CN104623688A - Therapeutic interleukin-4 vaccine capable of treating human or animal tumor diseases - Google Patents
Therapeutic interleukin-4 vaccine capable of treating human or animal tumor diseases Download PDFInfo
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Abstract
The invention discloses a therapeutic interleukin-4 vaccine capable of treating human or animal tumor diseases. The therapeutic interleukin-4 vaccine is an any-form protein vaccine or conjugated protein vaccine prepared by taking a natural or artificially-synthesized intact protein or protein fragment of interleukin-4 as an antigen or an any-form gene vaccine or fused gene vaccine prepared by taking an intact gene or gene fragment of interleukin-4 as an antigen gene or main antigen gene. According to the invention, a host is subjected to active immunotherapy by using an IL-4 vaccine, generally, the effective time of the first immunity can be up to about 2-3 months, the effective treatment time of the second immunity can be up to about 6 months, and the rehabilitation aim can be achieved through carrying out immunotherapy for 1-3 times. Compared with a method for treating human or animal tumor diseases by directly applying an IL-4 antibody, the therapeutic interleukin-4 vaccine has the characteristics of low application frequency, small dosage and the like, so that not only is the treatment cost greatly reduced, but also the possibility for generating anaphylactic reaction is greatly reduced.
Description
Technical field
This case is for the Chinese patent application (application number: divisional application 2011101749184) that " can be used for treating the interleukin-4 therapeutic vaccine of human or animal immune related diseases ".
Background technology
Up to the present, people still lack the chronic infectious disease caused by endotrophic pathogen (virus, antibacterial, parasite etc.) and more effectively treat way, and vaccine prevention effect is also undesirable.Theoretically, this kind of chronic infectious disease needs host to produce the reaction of a strong class (Th1) anti-infectious immunity could remove pathogen, but how to induce body to produce strong Th1 anti-infectious immunity to react and remain an individual difficult problem.The exploratory development long-term according to applicant finds, the clinical onset stage great majority of this kind of chronic infectious disease all show as very strong two classes (Th2) anti-infectious immunity reaction, namely react based on Th2 anti-infectious immunity; Subclinical infection then great majority all shows as very strong Th1 anti-infectious immunity reaction, namely reacts based on Th1 anti-infectious immunity.Further research proves, the generation of a class and the reaction of two para-immunities is not synchronous generation, complementary form, but mutually suppress, the form of rising one after another.IL-4 is that induction produces the immunoreactive major cytokine of Th2, if can reduce the content of IL-4 in host, can effectively suppress Th2 immunoreation, thus promote the immunoreactive generation of Th1.Experiment proves, injects anti-IL-4 antibody can reach this object to host.But due to the half-life of antibody shorter (about 21-28 days half-life of IgG), need repeated multiple times injection, not only increase cost, and easily cause allergic reaction.Be badly in need of a kind of with low cost and technological means that can not cause allergic reaction.
Summary of the invention
For above-mentioned prior art, the invention provides a class and prepare simple, the with low cost and safe and reliable interleukin-4 therapeutic vaccine that can be used for treating human or animal's immune related diseases.
The present invention is achieved by the following technical solutions:
Can be used for the interleukin-4 therapeutic vaccine for the treatment of human or animal's immune related diseases, be with the intact proteins of the natural of interleukin-4 or synthetic or protein fragments (polypeptide or oligopeptide, need before making vaccine further with other Antigenic macromolecules phase coupling) do any type of protein vaccine or coupling protein vaccine that antigen makes;
Or: do with the complete genome of interleukin-4 or genetic fragment any type of gene vaccine or fusion gene vaccine that antigen gene or major antigen gene make;
Or: with the complete genome of any carrier cloning IL-4 or genetic fragment, with the made gene vaccine of any expression system amplification gene, or express the intact proteins of IL-4 or protein fragments (polypeptide or oligopeptide) and do any type of protein vaccine or amalgamation protein vaccine that antigen makes; If selected expression vector belongs to live vector, also live vector recombinant vaccine can be made.
Medically conventional vaccine adjuvant can be added with in described vaccine.
Vaccine of the present invention not only can be used for treating the chronic infectious disease of human or animal and parasitic disease, also can be applied in anaphylaxis etc. and the Ia disease treatment of cancer, some type and go by further genralrlization.
Different according to prepared vaccine form, the embodiment of vaccine of the present invention can have multiple: as vaccines such as aluminium glue adjuvant, oily adjuvant, Liposome Adjuvants, can adopt the multitude of different ways such as subcutaneous injection, intradermal injection and intramuscular injection; If live vector vaccine, except above injection system, also can adopt the different modes such as oral, suction, collunarium, eye drip.
