple18-EcoRV/BAP carrier.Then, to cut through enzyme and identify that correct positive colony bacterium liquid send Beijing three rich polygala root biotechnology limited liability companys to check order, sequencing result shows and has obtained the correct pSimple18-EcoRV/BAP recombinant vectors that carries the IRES encoding sequence of sequence, with its called after pSimple18-EcoRV/BAP-IRES.
Three, the structure of eukaryotic DNA vaccine carrier pVAX1-IRES and evaluation
1, enzyme is cut
| PSimple18-Eco RV/BAP- | PVAX1 | |
| DNA | ||
| 10 * |
30 μ l, 5 μ l, 0.5 μ l, 1 μ l, 1 μ l, 12.5 μ l | 30 μ l, 5 μ l, 0.5 μ l, 1 μ l, 1 μ l, 12.5 μ l |
| |
50 |
50 μ l. |
2, enzyme is cut recovery, connection and the conversion of product
Double digestion product to step 1 carries out the detection of 1.2% agarose gel electrophoresis, (swimming lane M is DL 2000 DNA Marker to detected result as shown in Figure 3, swimming lane 1 is: the EcoR V of pVAX1 and Xho I double digestion product, swimming lane 2 is EcoR V and the Xho I double digestion product of pSimple18-EcoRV/BAP-IRES), pVAX1 cuts through enzyme and has obtained the big or small DNA band that is about 3000bp, pSimple18-EcoRV/BAP-IRES cuts the DNA band that has obtained 682bp through enzyme, and is consistent with expected results.With " the PCR segment quick glue reclaim test kit " of vast Tyke, Beijing biological gene technology limited liability company and reference reagent box specification sheets reclaims and purifying is cut the dna fragmentation and the carrier for expression of eukaryon pVAX1 of the IRES encoding sequence among the recombinant plasmid pSimple18-EcoRV/BAP-IRES that spends the night through enzyme.Goal gene IRES behind the purifying is connected with linearized vector pVAX1, and 16 ℃ connect 1h, and linked system is as follows:
Ligation Mix 10μl
Then, use the method identical will connect product transformed into escherichia coli DH5 α competent cell with step 2.
3, the screening of positive recombinant plasmid and evaluation
Picking is containing 6 of the single bacterium colonies (numbering 1-6) that grow on kantlex (50 μ g/mL) the LB resistant panel, be inoculated in respectively (5mL/ pipe) in the LB liquid nutrient medium that contains kantlex (50 μ g/mL), 37 ℃ of shaking table overnight incubation, the upgrading grain, carry out double digestion with restriction enzyme EcoR V and Xho I and identify, it is as follows that enzyme is cut system:
| |
10 * damping fluid | BSA | EcoR V | Xho I | DdH 2O | Altogether | |
| PVAX1- |
10 |
2 μ l | 0.5 μ l | 0.5 μ l | 0.5 μ l | 6.5 μ l | 20 μ l. |
Then, the double digestion product is carried out 1.2% agarose gel electrophoresis to be detected, (swimming lane M is DL 2000 DNA Marker to detected result as shown in Figure 4, swimming lane 1 is: the pVAX1 empty carrier, swimming lane 2 is the dna fragmentation of IRES, swimming lane 3-8 is the EcoR V and the Xho I double digestion product of the 1-6 mono-clonal plasmid selected), positive recombinant plasmid can obtain the DNA band of 3000bp and 682bp through double digestion, and wherein 4,5,7, No. 8 positive clones of mono-clonal, consistent with expected results, prove to have obtained sequence and all correct pVAX1 recombinant vectors that contains the IRES encoding sequence of on position, called after pVAX1-IRES, eukaryotic DNA vaccine carrier promptly of the present invention.
The expressive function checking of embodiment 2, eukaryotic DNA vaccine carrier pVAX1-IRES
Start the function that two kinds of genes carry out coexpression simultaneously for whether the eukaryotic DNA vaccine carrier pVAX1-IRES that checks embodiment 1 to make up has, at first from red fluorescent protein expression vector pDsRed1-N1 (Clontech company), amplify red fluorescent protein gene DsRed1 (EF 517319), from enhanced green fluorescence protein expression vector pEGFP-N1 (Clontech company), amplify enhanced green fluorescence protein gene EGFP (EF 587314), the upstream and downstream that the DsRed1 gene fragment that amplifies and EGFP gene fragment are inserted IRES encoding sequence in the pVAX1-IRES carrier respectively obtains recombinant expression plasmid pVAX1-IRES-DsRed1 then, pVAX1-IRES-EGFP, pVAX1-DsRed1-IRES-EGFP.With these three eukaryotic expression recombinant plasmid transient transfection human embryonic kidney cell 293T, analyze the expression of DsRed1, EGFP by flow cytometer and laser scanning co-focusing microscope, start the function that two kinds of genes carry out coexpression simultaneously thereby check constructed dna vaccine vector pVAX1-IRES whether to have
Concrete experimental technique is as follows:
6.PCR amplification DsRed1 gene and EGFP gene fragment
6. design of primers
According to red fluorescent protein gene DsRed1 (EF 517319) and enhanced green fluorescence protein EGFP gene (EF587314) design pcr amplification primer, primer sequence is as follows:
F-DsRed1 (upstream primer): 5 '-CCG
GGATCCACCGGTCGCCACCATGGTGC-3 ' (band underscore base is a restriction enzyme BamH I recognition site)
R-DsRed1 (downstream primer): 5 '-CCG
CGATCGCTACAGGAACAGGTGG-3 ' (band underscore base is a restriction enzyme Pvu I recognition site);
F-EGFP (upstream primer): 5 '-CCG
ATGCATCGCCACCATGGTGAGC-3 ' (band underscore base is a restriction enzyme Nsi I recognition site)
R-EGFP (downstream primer): 5 '-CCG
GCGCGCTTACTTGTACAGCTCG-3 ' (band underscore base is a restriction enzyme BssH II recognition site).
Primer is synthetic, be mixed with the use liquid of 20 μ mol/L behind the purifying with sterile purified water, packing, and-20 ℃ of preservations are standby.
2, the pcr amplification of DsRed1 gene fragment and recovery thereof, purifying
With plasmid pDsRed1-N1 (Clontech company) is template, under the guiding of primers F-DsRed1 and R-DsRed1, pcr amplification DsRed1 gene order on the GeneAmp PCR of Perkin Elmer company System 2400 type PCR instrument, 50 μ l PCR reaction systems are: 10 * Ex Taq damping fluid, 5 μ l, dNTPs (2.5mmol/L) 4 μ l, upstream primer (20 μ mol/L) 1 μ l, downstream primer (20 μ mol/L) 1 μ l, TaKaRa ExTaq (5U/ μ l, TaKaRa company) 1 μ l, template (100 times of pDsRed1-N1 plasmid dilutions) 1 μ l, sterile purified water 37 μ l.The PCR reaction conditions is: 95 ℃ of 5min of elder generation; 94 ℃ of 45s then, 62 ℃ of 45s, 72 ℃ of 2min circulate 35 times altogether; Last 72 ℃ are extended 10min.After reaction finishes, get 5 μ l pcr amplification products and carry out the detection of 1.2% agarose gel electrophoresis, (swimming lane M is DL 2000 DNA Marker to detected result shown in Fig. 5 A, swimming lane 1 is: the pcr amplification product of DsRed1 gene), obtained the DNA band of 723bp through amplification, consistent with the expection size.PCR product glue with vast Imtech reclaims test kit and the recovery of reference reagent box specification sheets and this purpose band of purifying.
3, the pcr amplification of EGFP gene fragment and recovery thereof, purifying
With plasmid pEGFP-N1 (Clontech company) is template, under the guiding of primers F-EGFP and R-EGFP, and pcr amplification EGFP gene order, reaction system and reaction conditions are identical with step 2.After reaction finishes, get 5 μ l pcr amplification products and carry out the detection of 1.2% agarose gel electrophoresis, (swimming lane M is DL 2000DNA Marker to detected result, and swimming lane 1 is: the pcr amplification product of EGFP gene) shown in Fig. 5 B, obtained the DNA band of 746bp through amplification, consistent with the expection size.PCR product glue with vast Imtech reclaims test kit and the recovery of reference reagent box specification sheets and this purpose band of purifying.
Two, the structure of pGEM-T Easy-DsRed1, pGEM-T Easy-EGFP recombinant cloning vector
1, pcr amplification product behind the purifying and pGEM-T Easy carrier are connected and connect the conversion of product
The goal gene purified product 6.0 μ l of pcr amplification
pGEM-T Easy 2.0μl
10×T
4 DNA Ligase buffer 1.0μl
T
4DNA Ligase(12μ/μl) 1.0μl
After reaction finishes, use with embodiment 1 step 2 in identical method with the connection product transformed into escherichia coli DH5 α competent cell of two genes.
