CN106632661A - Preparing broad-spectrum vaccine for preventing tumors and virus diseases from HLA-G molecule and antigen fragment of HLA-G molecule, and application of antigen fragment - Google Patents
Preparing broad-spectrum vaccine for preventing tumors and virus diseases from HLA-G molecule and antigen fragment of HLA-G molecule, and application of antigen fragment Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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Abstract
The invention creatively makes use of an HLA-G molecule and an antigen fragment of the HLA-G molecule to prepare a broad-spectrum vaccine for preventing various tumors and various viruses as well as invasion from unknown viruses to a human body or an animal body, thus enabling the human body or the animal body to get rid of harms caused by the tumors and the viruses.
Description
Technical field
The invention belongs to biomedicine technical field.It is prepared by the antigen fragment for particularly relating to HLA-G amino acid sequences
The application of broad-spectrum vaccine and method, the broad-spectrum vaccine can be used for the disease for preventing the tumour and virus of human body or animal body to cause.
Background technology
Malignant tumour is one of principal disease of threat human health and life, and its incidence of disease increases year by year and tends to year
Lightization.According to the annual report of the World Health Organization 1997, at that time the annual death toll of global tumor patient was up to 6,600,000, it is contemplated that arrive
The year two thousand twenty whole world tumor patient is up to 15,000,000.Updated statistics shows, over nearly 20 years, China is per in 4 to 5 dieds
Just there is one to die from cancer, cancer has become the killer of the mankind first.
Tumour is out of control, in popular anxiety, and before treatment tumour active drug is succeeded in developing, prevention of tumor becomes very big
Health demand.But the technology of the not yet effective prevention of tumor in the current whole world, research and develop the advanced method of prevention of tumor and skill
Art is and its urgent task.
New virus or unknown virus continuously emerges, and class is given people again and again and brings major disaster.Although the mankind are still
Do not develop the active drug for the treatment of virus infection, but how pre- preventing virus infection, particularly prevent unknown virus or all
The infection of virus is even more important.Although the mankind develop prevents some viral vaccines, prevention unknown virus or institute are developed
Virulent broad-spectrum vaccine is even more important, and so far non-people sets foot in.If the virulent epidemic disease of prevention unknown virus or institute can be developed
Seedling, will can resist the attack of unknown virus or all virus on human.The mankind expect the virulent epidemic disease of prevention unknown virus or institute
Seedling comes out early.
HLA-G is non-classical HLA I quasi-molecules.HLA-G was cloned and confirmed in 1987 by Geraghty etc..HLA-G
Molecule has the biological function for directly suppressing panimmunity competent cell, is one important immune tolerance molecule of body, in mother
Highly important effect is played in tire immunity, tumour immunity, trnasplantion immunity, autoimmunity and infection immunity.Research confirmation,
In tumour immunity, HLA-G can suppress NK cells, T cell and BMDC, make the immune system of body in suppression shape
State.Due to immunosuppressive actions of the HLA-G to body, tumour cell is set to escape immunosurveillance of the body to tumour cell,
Immunosurveillance escape is realized, tumour cell obtains the chance of free overpreading.
The immunohistochemical staining result of tumor biopsy shows, laryngocarcinoma, cancer of the esophagus, cancer of the stomach, colon and rectum carcinoma, lung cancer,
HLA-G can be expressed in the Several Kinds of Malignancy such as breast cancer, oophoroma, cancer of the uterus tissue.In addition, HBsAg, HCAg, HIV,
In the blood samples of virus carrier such as SARS, Ebola, stockaded village's card, the testing result of HLA-G is positive.Illustrate that HLA-G exists
Expression in the blood of tumor tissues and viral disease has generality so that HLA-G be expected to become all tumour cells and
The common target of virus, and be expected to be played a role in terms of prevention of tumor and viral disease as immunogene.But, adopt
The full molecules of HLA-G prepare broad-spectrum vaccine to be needed through the multiple tracks complicated procedures of forming such as gene recombination, expression, purifying, and early-stage work amount is big
And it is with high costs, it is unfavorable for the popularization production of vaccine and applies.
Immunogenic determinant position decides specificity of the antigenic substance in immune response, in each subject immunogenic molecule
Various or multiple determinants are there are, these determinants for being derived from the related HLA-G molecules of tumour or viral disease have
Prestige is applied among the active pharmaceutical ingredient of peptide vaccine using in the form of polypeptide fragment as immunogenic peptide.But, due to exempting from
Epidemic disease responsive cell has different degrees of compatibility, and the polypeptide fragment of immunogenic peptide to the determinant of different privileged sites
It is likely to cause immunogenicity to reduce with the reduction of structural complexity during vaccine is formed, the HLA- being currently known
G molecular antigens determinant can not provide accurately and effectively application message to prepare broad-spectrum vaccine.
The content of the invention
Present invention firstly provides a kind of antigen fragment, including following at least one amino acid sequences：
(1)YW EEE TRN TKA HA(SEQ ID NO:1)；
(2)RGY YNQ SEA SSH TL(SEQ ID NO:2)；
(3)PPK THV THH PVFD(SEQ ID NO:3)；
(4)PLM LRW KQS SL(SEQ ID NO:4)。
Wherein, in the antigen fragment and HLA-G amino acid sequences any one section include at least one above-mentioned amino acid sequence
Row (SEQ ID NO:Polypeptide fragment or the sequence of the homologue of the polypeptide fragment, analog or derivative 1-4) is identical.Should
The sequence of antigen fragment can be artificial synthesized；It may also be preferable that by HLA-G amino acid sequences by various modes
Shorten shearing and obtain, the HLA-G amino acid sequences can be native sequences Jing are manually separated or by genetic recombination and
The mode such as artificial synthesized of expression and obtain.