The present invention be directed to the defect existed in prior art: " injecting anti-IL-4 antibody to host; need repeated multiple times injection; not only increase cost, and easily cause allergic reaction ", and the technical scheme proposed, that is: the form of IL-4 vaccine is adopted to carry out active immunity treatment to host, generally, first immunisation can reach the effective time of about 2 ~ 3 months, and secondary immunity can reach effective treatment time of about half a year, through the immunization therapy of 1 ~ 3 time, the object of rehabilitation can be reached.With directly apply compared with anti-IL-4 Antybody therapy, have number of applications few, the features such as consumption is low, like this, not only greatly reduce treatment cost, and, greatly reduce and produce anaphylactoid probability.
Accompanying drawing explanation
Fig. 1: inoculate (see embodiment 1 below) the IL-4 gene recombinaton oil-adjuvant vaccine prepared by embodiment 1 respectively at first 3 weeks of induced hypersensitivity reaction and 1 week subcutaneous (s.c.) to experiment BALB/c mouse, separately establish pET-32 carrier oil Adjuvanted vaccines and normal saline immunized controls group.Low dosage (5 μ g) chicken ovalbumin (OVA) aluminium glue vaccine intraperitoneal inoculation is used respectively in the 0th week and the 3rd week, with sensitized mice, instilled by nostril, to induce generation anaphylaxis in the 5th week with high dose (80 μ g are dissolved in 50 μ l PBS) OVA.Result shows: IL-4 gene recombinaton oil-adjuvant vaccine immune group is compared with normal saline immunized controls group with pET-32 carrier oil Adjuvanted vaccines group, and IL-4 gene recombinaton oil-adjuvant vaccine significantly can reduce the generation of OVA-IgE specific antibody.
Fig. 2: give experiment BALB/c mouse respectively at before Li Shiman parasite inoculation 3 weeks and 1 week abdominal cavity (i.p.) inoculate (see embodiment 1 below) IL-4 gene recombinaton aluminium glue vaccine prepared by embodiment 1, separately establish pET-32 carrier aluminium glue vaccine and normal saline immunized controls group.Appropriate Li Shiman parasite subcutaneous (s.c.) injection inoculation is in mouse hind leg foot pad.Weekly by the pathological changes size at kind of calliper once pad place enough after inoculation, continue 8 ~ 10 weeks.Result shows, IL-4 genetic vaccine significantly can reduce the parasitic infection of Li Shiman.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1: prepared by structure and the vaccine of mice IL-4 genetic vaccine
According to the full genome coded sequence of mice IL-4, design a pair suitable primer, by RT-PCR method amplification IL-4cDNA sequence.Then, through determined dna sequence, after the cDNA sequence that proof increases is accurate, be cloned into the suitable position of pET-32 fusion expression vector again, and further recombiant plasmid and pET-32 fusion expression vector blank plasmid are proceeded in suitable expression coli strain, through scale fermentation, extraction purification IL-4 fusion rotein and the sulfur hydrogen reduction albumen expressed by vehicle Control.It is [different according to the content of purification IL-4 fusion rotein after sulfur hydrogen reduction albumen expressed by purification IL-4 fusion rotein or vehicle Control is mixed by proper proportion from oily adjuvant or aluminium glue adjuvant etc., to oil-adjuvant vaccine, the volume ratio of oil/protein solution can be controlled at (1:1) ~ (3:1); To aluminium glue Adjuvanted vaccines, the volume ratio of aluminium glue/protein solution can be controlled at (1:1) ~ (1:5); Require that the amount of IL-4 fusion rotein or vehicle Control sulfur hydrogen reduction albumen in every part vaccine is about 15-20 μ g; The volume of every part vaccine is about 0.1ml; Or according to the preparation of veterinary's pharmacopeia rule of operation], experiment vaccine is made in further homogenate emulsifying (being undertaken by the rule of operation of refiner used).
Described oily adjuvant is by the pale yellow oily liquid body formed after the Span-80 of 94% (V/V) mineral oil (No. 10 white oils), 6% (V/V), 2% (g/V) aluminium stearate heating for dissolving; Oil adjuvant is the milky water-in-oil type vaccine of the thickness that vaccine protein solution and oily adjuvant are formed through emulsifying.General employing injection inoculation, being product conventional in prior art, is in animal inactivated vaccine, use wider adjuvant at present.
Described aluminium glue adjuvant also uses wider adjuvant in animal inactivated vaccine.