2, the screening of positive recombinant plasmid and evaluation
Picking is containing the single bacterium colony that transforms DsRed1 gene and EGFP gene respectively each 6 (all numbering 1-6) that grows on penbritin (100 μ g/mL) the LB resistant panel, be inoculated in respectively (5mL/ pipe) in the LB liquid nutrient medium that contains penbritin (Amp+) (100 μ g/mL), 37 ℃ of shaking table overnight incubation, the upgrading grain, the plasmid (called after pGEM-T Easy-DsRed1) that carries the DsRed1 gene is carried out double digestion with restriction enzyme BamH I and Pvu I identify, it is as follows that enzyme is cut system:
| |
10 * damping fluid | BSA | BamH I | Pvu I | DdH 2O | Altogether | |
| PGEM-T Easy- |
10 |
2 μ l | 0.5 μ l | 0.5 μ l | 0.5 μ l | 6.5 μ l | 20 μ l. |
The plasmid (called after pGEM-T Easy-EGFP) that carries the EGFP gene is carried out double digestion with restriction enzyme BssH II and NsiI identify, it is as follows that enzyme is cut system:
| |
10 * damping fluid | BSA | BssH II | Nsi I | ddH 2O | Altogether |
| pGEM-T Easy-EGFP | 10μl | 2μl | 0.5μl | 0.5μl | 0.5μl | 6.5μl | 20μl。 |
The endonuclease reaction condition is 37 ℃ of water-bath enzymes and cuts and spend the night.Then, the double digestion product is carried out 1.2% agarose gel electrophoresis to be identified, wherein, the enzyme that carries the plasmid pGEM-T Easy-DsRed1 of DsRed1 gene is cut qualification result, and (swimming lane M is DL 2000 DNA Marker as shown in Figure 6A, swimming lane 1-6 is the BamH I and the Pvu I double digestion product of the 1-6 mono-clonal plasmid selected, swimming lane 7 is the DsRed1 gene fragment), positive recombinant plasmid can obtain the DNA band of 3015bp and 717bp through double digestion, wherein 1,3,4, No. 5 positive clones of mono-clonal, consistent with expected results, will cut through enzyme at last and identify that correct positive colony bacterium liquid send Beijing three rich polygala root biotechnology limited liability companys to check order, sequencing result shows and has obtained sequence and all correct recombination bacillus coli cloning vector pGEM-T Easy-DsRed1 that contains the DsRed1 gene of on position; The enzyme that carries the plasmid pGEM-T Easy-EGFP of EGFP gene is cut qualification result, and (swimming lane M is DL 2000 DNA Marker shown in Fig. 6 B, swimming lane 1-6 is the BssH II and the Nsi I double digestion product of the 1-6 mono-clonal plasmid selected), positive recombinant plasmid can obtain the DNA band of 3015bp and 740bp through double digestion, wherein 1,2,4,5, No. 6 positive clones of mono-clonal, consistent with expected results, to cut through enzyme at last and identify that correct positive colony bacterium liquid send Beijing three rich polygala root biotechnology limited liability companys to check order, sequencing result shows and has obtained sequence and all correct recombination bacillus coli cloning vector pGEM-T Easy-EGFP that contains the EGFP gene of on position.
Three, the structure of recombinant vectors pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP and pVAX1-DsRed1-IRES-EGFP
1, the structure of pVAX1-IRES-DsRed1
1) enzyme is cut
The positive recombinant plasmid pGEM-T Easy-DsRed1 that carries the DsRed1 gene of step 2 acquisition and the carrier for expression of eukaryon pVAX1-IRES of embodiment 1 structure are carried out double digestion with restriction enzyme BamH I and Pvu I, 37 ℃ of water-baths are spent the night, and it is as follows that enzyme is cut system:
| PGEM-T Easy-DsRed1 | PVAX1- | |
| DNA | ||
| 10 * damping fluid 100 * BSA BamH I Pvu I distilled |
30 μ l, 5 μ l, 0.5 μ l, 1 μ l, 1 μ l, 12.5 μ l | 30 μ l, 5 μ l, 0.5 μ l, 1 μ l, 1 μ l, 12.5 μ l |
| |
50 μ l. | 50μl。 |
Then, the double digestion product is carried out 1.2% agarose gel electrophoresis to be identified, enzyme is cut qualification result, and (swimming lane M is DL 2000 DNA Marker as shown in Figure 7, swimming lane 2 is BamH I and the Pvu I double digestion product that carries the positive recombinant plasmid pGEM-TEasy-DsRed1 of DsRed1 gene, swimming lane 4 is BamH I and the PvuI double digestion product of pVAX1-IRES), pGEM-T Easy-DsRed1 obtains the DNA band of 717bp through double digestion, pVAX1-IRES can obtain the DNA band that size is about 3600bp through double digestion, conforms to expected results.
2) enzyme is cut recovery, connection and the conversion of product
Reclaim test kit and reference reagent box specification sheets reclaims and the band of the linearization plasmid pVAX1-IRES of the DsRed1 gene of purifying 717bp and 3600bp with the PCR product glue of vast Imtech, then the goal gene DsRed1 behind the purifying is connected with linearized vector pVAX1-IRES, 16 ℃ connect 1h, and linked system is as follows:
PVAX1-IRES carrier 5 μ l
Ligation Mix 10μl
Then, use with embodiment 1 step 2 in identical method will be connected product transformed into escherichia coli DH5 α competent cell.
3) screening of positive recombinant plasmid and evaluation
Picking is containing 8 of the single bacterium colonies (numbering 1-8) that grow on kantlex (50 μ g/mL) the LB resistant panel, be inoculated in respectively (5mL/ pipe) in the LB liquid nutrient medium that contains kantlex (50 μ g/mL), 37 ℃ of shaking table overnight incubation, the upgrading grain, carry out double digestion with restriction enzyme BamH I and Pvu I and identify, it is as follows that enzyme is cut system:
| |
10 * damping fluid | BSA | BamH I | Pvu I | DdH 2O | Altogether | |
| PVAX1-IRES- |
10 |
2 μ l | 0.5 μ l | 0.5 μ l | 0.5 μ l | 6.5 μ l | 20 μ l. |
Enzyme is cut qualification result, and (swimming lane M is DL 2000 DNA Marker shown in Fig. 8 A, swimming lane 1-8 is the BamH I and the Pvu I double digestion product of the 1-8 mono-clonal plasmid selected), positive recombinant plasmid can obtain the DNA band of 3600bp and 717bp through double digestion, wherein 1,2, No. 4 positive clones of mono-clonal, consistent with expected results, to cut through enzyme at last and identify that correct positive colony bacterium liquid send Beijing three rich polygala root biotechnology limited liability companys to check order, sequencing result shows and has obtained sequence and all correct recombinant eukaryon expression vector that contains the DsRed1 gene of on position, called after pVAX1-IRES-DsRed1.
2, the structure of pVAX1-IRES-EGFP
1) enzyme is cut
The carrier for expression of eukaryon pVAX1-IRES that positive recombinant plasmid pGEM-T Easy-EGFP that carries the EGFP gene that step 2 is obtained and embodiment 1 make up carries out double digestion with restriction enzyme Nsi I and BssH II, and 37 ℃ of water-baths are spent the night, and it is as follows that enzyme is cut system:
| pGEM-T Easy-EGFP | pVAX1-IRES |
| |
30 μ l, 5 μ l, 0.5 μ l, 1 μ l, 1 μ l, 12.5 μ l | 30 μ l, 5 μ l, 0.5 μ l, 1 μ l, 1 μ l, 12.5 μ l |
| |
50 μ l. | 50μl。 |
Then, the double digestion product is carried out 1.2% agarose gel electrophoresis to be identified, enzyme is cut qualification result, and (swimming lane M is DL 2000 DNA Marker as shown in Figure 7, swimming lane 1 is Nsi I and the BssH II double digestion product that carries the positive recombinant plasmid pGEM-T Easy-EGFP of EGFP gene, swimming lane 3 is Nsi I and the BssH II double digestion product of pVAX1-IRES), pGEM-T Easy-EGFP can obtain the DNA band of 740bp through double digestion, pVAX1-IRES can obtain the DNA band of 3600bp through double digestion, conforms to expected results.
2) enzyme is cut recovery, connection and the conversion of product
Reclaim test kit and reference reagent box specification sheets reclaims and the band of the linearization plasmid pVAX1-IRES of the EGFP gene of purifying 740bp and 3600bp with the PCR product glue of vast Imtech, then the goal gene EGFP behind the purifying is connected with linearized vector pVAX1-IRES, 16 ℃ connect 1h, and linked system is as follows:
PVAX1-IRES carrier 5 μ l
Ligation Mix 10μl
Then, use with embodiment 1 step 2 in identical method will be connected product transformed into escherichia coli DH5 α competent cell.
3) screening of positive recombinant plasmid and evaluation
Picking is containing 3 of the single bacterium colonies (numbering 1-3) that grow on kantlex (50 μ g/mL) the LB resistant panel, be inoculated in respectively (5mL/ pipe) in the LB liquid nutrient medium that contains kantlex (50 μ g/mL), 37 ℃ of shaking table overnight incubation, the upgrading grain, carry out double digestion with restriction enzyme Nsi I and BssH II and identify, it is as follows that enzyme is cut system:
| |
10 * damping fluid | BSA | Nsi I | BssH II | DdH 2O | Altogether | |
| PVAX1-IRES- |
10 |
2 μ l | 0.5 μ l | 0.5 μ l | 0.5 μ l | 6.5 μ l | 20 μ l. |
Enzyme is cut qualification result, and (swimming lane M is DL 2000 DNA Marker shown in Fig. 8 B, swimming lane 1-3 is the Nsi I and the BssH II double digestion product of the 1-3 mono-clonal plasmid selected), positive recombinant plasmid can obtain the DNA band of 3600bp and 740bp through double digestion, No. 3 positive clones of mono-clonal wherein, consistent with expected results, to cut through enzyme at last and identify that correct positive colony bacterium liquid send Beijing three rich polygala root biotechnology limited liability companys to check order, sequencing result shows and has obtained sequence and all correct recombinant eukaryon expression vector that contains the EGFP gene of on position, called after pVAX1-IRES-EGFP.