Further, the HLA-G amino acid sequences include HLA-G molecule Isomers amino acid sequences and the HLA-G
The examples of conservative variations of molecule Isomers amino acid sequence, bioactive fragment or derivative.At present it is known that HLA-G it is natural
Molecule has 7 kinds of isomers, and this has 4 kinds of different amino acid sequences (SEQ ID NO in 7 in isomers:5-8), this 4 kinds not
Same amino acid sequence (SEQ ID NO:5-8) can select as one kind of the HLA-G amino acid sequences is preferred.
Further, the HLA-G amino acid sequences and amino acid sequence SEQ ID NO:One of 5-8 is with least 15%
Homology, preferably at least with 35% homology, also preferably at least have 60%, 70%, 80% or 90% it is homologous
Property, more preferably at least with 95%, 96%, 97%, 98%, 99% homology.
Further, the antigen fragment is preferably sequence SEQ ID NO:One of 1-4.
Further, the antigen fragment is preferably sequence SEQ ID NO:One of 9-12.
The present invention is also claimed application of the above-mentioned antigen fragment in the broad-spectrum vaccine of prevention of tumor and virosis is prepared.
Wherein, the tumour and virosis include that all tumours and viral disease of HLA-G can be expressed, including unknown
Tumour and virus.These tumours include：(1) squamous cell carcinoma from epithelial tissue, basal-cell carcinoma, gland cancer, mamillary
Gland cancer, cystadenocarcinoma, malignant pleomorphic adenoma and Transitional epithelial cancer etc.；(2) fibrosarcoma from mesenchymal tissue, malignant fibrous
Histocyte cancer, embryonal-cell lipoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, lymphangioendothelial sarcoma, osteosarcoma, cartilage meat
Knurl, synovial sarcoma and malignant mesothelioma etc.；(3) from the lymthoma and leukaemia etc. of lymphohematopoietic tissue delivered；(4) from god
Neurofibrosarcoma, malignant schwannoma, glioblastoma cytoma, medulloblastoma, malignant meningioma and nerve that Jing is organized
Blastoma etc.；(5) from gastral esophageal squamous cell carcinoma cytoma, esophageal adenocarcinoma cells knurl, cancer of the stomach adenoma, stomach squamous carcinoma, liver
Cytoma, the carcinoma of the rectum, colon cancer, cancer of pancreas；(6) from the non-small cell lung cancer of respiratory tract；(7) from its hetero-organization
Melanoma, chorioepithelioma, ovarian cancer cell knurl, laryngocarcinoma, breast cancer, seminoma, prostate cancer, cervical carcinoma, nothing
Property cytoma, embryonal carcinoma and malignant teratoma etc..These viruses include：(1) Poxviridae, iris disease in double-stranded DNA virus
Malicious section, Rhabdoviridae, herpetoviridae, Adenoviridae, Papillomaviridae, polyomavirus section, polydnavirus section, vesica
Viraceae, Fei Zhou CSFVs section etc.；(2) Geminiviridae, Circoviridae, Parvoviridae in single-stranded DNA viruses etc.；
(3) Hepadnaviridae, Retroviridae, Pseudoviridae, Metaviridae in DNA and RNA retrovirus etc.；(4)
Reoviridae, birnavirus section in diplornavirus, the spherical mycoviruses section of bi-component double-stranded RNA, attenuated virus
Section etc.；(5) Narnaviridae in 20S RNA；(6) Paramyxoviridae, the rhabdovirus in single strand RNA virus is born
Section, filamentous virus section, Bornaviridae, orthomyxoviridae family, bunyaviridae, salad Viraceae etc.；(7) positive single stranded RNA
Levirividae, Arteriviridae in virus, coronaviridae, Picornaviridae.
Preferably, these tumours include：Laryngocarcinoma, the cancer of the esophagus, cancer of the stomach, liver cancer, cancer of pancreas, colon cancer, prostate cancer, lung
Cancer, breast cancer, oophoroma, cervical carcinoma；These viruses include：HBsAg, HCAg, HIV, SARS, Ebola, stockaded village's card, Dengue pyreticosis
Poison, avian influenza virus.
Further, the vaccine is produced in vivo the HLA-G that can be combined with HLA-G by the antigen fragment and is resisted
Body and play a role.
Present invention also offers the method for preparing the broad-spectrum vaccine of prevention of tumor and virosis, including using above-mentioned antigen slide
The immunogenic steps of Duan Zuowei.
Further, methods described also includes for the antigen fragment and carrier protein being combined into compound, then will be compound
The step of thing is purified.
Further, the antigen fragment can be obtained with the compound of carrier protein by being combined using coupling reagent
, such as adopt carbodiimides, glutaraldehyde and two isocyanide acid compounds, it is also possible to by gene fusion construct Jing organism surfaces
Up to and obtain.
Wherein, the carrier protein is preferably hemocyanin, and the coupling reagent is preferably glutaraldehyde.