Embodiment 2: safety testing and the anaphylaxis of mice IL-4 genetic vaccine are tested
By the method for embodiment 1, after suitably being diluted by the sulfur hydrogen reduction albumen normal saline expressed by purification IL-4 fusion rotein or vehicle Control, be mixed with into IL-4 amalgamation protein vaccine or vehicle Control sulfur hydrogen reduction protein vaccine with oily adjuvant or aluminium glue adjuvant by the volume ratio of 1:1.The IL-4 amalgamation protein vaccine prepared divides low (0.1ml, about 1 part), in (0.25ml, about 2.5 parts), high (0.5ml, about 5 parts) three various dose groups immune BALB/c mouse respectively, separately establish pET-32 carrier bacterin and saline control group, often organize 5 mices, subcutaneous multi-point injection.3 weeks, interval, carries out second time and third time inoculation respectively.Observe after vaccine injection that mice has no adverse reaction, the phenomenon such as irritated, dead respectively.Result shows: physically well develop after mouse inoculation vaccine, and the mental status and appetite are all normal, and inoculation position has the untoward reaction such as transient swelling, heating, especially more obvious in high dose group performance.For oily adjuvant Seedling, high dose group mice, because vaccine absorbs comparatively slow, has slight granuloma to react.
Embodiment 3: mice IL-4 genetic vaccine strengthens the test of anti-tumor activity
By the method for embodiment 1, after the sulfur hydrogen reduction albumen normal saline expressed by the IL-4 fusion rotein of purification or vehicle Control is suitably diluted and aluminium glue adjuvant be mixed with in the ratio of 1:1 and test or control vaccine.Require that the amount of IL-4 fusion rotein or vehicle Control sulfur hydrogen reduction albumen in every part vaccine is about 15 ~ 20 μ g; The volume of every part vaccine is about 0.1ml.To experiment mice respectively at tumor cell attack first 3 weeks and 1 week abdominal cavity (i.p.) inoculate IL-4 gene recombinaton aluminium glue vaccine prepared by embodiment 1, separately establish pET-32 carrier aluminium glue vaccine and normal saline immunized controls group.Check a tumor growth situation every 3 days after tumor inoculation, test end in 60 days.Result shows, IL-4 genetic vaccine significantly can reduce the incidence rate (see table 1) of tumor.
Table 1: mice IL-4 genetic vaccine strengthens the result of the test (often organize 10, data are survival rate %) of anti-tumor activity
Embodiment 4: the test of relapse rate after mice IL-4 genetic vaccine reduction tumor operation
By the method for embodiment 1, after the sulfur hydrogen reduction albumen normal saline expressed by the IL-4 fusion rotein of purification or vehicle Control is suitably diluted and aluminium glue adjuvant be mixed with in the ratio of 1:1 and test or control vaccine.Require that the amount of IL-4 fusion rotein or vehicle Control sulfur hydrogen reduction albumen in every part vaccine is about 15-20 μ g; The volume of every part vaccine is about 0.1ml.To experiment mice respectively at the IL-4 gene recombinaton aluminium glue vaccine when same day after tumor operation and the 14th day prepared by intraperitoneal inoculation embodiment 1, separately establish pET-32 carrier aluminium glue vaccine and normal saline immunized controls group.Checked a tumor growth situation every 3 days after tumor operation, test and terminate after 100 days.Result shows, IL-4 genetic vaccine significantly can reduce the relapse rate (see table 2) of tumor.Table 2: mice IL-4 genetic vaccine strengthens the result of the test (often organize 10, data are survival rate %) of anti-tumor activity
Embodiment 5: mice IL-4 genetic vaccine suppresses to breathe the anaphylactoid test of channel type
By the method for embodiment 1, be mixed with in the ratio of 1:1 with oily adjuvant after the sulfur hydrogen reduction albumen normal saline expressed by the IL-4 fusion rotein of purification or vehicle Control is suitably diluted and test or control vaccine.Require that the amount of IL-4 fusion rotein or vehicle Control sulfur hydrogen reduction albumen in every part vaccine is about 15-20 μ g; The volume of every part vaccine is about 0.1ml.Inoculate the IL-4 gene recombinaton oil-adjuvant vaccine prepared by embodiment 1 respectively at first 3 weeks of induced hypersensitivity reaction and 1 week subcutaneous (s.c.) to experiment BALB/c mouse, separately establish pET-32 carrier oil Adjuvanted vaccines and normal saline immunized controls group.Low dosage (5 μ g) chicken ovalbumin (OVA) aluminium glue vaccine intraperitoneal inoculation is used respectively in the 0th week and the 3rd week, with sensitized mice, instilled by nostril, to induce generation anaphylaxis in the 5th week with high dose (80 μ g are dissolved in 50 μ l PBS) OVA.Result shows: IL-4 gene recombinaton oil-adjuvant vaccine immune group is compared with normal saline immunized controls group with pET-32 carrier oil Adjuvanted vaccines group, IL-4 gene recombinaton oil-adjuvant vaccine significantly can reduce the quantity (see table 3) of the oxyphil cell in the generation (see accompanying drawing 1) of OVA-IgE specific antibody and pulmonary branches trachea folliculus flushing liquor, and significantly suppresses the hypertrophy of goblet cell and the degree of histiocyte inflammation.