3, the structure of pVAX1-DsRed1-IRES-EGFP
1) enzyme is cut
The carrier for expression of eukaryon pVAX1-IRES-DsRed1 that carries the DsRed1 gene that step 1 is obtained carries out double digestion with restriction enzyme Nsi I and BssH II, and 37 ℃ of water-baths are spent the night, and it is as follows that enzyme is cut system:
| PVAX1-IRES- | |
| DNA | |
| 10 * damping fluid 100 * BSA Nsi I BssH II distilled |
30 μ l, 5 μ l, 0.5 μ l, 1 μ l, 1 μ l, 12.5 μ l |
| |
50 μ l. |
Then, the double digestion product is carried out 1.2% agarose gel electrophoresis identify, can obtain the DNA band that size is about 4300bp, conform to expected results through double digestion.
2) enzyme is cut recovery, connection and the conversion of product
PCR product glue with vast Imtech reclaims the band that test kit and the recovery of reference reagent box specification sheets and purifying size are about the linearization plasmid pVAX1-IRES-DsRed1 of 4300bp, then the EGFP gene behind the purifying is connected with linearization plasmid pVAX1-IRES-DsRed1,16 ℃ connect 1h, and linked system is as follows:
pVAX1-IRES-DsRed1 5μl
Ligation Mix 10μl
Then, use with embodiment 1 step 2 in identical method will be connected product transformed into escherichia coli DH5 α competent cell.
3) screening of positive recombinant plasmid and evaluation
Picking is containing 3 of the single bacterium colonies (numbering 1-3) that grow on kantlex (50 μ g/mL) the LB resistant panel, be inoculated in respectively (5mL/ pipe) in the LB liquid nutrient medium that contains kantlex (50 μ g/mL), 37 ℃ of shaking table overnight incubation, the upgrading grain, carry out double digestion with restriction enzyme Xho I and BamH I and identify, it is as follows that enzyme is cut system:
| |
10 * damping fluid | BSA | Xho I | BamH I | ddH 2O | Altogether |
| pVAX1-DsRed1-IRES-EGFP | 10μl | 2μl | 0.5μl | 0.5μl | 0.5μl | 6.5μl | 20μl。 |
Enzyme is cut qualification result, and (swimming lane M is DL 2000 DNA Marker shown in Fig. 8 C, the mono-clonal plasmid of swimming lane 1 for selecting, swimming lane 2 is the Xho I and the BamH I double digestion product of the mono-clonal plasmid selected), positive recombinant plasmid can obtain DNA band about 3600bp and 2100bp through double digestion, consistent with expected results, to cut through enzyme at last and identify that correct positive colony bacterium liquid send Beijing three rich polygala root biotechnology limited liability companys to check order, sequencing result shows and has obtained sequence and all correct recombinant eukaryon expression vector that contains DsRed1 gene and EGFP gene of on position, called after pVAX1-DsRed1-IRES-EGFP.
4) a large amount of upgrading grains
With identifying that correct transfection respectively has the recombinant clone bacterium liquid incubated overnight of plasmid pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP and pVAX1-DsRed1-IRES-EGFP, extract plasmid according to the Wizard PlusMinipreps DNA Purification system of Promega company and with reference to product description.
Four, eukaryotic expression recombinant plasmid transient transfection 293T cell and expression level detect
1, transfection 293T cell
Three kinds of carrier for expression of eukaryon pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP that adopt that liposome method makes up step 3 and pVAX1-DsRed1-IRES-EGFP be transfection human embryo kidney (HEK) 293T cell respectively, concrete grammar is: 293T cell (available from consonance medical university cell centre) is incubated in the RPMI1640 substratum (Gibco BRL company) that contains 10% foetal calf serum, the 293T cell of taking the logarithm vegetative period, go in 6 well culture plates, with the conventional digestion of 0.25% pancreatin (or EDTA) back inoculation in 37 ℃, 50mL/L CO
2Cultivate 24h (the cell stand density is 80-90%) in the incubator, then that positive control plasmid pEGFP-N1, pDsRed1-N1 and evaluation is correct recombinant plasmid pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP and pVAX1-DsRed1-IRES-EGFP are respectively at the transfection reagent Lipofectamine of Invitrogen company
TMTransfection 293T cell under 2000 the mediation, concrete operations reference reagent box specification sheets, the blank group only adds the blank liposome of equivalent.
2, laser scanning co-focusing microscope detects various recombinant plasmids at the intracellular expression of 293T
Radiance 2100 with Bio-Rad company
TMLaser scanning co-focusing microscope detects the expression of various recombinant plasmids in the 293T transfectional cell of step 1, easy to detect may further comprise the steps:
A. behind the transfection 48h with 0.25% trysinization 293T transfectional cell, piping and druming is prepared into single cell suspension gently;
B. the PBS that contains 2% newborn calf serum with the ice bath precooling cleans cell 3 times;
C. use 1% Paraformaldehyde 96 re-suspended cell, adjust cell concn, cell suspension is dripped on the slide glass, mountant is sealed up for safekeeping;
D. detect the expression of various recombinant plasmids with laser scanning co-focusing microscope.
The laser scanning co-focusing microscope detected result of the 293T cell of transient transfection after 48 hours is (a:pDsRed1-N1 as shown in Figure 9; B:pEGFP-N1; C:pVAX1-IRES-DsRed1; D:pVAX1-IRES-EGFP; E-g:pVAX1-DsRed1-IRES-EGFP; H: blank), the 293T cell of transfection positive control plasmid pDsRed1-N1 and pEGFP-N1 all has redness or green fluorescence to present that (a b), proves that rotaring redyeing system sets up successfully; The 293T cell of transfection recombinant plasmid pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP also send redness or green fluorescence (c, d), prove the DsRed1 that is connected into, EGFP gene all can be in the pVAX1-IRES carrier that has made up normal expression; Excite the 293T cell of observing transfection recombinant plasmid pVAX1-DsRed1-IRES-EGFP down at 575nm or two kinds of wavelength of 518nm respectively, red color visible or green fluorescence present (e, f), when exciting jointly with 575nm and two kinds of wavelength of 518nm, as seen some 293T cell is yellow (g), proof is connected into the DsRed1 gene of IRES encoding sequence upstream in the pVAX1-IRES carrier and the EGFP gene in downstream not only can be expressed in the 293T cell respectively, and can in same cell, the effect by IRES carry out coexpression, prove that dna vaccine vector pVAX1-IRES of the present invention has and start the function that two kinds of genes carry out coexpression simultaneously.
3, the transient expression efficient of the various recombinant plasmids of cells were tested by flow cytometry
With FACSCalibur cells were tested by flow cytometry step 1 transient transfection of the BD company transient expression efficient of various recombinant plasmids in the 293T transfectional cell after 48 hours, concrete grammar is as follows:
A. behind the transfection 48h with 0.25% trysinization 293T transfectional cell, piping and druming is prepared into single cell suspension gently;
B. the PBS that contains 2% newborn calf serum with the ice bath precooling cleans cell 3 times;
C. use 0.5mL 1% Paraformaldehyde 96 re-suspended cell, use the expression of the various recombinant plasmids of cells were tested by flow cytometry in the 293T transfectional cell at last.
The various recombinant plasmids of transient transfection after 48 hours are at the flow cytometer detected result of the intracellular transient expression efficient of 293T (A: blank as shown in figure 10; B:pDsRed1-N1; C:pEGFP-N1; D:pVAX1-IRES-DsRed1; E:pVAX1-IRES-EGFP; F:pVAX1-DsRed1-IRES-EGFP (UL represents up-left, the upper left corner, 575nm, reaction be a cell of single expression red fluorescent protein; LR is low-right, the lower right corner, 518nm, reaction be a cell of single expression green fluorescent protein; UR is up-right, the upper right corner, and 575nm+518nm, double expression(DE) is not only to have expressed red fluorescent protein but also expressing green fluorescent protein in the same cell; LL is low-left, the lower left corner, what represent is the cell of jack to jack adapter), as seen DsRed1 expression of gene situation is (10.24%+18.86%=29.10%) among the plasmid pVAX1-DsRed1-IRES-EGFP, the situation (18.86%) that EGFP expression of gene situation (5.24%+18.86%=24.10%), DsRed1 gene and EGFP gene are expressed simultaneously.Show that the DsRed1 gene that is connected into IRES encoding sequence upstream in the pVAX1-IRES carrier and the EGFP gene in downstream not only can express respectively in the 293T cell, and can in same cell, the effect by IRES carry out coexpression, prove further that dna vaccine vector pVAX1-IRES of the present invention has and start the function that two kinds of genes carry out coexpression simultaneously.