Antigen fragment (the amino acid sequence SEQ ID NO that the present invention is provided:1-4 and including at least SEQ ID NO:1-4 its
One of other antigen fragments), the structural conservation with height and higher immunocompetence can pass through organism immune
Reaction produces HLA-G antibody (HLA-G specific antibodies), and the HLA-G antibody can prevent HLA-G pair by being combined with HLA-G
The immunosuppressive action of body, cut-out tumour and virus immunity are escaped, and make tumour cell and virus easily clear by human immune system
Except and eliminate.The antigen fragment that the present invention is provided only containing the amino acid residue of as little as 10 or so, is keeping its immunocompetence
On the premise of significantly reduce synthesis cost and vaccine prepare early-stage work amount, and due to the well-conserved of structure so that
It has the combination effect for being no less than, being even better than general immunologic determinants sequence, from the point of view of experimental data, from ammonia of the present invention
Base acid sequence SEQ ID NO:The vaccine that one of 1-4 is prepared as vaccine raw material, the titre of gained antibody Jing after immune animal
Up to 1 to 10 more than ten thousand (ELISA method).
Following term used in description of the invention and claims, typically contains unless otherwise indicated with following
Justice, and following implications are considered as within the ken of those skilled in the art：
" HLA-G amino acid sequences " is interpreted broadly, and is not only interpreted as including the amino of HLA-G natural molecule Isomers
Acid sequence is (such as SEQ ID NO:5-8), be further appreciated that be include HLA-G molecule Isomers amino acid sequences examples of conservative variations,
Bioactive fragment or derivative, the examples of conservative variations of the HLA-G molecule Isomers amino acid sequences, bioactive fragment or
Include sequence SEQ ID NO in derivative:At least one of 1-4；These broadly understood HLA-G amino acid sequences itself or
Its any one section includes sequence SEQ ID NO:The fragment of at least one of 1-4 can become coming for antigen fragment of the present invention
Source, or, these HLA-G amino acid sequences that broadly understood itself or its any one section are comprising sequence SEQ ID NO:In 1-4
At least one fragment is respectively provided with and antigen fragment identical amino acid sequence of the present invention.
" amino acid sequence " only relates to the primary structure (prlmary structure of protein) of amino acid residue arrangement, does not expand understanding
It is comprising amino acid sequence steric two grades and tertiary structure.
" polypeptide " refers to the peptide chain with amino acid sequence, including polypeptide (20 amino acid residues of oligopeptides, peptide, narrow sense
Peptide above) or protein sequence and its fragment or part, can be glycosylation or nonglycosylated, one in amino acid sequence
Or more amino acid can be modified or not be modified.When its amino acid sequence is related to a kind of naturally occurring protein point
Son amino acid sequence when, this " polypeptide " or " protein " do not mean that amino acid sequence is limited to it is naturally occurring with this
The related complete or on all four natural amino acid of protein molecule or its sequence.
It is " homologous " to include complete homologous and homeologous, there is relatively broad implication in the present invention, it is anti-in description
When former fragment, polypeptide or amino acid sequence, refer to same or analogous structure or function, or with similar amino acid sequence
Row, including the variant of sequence, analog and derivative；When percentage is related to, have in the arrangement for referring to sequence amino acid residue
The homogeny of certain percentage.
" analog " refers to the polypeptide for being kept substantially antigen fragment activity of the present invention, as long as the analog can pass through SEQ
ID NO:At least one of 1-4 produces the HLA-G antibody that can be combined with HLA-G.The polypeptide " analog " of the present invention can be wrapped
Include：(I) in the sequence continuous or compartment of terrain is inserted, lacks, replacing one or more amino acid residues, and one or more of
Insertion, disappearance, the replacement of amino acid residue can simultaneously or asynchronously exist in same sequence；(II) in sequence one or more
The group of amino acid residue is replaced or lacked by other groups；(III) one or more amino acid residues are modified in sequence.
" derivative " refers to by former polypeptide, protein or amino acid sequence when polypeptide, protein or amino acid sequence is described
Association polypeptide, protein or the amino acid sequence being derived.The polypeptide of the present invention, antigen fragment or amino acid sequence include this
The derivative of sample：SEQ ID NO can be passed through:At least one of 1-4 produces the HLA-G antibody that can be combined with HLA-G.This
The not limiting example of bright " derivative " can include：(IV) mature polypeptide and another kind of compound fusion, or (V) is in amino
Merge or insert additional amino acid sequence (linker, protein purification mark sequence, restriction enzyme site etc.) in acid sequence.
" conservative " refers to that involved amino acid sequence has higher similitude or homogeneity with original series, is able to maintain that
The basic structure of its original series, BA or function, can typically pass through similar amino acid residue replacement etc. and obtain,
For example, replacement between lysine and arginine, leucine and isoleucine etc..
" variant " refers to the amino acid sequence with one or more amino acid changes, and the change may include amino acid sequence
Or the insertion of amino acid or nucleotides, disappearance or replacement in nucleotide sequence.Variant can be sexually revised with conservative, wherein the ammonia replaced
Base acid has similar structure or chemical property to original acid, and the such as replacement between leucine and isoleucine also can have
Non-conservation changes.
