CN105906699A - Lung cancer antigen protein, coding gene thereof and preparation method of therapeutic lung cancer vaccine - Google Patents
Lung cancer antigen protein, coding gene thereof and preparation method of therapeutic lung cancer vaccine Download PDFInfo
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Abstract
The invention provides a lung cancer antigen protein, a coding gene thereof and a preparation method of a therapeutic lung cancer vaccine. An MUC1 polypeptide epitope target is selected firstly, normal cells do not have the target which is only specifically expressed by cancer cells and precancer cells, and good safety is achieved. Secondly, a selected adjuvant Cs is directly coupled with MNR by gene fusion recombinant expression, and immunogenicity of the MNR protein is improved.
Description
Technical field
The present invention relates to the antigen protein of a kind of pulmonary carcinoma and encoding gene thereof and the preparation of therapeutic lung cancer vaccine
Method.
Background technology
The annual new cancer patient of China there are about 2,500,000, and because of cancer, the number of death is about 140
Ten thousand.Following air, water pollution, environmental disruption may improve sickness rate and the case fatality rate of cancer further.
In terms of disease kind, occupying national malignant tumor morbidity primary is pulmonary carcinoma, is secondly gastric cancer, colorectal cancer, liver
Cancer and the esophageal carcinoma, the death of the whole nation, residence is primary is still pulmonary carcinoma, is secondly hepatocarcinoma, gastric cancer, the esophageal carcinoma and knot
Rectal cancer.
The research of cancer treatment drugs is current international study hotspot.Treatment of cancer is except operation, change
Outside treatment, radiotherapy, biological immune treatment development in recent years is rapid, cell therapy, Antybody therapy and vaccine therapy
The gesture that formation is stood like the legs of a tripod, has greatly and encloses cancer and annihilate possible.Vaccine therapy cancer applications is convenient, effect
Significantly, deeply welcome by extensive patients.Inoculated tumour vaccine is the one in immunotherapy method, and its principle is
Tumor cell or tumor antigen material induction body is utilized to produce specific cellular immunity and humoral immune reaction,
I.e. activate the immune system of patient self, carry out the anti-cancer ability of enhancing body, so stop tumor growth,
Diffusion and recurrence, be finally reached the purpose controlling even to remove tumor.Tumor is not external, but autologous
Cytogenetic, therefore it is reacted not as strong to antibacterial, viral reaction by body, so that early
Phase symptom is inconspicuous, and the when of discovery, tumor has reached an advanced stage the stage, and tumor cell there occurs transfer,
All of Therapeutic Method is all unable to reach the purpose of healing.Inoculated tumour vaccine then can be tumorigenic
Just can remove tumor efficiently, specifically in early days, and untoward reaction is little.Additionally, tumor vaccine therapy is also
Can combine with operative treatment, radiotherapy and change furuncle, in Comprehensive Treatment tumor, occupy critical role.
Tumor vaccine derives from autologous or allogeneic tumor cell or its crude extract, resists with tumour-specific
Former or tumor associated antigen.It can attack tumor cell by exciting specific immune function, overcomes tumor
Immunosuppressive condition caused by product, strengthens the immunogenicity of tumor associated antigen, improves autoimmunity
Carry out tumors destroyed.The immunization therapy of tumor specific antigen can start drenches with tumor-specific cytotoxicity T
Bar cell effect is main Graft Versus Tumor, effectively hits tumor, prevents transfer, recurrence and does not injure normal
Tissue, its antineoplastic specificity and immunological memory are that additive method can not be compared, have curative effect height,
The advantage such as high specificity, untoward reaction are little.According to the particular use of tumor vaccine, two kinds can be divided into: a kind of
It is preventative vaccine, as prepared vaccine with the gene relevant with some specific tumor generation or antigen, is inoculated in
There is the healthy population of genetic predisposition, and then the generation of tumor can be controlled.Another kind is therapeutic vaccine,
It is based on tumor associated antigen.
Occur that cancer, cancerous cell locally or systemically go out when immune system is to the immunosurveillance failure of malignant cell
Existing immunosuppressive condition.The change of heredity and epigenetic occurs during cell deterioration, synthesizes some sudden changes
Albumen or change the expression of some genes；Cancerous tumor cell genome is unstable, is more easy to accumulation hereditary change,
Add potential antigen；Factor in cancer cell and in microenvironment can promote that cancerous cell produces adaptability
Reaction, protein translation and degraded change, and the change of immune peptide group occur at cell surface, express and
The quality and quantity that normal cell is expressed is different tumor associated antigen.
The immunoreation utilizing tumor associated antigen induced tumor special has important value, and its principle is logical
Crossing activation patient's self immune system, the specificity utilizing tumor cell or tumor antigen material induction body is thin
Born of the same parents' immunity and humoral immune reaction, the anti-cancer ability of enhancing body, stop the growth of tumor, spread and recur,
To reach to remove or control the purpose of tumor.A kind of method is exactly the tumor associated antigen by purification or restructuring
Inject together with adjuvant, induction humoral immunization and cellular immunization.Cellular immunization acts on act foot in tumor-killing
Weight, conventional method is that costimulatory molecules or immunity chemotherapy, radiotherapy and immunologic surveillance are used in combination
Point blocker.Clinical trial can use one or more MHC of injection (major histocompatibility antigen)
Restricted tumor associated antigen source epitope polypeptide, this epi-position can be with antigen presenting cell surface
MHC molecule combines, and is directly presented to T cell.Clinical trial can use total length tumor associated antigen,
Epi-position synthesizes long polypeptide or glycated polypeptides analogue body.Epi-position synthesizes long polypeptide on using not by patient MHC
Limiting, it is by antigen presenting cell processing such as DC and presents induction immunoreation.Glycated polypeptides analogue body can
Treatment for tumors such as hematopathys.Polypeptide cancer vaccine typically adds Montanide ISA-51 as adjuvant,
Subcutaneous or intra-tumoral injection, it is also possible to TLR agonist Picibanil, Hiltonol be used in combination, or α-dry
Disturb the cytokines such as element, interleukin-2, GM-CSF, or immunostimulation antibody such as PD-1 antibody is such as
Nivolumab, or increase PADREs (the pan-DR binding peptide epitopes) factor helping signal
Such as interferon-alpha, or cell therapy, or hormone therapy, or immunogenicity chemotherapy or classic chemotherapy.