The structure and the eukaryotic expression of embodiment 3, multitarget composite antigen Antioncogene vaccine
One, the design of antigenic compound 2PAG fusion gene
The main activated cell immune response of Survivin, the mRNA of Survivin can produce three kinds of isomer: Survivin, Survivin-2B and Survivin-EX3 through different cut modes in vivo, is that the anti-tumor vaccine of specificity target spot has entered the II clinical trial phase stage with Survivin-2B from aminoterminal 80-88 amino acids residue wherein.Find through detailed investigation of related literatures, therefore the T cell antigen epitope of Survivin-2B mainly concentrates between aminoterminal 5-28 and 80-121 amino acids residue, chooses these two zones as one of target spot of multitarget composite antigen Antioncogene vaccine of the present invention.From Genebank, search the base sequence (134-205 that corresponds to above-mentioned two zones among the people Survivin 2B (NM-001012271), 359-513), again by connecting arm " nG-; wherein n can be K (Methionin), R (arginine), C (halfcystine), Q (glutamine), G (glycine) or N (l-asparagine); present embodiment n is N (l-asparagine) " thus encoding sequence connect to constitute the Sur fusion gene with two segment DNA sequences, its nucleotide sequence is shown in the sequence in the sequence table 1, total length 273bp, 91 amino-acid residues of encoding.
CTP37 is 37 peptides that are positioned at hCG β chain carboxy-terminal 109-145 amino acids residue, has the specific antigen epi-position of hCG.The CTP37 of people and monkey is united use structure composition target antigen, and method is: the encoding sequence [people: NM_033043 who searches hCG β-CTP37 of people and monkey from Genebank; Monkey: NM_001032928 (homology comparative result, monkey: people=76%; Monkey: mouse=100%), (n can be K, R, C, Q, G or N by connecting arm-nG-, present embodiment n is N) the CTP37 encoding sequence of two species is connected to form fusion gene hmCTP, its nucleotide sequence is shown in the sequence in the sequence table 2, total length is 228bp, 76 amino-acid residues of encoding.
3 ' the end of fusion gene Sur and the 5 ' end of fusion gene hmCTP are connected and promptly obtain antigenic compound 2PAG fusion gene, and its structural formula is intended to as shown in figure 11.
Two, the pcr amplification of antigenic compound 2PAG fusion gene
1, design of primers
Two pairs of primers of nucleotide sequence design (band underscore base is the restriction enzyme enzyme recognition site) according to Sur and hmCTP gene, wherein 5 ' of Sur-F1 end contains restriction enzyme Xho I recognition site, 5 ' the end of hmCTP-R1 contains restriction enzyme EcoR I recognition site, Sur-SC-R1 and Sur-SC-F1 have the complementary region of 20bp, and concrete primer sequence is as follows:
Sur-F1：5’-CCCGGCTCGAGACATTGCCCCCTG-3’
Sur-SC-R1：5’-CATCACAGGTCTTCTTATTGTTGG-3’
Sur-SC-F1：5’-CAATAAGAAGACCTGTGATGACCCCCG-3’
hmHCG-R1：5’-GGCCCGAATTCTTGTGGAAGGAAAGGG-3’。
2, the pcr amplification of antigenic compound 2PAG fusion gene
At first, hold up synthetic above-mentioned two fusion gene Sur of day Bioisystech Co., Ltd and hmCTP by Beijing, and be connected to respectively among the T-easy carrier pGEM-T Easy (TaKaRa company), the recombinant vectors called after Sur-T-easy that will contain fusion gene Sur will contain the recombinant vectors called after hmCTP-T-easy of fusion gene hmCTP.Then, be template with plasmid Sur-T-easy and the hmCTP-T-easy that contains synthetic gene respectively, pcr amplification goes out fusion gene Sur-SC and SC-hmCTP.Wherein, the 5 ' end of 3 ' of Sur-SC end and SC-hmCTP contains the complementary sequence of 20bp.The pcr amplification system is: 10 * PCR damping fluid, 5 μ l, dNTPs Mixture (2.5mM) 5 μ l, each 1 μ l of primer (20pM) Sur-F1 and Sur-SC-R1 (or Sur-SC-F1 and hmCTP-R1), template Sur-T-easy (or hmCTP-T-easy) 1 μ l, Taq archaeal dna polymerase (5U/ μ l) 1 μ l adds deionized water to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 58 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 5 circulations; Last 72 ℃ are continued to extend 10min.After reaction finishes, get 5 μ l pcr amplification products and carry out the detection of 1% agarose gel electrophoresis, (swimming lane M is DL 2000 DNA Marker to detected result as shown in figure 12, swimming lane 1 is: fusion gene Sur-SC, swimming lane 2 is: fusion gene SC-hmCTP), obtained the DNA band of 273bp and 228bp through amplification, consistent with the expection size.Reclaim the purpose band of test kit and the recovery of reference reagent box specification sheets and purifying fusion gene Sur-SC (273bp) and fusion gene SC-hmCTP (228bp) with the PCR product glue of vast Imtech.Be template jointly respectively to get 1 μ l after 50 times of the fusion gene Sur-SC of pcr amplification and the SC-hmCTP dilutions again, method expansion of antigen mixture 2PAG fusion gene with bridging PCR, under the guiding of primer Sur-F1 and hmCTP-R1, carry out pcr amplification, after reaction finishes, get 5 μ l pcr amplification products and carry out the detection of 1% agarose gel electrophoresis, (swimming lane M is DL 2000 DNA Marker (available from TaKaRa company) to detected result as shown in figure 12, swimming lane 3 is the antigenic compound 2PAG fusion gene of pcr amplification), obtained the DNA band of 523bp through amplification, consistent with the expection size.Reclaim the purpose band of test kit and the recovery of reference reagent box specification sheets and this antigenic compound of purifying 2PAG fusion gene with the PCR product glue of vast Imtech.
Three, the acquisition of human GM-CSF and B7.1 fusion gene
The encoding sequence of going up disclosed human GM-CSF (NM-000758) and B7.1 (NM-005191) by GeneBank designs its pcr amplification primer sequence, and sequence is as follows:
GMF1 (upstream primer): 5 '-CCG
ATGCATATGTGGCTGCAGAGCCTG-3 ' (band underscore base is a restriction enzyme Nsi I recognition site)
GMR1 (downstream primer): 5 '-GCCGCCGCGTCGACCCCTGCCGCCCTCCTGGACTGGCTCCCAGC-3 ';
B7 F1 (upstream primer): 5 '-GGCGGCAGGGGTCGACGCGGCGGCGGTCTTTCTCACTTCTGTTC-3 '
B7 R1 (downstream primer): 5 '-CCG
GCGCGCTTATACAGGGCGTACACTTTCCC-3 ' (band underscore base is a restriction enzyme BSSH II recognition site).
At first, hold up the encoding sequence of synthetic human GM-CSF of day Bioisystech Co., Ltd and B7.1 by Beijing, and be connected to respectively in the pGEM-T Easy carrier (available from TaKaRa company), the recombinant vectors called after pGEM-TEasy-GM-CSF that will contain the human GM-CSF encoding sequence will contain the recombinant vectors called after pGEM-TEasy-B7.1 of B7.1 encoding sequence.Then, be template with plasmid pGEM-TEasy-GM-CSF and the pGEM-TEasy-B7.1 that contains synthetic gene respectively, pcr amplification GM-CSF full length gene sequence under the guiding of GMF1 and GMR1, pcr amplification B7.1 full length gene sequence under the guiding of B7 F1 and B7 R1, respectively get 1 μ l jointly as template, pcr amplification GM-CSF and B7.1 fusion gene under the guiding of primer GMF1 and B7 R1 after general's PCR product separately dilutes 50 times then.Concrete reaction system and reaction conditions are referring to step 2.After reaction finishes, get 5 μ l pcr amplification products and carry out the detection of 1% agarose gel electrophoresis, (swimming lane M is DL 2000 DNA Marker to detected result as shown in figure 13, swimming lane 1 is: the human GM-CSF gene, swimming lane 2 is: the B7.1 gene, swimming lane 3 is the human GM-CSF factor and the B7.1 fusion gene of pcr amplification), obtained the DNA band of 1245bp through amplification, consistent with the expection size.Reclaim the purpose band of test kit and the recovery of reference reagent box specification sheets and this human GM-CSF of purifying and B7.1 fusion gene with the PCR product glue of vast Imtech.The human GM-CSF gene is connected by 8 peptide-coding sequences with the B7.1 gene, can make the human GM-CSF factor and B7.1 molecular separation under the effect of ubiquitous in vivo Furin proteolytic enzyme after the expression, thereby do not influence their space conformation, be beneficial to bring into play function separately.