" bioactive fragment " refers to that the fragment can substantially maintain its original activity and function, piece in biomolecule
Section can be identical with original segments, it is also possible to changes on the basis of original segments, the position of change generally exists
The nonfunctional area of original segments or the conservative of non-determinant region (such as linker areas) sexually revise (examples of conservative variations), but are not excluded for
There is the non-primary fragment that non-conservation changes, the non-primary fragment can be used as by SEQ ID NO:At least one of 1-4
The source of the antigen fragment of the present invention of HLA-G antibody is produced, or, the non-primary fragment itself or its any one section include sequence
SEQ ID NO:The fragment of at least one of 1-4 is respectively provided with and antigen fragment identical amino acid sequence of the present invention.
The antigen fragment or sequence SEQ ID NO of " activity " in the description present invention:1-4 for the moment, refer to the antigen fragment or
Sequence SEQ ID NO:One of 1-4 can be produced the HLA-G that can be combined with HLA-G by the immune response of human body or animal and be resisted
Body.
Specific embodiment
In addition to being defined, technical term used has and the invention one of ordinary skill in the art in following examples
The identical meanings being commonly understood by.Test reagent used in following examples, if no special instructions, is routine biochemistry reagent；
The experimental technique, if no special instructions, is conventional method.
The invention is described in detail with reference to embodiment.In following embodiments, the immunogene for preparing vaccine is equal
It is commercial or synthesized by commercial synthetic method and obtained.
Embodiment 1
Broad-spectrum vaccine is prepared with HLA-G 1, HLA-G 5, HLA-G 7 respectively as immunogene.HLA-G 1、HLA-G5、
HLA-G 7 is three isomers of HLA-G molecules, with identical amino acid sequence (SEQ ID NO:5), it prepares vaccine
Process is identical, and by taking HLA-G 1 as an example, the preparation process of vaccine is as follows.
Take 33mg HLA-G 1 and be dissolved in 50ml pH7.4, in 0.01M/L PBS solutions.The concentration of HLA-G 1 is 660mg/
L.The solution is placed on magnetic stirrer, a stirrer is placed in the solution, opened magnetic stirrer and make stirrer slowly
The stirring solution of HLA-G 1.Then KAL (the SO that 3ml concentration is 1mMol/L are slowly added into4)2-12H2O solution, is being continuously agitated
Lower dropwise addition 1ml concentration is the NaOH solution of 1mMol/L, continues to stir 10 minutes, stops stirring, is adjusted with the HCL of 0.1Mol/L
The pH value of solution is sterile filtered rearmounted 4 DEG C to 7.4, Jing and preserves.
Embodiment 2
Broad-spectrum vaccine is prepared with HLA-G 2, HLA-G 6 respectively as immunogene.HLA-G 2, HLA-G 6 are HLA-G point
Two isomers of son, with identical amino acid sequence (SEQ ID NO:6), its prepare vaccine process it is identical, with HLA-G
As a example by 2, the preparation process of vaccine is as follows.
Take 24mg HLA-G 2 and be dissolved in 50ml pH7.4, in 0.01M/L PBS solutions.The concentration of HLA-G 2 is 480mg/
L.The solution is placed on magnetic stirrer, a stirrer is placed in the solution, opened magnetic stirrer and make stirrer slowly
The stirring solution of HLA-G 2.Then KAL (the SO that 2.1ml concentration is 1mMol/L are slowly added into4)2-12H2O solution, is ceaselessly stirring
The NaOH solution that lower dropwise addition 0.72ml concentration is 1mMol/L is mixed, continues to stir 10 minutes, stop stirring, with the HCL of 0.1Mol/L
The pH value of adjustment solution is sterile filtered rearmounted 4 DEG C to 7.4, Jing and saves backup.
Embodiment 3
Using (the SEQ ID NO of HLA-G 3:7) broad-spectrum vaccine is prepared as immunogene：Take 14mg HLA-G 3 and be dissolved in 50ml
In pH7.4,0.01M/L PBS solution.The concentration of HLA-G 3 is 279mg/L.The solution is placed on magnetic stirrer, molten
A stirrer is placed in liquid, magnetic stirrer is opened and is made stirrer slowly stir the solution of HLA-G 3.Then it is slowly added into
1.3ml concentration is the KAL (SO of 1mMol/L4)2-12H2O solution, is being continuously agitated lower dropwise addition 0.42ml concentration for 1mMol/L
NaOH solution, continue to stir 10 minutes, stop stirring, the pH value for adjusting solution with the HCL of 0.1Mol/L is aseptic to 7.4, Jing
Filter rearmounted 4 DEG C to save backup.
Embodiment 4
Using (the SEQ ID NO of HLA-G 4:8) broad-spectrum vaccine is prepared as immunogene：Take 23mg HLA-G 4 to be dissolved in
In 100ml pH7.4,0.01M/L PBS solution.The concentration of HLA-G 4 is 459mg/L.The solution is placed in into magnetic stirrer
On, a stirrer is placed in the solution, open magnetic stirrer and make stirrer slowly stir the solution of HLA-G 4.Then slowly
2.1ml concentration is added for the KAL (SO of 1mMol/L4)2-12H2O solution, be lower dropwise addition 0.7ml concentration is continuously agitated
The NaOH solution of 1mMol/L, continues to stir 10 minutes, stops stirring, and the pH value of solution is adjusted to 7.4 with the HCL of 0.1Mol/L,
Jing is sterile filtered rearmounted 4 DEG C and saves backup.