Research shows, has a kind of molecule MUC1 and relation between tumor close, has become as immunotherapy of tumors
Target spot.Evidence comes from following 7 aspects: (1) monoclonal antibody research finds, normal epithelium cell is deposited
In the low expression of MUC1, and during cancer, (such as mammary gland, ovary and kidney) MUC1 high expressed (increases by 100
Times)；When cancer occurs, MUC1 is present in whole cell surface, and normal structure is confined to secretory cell
Top end surface.(2) have been found that MUC1 has the highest immunogenicity in Mus, as by by human carcinomas
Disease cellular immunization Mus manufactures monoclonal antibody, almost all of antibody all with high immunogenicity VNTR
(PDTRPAPGSTAPPAHGVTSA) 40-80 reaction, be in fact and APDTR in this region
Aminoacid reacts.(3) there is Aberrant glycosylation in cancer cell surfaces saccharide, forms new sugared epi-position (Tn, STn
And TF) and the exposure (such as at the APDTRPA of VNTR) of peptide epitopes.(4) have turned out from mammary gland
The T cell obtained in the draining lymph node of cancer and Patients with Pancreatic Cancer is to know in the restrictive mode of non-MHC
Other MUC1 cancerous cell.(5) in breast cancer patients, the cytotoxic T lymphocyte that MHC limits is found,
The antibody for MUC1 VNTR district is obtained with cancer patient.(6) head/neck cancer (tongue, larynx,
Pharynx and amygdaline), the squamous cell carcinoma of esophagus, carcinoma of gallbladder, hepatocarcinoma, carcinoma of urethra, thyroid carcinoma,
Carcinoma of endometrium, multiple myeloma, acute myelogenous leukemia, acute/chronic lymphocytic leukemia,
In hairy cell leukemia, follicular lymphoma, plasmocytoma and diffusivity large B cell lymphoid tumor it has also been found that
MUC1 process LAN.(7) MUC1 expresses cancer ancestral/stem cell.All these features defines swollen
The basis that in tumor immune Research, MUC1 studies as target spot.
External peptide based on the VNTR of MUC1 combines or mixes the intrinsic of multiple inherent immunity system
The vaccine of stimulus object has carried out I clinical trial phase.Adjuvant includes SB-AS2, incomplete Freund's adjuvant and keyhole
Worm relative hemocyanin (KLH) etc..Two nonglycosylated VNTR dosage forms enter III the most like a bomb
Clinical trial phase.First is Stimuvax (Merck, NJ, USA), and it is 25 aggressiveness
VNTR MUC1 peptide is by liposome, and a Toll-like receptor-4 agonist (lipid A) is unified in parallel connection, existing
Carrying out three III clinical trial phases: two at nonsmall-cell lung cancer (NSCLC), one at estrogen
The breast carcinoma associating hormone therapy of receptor positive.These tests study acquisition based on the IIB phase challenging
As a result, i.e. demonstrate a group patient being in IIIB stage NSCLC (being limited to the disease of local), connect
The median survival interval nursed by Stimuvax and highest standard is 30.6 months, and only accepts highest standard nursing
The median survival interval of patient be 13.3 months.The III phase lung cancer therapy test of nearest people more than 1,000 shows cancer
Vaccine overall treatment effect is the most inconspicuous, may with enter group be that Patients with Advanced Lung Cancer has relation.By packet
Finding following rule: the therapeutic effect of smoking patients (n=762) is better than the patient of non-smoking, median is raw
Time of depositing extended to 32.1 months (p=0.0058) from 20.6 months；The median of female patient (n=269)
Life span extended to from 20.6 months 36.8 months (p=0.0370), the middle position of non-adenocarcinoma patients (n=496)
Number life span extended to 29.6 months (p=0.0435) from 19.6 months；Merge the patient of chemotherapy simultaneously
(n=806) median life span extended to the research of 30.8 months (p=0.0175) and shows from 20.6 months,
Relative to using the patient of vaccine after chemotherapy, the patient that only chemotherapy and vaccine use simultaneously is benefited.Second
Being that MUC1 (100 aggressiveness) combines oxidized mannan, it is used a lot of years, is facing
Before Chuan, I phase and the II phase tests and the III phase tests research demonstrate effectiveness.31 menopause altogether
After breast carcinoma II phase patient in 36 weeks by immunity mannan-MUC1 9 times, and combine him
Former times is not fragrant.15 patients of placebo group have 4 recurrences, and with aoxidizing the 16 of mannan-MUC1 treatment
Name patient does not recur (p=0.0292).In 13 patients accepting vaccine, 9 detect MUC1
Antibody, in 10 patients accepting vaccine, 4 detect that MUC1 specific T-cells reacts.Placebo group
MUC1 specific immune response does not occurs.12-15 follows up a case by regular visits to III phase clinical research and shows: the breast of placebo group
Adenocarcinoma relapse rate is 60% (6/15), and the patient's breast cancer relapse rate using cancer vaccine is 12.5% (2/16),
Life-span significantly extends, and after overall use cancer vaccine, the percentage ratio pole without cancer patient dramatically increases；Patient is never
The toxicity that causes of vaccine and autoimmune disease occur, vaccine injection ensure these patient with breast cancers in 7 years without cancer
Disease is recurred.
Clinical trial also shows that cancer vaccine is all safe to 6 Pancreas cancer patients, wherein 1 disease
Feelings are stable.The II clinical trial phase comprising 40 patients with prostate cancer shows, cancer vaccine improves 13 trouble
The PSA doubling time of person, wherein 10 PSA stabilize 8 months.Research to 11 ovarian cancer patients shows,
Cancer vaccine can be safe with the generation of induction of antibodies.The II phase comprising 37 renal cell carcinoma patients is clinical
Test shows, some patients reaches more than 6 months the stable disease time.According to hepatocarcinoma, colorectal cancer, first
The antigen presentation situation of shape adenocarcinoma, cancer vaccine may be suitable for the treatment of this Partial tumors patient.1 example can not
The recurrence patients with gastric cancer of operation uses the treatment of antigen induction DC cell local, does not occurs multiple in 30 months
Send out.I/II clinical trial phase research to 15 multiple myeloma patients shows, applies the big of cancer vaccine
, there is the specific immunity of T cell and B cell in some patients stable disease (at least 17.5～41.3 months).