Four, the structure of dna vaccination pVAX1-2PFcGB
1, the structure that contains the eukaryon expression plasmid pCI-2PAG-Fc-GPI of antigenic compound 2PAG fusion gene
The antigenic compound 2PAG fusion gene that step 2 is obtained carries out behind the double digestion with restriction enzyme Xho I and EcoR I and contains people Igk chain targeting signal peptide (sig) through the same enzyme double digestion, the carrier for expression of eukaryon pCI-GPI of people Ig G-Fc and glycosyl-phosphatidyl inositol (GPI) grappling signal peptide gene sequence (makes up on the basis of the eukaryon expression plasmid pCI-neo+ of Promega company and forms, construction process is: insert universal sequence and make up and form between the restriction enzyme Nhe I at the former multiple clone site of pCI-neo+ place and Xba I, the gene order of this universal sequence is the encoding sequence-Xba I-3 ' of 5 '-Nhe I-people Igk chain targeting signal peptide (sig) encoding sequence-Xho I-EcoR V-EcoR I-human IgG Fc section encoding sequence-glycosylation phosphatidylinositols (GPI) grappling signal peptide, and the foreign immunologic molecular gene can select proper restriction site to insert between Nhe I-EcoR I.PCI-GPI is except that the encoding sequence that contains human IgG Fc section encoding sequence and glycosylation phosphatidylinositols (GPI) grappling signal peptide, the main skeleton structure that also contains the pCI carrier is as CMV early promoter, chimeric intron, Neo selection markers, SV40 enhanser and ammonia benzyl resistant gene Amp
R+Deng) connect, to connect product and be converted into the bacillus coli DH 5 alpha competent cell, screening positive clone, carrying out bacterium colony PCR with primer Sur-F1 and hmCTP-R1 identifies, upgrading grain then, carry out double digestion with restriction enzyme Nhe I and Not I and identify that PCR and enzyme are cut qualification result (swimming lane M:DL 2000 DNA Marker as shown in figure 14; The Xho I+EcoR I enzyme of swimming lane 1:2PAG fusion gene is cut product; The Xho I+EcoR I enzyme of swimming lane 2:pCI-GPI is cut product; Swimming lane 3: bacterium colony PCR qualification result; Swimming lane 4:pCI-neo-2PAG-Fc-GPI plasmid; The Nhe I+Not I enzyme of swimming lane 5:pCI-neo-2PAG-Fc-GPI is cut product), the positive colony plasmid is cut the DNA band that can obtain 5472bp and 2473bp through enzyme, to identify that correct positive colony bacterial strain is checked order by Beijing three rich biological company limiteds, sequencing result shows that acquisition sequence and on position all correctly contain the eukaryon expression plasmid of antigenic compound 2PAG fusion gene, called after pCI-2PAG-Fc-GPI.
2, the structure of eukaryon expression plasmid pVAX-sig-2P-FC-GPI-IRES
With the correct positive recombinant plasmid pCI-2PAG-Fc-GPI of order-checking earlier with restriction enzyme Not I carry out single endonuclease digestion also purified after, mend the sticky end that produces behind the flat single endonuclease digestion with Klenow enzyme (TaKaRa company) and reference reagent box specification sheets, then the product behind the purifying is carried out single endonuclease digestion with restriction enzyme Nhe I again, cut glue subsequently and reclaim the purpose fragment (5 ' end is cut the sticky end that the back produces for Nhe I enzyme, 3 ' the end flush end that flat back produces for Klenow mends) that contains the linearizing sig-2PAG-FC-GPI of 2473bp.Then, the carrier for expression of eukaryon pVAX1-IRES that embodiment 1 is made up carries out being connected with sig-2PAG-Fc-GPI behind double digestion and the purifying with restriction enzyme Nhe I and EcoR V, to connect product and be converted into the bacillus coli DH 5 alpha competent cell, screening positive clone, carrying out bacterium colony PCR with primer Sur-F1 and hmCTP-R1 identifies, upgrading grain then, the upgrading grain, carry out double digestion with restriction enzyme Nhe I and Cla I and identify that bacterium colony PCR and enzyme are cut qualification result (swimming lane M:DL 2000 DNAMarker as shown in figure 15; Swimming lane 1:sig-2PAG-Fc-GPI/Not I → Klenow → Nhe I; The NheI+EcoR V enzyme of swimming lane 2:pVAX1-IRES is cut product; Swimming lane 3: bacterium colony PCR qualification result; Swimming lane 4:pVAX1-sig-2PAG-Fc-GPI-IRES plasmid; The Nhe I+Cla I enzyme of swimming lane 5:pVAX1-sig-2PAG-Fc-GPI-IRES is cut product), the positive colony plasmid is cut the DNA band that can obtain 3600bp and 2473bp through enzyme, to identify that correct positive colony bacterial strain is checked order by Beijing three rich biological company limiteds, sequencing result shows that acquisition sequence and on position all correctly contain the eukaryon expression plasmid of sig-2PAG-FC-GPI dna fragmentation, called after pVAX-sig-2P-FC-GPI-IRES.
3, the structure of pVAX1-IRES-GM/B7
With the GM-CSF of step 3 amplification and B7.1 fusion gene with restriction enzyme Nsi I be connected after BSSH II carries out double digestion with through the carrier pVAX1-IRES of enzyme double digestion equally, to connect product and be converted into the bacillus coli DH 5 alpha competent cell, screening positive clone, the upgrading grain, carry out double digestion with restriction enzyme Nsi I and BSSHII and identify that enzyme is cut qualification result (swimming lane M:DL 2000 DNA Marker as shown in figure 16; Swimming lane 1:pVAX1-IRES-GM/B7 plasmid; Swimming lane 2:GM-CSF and B7.1 fusion gene; The Nsi I+BSSHII enzyme of swimming lane 3:pVAX1-IRES-GM/B7 plasmid is cut product), the positive colony plasmid is cut the DNA band that can obtain 3600bp and 1245bp through enzyme, to identify that correct positive colony bacterial strain is checked order by Beijing three rich biological company limiteds, sequencing result shows that acquisition sequence and on position all correctly contain the eukaryon expression plasmid of GM-CSF and B7.1 fusion gene, called after pVAX1-IRES-GM/B7.
4, the acquisition of dna vaccination pVAX1-2PFcGB
After positive recombinant plasmid pVAX1-sig-2PAG-Fc-GPI-IRES after identifying step 2 correctly carries out double digestion with restriction enzyme Nhe I and Cla I, enzyme is cut product carry out the detection of 1% agarose gel electrophoresis, reclaim the also purpose fragment that contains sig-2PAG-FC-GPI of purifying 2473bp, and then be connected with the eukaryon expression plasmid pVAX1-IRES-GM/B7 of Cla I double digestion through Nhe I, to connect product and be converted into the bacillus coli DH 5 alpha competent cell, screening positive clone, the upgrading grain, carry out double digestion with restriction enzyme Nsi I and BSSH II and identify that enzyme is cut qualification result (swimming lane M:DL 2000 DNA Marker as shown in figure 17; The NheI+Cla I enzyme of swimming lane 1:sig-2PAG-Fc-GPI is cut product; The Nhe I+Cla I enzyme of swimming lane 2:pVAX1-IRES-GM/B7 is cut product; Swimming lane 3:PCR identifies; Swimming lane 4:pVAX1-sig-2PAG-Fc-GPI-GM/B7 plasmid; The Nhe I+Cla I enzyme of swimming lane 5:pVAX1-sig-2PAG-Fc-GPI-IRES is cut product), the positive colony plasmid is cut through enzyme can obtain the DNA band that size is about 2000bp, to identify that correct positive colony bacterial strain is checked order by Beijing three rich biological company limiteds, sequencing result shows acquisition sequence and all correct eukaryon expression plasmid of on position, be multitarget composite antigen Antioncogene vaccine of the present invention, called after pVAX1-sig-2PAG-Fc-GPI-GM/B7 (being called for short pVAX1-2PFcGB).
Five, the eukaryotic expression of multitarget composite antigen Antioncogene vaccine
1, the eukaryotic expression of dna vaccination pVAX1-2PFcGB
Adopt with embodiment 2 in identical liposome method with the dna vaccination pVAX1-2PFcGB transfection human embryo kidney (HEK) 293T cell of step 4 structure, set up three experimental group and blank group simultaneously, the blank group only adds the blank liposome of equivalent, is respectively applied for flow cytometer detection and immunofluorescence and detects.
2, flow cytometer detects
Adopt with embodiment 2 in FACSCalibur cells were tested by flow cytometry step 1 transient transfection of the identical method usefulness BD company transient expression efficient of pVAX1-2PFcGB in the 293T transfectional cell after 48 hours, in pVAX1-2PFcGB, the carboxyl terminal of 2PAG fusion gene has merged human IgG Fc fragment gene, can be used as the label that detects expressing fusion protein, therefore use the rabbit anti-human igg (available from China fir Golden Bridge Technology, Inc. in Beijing) of FITC mark respectively, the antibody of the anti-people B7 of the rabbit of PE mark (EBioScience company) is hatched with the cell of transfection after 48 hours, insert the expression of GM-CSF/B7.1 fusion gene in the 293T cell in fragment 2PAG-Fc-GPI and the carrier in order to detect, the flow cytometer detected result is (A: blank group (FITC) as shown in figure 18; B: experimental group (FITC); C: blank group (PE); D: experimental group (PE)), transfection is after 48 hours, and the positive expression rate of inserting fragment 2PAG-Fc-GPI in the 293T cell is 44.46%, and average fluorescent strength is 334.17; The positive expression rate of GM-CSF/B7.1 is 52.26% in the carrier, average fluorescent strength is 497.86, shows that the GM-CSF/B7.1 fusion gene in the 2PAG-Fc-GPI gene fragment that connects into IRES encoding sequence upstream in the pVAX1-IRES carrier and downstream all obtains to express.