Embodiment 5
If preparing broad-spectrum vaccine as combined immunization original using the isomers of two or more HLA-G, its operation
On the basis of embodiment 1 33mg HLA-G 1 and 3ml concentration for 1mMol/L KAL (SO4)2-12H2O solution and 1ml are dense
33mg, 3ml, the 1ml spent in the NaOH solution for 1mMol/L is multiplied by n, and n is represented using the species number of HLA-G isomers.
Such as two kinds isomers are combined as immunogene, and n=2, three kinds of isomers are former as combined immunization, n=3.By that analogy.
Embodiment 6
Using SEQ ID NO:1 polypeptide (small peptide) prepares broad-spectrum vaccine as immunogene.The dissolving of 33mg hemocyanin
In 50ml pH7.4, the PBS solution of 0.01mol, under electromagnetic agitation, then 3.3mg immunogene small peptides are dissolved in, continue to stir 10
Minute, 5% glutaraldehyde water solution is taken, 1000 times are diluted with above-mentioned PBS solution, the glutaraldehyde solution 0.15ml after dilution is taken,
It is added dropwise under the stirring of magnetic stirrer in above-mentioned solution, is further continued for stirring 30 minutes, places 4 DEG C of refrigerator overnights.
Solution prepared by the day before yesterday is crossed sephadex-G25 chromatographic columns by next day, collects 280 nanometers of absworption peaks, is removed and is not participated in
With reference to glutaraldehyde and immunogene small peptide.Then after anti-HLA-G affinity columns, uncombined immunogene small peptide is removed
Hemocyanin, with pH3.5, what the glycine-HCL buffer solutions desorption hemocyanin of 0.05Mol was combined with immunogene small peptide is combined
Thing, and with 0.05Mol NaOH neutralize the stripping liquid at once, Jing after appropriate concentration again through embodiment 1 method process obtain by
The broad-spectrum vaccine that immunogene small peptide is prepared into.
Embodiment 7
Using SEQ ID NO:2 polypeptide (small peptide) prepares broad-spectrum vaccine as immunogene.The dissolving of 33mg hemocyanin
In 50ml pH7.4, the PBS solution of 0.01mol, under electromagnetic agitation, then 3.3mg immunogene small peptides are dissolved in, continue to stir 10
Minute, 5% glutaraldehyde water solution is taken, 1000 times are diluted with above-mentioned PBS solution, the glutaraldehyde solution 0.15ml after dilution is taken,
It is added dropwise under the stirring of magnetic stirrer in above-mentioned solution, is further continued for stirring 30 minutes, places 4 DEG C of refrigerator overnights.
Solution prepared by the day before yesterday is crossed sephadex-G25 chromatographic columns by next day, collects 280 nanometers of absworption peaks, is removed and is not participated in
With reference to glutaraldehyde and immunogene small peptide.Then after anti-HLA-G affinity columns, uncombined immunogene small peptide is removed
Hemocyanin, with pH3.5, what the glycine-HCL buffer solutions desorption hemocyanin of 0.05Mol was combined with immunogene small peptide is combined
Thing, and with 0.05Mol NaOH neutralize the stripping liquid at once, Jing after appropriate concentration again through embodiment 1 method process obtain by
The broad-spectrum vaccine that immunogene small peptide is prepared into.
Embodiment 8
Using SEQ ID NO:3 polypeptide (small peptide) prepares broad-spectrum vaccine as immunogene.Due to SEQ ID NO:3 with
SEQ ID NO:1 amino acid number is identical, is 13 amino acid, and the two mass difference is very few, therefore concrete operations are same
Embodiment 6.
Embodiment 9
Using SEQ ID NO:4 polypeptide (small peptide) prepares broad-spectrum vaccine as immunogene.The dissolving of 33mg hemocyanin
In 50ml pH7.4, the PBS solution of 0.01mol, under electromagnetic agitation, then 3.3mg immunogene small peptides are dissolved in, continue to stir 10
Minute, 5% glutaraldehyde water solution is taken, 1000 times are diluted with above-mentioned PBS solution, the glutaraldehyde solution 0.15ml after dilution is taken,
It is added dropwise under the stirring of magnetic stirrer in above-mentioned solution, is further continued for stirring 30 minutes, places 4 DEG C of refrigerator overnights.
Solution prepared by the day before yesterday is crossed sephadex-G25 chromatographic columns by next day, collects 280 nanometers of absworption peaks, is removed and is not participated in
With reference to glutaraldehyde and immunogene small peptide.Then after anti-HLA-G affinity columns, uncombined immunogene small peptide is removed
Hemocyanin, with pH3.5, what the glycine-HCL buffer solutions desorption hemocyanin of 0.05Mol was combined with immunogene small peptide is combined
Thing, and with 0.05Mol NaOH neutralize the stripping liquid at once, Jing after appropriate concentration again through embodiment 1 method process obtain by
The broad-spectrum vaccine that immunogene small peptide is prepared into.