The research of I clinical trial phase shows, hepatic metastasis colorectal cancer patients uses expression after undergoing hepatectomy for liver metastases
The xenogenesis cancer cell vaccine of GM-CSF, vein low dosage gives cyclophosphamide simultaneously, and patient produces anti-MUC1
Antibody, in 9 patients, 6 time-to-live were more than 3 years, 4 again without recurrence.II phase clinical research finds,
74 patients use DC cell therapy and vaccine therapy, and 2 years of colorectal cancer patients are close without recurrence survival rate
(respectively 47% and 55%), is all better than nonimmune group.39 adenoma of colon in late periods (Precancerous Lesion of Colon)
After inoculation cancer vaccine, 17 antibody producing anti-MUC1 and long term immune memory, and residue does not produces anti-
The medullary system suppression cell quantity that 22 patients of body are because before vaccination in blood is more relevant.For urgency
Property the disease such as myelomatosis, use immunological adjuvant Montanide ISA-51 to be probably harmful.
Although the life of patient is not reaching to clinically desirable by therapeutic cancer vaccine Stimuvax, but
It is that the research for therapeutic cancer vaccine does not terminate with regard to this, it is contemplated that by changing therapeutic cancer
Vaccine molecules structure, increases the immunogenicity of vaccine, thus the clinical practice for cancer vaccine creates conditions.
Summary of the invention
It is an object of the invention to provide the antigen protein of a kind of pulmonary carcinoma and encoding gene thereof and therapeutic pulmonary carcinoma epidemic disease
The preparation method of Seedling.
First aspect, the present invention provides the antigen protein of a kind of pulmonary carcinoma, including following (a) or (b)
Protein:
A protein that () is made up of aminoacid sequence shown in SEQ ID NO:1,
B () aminoacid sequence in (a) is through replacing, lacking or add one or several aminoacid
And there is the protein derivative by (a) of identical function.
Second aspect, the present invention provides the encoding gene of a kind of antigen protein encoded in first aspect.
In the first mode in the cards of second aspect, the upstream and downstream of described DNA molecular
Respectively further comprising a restriction enzyme site, the restriction enzyme site of upstream is NdeI restriction enzyme site；The enzyme action position in downstream
Point is NcoI restriction enzyme site.
The third aspect, the present invention provides a kind of the first possible realization containing second aspect or second aspect
The recombinant expression carrier of the encoding gene described in mode.
In the first mode in the cards of the third aspect, the carrier that sets out of described recombinant expression carrier
For pMAL-p5X.
Fourth aspect, the present invention provides a kind of recombinant bacterium containing the recombinant expression carrier described in the third aspect
Or transgenic cell line.
5th aspect, the present invention provides the preparation method of a kind of therapeutic lung cancer vaccine, including:
(1) activate with recombinant expression carrier described in the third aspect by the culture medium added with ampicillin
DH5 α bacterial strain, is subsequently adding IPTG abduction delivering, centrifugal collection bacteria culture media；
(2) add ammonium sulfate solution precipitation rear overhang to bacteria culture media to rise and carry out affinity purification and maltose
Aqueous solution eluting, obtains the protein of purification；
(3) protein of purification is concentrated by ultrafiltration, obtains destination protein matter；
(4) destination protein matter detoxification is processed, be subsequently adding adjuvant mixing, obtaining medical treatment property lung cancer vaccine.
In the first mode in the cards of the 5th aspect, in step (2), the quality of ammonium sulfate is dense
Degree is 26.3%-53.0%.
In the second mode in the cards of the 5th aspect, in step (2), maltose solution is dense
Degree is 10mmol/L.
In the third mode in the cards of the 5th aspect, described adjuvant is aluminum hydroxide adjuvant.
The antigen protein of a kind of pulmonary carcinoma that the present invention provides and encoding gene thereof and the system of therapeutic lung cancer vaccine
Preparation Method, the MUC1 polypeptide epitope target spot first selected (for convenience of describing, named MNR), this target
Point is that normal cell does not has, only cancer cell and cell specific expression precancer, has well peace
Quan Xing.Secondly the adjuvant Cs selected is directly and MNR is by the recombinant expressed coupling of gene fusion, adds
The immunogenicity of MNR albumen.Then, select the aluminium adjuvant that clinic is commonly used as adjuvant, increase vaccine
Action time.The cancer vaccine finally obtained passes through intramuscular injection, and zoopery shows to have well peace
Full property and effectiveness.
Accompanying drawing explanation
Fig. 1 is the electrophoretogram that antigen protein provided by the present invention is recombinant expressed in escherichia coli；
Fig. 2 is antigen protein provided by the present invention electrophoretogram of purification from bacteria culture media；
Fig. 3 is the electrophoretogram after antigen protein provided by the present invention is concentrated by ultrafiltration；
Fig. 4 is MBP and antigen protein provided by the present invention contrasts electrophoresis from the purification of bacteria culture media
Figure；
Fig. 5 is that the enzyme action of antigen protein provided by the present invention identifies electrophoretogram；
Fig. 6 is the electrophoretogram after antigen protein prick post provided by the present invention；
Fig. 7 is the electrophoretogram after antigen protein affinity purification provided by the present invention；
Fig. 8 is test example 1 result schematic diagram；
Fig. 9 is the result schematic diagram of first test in test example 2；
Figure 10 is the result schematic diagram of second test in test example 2；
Detailed description of the invention
In order to make those skilled in the art be more fully understood that the present invention program, below in conjunction with the accompanying drawings and specifically
The present invention is described in further detail for embodiment.