3, immunofluorescence detects
Adopt with embodiment 2 in identical method handle cell, under fluorescent microscope, directly carry out the immunofluorescence detection after dripping sheet.Hatch with antibody and the cell of transfection after 48 hours of the anti-people B7 of rabbit of the rabbit anti-human igg of FITC mark, PE mark respectively, insert the expression of GM-CSF/B7.1 fusion gene in the 293T cell in fragment 2PAG-Fc-GPI and the carrier in order to detect, the immunofluorescence detected result is (A: through the 293T of FITC-IgG antibody incubation cell as shown in figure 19; B: through the 293T of PE-B7 antibody incubation cell), find through fluorescent microscope observation, 293T cell behind the transient transfection pVAX1-2PFcGB 48h all has fluorescence to show, and mainly be distributed in (Fig. 6) on the cytolemma, and the 293T cell detection results of untransfected recombinant plasmid pVAX1-2PFcGB is all negative, proves that the GM-CSF/B7.1 fusion gene all can effective expression in insertion fragment 2PAG-Fc-GPI and the carrier.
The antitumor activity of embodiment 4, multitarget composite antigen Antioncogene vaccine pVAX1-2PFcGB detects
Detect the antitumor activity of multitarget composite antigen Antioncogene vaccine pVAX1-2PFcGB of the present invention with following method, plasmid pVAX1-2PAG, pVAX1-2PAG-Fc-GPI (structural representation is seen Figure 29) and the pVAX1-IRES-GM/B7 that makes up with embodiment 3 is contrast simultaneously, and detection method is as follows:
One, multitarget composite antigen Antioncogene vaccine pVAX1-2PFcGB immune mouse
1, immunity a large amount of extraction and purifications of plasmid DNA
Prepare immunity in a large number with vast Tyke, Beijing biological gene technology limited liability company " a large amount of rapid extraction test kits of the ultrapure plasmid of Type B " and reference reagent box specification sheets and use plasmid DNA, concrete grammar may further comprise the steps:
A. 200mL is carried 37 ℃ of cultivations of bacillus coli DH 5 alpha reorganization bacterium of pVAX1-2PFcG and collect thalline after 12 hours, add 7mL solution 1, vibration is to thoroughly suspension;
B. add 10.5mL solution 2, put upside down mixing gently, make the abundant cracking of thalline, placed on ice subsequently 5 minutes;
C. add 10.5mL solution 3, put upside down mixing gently, room temperature was placed 10 minutes, and 4 ℃, 12, the centrifugal 15min of 000rpm;
D. behind the Virahol mixing with supernatant and 0.6 times of volume, 4 ℃, 10, the centrifugal 20min of 000rpm;
E. collecting precipitation is dissolved in the 2mL distilled water and adds 80 μ l 10mg/mL RNase A, 37 ℃ of insulations 30 minutes;
F. add isopyknic binding buffer liquid, move into adsorption column behind the mixing, room temperature, 12, the centrifugal 1min of 000rpm;
G. outwell waste liquid, add 500 μ l and remove the intracellular toxin damping fluid, room temperature, 2, the centrifugal 1min of 000rpm, repeat 1 time after, once more in room temperature, 12, the centrifugal 3min of 000rpm; Remove the intracellular toxin damping fluid as far as possible;
H. outwell waste liquid, add 800 μ l rinsing damping fluids, leave standstill after 1 minute 12, the centrifugal 1min of 000rpm, repeat 2 times after, once more 12, the centrifugal 2min of 000rpm removes the rinsing damping fluid as far as possible;
I. adsorption column is put into a clean centrifuge tube, added 50 μ l sterilized waters, leave standstill 2-5min under the room temperature in the central authorities of adsorption film, 12, the centrifugal 2min of 000rpm collects elutriant, and-20 ℃ of storages are standby.
The collection elutriant is the plasmid DNA of purification, adds 10 * PBS and makes plasmid DNA be dissolved in PBS solution.Determined by ultraviolet spectrophotometry dna content and purity, dna content is about 1 μ g/ μ l as a result, and purity reaches 90% ,-20 ℃ of preservations.
2, animal grouping
The female Balb/c mouse (available from Military Medical Science Institute's Experimental Animal Center) of 6 ages in week, body weight 18-20g is divided into 6 groups at random, every group 9, the A group is the PBS control group, the B group is pVAX1 empty carrier group, the C group is the pVAX1-IRES-GM/B7 group, the D group is the pVAX1-2PAG group, and the E group is pVAX1-2PFcGB group (gene vaccine pVAX1-2PFcGB) for pVAX1-2PAG-Fc-GPI group, F group.
3, the subcutaneous transplantation knurl is attacked
The mouse mastopathy cell EMT6 (available from Shanghai cell resource center of the Chinese Academy of Sciences) of results logarithmic phase, the preparation single cell suspension is washed 2 times with PBS, adjusts cell concn to 2 * 10
6Individual/mL, in-d5 days at mouse back right side subcutaneous vaccination 100 μ l cell suspensions promptly 2 * 10
5Individual/only.
4, the immunity of Balb/c mouse
Immunization protocol as shown in figure 20, the Balb/c mouse after the subcutaneous transplantation knurl is attacked 5 days, the beginning vaccination.Immunization ways adopts the injection of quadriceps muscle of thigh muscle, and A organizes injection 100 μ l PBS, and all the other each groups are only injected corresponding D NA plasmid 100 μ g/ respectively, are total to immunity 4 times respectively at d0, d5, d15 and d20.Before each immunity, at first inject 100 μ l, 0.25% bupivacaine pre-treatment inoculation position, behind the 72h in the same area vaccination.The 5th day (d25) detects the production of mouse internal antibody and collects the splenocyte of every group of 2 mouse after the last immunity, prepares the CTL killing experiments; The neck that broke in d30 days is put to death every group of all the other each mouse and is won tumor tissues and weigh.
Two, immune effect detects
1, vaccine immunity protection effect observation and detection
1) the one-tenth knurl time of immune mouse
At first give Balb/c mouse subcutaneous injection inoculation 2 * 10
5Individual EMT6 cell, 5d behind the inoculating cell begins to inject corresponding plasmid DNA or PBS respectively to each group mouse quadriceps muscle of thigh, observes and the tumor nodule formation time (can lay one's hand on and, the about 2-3mm of major diameter) of mouse respectively organized in record.
Each organizes the one-tenth knurl time statistics such as the table 1 and shown in Figure 21 of immune mouse subcutaneous transplantation knurl, and as can be seen, there is difference in various degree in the tumor nodule formation time of each immune group.The one-tenth knurl time of A group the earliest, about about 6 days; The one-tenth knurl time phase difference of B group, C group is few, no significance difference (P＞0.05).D, the E that contains antigenic compound 2PAG becomes the knurl time all to be later than control group with F group mouse, morning one-tenth knurl time of D group mouse wherein, about about 8 days; The E group is taken second place, and the one-tenth knurl time of F group mouse the latest, just can detect the formation of tumor nodule up to about 12 days.The E group is compared with control group with the F group, and the one-tenth knurl time difference of mouse is (P＜0.05) significantly.
Table 1 is respectively organized the one-tenth knurl time of immune mouse subcutaneous transplantation knurl
| The average one-tenth knurl time (my god) | |
| The A group, (PBS) B group, (pVAX1 empty carrier) C group, (pVAX1-IRES-GM/B7 empty carrier) D group, (pVAX1-2PAG) E group, (pVAX1-2PAG-Fc-GPI) F group, (pVAX1-2PFcGB) | 6±1.01 6.28±0.81 6.57±1.13 8.14±0.99 9.57±1.23 ※ 12.29±1.26 ※ |
Annotate:
※Compare with control group, this group mouse becomes knurl prolongation of latency (P＜0.05)
2) the growth of xenografted situation detects in the immune mouse body
7 mouse in each immune group of chosen in advance, inoculated with subcutaneous injections 2 * 10
5Beginning vaccination in the 5th day behind the individual/EMT6 cell.From beginning after the mouse subcutaneous transplanting knurl is attacked to putting to death in 35 days of mouse, per 2 days vertical major diameters and vertical minor axis with a tumour of vernier caliper measurement, and calculate the average-volume of every group of mouse interior tumor by following formula, draw its tumor growth curve, tumor growth curve is carried out linear regression and its slope is done the t check, and the calculation formula of the average-volume of mouse interior tumor is as follows:
In the immune mouse body growth curve of transplanted tumor as shown in figure 22, pVAX1 empty carrier group (B group) and the pVAX1-IRES-GM/B7 that does not contain complex antigen 2PAG organize between intravital interior establiss situation of (C group) mouse and the PBS control group does not have marked difference (P＞0.05); Through D, the E of vaccine therapy and the mouse in the F group, its no knurl all prolongs than control group latent period, and growth of tumor is all than control group slow (P＜0.05).Wherein, the E group is compared with the PBS control group with the F group, and difference is (P＜0.01) extremely significantly.The no knurl of mouse is the longest latent period in the F group, its growth of tumor speed is the slowest before d22, compared significant difference (P＜0.05) with the E group, but difference not significantly (P＞0.05) after d22, this may with for the second time and for the third time between the immunity prolongation of time relevant, fail in time to restrain growth of tumor.