Embodiment 10
Using SEQ ID NO:9 polypeptide prepares broad-spectrum vaccine as immunogene.Take 3.3mg immunogene polypeptides and be dissolved in 50ml
In pH7.4,0.01M/L PBS solution.The concentration of immunogene polypeptide is 66mg/L.The solution is placed on magnetic stirrer,
A stirrer is placed in solution, magnetic stirrer is opened and is made stirrer slowly stir the solution of immunogene polypeptide.Then slowly
0.3ml concentration is added for the KAL (SO of 1mMol/L4)2-12H2O solution, be lower dropwise addition 0.1ml concentration is continuously agitated
The NaOH solution of 1mMol/L, continues to stir 10 minutes, stops stirring, and the pH value of solution is adjusted to 7.4 with the HCL of 0.1Mol/L,
Jing is sterile filtered rearmounted 4 DEG C and preserves.
Embodiment 11
Using SEQ ID NO:10 polypeptide prepares broad-spectrum vaccine as immunogene.Take 5.3mg immunogene polypeptides to be dissolved in
In 50ml pH7.4,0.01M/L PBS solution.The concentration of immunogene polypeptide is 106mg/L.The solution is placed in into magnetic stirrer
On, a stirrer is placed in the solution, open magnetic stirrer and make stirrer slowly stir immunogene polypeptide solution.Then delay
Slow KAL (the SO for adding 0.48ml concentration for 1mMol/L4)2-12H2O solution, be lower dropwise addition 0.16ml concentration is continuously agitated
The NaOH solution of 1mMol/L, continues to stir 10 minutes, stops stirring, and the pH value of solution is adjusted to 7.4 with the HCL of 0.1Mol/L,
Jing is sterile filtered rearmounted 4 DEG C and preserves.
Embodiment 12
Using SEQ ID NO:11 polypeptide prepares broad-spectrum vaccine as immunogene.Take 5.9mg immunogene polypeptides to be dissolved in
In 50ml pH7.4,0.01M/L PBS solution.The concentration of immunogene polypeptide is 119mg/L.The solution is placed in into magnetic stirrer
On, a stirrer is placed in the solution, open magnetic stirrer and make stirrer slowly stir immunogene polypeptide solution.Then delay
Slow KAL (the SO for adding 0.54ml concentration for 1mMol/L4)2-12H2O solution, be lower dropwise addition 0.18ml concentration is continuously agitated
The NaOH solution of 1mMol/L, continues to stir 10 minutes, stops stirring, and the pH value of solution is adjusted to 7.4 with the HCL of 0.1Mol/L,
Jing is sterile filtered rearmounted 4 DEG C and preserves.
Embodiment 13
Using SEQ ID NO:12 polypeptide prepares broad-spectrum vaccine as immunogene.Take 11.6mg immunogene polypeptides to be dissolved in
In 50ml pH7.4,0.01M/L PBS solution.The concentration of immunogene polypeptide is 231mg/L.The solution is placed in into magnetic stirrer
On, a stirrer is placed in the solution, open magnetic stirrer and make stirrer slowly stir immunogene polypeptide solution.Then delay
Slow KAL (the SO for adding 1.1ml concentration for 1mMol/L4)2-12H2O solution, be lower dropwise addition 0.35ml concentration is continuously agitated
The NaOH solution of 1mMol/L, continues to stir 10 minutes, stops stirring, and the pH value of solution is adjusted to 7.4 with the HCL of 0.1Mol/L,
Jing is sterile filtered rearmounted 4 DEG C and preserves.
The sensitization rabbit prevention of tumor of embodiment 14 is tested
Experimental group takes healthy adult rabbit 9, point a, b, c3 group, and 3 per group, control group takes normal adult rabbit 9,
It is divided into a, b, c3 group, 3 per group.Experimental group broad-spectrum vaccine immunity.The vaccine immunity that a groups are obtained with embodiment 1, b groups are real
Apply the vaccine immunity of the acquisition of example 6, the vaccine immunity that c groups are obtained with embodiment 10,20 micrograms of immunizing dose/kg, the immunity 1 per 10 days
Secondary hypodermic injection, altogether immunity 3 times check that antibody titer reaches 1:More than 10000 (ELISA methods) stop immunity.Control group is used
The immunity of pH7.4,0.01mol/L PBS control.1 milliliter every time, co-injection 3 times.
Inoculated tumour test cell line：It is inoculated with the ovarian cancer cell of equal number to experimental group and the subcutaneous multiple spot of control group respectively
Strain A2780,2000/ml, 3 points of injection 0.5ml.Through observation in 1-2 month, it is found that experimental group does not grow tumour, protective rate
100%, and control group all grown tumour.Experiment proves effective to pre- anti-cancer using HLA-G broad-spectrum vaccines.
Embodiment 15
Prevention experiment is carried out to other tumours using the identical method of same embodiment 14.Experimentation and reagent dosage can
Conventional adjustment is carried out with the needs set up according to different tumor models, this is easily to realize for those skilled in the art.
The various vaccines obtained in embodiment of the present invention 1-13 both participate in the prevention experiment no less than 5 kinds of tumor models, and experimental result is equal
After showing the vaccine immunity using the present invention, experimental group rabbit does not find tumour growth, and control group rabbit produces tumour.