Embodiment 1
Antigen protein synthesizes: use Gene Designer software to carry out codon optimized to encoding gene,
And introducing NdeI restriction enzyme site at upstream region of gene, downstream introduces NcoI restriction enzyme site, synthetic gene
It is cloned into pUC57 carrier, ammonia benzyl resistance.With NdeI and NcoI enzymes double zyme cutting after gene chemical synthesis,
Connect into the pMAL-p5X plasmid through same enzyme action, and connect with DNA ligase, be transformed into sense
By state DH5 α antibacterial, screen with on the agar plate containing ammonia benzyl (AMP), select monoclonal and use
LB-AMP culture medium culturing, extracts plasmid enzyme restriction and identifies, then carries out order-checking and identifies.Will be containing coding
The pMAL-p5X plasmid (for convenience of describing, by named for this plasmid pMAL-p5X-MNR) of gene
DH5 α antibacterial with LB-AMP cultivate, treat that OD600nm reaches the 0.5 final concentration of 1mM of addition
IPTG, abduction delivering 12h.Same abduction delivering with pMAL-p5X DH5 α antibacterial as
Comparison.Taking the bacterium solution before and after induction and carry out protein electrophorese, coomassie brilliant blue R250 dyes.DH5α
-pMAL-p5X-MNR gives expression to the protein (shown in finger arrow as right in Fig. 1) of about 65KD, and compares
Antibacterial DH5 α-pMAL-p5X gives expression to the protein (shown in finger arrow as left in Fig. 1) of about 48KD.
After testing, the primary structure of antigen protein is as shown in sequence table SEQ ID NO:1.Its gene is compiled
Code sequence is as shown in sequence table SEQ ID NO:2.
Embodiment 2
The derived protein synthesis of antigen protein: use Gene Designer software to carry out close to encoding gene
Numeral optimizes, and introduces BamHI restriction enzyme site at upstream region of gene, and downstream introduces EcoRI restriction endonuclease position
Point, synthetic gene is cloned into pUC57 carrier, ammonia benzyl resistance.With BamHI and EcoRI after gene chemical synthesis
Enzymes double zyme cutting, connects into the pMAL-p5X plasmid through same enzyme action, and uses DNA ligase
Connect, be transformed into competence DH5 α antibacterial, screen with on the agar plate containing ammonia benzyl (AMP),
Select monoclonal LB-AMP culture medium culturing, extract plasmid enzyme restriction and identify, then carry out order-checking and identify.
The DH5 α antibacterial of the pMAL-p5X plasmid containing encoding gene LB-AMP is cultivated, treats
OD600nm reaches the IPTG, abduction delivering 12h of the 0.5 final concentration of 1mM of addition.Before and after taking induction
Bacterium solution carry out protein electrophorese, coomassie brilliant blue R250 dyes.After testing, the one-level of antigen protein
Structure is as shown in sequence table SEQ ID NO:3.Its gene coded sequence such as sequence table SEQ ID NO:
Shown in 4.
Embodiment 3
The derived protein synthesis of antigen protein: use Gene Designer software to carry out close to encoding gene
Numeral optimizes, and introduces NcoI restriction enzyme site at upstream region of gene, and downstream introduces EcoRI restriction endonuclease position
Point, synthetic gene is cloned into pUC57 carrier, ammonia benzyl resistance.With NcoI and EcoRI after gene chemical synthesis
Enzymes double zyme cutting, connects into the pMAL-p5X plasmid through same enzyme action, and uses DNA ligase
Connect, be transformed into competence DH5 α antibacterial, screen with on the agar plate containing ammonia benzyl (AMP),
Select monoclonal LB-AMP culture medium culturing, extract plasmid enzyme restriction and identify, then carry out order-checking and identify.
The DH5 α antibacterial of the pMAL-p5X plasmid containing encoding gene LB-AMP is cultivated, treats
OD600nm reaches the IPTG, abduction delivering 12h of the 0.5 final concentration of 1mM of addition.Before and after taking induction
Bacterium solution carry out protein electrophorese, coomassie brilliant blue R250 dyes.After testing, the one-level of antigen protein
Structure is as shown in sequence table SEQ ID NO:5.Its gene coded sequence such as sequence table SEQ ID NO:
Shown in 6.
Embodiment 4
1. antigen protein purification
The DH5 α with pMAL-p5X-MNR is activated with the LB culture medium culturing added with ampicillin
Bacterial strain, is transferred to the big bottle LB culture medium culturing added with ampicillin, treats OD600nmReach 0.5 addition
The IPTG of final concentration of 1mmol/L, abduction delivering 12h.Centrifugal collection bacterial precipitation and culture medium.
The process of bacterial precipitation: bacterial precipitation Tris buffer (proportioning is 20mmol/L Tris-HCl,
0.2mmol/LNaCl, 10mmol/L beta-mercaptoethanol, 1mmol/L EDTA) hang, ultrasonication
10min, centrifugal collection supernatant, by 8 post bed Tris buffer balances upper after filtering with microporous membrane
Amylose resin, after 12 post bed Tris buffer solution, then with the Tris containing 10mM maltose
Buffer solution elution, it is thus achieved that destination protein matter.Upper polyacrylamide gel electricity after taking the protein denaturation of eluting
Swimming, coomassie brilliant blue R250 dyes.
The process of bacteria culture media: bacteria culture media ammonium sulfate precipitation rear overhang plays amylose resin,
With 10mmol/L maltose eluting.In bacterial precipitation processes, same method carries out electrophoresis and dyeing mirror
Determine purified fractions.Using Brandford method to measure protein concentration, the antigen protein obtained by purification adds
In Millipore ultra-filtration centrifuge tube (MWCO 10000), 4000 × g is centrifuged 15min, protein
Concentrated.The protein same method concentrated is measured concentration, and carries out protein after diluting 20 times
Electrophoresis.Antibacterial bacterium solution is degerming through inner pressed membrane filtration, and filtrate concentrates through external-compression type filter membrane again.Concentrated solution
Dialysing 2 times through 10K filter membrane, after being centrifuged, the upper affine resin purification of Amylose obtains destination protein, so
After detecting with G250 afterwards, upper SDS-PAGE analyzes.
Comparison bacterial expression: with the LB culture medium culturing added with ampicillin with pMAL-p5X's
DH5 α bacterial strain, treats OD600nmReach the IPTG, abduction delivering 12h of the 0.5 final concentration of 1mmol/L of addition.
Centrifugal collection culture medium, plays upper mylose resin, 10mM maltose eluting with ammonium sulfate precipitation rear overhang.
Electrophoresis and dyeing purification Identification composition.