3) the subcutaneous transplantation knurl is attacked the survival condition of back 35 days mouse
Inoculated with subcutaneous injections 2 * 10
5Behind the individual/EMT6 cell in 35 days, it is as shown in table 2 that each organizes the survival condition of mouse (7/group), PBS control group and pVAX1 empty carrier group all have 1 mouse natural death, and 7 mouse of all the other each groups all survive, and show that gene vaccine of the present invention has higher security.
Table 2 subcutaneous transplantation knurl is attacked the back survival condition of respectively organizing immune mouse in 35 days
| Death toll (only/7) | Survival rate (%) | |
| The A group, (PBS) B group, (pVAX1 empty carrier) C group, (pVAX1-IRES-GM/B7 empty carrier) D group, (pVAX1-2PAG) E group, (pVAX1-2PAG-Fc-GPI) F group, (pVAX1-2PFcGB) | 1 1 0 0 0 0 | 85.7 85.7 100 100 100 100 |
4) immune mouse is to the restraining effect of subcutaneous transplantation knurl
Inoculated with subcutaneous injections 2 * 10
535 days disconnected necks are put to death the interior survival mice of each group behind the individual/EMT6 cell, win tumor tissues, and take pictures.After the knurl weight was respectively organized in weighing, the inhibiting rate of vaccine to tumour respectively organized in calculating according to formula, and the calculation formula of tumor control rate is as follows:
Subcutaneous vaccination EMT6 cell and through vaccine immunity after 30 days, the stripped gross tumor volume comparative result of each immune group mouse is (A:PBS blank group as shown in figure 23; The B:pVAX1 group; The C:pVAX1-IRES group; The D:pVAX1-2PAG group; The E:pVAX1-2PAG-Fc-GPI group; The F:pVAX1-2PFcGB group), it is heavy as shown in figure 24 that the subcutaneous transplantation knurl is attacked the average knurl of respectively organizing mouse after 35 days, the statistics of the tumor control rate of each immune group such as table 3 and shown in Figure 25, and the result shows, the average knurl heavy phase of A group, B group and C group is near, does not have significant difference (P＞0.05); Compare with control group, the average knurl weight average of D group, E group and F group obviously reduces, and shows that the gene vaccine that the present invention contains complex antigen gene 2PAG has tumors of higher inhibiting rate (P＜0.01); Wherein the average knurl of F group is heavy minimum, has the highest tumor control rate, with the difference of control group extremely significantly (P＜0.001).
Table 3 is respectively organized the restraining effect of immune mouse to tumour
| Average knurl heavy (g) | Tumor control rate (%) | |
| The A group, (PBS) B group, (pVAX1 empty carrier) C group, (pVAX1-IRES-GM/B7 empty carrier) D group, (pVAX1-2PAG) E group, (pVAX1-2PAG-Fc-GPI) F group, (pVAX1-2PFcGB) | 3.78±0.59 3.50±0.44 3.33±0.51 1.98±0.75 1.41±0.33 0.86±0.31 | - 7.4 11.9 47.6 ※ 62.7 ※ 77.27 ※※ |
Annotate:
※Compare with control group, this group mouse is to the restraining effect highly significant (P＜0.01) of tumour;
※ ※P＜0.001.
2, the ELISA of serum antibody detects
After the last immunity the 5th day (d25), get the tail vein of D, E and F group immune mouse, the centrifugal 10min of 3000rpm behind the room temperature placement 3h, get upper serum, with Sur fusion rotein wrapper sheet, detect antibody titers with the ELISA method, set up the negative control group of the preceding mouse serum of immunity simultaneously, detection method may further comprise the steps:
A. wrap quilt: it is 3 μ g/mL that albumen is diluted to final concentration with coating buffer, and bag is by elisa plate, and 4 ℃ are spent the night;
B. washing: get rid of and abandon coating buffer in the elisa plate hole, PBST washing 1 time;
C. sealing: 2% casein room temperature sealing 4h, discard confining liquid, pat dry elisa plate;
D. detect: immune mouse serum adds each hole, 100ul/ hole, 37 ℃, 30min after using the PBS doubling dilution;
E. mark: PBST washing back adds the sheep anti-mouse igg (available from China fir Golden Bridge Technology, Inc. in Beijing) of HRP mark, 37 ℃ of reaction 20min;
F. colour developing: after the PBST washing, the TMB 10min that develops the color, 2M sulfuric acid termination reaction.Detect the OD value of 450nm with SPECTRAIII enzyme connection instrument.The result judges: the OD value (P) of testing sample is positive more than or equal to 2.1 (P/N 〉=2.1) with the ratio of the OD value (N) of negative control, shows that the maximum antibody dilution of positive findings is the antibody titer of immune serum.
The ELISA detected result of the antibody titer of D, E and F group immune serum all has specific antibody to occur in D, E and the F group immune serum as shown in figure 26, and the serum antibody titer of D group is 1: 3200, and the E group is identical with the F group, is 1: 6400.Above-mentioned detected result shows that gene vaccine of the present invention has better immunogenicity.
3, the mensuration of immune mouse CTL killing activity
Measure immune mouse CTL killing activity, concrete grammar may further comprise the steps:
1) preparation of immune mouse spleen mononuclearcell suspension
After the last immunity the 5th day (d25), disconnected neck is put to death remaining two mouse in each group, and the spleen of getting mouse under aseptic condition prepares splenocyte suspension, and concrete operations are as follows:
A. open the ultra violet lamp 30 minutes of Bechtop, get mouse and draw neck to put to death, be soaked in 5min in 75% ethanol after the tap water flushing, put into super clean bench mouse dissection plate upper left side clinostatism immediately;
B. use 75% ethanol disinfection mouse web portion, aseptic operation is opened the abdominal cavity and is taken out spleen, remove fat and reticular tissue, be cut into (5-7 section) and be put in 200 order stainless steel mesh screens, mesh screen is placed the plate of putting into 2-3mL serum-free RPMI-1640 nutrient solution in advance, squeeze out splenocyte with grinding rod, extruding is clean as far as possible;
C. with an amount of serum-free RPMI-1640 nutrient solution flushing steel mesh, leave standstill 3-5min in the ice bath, get supernatant and partly move in the centrifuge tube, add nutrient solution to 10mL;
D. prepare two transparent scale centrifuge tubes, add the 4mL lymphocyte separation medium respectively, splenocyte suspension slowly is added in parting liquid top, the centrifugal 20min of 1500rpm;
E. get tunica albuginea after centrifugal, add the 10mL substratum and wash (the centrifugal 10min of 1000rpm) 2 times;
F. at last cell is resuspended in the lymphocytes culture medium and (not exclusively adds 20%FCS in the substratum).Count with lymphocyte count liquid pair cell, in general, can gather in the crops 108 cell in the splenocyte suspension, after lymphocyte separation medium separates, can obtain 10
7Cell, adjust cell concn to 1 * 10
6/ mL adds the cytokine PHA-P 2 μ g/mL and the IL-2 20IU/mL that keep lymphocyte growth, puts into CO
2Cultivate in the incubator.
2) effector cell's preparation
Get the spleen mononuclearcell suspension of immunity back mouse, add PHA-P 2 μ g/mL and IL-2 10IU/mL and keep lymphocyte growth, be the detection specificity lethal effect, the former nucleoprotein of reorganization Sur that adds the ordinary method preparation, working concentration 25 μ g/mL, with cell co-stimulatory, CO
2Cultivate in the incubator after 3 days as the effector cell.
3) preparation of target cell
With mouse mastopathy cell EMT6 at CO
2Cultivate standby in the incubator.
4) serum lactic dehydrogenase (LDH)-method for releasing is surveyed killing activity
The foundation of A, reaction system
Imitate target cell and hatch the process of killing and wounding altogether, carry out in 96 hole circle floor cells culture plates, whole reaction system will be provided with strict control wells and test holes, establishes 3 multiple holes for every group, is 200 μ l systems, comprises following content:
A. different concns effector cell LDH nature release aperture (forming): collection effector cell, adjustment cell concn doubling dilution, every hole 50 μ l by effector cell and serum-free RPMI-1640 substratum;
B. target cell LDH nature release aperture (target cell is formed with serum free medium): every hole adding target cell (1 * 10
4/ mL) 50 μ l complement to 100 μ l with serum free medium;
C. the maximum release aperture of target cell (forming): every hole adding target cell (1 * 10 by target cell, serum-free RPMI-1640 substratum and 10 * lysate of adding subsequently
4/ mL) 50 μ l supply 100 μ l with serum free medium;
D. substratum background value hole: add 100 μ l serum-free RPMI-1640 substratum, be used for proofreading and correct discharging naturally of substratum LDH;
E. volume correction hole: add 100 μ l serum-free RPMI-1640 substratum and 10 * lysate (the CytoTox 96@ Non-Radioactive Cytotoxicity Assay test kit of Promega company provides), proofread and correct the volumetric errors that 10 μ l lysates cause;
F. experimental port: every hole adds target cell (5 * 10
4/ mL) 50 μ l add the good effector cell 50 μ l of doubling dilution, and making and imitating the target ratio is 60: 1; 30: 1; 15: 1; 7.5: 1.
After institute responds and sets up, in the centrifugal 4min of room temperature 300rpm.