These tumours include：(1) squamous cell carcinoma from epithelial tissue, basal-cell carcinoma, gland cancer, mamillary gland
Cancer, cystadenocarcinoma, malignant pleomorphic adenoma and Transitional epithelial cancer etc.；(2) fibrosarcoma from mesenchymal tissue, malignant fibrous group
Knit cell cancer, embryonal-cell lipoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, lymphangioendothelial sarcoma, osteosarcoma, chondrosarcoma,
Synovial sarcoma and malignant mesothelioma etc.；(3) from the lymthoma and leukaemia etc. of lymphohematopoietic tissue delivered；(4) from nerve
The neurofibrosarcoma of tissue, malignant schwannoma, glioblastoma cytoma, medulloblastoma, malignant meningioma and nerve are female
Cytoma etc.；(5) it is thin from gastral esophageal squamous cell carcinoma cytoma, esophageal adenocarcinoma cells knurl, cancer of the stomach adenoma, stomach squamous carcinoma, liver
Born of the same parents' knurl, the carcinoma of the rectum, colon cancer, cancer of pancreas；(6) from the non-small cell lung cancer of respiratory tract；(7) from the black of its hetero-organization
It is melanoma, chorioepithelioma, ovarian cancer cell knurl, laryngocarcinoma, breast cancer, seminoma, prostate cancer, cervical carcinoma, asexual
Cytoma, embryonal carcinoma and malignant teratoma etc..
The sensitization rabbit of embodiment 16 prevents viral challenge
Experimental group takes healthy adult rabbit 9, point a, b, c3 group, and 3 per group, control group takes normal adult rabbit 9.It is real
Test group with broad-spectrum vaccine immunity, wherein, the vaccine immunity that a groups are obtained with embodiment 2, the vaccine of the acquisition of b groups embodiment 7 is exempted from
Epidemic disease, the vaccine immunity that c groups are obtained with embodiment 11 checks that antibody titer reaches 1:More than 50000 (ELISA methods) stop immunity.It is right
PH7.4, the immunity of 0.01mol/L PBS controls are used according to group.
Experimental group and control group are inoculated with respectively the avian influenza virus (0.05-1.0 micrograms/kg/ time) of same dose by drop
Eye or collunarium are infected, and once a day, coinfection 2 times is observed 5-20 days.Statistical experiment group and control group morbidity rabbit are only
Number.Experimental result, 9 rabbit of experimental group all fail ill protective rate 100%, and 9 rabbit of control group are all ill.Experiment knot
Fruit confirms that this vaccine has the function of prevention avian flu virus infection.
Embodiment 17
Prevention experiment is carried out to other viruses using the identical method of same embodiment 16.Experimentation and reagent dosage can
Conventional adjustment is carried out with the needs set up according to different virus infection model, this is easily to realize for those skilled in the art
's.The various vaccines obtained in embodiment of the present invention 1-13 both participate in the prevention experiment no less than 5 kinds of viral infection models, experiment
As a result after showing the vaccine immunity using the present invention, experimental group rabbit does not find infection virus, and control group rabbit is felt
Catch an illness viral disease.
These viruses include：(1) Poxviridae, Iridoviridae, Rhabdoviridae, herpesviral in double-stranded DNA virus
Section, Adenoviridae, Papillomaviridae, polyomavirus section, polydnavirus section, Ascoviridae, Fei Zhou CSFVs section etc.；
(2) Geminiviridae, Circoviridae, Parvoviridae in single-stranded DNA viruses etc.；(3) in DNA and RNA retrovirus
Hepadnaviridae, Retroviridae, Pseudoviridae, Metaviridae etc.；(4) reovirus in diplornavirus
Section, birnavirus section, the spherical mycoviruses section of bi-component double-stranded RNA, Hypoviridae etc.；(5) it is naked in 20S RNA
Dew RNA virus section；(6) Paramyxoviridae, Rhabdoviridae, filamentous virus section, the borna virus in single strand RNA virus is born
Section, orthomyxoviridae family, bunyaviridae, salad Viraceae etc.；(7) Levirividae, the artery in positive single strand RNA virus
Scorching Viraceae, coronaviridae, Picornaviridae.
The preferred embodiment of the invention is the foregoing is only, it is all at this not to limit the invention
Within the spirit and principle of innovation and creation, any modification, equivalent substitution and improvements made etc. should be included in the invention
Protection domain within.
Claims (10)
1. a kind of antigen fragment, including with SEQ ID NO:The amino acid sequence of at least one of 1-4.
2. antigen fragment according to claim 1, it is characterised in that in the antigen fragment and HLA-G amino acid sequences
Any one section includes SEQ ID NO:The homologue of the polypeptide fragment of at least one of 1-4 or the polypeptide fragment, analog spread out
Biological sequence is identical.
3. antigen fragment according to claim 2, it is characterised in that the HLA-G amino acid sequences include HLA-G molecules
Examples of conservative variations, the bioactive fragment of Isomers amino acid sequence and the HLA-G molecule Isomers amino acid sequences
Or derivative.
4. antigen fragment according to claim 2, it is characterised in that the HLA-G amino acid sequences and amino acid sequence
SEQ ID NO:One of 5-8 with least 15% homology, preferably at least with 35% homology, also preferably at least
With 60%, 70%, 80% or 90% homology, more preferably at least with 95%, 96%, 97%, 98%, 99% it is same
Source property.
5. antigen fragment according to claim 1, it is characterised in that the antigen fragment is sequence SEQ ID NO:1-4
One of.
6. antigen fragment according to claim 1, it is characterised in that the antigen fragment is sequence SEQ ID NO:9-12
One of.
7. the antigen fragment described in any one of claim 1-6 in the broad-spectrum vaccine for preparing prevention of tumor and virosis should
With.