Conclusion:
(1) bacteria culture media is used 26.3% ammonium sulfate precipitation, molecular weight can be obtained and be about 65KD's
Protein；Strengthen the concentration of ammonium sulfate to 33.3% and 53.0%, it is also possible to precipitate considerable amount of molecular weight
It is about the protein of 65KD.More higher than the protein impurities of 53.0% ammonium sulfate precipitation.The egg of 65KD
White matter can be obtained, by Amylose resin-bonded and 10mmol/L maltose eluting, the albumen that concentration is higher
Matter, its molecular weight and MUC1-N variable in-line repeat MBP fusion protein molecule amount theoretical value 64.9KD
Unanimously.(as shown by the arrows in figure 2)
(2) protein of purification is concentrated (see Fig. 3 arrow after 10KD super filter tube centrifugal filtration
Head indication), there is a small amount of protein to spill, the protein spilt still can in conjunction with Amylose resin-bonded also
By 10mM maltose eluting.The protein concentration being concentrated by ultrafiltration reaches 5mg/ml.-20 DEG C preserve one week
After, protein has Partial digestion.
(3) being the destination protein with MNR gene to confirm the protein of purification, we will compare
The DH5 α that plasmid (containing only MBP gene) converts carries out abduction delivering, and carries out the sulphuric acid of culture medium
Ammonium precipitation and Amylose resin purification.Find the protein molecular weight from culture medium purification and MBP mono-
Cause, less than MNR-MBP molecular weight.The former is big in the albumen precipitation amount that ammonium sulfate concentrations is 50-67%
In the albumen precipitation amount of 20-50%, the latter's ammonium sulfate concentrations is that the albumen precipitation amount of 20-50% is more than
The albumen precipitation amount of 50-67%, is shown in Fig. 4.
(4) protein enzymolysis substantially after room temperature 3h processes, enzymolysis completely after 21h.
Obtain the protein of molecular weight about 17KD, and the molecular weight ratio of MNR is closer to, and (160 aminoacid are residual
Base, is shown in Fig. 5 arrow indication).The protein of 17KD is obtained in Q-Sepharose purification, prick post liquid
(see Fig. 6 arrow indication)
(5) SDS-PAGE analyze show, antibacterial bacterium solution after ＜ 200K type inner pressed membrane filtration on
Clear liquid contains destination protein matter, concentrates nearly 3 times, through Amylose after CLW-002 type external-compression type filter membrane
Affine resin purification obtains the destination protein matter that concentration is higher, sees Fig. 7.
2. destination protein (named MNR-MBP) removes endotoxin
MNR-MBP after ultrafiltration concentration through Tris buffer (20mmol/L Tris-HCl,
0.2mol/LNaCl, 10mmol/L beta-mercaptoethanol, 1mmol/L EDTA) balance, use microporous filter membrane
After filtration, the Amylose resin of upper 8 post bed Tris buffer balances, washes with 12 post bed Tris buffer
By the Tris buffer solution elution containing 10mmol/L maltose after washing, it is thus achieved that destination protein matter MNR-MBP.
Removing endotoxin with Q-sepharose prick post, post bed 1.6ml, after balancing with the Tris buffer of pH 7.4
Loading, it is degerming that prick post liquid crosses 0.22 μm filter membrane, measures protein content by Braford method, uses tachypleus amebocyte lysate
Measuring endotoxin content, being qualified when endotoxin content is less than 500EU/mg, if not reaching criterion of acceptability
Repeating removal endotoxin step until qualified, dilution destination protein MNR-MBP solution arrives
1mg/ml preserves.
Experiment uses various ways to remove the endotoxin of destination protein MNR-MBP, including ammonium sulfate
Precipitation secondary, cross after Amylose resin ultrafiltration again, Octyl-sepharose prick post, Q-sepharose after eluting
Cross affinity column after prick post, only ultrafiltration to add Q-sepharose prick post purification and can reach requirement, endotoxic
Content is less than 500Eu/mg.
3. the preparation of lung cancer vaccine
1% gelatin, 5% sucrose, the AlCl of NaCl, 0.9mg/ml of 0.65% is added in distilled water3,
PH to 5.8 is regulated with 10N NaOH.110 DEG C of autoclaving 20min, cross 0.22 μm after cooling
Filter membrane, collection filter liquor is aluminum hydroxide adjuvant, and 4 DEG C save backup.Take 0.5ml aluminum hydroxide adjuvant to add
Entering 0.5mg MNR-MBP destination protein, mix homogeneously becomes lung cancer vaccine.This lung cancer vaccine be transparent,
Odorless, liquid with no taste.PH when 25 ± 0.5 DEG C is 6.0～8.0.
Generally, lung cancer vaccine preparation process of the present invention is as follows: expressed in Host Strains by genetic engineering
MNR-MBP, after obtain the other MNR-MBP of injection stage through multiple purification, detoxification, filtration sterilization,
Requirement is reached through the detection of tachypleus amebocyte lysate endotoxin.Synthetic route: prepared by gene → plasmid construction → recombinant expressed
→ ultrafiltration purification → affinity purification → detoxification → filtration sterilization → adjuvant interpolation → finished product packing.Technique describes:
Registering batch to criticize as 12g/, the batch of drafting of the big production batch of commercialization is 750/ batch.To register batch as generation
The technique of table is described as follows: protein crude product Tris buffer (20mmol/L Tris-HCl, 0.2M
NaCl, 1mmol/L EDTA) dialysis, by 8 post bed Tris buffer balances upper after filtering with microporous membrane
Amylose resin, with after 12 post bed Tris buffer solution with the Tris containing 10mmol/L maltose
Buffer solution elution, it is thus achieved that destination protein matter.
Test example 1
Vaccine animal immune is tested
Take 10 6-8 week old male Balb/C mouses, be divided into experimental group and matched group.Before the experimental group right side
Leg muscle inject 100 μ L (50 μ g) vaccine (aluminium hydroxide is adjuvant, is dissolved in 20mmol/L Tris-HCl,
0.2mol/L NaCl, 1mmol/L EDTA, pH value is 7.4), the same injection location of matched group 100
μ L aluminum hydroxide adjuvant.Twice a week, totally 3 times.Immunity before and rear 2 eye sockets of immunity take blood for
The detection of antibody.ELISA detects discovery: there is not MNR before vaccine immunity in Balb/C mice serum
Antibody, occur a small amount of for the antibody at MNR after 7 days, about many MNR occurred after 14 days
Antibody, there is not the antibody of MNR, sees Fig. 8 in an injecting immune adjuvant.