Table 3: mice IL-4 genetic vaccine suppresses the secretion of oxyphil cell in pulmonary branches trachea folliculus
Embodiment 6: the test of mice IL-4 genetic vaccine treatment murine chronic leishmaniasis
By the method for embodiment 1, after the sulfur hydrogen reduction albumen normal saline expressed by the IL-4 fusion rotein of purification or vehicle Control is suitably diluted and aluminium glue adjuvant be mixed with in the ratio of 1:1 and test or control vaccine.Require that the amount of IL-4 fusion rotein or vehicle Control sulfur hydrogen reduction albumen in every part vaccine is about 15-20 μ g; The volume of every part vaccine is about 0.1ml.Give experiment BALB/c mouse respectively at before Li Shiman parasite inoculation 3 weeks and 1 week abdominal cavity (i.p.) inoculate IL-4 gene recombinaton aluminium glue vaccine prepared by embodiment 1, separately establish pET-32 carrier aluminium glue vaccine and normal saline immunized controls group.Appropriate Li Shiman parasite subcutaneous (s.c.) injection inoculation is in mouse hind leg foot pad.Weekly by the pathological changes size at kind of calliper once pad place enough after inoculation, continue 8 ~ 10 weeks.Result shows, IL-4 genetic vaccine significantly can reduce the parasitic infection of Li Shiman (see accompanying drawing 2).
Embodiment 7: mice IL-4 genetic vaccine strengthens the test lungy of mice preventing chronic
By the method for embodiment 1, after the sulfur hydrogen reduction albumen normal saline expressed by the IL-4 fusion rotein of purification or vehicle Control is suitably diluted and aluminium glue adjuvant be mixed with in the ratio of 1:1 and test or control vaccine.Require that the amount of IL-4 fusion rotein or vehicle Control sulfur hydrogen reduction albumen in every part vaccine is about 15-20 μ g; The volume of every part vaccine is about 0.1ml.To experiment mice respectively at first 3 weeks of bacillus calmette-guerin vaccine (BCG) inoculation and 1 week abdominal cavity (i.p.) inoculate IL-4 gene recombinaton aluminium glue vaccine prepared by embodiment 1, separately establish pET-32 carrier aluminium glue vaccine and normal saline immunized controls group.Appropriate bacillus calmette-guerin vaccine is to mice subcutaneous (s.c.) or vein (i.v.) injection inoculation.Within after inoculation 12 ~ 16 weeks, attack (i.v. inoculation) with high dose recombinant BCG, then, within 10 ~ 12 weeks, get mouse spleen, grinding, filter, centrifugal, get appropriate single-cell suspension liquid to be taped against in agar version, insert in 37 DEG C of incubators and cultivate about 3 weeks, count colony counts on each flat board.Result shows, IL-4 genetic vaccine significantly can reduce the infection (see table 4) of BCG.
Table 4: mice IL-4 genetic vaccine strengthens mice preventing chronic result of the test lungy (often organize 10, data are bacterium colony average on each flat board)
Claims (2)
1. the application of interleukin-4 therapeutic vaccine in the medicine of preparation treatment tumor, described interleukin-4 therapeutic vaccine is: 1. do with the intact proteins of the natural of interleukin-4 or synthetic or protein fragments any type of protein vaccine or coupling protein vaccine that antigen makes; Or: 2. express the intact proteins of IL-4 or protein fragments with any expression system and do any type of protein vaccine or amalgamation protein vaccine that antigen makes;
Described protein fragments is polypeptide or oligopeptide, and takes a step forward and other Antigenic macromolecules phase coupling making vaccine.
2. application according to claim 1, is characterized in that: be added with medically conventional vaccine adjuvant in described vaccine.
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| CN201510010040.9A CN104623688B (en) | 2011-06-27 | 2011-06-27 | It can be used for treating the interleukin-4 therapeutic vaccine of human or animal's tumor disease |
| CN201110174918.4A CN102266551B (en) | 2011-06-27 | 2011-06-27 | Interleukin-4 therapeutic vaccine for treating immune related diseases of humans or animals |
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| CN1692942A (en) * | 2004-09-17 | 2005-11-09 | 四川大学 | Prepn. and application tech. for pig interleukin-4 gene anti-diseases prepns. |
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| CN1692942A (en) * | 2004-09-17 | 2005-11-09 | 四川大学 | Prepn. and application tech. for pig interleukin-4 gene anti-diseases prepns. |
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