8. according to it is inner require 7 described in application, it is characterised in that the tumour and virosis are the tumour that can express HLA-G
And viral disease.
These tumours include：(1) squamous cell carcinoma from epithelial tissue, basal-cell carcinoma, gland cancer, papillary adenocarcinoma, capsule
Gland cancer, malignant pleomorphic adenoma and Transitional epithelial cancer etc.；(2) fibrosarcoma from mesenchymal tissue, malignant fibrous histiocytoma are thin
Born of the same parents' cancer, embryonal-cell lipoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, lymphangioendothelial sarcoma, osteosarcoma, chondrosarcoma, synovial membrane
Sarcoma and malignant mesothelioma etc.；(3) from the lymthoma and leukaemia etc. of lymphohematopoietic tissue delivered；(4) from nerve fiber
Neurofibrosarcoma, malignant schwannoma, glioblastoma cytoma, medulloblastoma, malignant meningioma and neuroblast
Knurl etc.；(5) from gastral esophageal squamous cell carcinoma cytoma, esophageal adenocarcinoma cells knurl, cancer of the stomach adenoma, stomach squamous carcinoma, hepatoma,
The carcinoma of the rectum, colon cancer, cancer of pancreas；(6) from the non-small cell lung cancer of respiratory tract；(7) from the melanin of its hetero-organization
Knurl, chorioepithelioma, ovarian cancer cell knurl, laryngocarcinoma, breast cancer, seminoma, prostate cancer, cervical carcinoma, asexual cell
Knurl, embryonal carcinoma and malignant teratoma etc..
These viruses include：(1) Poxviridae, Iridoviridae, Rhabdoviridae, herpetoviridae, gland in double-stranded DNA virus
Viraceae, Papillomaviridae, polyomavirus section, polydnavirus section, Ascoviridae, Fei Zhou CSFVs section etc.；(2) it is single
Geminiviridae, Circoviridae, Parvoviridae in chain DNA virus etc.；(3) the thermophilic liver in DNA and RNA retrovirus
DNA virus section, Retroviridae, Pseudoviridae, Metaviridae etc.；Reoviridae (4) in diplornavirus,
Birnavirus section, the spherical mycoviruses section of bi-component double-stranded RNA, Hypoviridae etc.；(5) it is exposed in 20S RNA
RNA virus section；(6) bear single strand RNA virus in Paramyxoviridae, Rhabdoviridae, filamentous virus section, Bornaviridae,
Orthomyxoviridae family, bunyaviridae, salad Viraceae etc.；(7) Levirividae, the arteritis in positive single strand RNA virus
Viraceae, coronaviridae, Picornaviridae.
Preferably, these tumours include：Laryngocarcinoma, the cancer of the esophagus, cancer of the stomach, liver cancer, cancer of pancreas, colon cancer, prostate cancer, lung cancer, breast
Gland cancer, oophoroma, cervical carcinoma；These viruses include：HBsAg, HCAg, HIV, SARS, Ebola, stockaded village's card, dengue fever virus, fowl
Influenza virus.
Preferably, the vaccine HLA-G antibody that can be combined with HLA-G is produced in vivo by the antigen fragment and
Play a role.
9. a kind of method of the broad-spectrum vaccine for preparing prevention of tumor and virosis, including using described in any one of claim 1-6
Antigen fragment as immunogenic step.
10. method according to claim 9, it is characterised in that methods described is also included the antigen fragment and carrier
Protein combination into compound, the step of then compound is purified.
Preferably, the antigen fragment can be obtained with the compound of carrier protein by being combined using coupling reagent,
Such as adopt carbodiimides, glutaraldehyde and two isocyanide acid compounds, it is also possible to by gene fusion construct Jing organism expressings
And obtain.
It is furthermore preferred that the carrier protein is preferably hemocyanin, the coupling reagent is preferably glutaraldehyde.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112351795A (en) * | 2018-02-23 | 2021-02-09 | 安贝科思生物制剂公司 | Combination anticancer therapy using anticancer agents and antibodies targeting complexes containing atypical HLA-I and neoantigens |
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| CN103415534A (en) * | 2009-03-10 | 2013-11-27 | 贝勒研究院 | Antigen presenting cell targeted cancer vaccines |
| CN104628858A (en) * | 2008-09-16 | 2015-05-20 | 加尼梅德药物公司 | Monoclonal antibodies for treatment of cancer |
| WO2016160622A3 (en) * | 2015-03-27 | 2016-12-15 | University Of Southern California | Hla-g as a novel target for car t-cell immunotherapy |
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2017
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|---|---|---|---|---|
| CN104628858A (en) * | 2008-09-16 | 2015-05-20 | 加尼梅德药物公司 | Monoclonal antibodies for treatment of cancer |
| CN103415534A (en) * | 2009-03-10 | 2013-11-27 | 贝勒研究院 | Antigen presenting cell targeted cancer vaccines |
| WO2016160622A3 (en) * | 2015-03-27 | 2016-12-15 | University Of Southern California | Hla-g as a novel target for car t-cell immunotherapy |
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| CN112351795A (en) * | 2018-02-23 | 2021-02-09 | 安贝科思生物制剂公司 | Combination anticancer therapy using anticancer agents and antibodies targeting complexes containing atypical HLA-I and neoantigens |
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