Test example 2
The pharmacodynamic experiment of vaccine
2.1 take 49 6-8 week old male C 57 BL/6 J mouses, be divided into normal saline group (matched group),
50 μ g experimental grouies (vaccine of the present invention), often group 7.Injection 1 time every two weeks, totally 3 times.The 3rd
After secondary immunity the 4th day, oxter, right side injection 1 × 106 (50 μ L) lung carcinoma cell, measure little after 14 days
The volume of Mus tumor.Found that compare with matched group, the tumor of 50 μ g experimental grouies significantly diminishes,
See Fig. 9.
2.2 take 28 6-8 week old male C 57 BL/6 J mouses, are divided into 4 groups and inject 50 μ g experimental grouies
(vaccine of the present invention), often group 7.Injection 1 time, injects 2,4,6,8 times respectively every two weeks.?
After last immunity the 4th day, oxter, right side injection 1 × 106 (50 μ L) lung carcinoma cell, after 14 days
Measure the volume of mouse tumor.Found that immune time is the most, the volume of tumor is the least, sees below Figure 10.
2.3 take 10 6-8 week old male C 57 BL/6 J mouses, oxter, right side injection 1 × 106 (50 μ L)
Melanoma cell, measures the volume of mouse tumor after the 8th day.It is divided into 2 groups of injecting normal salines and 50
μ g experimental group (vaccine of the present invention), often group 5.Injection 2 times weekly, and the 12nd, 14,16
It measures tumor size.Found that the 14th of tumor growth the day, vaccine injection of the present invention inhibits black
The growth of element tumor, is shown in Table 1.
The growth of table 1 vaccine injection of the present invention suppression melanoma
Test example 3
Vaccine animal toxicology is tested
Take 10 6-8 week old male Balb/C mouses, be divided into experimental group and matched group.Before the experimental group right side
Leg muscle injects 100 μ L (50 μ g) vaccine of the present invention, and (aluminium hydroxide is adjuvant, is dissolved in 20mmol/L
Tris-HCl, 0.2mol/L NaCl, 1mmol/L EDTA, pH value is 7.4), the same position of matched group
Inject 100 μ L aluminum hydroxide adjuvants.Twice a week, totally 3 times.Within after animal inoculation vaccine 14 days, put to death
Animal is observed animal and has no the organic disease of the internal organs such as heart, liver, spleen, lung, kidney.
Test example 1-3 is to prepare the test of lung cancer vaccine for antigen protein of the present invention, through same examination
Test means detection, find that the vaccine that the derived protein that embodiment 2 and 3 provides prepares also has identical effect
Really, do not repeat them here.
Application Example 1
Vaccine clinical application process
First 1 year course for the treatment of, 1-4 pin is spaced one week, and 5-15 pin is spaced January.Cancer is suffered from early days
Person uses 1 year；Cancer patient in mid-term increases by a course for the treatment of, and 16-27 pin is spaced January, and cancer is suffered from early days
Person uses 2 years.Bilateral upper arm muscle injection.
Above to the antigen protein of a kind of pulmonary carcinoma provided by the present invention and encoding gene thereof and therapeutic lung
The preparation method of Theratope is described in detail.The specific case principle to the present invention used herein
And embodiment is set forth, the explanation of above example is only intended to help to understand the core of the present invention
Thought.It should be pointed out that, for those skilled in the art, former without departing from the present invention
On the premise of reason, it is also possible to the present invention is carried out some improvement and modification, these improve and modification also falls into
In the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>Chinese Academy of Medical Sciences Beijing Union Medical College Hospital
<120>antigen protein of pulmonary carcinoma and encoding gene thereof and the preparation method of therapeutic lung cancer vaccine
<130> P20160046
<160> 6
<170> PatentIn version
3.5
<210> 1
<211> 573
<212> PRT
<213>artificial sequence
<400> 1
Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15
Thr Met Met
Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys
20 25 30
Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45
Val Gly Lys
Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu
50 55 60
His Pro Asp
Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80
Asp Gly Pro
Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr
85 90 95
Ala Gln Ser
Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln
100 105 110
Asp Lys Leu
Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125
Leu Ile Ala
Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn
130 135 140
Lys Asp Leu
Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala
145 150 155 160
Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175
Leu Gln Glu
Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly
180 185 190
Tyr Ala Phe
Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205
Val Asp Asn
Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu
210 215 220
Ile Lys Asn
Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu
225 230 235 240
Ala Ala Phe
Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp
245 250 255
Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270
Leu Pro Thr
Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu
275 280 285
Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300
Phe Leu Glu
Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn
305 310 315 320
Lys Asp Lys
Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu
325 330 335
Leu Val Lys
Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys
340 345 350
Gly Glu Ile
Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala
355 360 365
Val Arg Thr
Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp
370
375 380
Glu Ala Leu
Lys Asp Ala Gln Thr Asn Ser Ser Ser Asn Asn Asn Asn
385 390 395 400
Asn Asn Asn
Asn Asn Asn Leu Gly Ile Glu Gly Arg Ile Ser His Met
405 410 415
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
420 425 430
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
435 440 445
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
450 455 460
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
465 470 475 480
Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
485 490 495
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
500 505 510
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
515 520 525
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
530 535 540
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Ser Met Gly Gly
545 550 555 560
Arg Asp Ile
Val Asp Gly Ser Glu Phe Pro Ala Gly Asn
565 570
<210> 2
<211> 420
<212> DNA
<213>artificial sequence
<400> 2
ggtgttactt
ctgctcctga tactcgtcct gctcctggtt ctactgcacc gccagcacat 60
ggcgtgacgt
ctgcgccaga tacccgtccg gcaccgggtt ccaccgcccc accggcacac 120
ggcgtaacct
ccgcgccaga cacccgtcca gcgccaggtt ctaccgctcc gcctgctcat 180
ggtgttacct
ctgccccgga cactcgtccg gctccaggtt ctactgcccc gccagctcat 240