B, cell cultures and results supernatant
96 well culture plates are put into CO
2Cultivated 4 hours in the incubator, cultivate and finish preceding 45min, in the maximum release aperture of target cell, add 10 * lysate, continue to cultivate and examine under a microscope the cracking situation of target cell in the maximum release aperture, if cracking not exclusively can be added into 10 * lysate.After finish cultivating, the centrifugal 4min of 250rpm at room temperature.
C, LDH measure
Measure the killing activity of each immune group mouse boosting cell with active detection kit CytoTox 96@ Non-RadioactiveCytotoxicity Assay of the CTL of Promega company and reference reagent box specification sheets, method is: prepare another piece 96 hole elisa plates, get supernatant 50 μ l at the bottom of 96 hole circles the culture plate and move into opposite position in the elisa plate, add 50 μ l substrate solutions in every hole, after building room temperature lucifuge reaction 30min, add 50 μ l stop buffers again, 490nm reads light absorption value in the place.
D, experiment with computing result
Obtain the mean value of respectively organizing the set multiple hole of experimental data earlier, calculate the correction value of respectively organizing data then, promptly experimental group and natural release group deduct substratum background releasing value; Maximum release group deducts volume and corrects releasing value, uses the correction value of respectively organizing experimental data, is calculated as follows:
The kill rate statistics of each immune group mouse boosting cell such as table 4 and shown in Figure 28, experimental result shows, all produced cell immune response in various degree in D, E and the F group immune mouse body, along with the increase of imitating the target ratio, CTL specific killing rate also rises gradually, have significant difference (P＜0.05) with control group, wherein the CTL kill rate of F group is the highest, is all to have extremely significant difference (P＜0.01) with control group at 60: 1,30: 1 and 15: 1 o'clock imitating the target ratio; The CTL kill rate of D group and E group is basic identical.
The different killing activities of imitating each immune group mouse boosting cell of target ratio of table 4
| The average kill rate of CTL (%) | ||||
| 60∶1 | 30∶1 | 15∶1 | 7.5∶1 | |
| The A group, (PBS) B group, (pVAX1 empty carrier) C group, (pVAX1-IRES-GM/B7 empty carrier) D group, (pVAX1-2PAG) E group, (pVAX1-2PAG-Fc-GPI) F group, (pVAX1-2PFcGB) | 7.73 12.09 17.57 40.75 ※ 48.56 ※ 68.74 ※※ | 6.03 8.05 14.36 31.21 ※ 30.97 ※ 44.39 ※※ | 5.46 6.06 11.01 28.04 ※ 24.56 ※ 30.99 ※※ | 2.69 4.48 9.71 23.99 ※ 18.48 ※ 20.60 ※ |
Annotate:
※Compare with control group, this group mouse is to the restraining effect highly significant (P＜0.05) of tumour;
※ ※P＜0.01.
Sequence table
<160>2
<210>1
<211>273
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
acattgcccc ctgcctggca gccctttctc aaggaccacc gcatctctac attcaagaac 60
tggcccttct tgaacggcgc ttacgcctgt aataccagca ctttgggagg ccgaggcggg 120
cggatcacaa gagaggaaca taaaaagcat agctccggtt gcgctttcct ttctgtcaag 180
aagcagtttg aagaactgac ccttggtgaa tttttgaaac tggacagaga aagagccaag 240
aacaaaattg caaaggaaac caacaataag aag 273
<210>2
<211>228
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
acctgtgatg acccccgctt ccaggactcc tcttcctcaa aggcccctcc ccccagcctt 60
ccaagtccat ccagactccc tgggcccagc gacaccccta tcctcccaca aaacggcacc 120
tgtgatgacc cccacctcca ggcctcctct tcctcaaagg accctccccc cagccctcca 180
agtccatccg gactcctgga gccagcagac aaccctttcc ttccacaa 228
Claims (10)
1, a kind of dna vaccination carrier for expression of eukaryon is to add the reorganization pVAX1 carrier for expression of eukaryon that the internal ribosome entry site encoding sequence obtains in the pVAX1 carrier framework.
2, dna vaccination carrier for expression of eukaryon according to claim 1 is characterized in that: the two ends of the internal ribosome entry site encoding sequence in described dna vaccination carrier for expression of eukaryon also are added with one or more rare restriction enzyme enzyme recognition sites; Described rare restriction enzyme comprises EcoR V, Cla I, BstB I, Pvu IPsi I, BssH II, Swa I, Pac I, Nsi I and Xho I.
3, dna vaccination carrier for expression of eukaryon according to claim 2 is characterized in that: described dna vaccination carrier for expression of eukaryon is pVAX1-IRES.
4, the application of each described dna vaccination carrier for expression of eukaryon of claim 1-3 in the preparation gene vaccine.
5, application according to claim 4 is characterized in that: described gene vaccine is antitumor, anti influenza, anti-AIDS, antiviral hepatitis, tuberculosis, rabies or antimalarial gene vaccine.
6, application according to claim 5, it is characterized in that: the activeconstituents of described Antioncogene vaccine contains the fusion gene of Survivin-2B from aminoterminal 5-28 position and 80-121 amino acids residue encoding sequence formation for the upstream of ribosome entry site(RES) encoding sequence in the carrier framework of each described dna vaccination carrier for expression of eukaryon of claim 1-3, and the recombinant eukaryon expression vector of the fusion gene of the core fragment CTP37 regional code sequence of the chorion gonadotrophic hormone beta chain of people and monkey formation.
7, application according to claim 6, it is characterized in that: described Survivin-2B is connected by connecting arm " nG " with encoding sequence from aminoterminal 80-121 amino acids residue from the encoding sequence of aminoterminal 5-28 amino acids residue, wherein, n is K, R, C, Q, G or N; The core fragment CTP37 regional code sequence of the chorion gonadotrophic hormone beta chain of described people and monkey is connected by connecting arm " nG ", and wherein, n is K, R, C, Q, G or N.
8, application according to claim 6 is characterized in that: the upstream of internal ribosome entry site encoding sequence also is connected with people Ig κ chain targeting signal peptide-coding sequence and/or human IgG-Fc section encoding sequence and/or glycosyl-phosphatidyl inositol anchor and decides signal coding sequence in the carrier framework of the carrier for expression of eukaryon of described Antioncogene vaccine.
9, according to claim 6 or 7 or 8 described application, it is characterized in that: the downstream of internal ribosome entry site encoding sequence also is connected with human GM-CSF and/or B7.1 costimulatory molecules encoding sequence in the carrier framework of the carrier for expression of eukaryon of described Antioncogene vaccine, and the encoding sequence of interleukin-, Interferon, rabbit and/or tumour necrosis factor.
10, application according to claim 9, it is characterized in that: the described Survivin-2B of containing is from the fusion gene of aminoterminal 5-28 position and 80-121 amino acids residue encoding sequence formation, and the recombinant eukaryon expression vector recombinant eukaryon expression vector of the fusion gene of the core fragment CTP37 regional code sequence of people and monkey chorion gonadotrophic hormone beta chain formation is pVAX1-2PAG or pVAX1-2PAG-Fc-GPI or pVAX1-2PFcGB.
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| CNA2007100998874A CN101096680A (en) | 2007-05-31 | 2007-05-31 | NDA vaccine eucaryon expression carrier and application in preparation of gene vaccine |
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| CN102391377A (en) * | 2011-11-01 | 2012-03-28 | 孙嘉琳 | Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein |
| CN103068985A (en) * | 2010-06-24 | 2013-04-24 | 美国政府(由卫生和人类服务部、疾病控制和预防中心的部长所代表) | Pan-lyssavirus vaccines against rabies |
| CN105400798A (en) * | 2008-11-17 | 2016-03-16 | Vgx药品有限责任公司 | Antigens that elicit immune response against flavivirus and methods of using same |
| CN107281475A (en) * | 2017-06-15 | 2017-10-24 | 杭州贝罗康生物技术有限公司 | A kind of gene vaccine for preventing and treating tumour and its production and use |
| WO2021155501A1 (en) * | 2020-02-05 | 2021-08-12 | 瓦克斯恩有限公司 | Fusion of survivin and gm-csf, coding dna, recombinant expression vector, anti-tumor vaccine, and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105400798A (en) * | 2008-11-17 | 2016-03-16 | Vgx药品有限责任公司 | Antigens that elicit immune response against flavivirus and methods of using same |
| CN103068985A (en) * | 2010-06-24 | 2013-04-24 | 美国政府(由卫生和人类服务部、疾病控制和预防中心的部长所代表) | Pan-lyssavirus vaccines against rabies |
| CN102391377A (en) * | 2011-11-01 | 2012-03-28 | 孙嘉琳 | Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein |
| CN102391377B (en) * | 2011-11-01 | 2013-11-06 | 孙嘉琳 | Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein |
| CN107281475A (en) * | 2017-06-15 | 2017-10-24 | 杭州贝罗康生物技术有限公司 | A kind of gene vaccine for preventing and treating tumour and its production and use |
| CN107281475B (en) * | 2017-06-15 | 2020-12-25 | 杭州贝罗康生物技术有限公司 | Gene vaccine for preventing and treating tumor and preparation method and application thereof |
| WO2021155501A1 (en) * | 2020-02-05 | 2021-08-12 | 瓦克斯恩有限公司 | Fusion of survivin and gm-csf, coding dna, recombinant expression vector, anti-tumor vaccine, and application thereof |
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