ggcgtcactt
ccgccccgga tacccgtcct gccccgggct ctactgcgcc tccggctcac 300
ggcgttacct
ctgcaccgga tactcgtccg gctccgggct ctaccgcacc acctgctcat 360
ggcgtaacga
gcgctcctga tacccgtccg gctccgggtt ccactgcacc tccggcccac 420
<210> 3
<211> 713
<212> PRT
<213>artificial sequence
<400> 3
Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15
Thr Met Met
Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys
20 25 30
Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45
Val Gly Lys
Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu
50 55 60
His Pro Asp
Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80
Asp Gly Pro
Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr
85 90 95
Ala Gln Ser
Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln
100 105 110
Asp Lys Leu
Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125
Leu Ile Ala
Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn
130 135 140
Lys Asp Leu
Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala
145 150 155 160
Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175
Leu Gln Glu
Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly
180 185 190
Tyr Ala Phe
Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205
Val Asp Asn
Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu
210 215 220
Ile Lys Asn
Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu
225 230 235 240
Ala Ala Phe
Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp
245 250 255
Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270
Leu Pro Thr
Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu
275 280 285
Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300
Phe Leu Glu
Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn
305 310 315 320
Lys Asp Lys
Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu
325 330 335
Leu Val Lys
Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys
340 345 350
Gly Glu Ile
Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala
355 360 365
Val Arg Thr
Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp
370 375 380
Glu Ala Leu
Lys Asp Ala Gln Thr Asn Ser Ser Ser Asn Asn Asn Asn
385 390 395 400
Asn Asn Asn Asn
Asn Asn Leu Gly Ile Glu Gly Arg Ile Ser His Met
405 410 415
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
420 425 430
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
435 440 445
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
450 455 460
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
465 470 475 480
Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
485 490 495
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
500 505 510
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
515 520 525
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
530 535 540
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
545 550 555 560
Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
565 570 575
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
580 585 590
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
595 600 605
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
610 615 620
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
625 630 635 640
Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
645 650 655
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
660 665 670
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
675 680 685
Gly Ser Thr
Ala Pro Pro Ala His Ser Met Gly Gly Arg Asp Ile Val
690 695 700
Asp Gly Ser
Glu Phe Pro Ala Gly Asn
705 710
<210> 4
<211> 840
<212> DNA
<213>artificial sequence
<400> 4
ggtgttactt
ctgctcctga tactcgtcct gctcctggtt ctactgcacc gccagcacat 60
ggcgtgacgt
ctgcgccaga tacccgtccg gcaccgggtt ccaccgcccc accggcacac 120
ggcgtaacct
ccgcgccaga cacccgtcca gcgccaggtt ctaccgctcc gcctgctcat 180
ggtgttacct
ctgccccgga cactcgtccg gctccaggtt ctactgcccc gccagctcat 240
ggcgtcactt
ccgccccgga tacccgtcct gccccgggct ctactgcgcc tccggctcac 300
ggcgttacct
ctgcaccgga tactcgtccg gctccgggct ctaccgcacc acctgctcat 360
ggcgtaacga
gcgctcctga tacccgtccg gctccgggtt ccactgcacc tccggcccac 420
ggtgttactt
ctgctcctga tactcgtcct gctcctggtt ctactgcacc gccagcacat 480
ggcgtgacgt
ctgcgccaga tacccgtccg gcaccgggtt ccaccgcccc accggcacac 540
ggcgtaacct
ccgcgccaga cacccgtcca gcgccaggtt ctaccgctcc gcctgctcat 600
ggtgttacct
ctgccccgga cactcgtccg gctccaggtt ctactgcccc gccagctcat 660
ggcgtcactt
ccgccccgga tacccgtcct gccccgggct ctactgcgcc tccggctcac 720
ggcgttacct
ctgcaccgga tactcgtccg gctccgggct ctaccgcacc acctgctcat 780
ggcgtaacga
gcgctcctga tacccgtccg gctccgggtt ccactgcacc tccggcccac 840
<210> 5
<211> 853
<212> PRT
<213>artificial sequence
<400> 5
Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15
Thr Met Met
Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys
20 25 30
Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45
Val Gly Lys
Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu
50 55 60
His Pro Asp
Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80
Asp Gly Pro
Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr
85 90 95
Ala Gln Ser
Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln
100 105 110
Asp Lys Leu
Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125
Leu Ile Ala
Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn
130 135 140
Lys Asp Leu
Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala
145 150 155 160
Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175
Leu Gln Glu
Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly
180 185 190
Tyr Ala Phe
Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205
Val Asp Asn
Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu
210 215 220
Ile Lys Asn
Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu
225 230 235 240
Ala Ala Phe
Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp
245 250 255
Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270
Leu Pro Thr
Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu
275 280 285
Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300
Phe Leu Glu
Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn
305 310 315 320
Lys Asp Lys
Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu
325 330 335
Leu Val Lys
Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys
340 345 350
Gly Glu Ile
Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala
355 360 365
Val Arg Thr
Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp
370 375 380
Glu Ala Leu
Lys Asp Ala Gln Thr Asn Ser Ser Ser Asn Asn Asn Asn
385 390 395 400
Asn Asn Asn Asn
Asn Asn Leu Gly Ile Glu Gly Arg Ile Ser His Met
405 410 415
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
420 425 430
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
435 440 445
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
450 455 460
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
465 470 475 480
Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
485 490 495
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
500 505 510
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
515 520 525
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
530 535 540
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
545 550 555 560
Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
565 570 575
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
580 585 590
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
595 600 605
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
610 615 620
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
625 630 635 640
Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
645 650 655
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
660 665 670
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
675 680 685
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
690 695 700
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
705 710 715 720
Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
725 730 735
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
740 745 750
Pro Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
755 760 765
Gly Ser Thr
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
770 775 780
Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
785 790 795 800
Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
805 810 815
Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
820 825 830
Pro Pro Ala
His Ser Met Gly Gly Arg Asp Ile Val Asp Gly Ser Glu
835 840 845
Phe Pro Ala
Gly Asn
850
<210> 6
<211> 1260
<212> DNA
<213>artificial sequence
<400> 6
ggtgttactt
ctgctcctga tactcgtcct gctcctggtt ctactgcacc gccagcacat 60
ggcgtgacgt
ctgcgccaga tacccgtccg gcaccgggtt ccaccgcccc accggcacac 120
ggcgtaacct
ccgcgccaga cacccgtcca gcgccaggtt ctaccgctcc gcctgctcat 180
ggtgttacct
ctgccccgga cactcgtccg gctccaggtt ctactgcccc gccagctcat 240
ggcgtcactt
ccgccccgga tacccgtcct gccccgggct ctactgcgcc tccggctcac 300
ggcgttacct
ctgcaccgga tactcgtccg gctccgggct ctaccgcacc acctgctcat 360
ggcgtaacga
gcgctcctga tacccgtccg gctccgggtt ccactgcacc tccggcccac 420
ggtgttactt
ctgctcctga tactcgtcct gctcctggtt ctactgcacc gccagcacat 480
ggcgtgacgt
ctgcgccaga tacccgtccg gcaccgggtt ccaccgcccc accggcacac 540
ggcgtaacct
ccgcgccaga cacccgtcca gcgccaggtt ctaccgctcc gcctgctcat 600
ggtgttacct
ctgccccgga cactcgtccg gctccaggtt ctactgcccc gccagctcat 660
ggcgtcactt
ccgccccgga tacccgtcct gccccgggct ctactgcgcc tccggctcac 720
ggcgttacct
ctgcaccgga tactcgtccg gctccgggct ctaccgcacc acctgctcat 780
ggcgtaacga
gcgctcctga tacccgtccg gctccgggtt ccactgcacc tccggcccac 840
ggtgttactt
ctgctcctga tactcgtcct gctcctggtt ctactgcacc gccagcacat 900
ggcgtgacgt
ctgcgccaga tacccgtccg gcaccgggtt ccaccgcccc accggcacac 960
ggcgtaacct
ccgcgccaga cacccgtcca gcgccaggtt ctaccgctcc gcctgctcat 1020
ggtgttacct
ctgccccgga cactcgtccg gctccaggtt ctactgcccc gccagctcat 1080
ggcgtcactt
ccgccccgga tacccgtcct gccccgggct ctactgcgcc tccggctcac 1140
ggcgttacct
ctgcaccgga tactcgtccg gctccgggct ctaccgcacc acctgctcat 1200
ggcgtaacga
gcgctcctga tacccgtccg gctccgggtt ccactgcacc tccggcccac 1260
Claims (10)
1. an antigen protein for pulmonary carcinoma, including the protein of following (a) or (b):
A protein that () is made up of aminoacid sequence shown in SEQ ID NO:1,
(b) aminoacid sequence in (a) through replacement, lack or add one or several aminoacid and
There is the protein derivative by (a) of identical function.
2. the encoding gene of antigen protein described in coding claim 1.
The encoding gene of antigen protein the most according to claim 2, it is characterised in that described DNA
The upstream and downstream of molecule respectively further comprises a restriction enzyme site, and the restriction enzyme site of upstream is NdeI enzyme action position
Point；The restriction enzyme site in downstream is NcoI restriction enzyme site.
4. contain the recombinant expression carrier of encoding gene described in Claims 2 or 3.
Recombinant expression carrier the most according to claim 4, it is characterised in that described recombinant expressed load
The carrier that sets out of body is pMAL-p5X.
6. contain recombinant bacterium or the transgenic cell line of recombinant expression carrier described in claim 5.
7. a preparation method for therapeutic lung cancer vaccine, including:
(1) have the right recombinant expression carrier described in requirement 4 with the culture medium activation zone added with ampicillin
DH5 α bacterial strain, be subsequently adding IPTG abduction delivering, centrifugal collect bacteria culture media；
(2) add ammonium sulfate solution precipitation rear overhang to bacteria culture media to rise and carry out affinity purification and maltose
Aqueous solution eluting, obtains the protein of purification；
(3) protein of purification is concentrated by ultrafiltration, obtains destination protein matter；
(4) destination protein matter detoxification is processed, be subsequently adding adjuvant mixing, obtaining medical treatment property lung cancer vaccine.
The preparation method of therapeutic lung cancer vaccine the most according to claim 7, it is characterised in that step
Suddenly, in (2), the mass concentration of ammonium sulfate solution is 26.3%-53.0%.
The preparation method of therapeutic lung cancer vaccine the most according to claim 7, it is characterised in that step
Suddenly, in (2), maltose solution concentration is 10mmol/L.
The preparation method of therapeutic lung cancer vaccine the most according to claim 7, it is characterised in that institute
Stating adjuvant is aluminum hydroxide adjuvant.
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| CN201610284535.5A CN105906699A (en) | 2016-04-29 | 2016-04-29 | Lung cancer antigen protein, coding gene thereof and preparation method of therapeutic lung cancer vaccine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114316066A (en) * | 2021-11-11 | 2022-04-12 | 元本(珠海横琴)生物科技有限公司 | MNR2 protein purification method |
| CN116059387A (en) * | 2021-11-04 | 2023-05-05 | 元本(珠海横琴)生物科技有限公司 | Vaccine for resisting pancreatic cancer and medical application thereof |
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| WO2002062319A2 (en) * | 2001-01-19 | 2002-08-15 | Corixa Corporation | Microsphere delivery of mucin peptides |
| CN1513556A (en) * | 2003-02-19 | 2004-07-21 | 台桂香 | Recombination human Mucl-MBP fusion protein antitumour vaccine and production technology |
| CN105002203A (en) * | 2015-07-14 | 2015-10-28 | 北京嘉万生物技术有限公司 | Human pepsinogen I (PGI)-maltose-binding protein (MBP) fusion protein soluble secretory expression and use |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116059387A (en) * | 2021-11-04 | 2023-05-05 | 元本(珠海横琴)生物科技有限公司 | Vaccine for resisting pancreatic cancer and medical application thereof |
| WO2023077924A1 (en) * | 2021-11-04 | 2023-05-11 | 元本(珠海横琴)生物科技有限公司 | Vaccine against pancreatic cancer, and medical use thereof |
| CN116059387B (en) * | 2021-11-04 | 2023-09-22 | 元本(珠海横琴)生物科技有限公司 | Vaccine for resisting pancreatic cancer and medical application thereof |
| CN114316066A (en) * | 2021-11-11 | 2022-04-12 | 元本(珠海横琴)生物科技有限公司 | MNR2 protein purification method |
| CN114316066B (en) * | 2021-11-11 | 2023-07-14 | 元本(珠海横琴)生物科技有限公司 | Purification method of MNR2 protein |
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