CN102191271A - Anti-tumor recombinant plasmid as well as gene vaccine, preparation method and application thereof - Google Patents
Anti-tumor recombinant plasmid as well as gene vaccine, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an anti-tumor recombinant plasmid as well as a gene vaccine and application thereof. The recombinant plasmid comprises a cDNA fragment of a Cyp26a1 gene and a eukaryon expression vector pVAX1; the base sequence of the recombinant plasmid is shown as SEQ ID NO.1; and the recombinant plasmid is constructed by the following steps: designing a specificity primer by extracting and purifying the total RNA of a rat gravid uterus, cloning and augmenting the cDNA fragment of the Cyp26a1 gene of the rat uterus by adopting a temperature dropping reverse transcription-polymerase chained augmentation method; and recombining the cDNA fragment into the eukaryon expression vector to form the recombination plasmid. The prepared gene vaccine can be used for preparing medicines used for preventing and/or treating mammary cancers of mammals, has the advantages of safety, long acting, stability and convenience for operation, can simultaneously stimulate humoral immunity and cell immunity in high efficiency; and compared with other immunity means, the gene vaccine has strong immunity efficacy, needs a small immunity amount and can effectively inhibit the growth and the transference of tumors.
Description
Technical field
The invention belongs to biological and modern agricultural technology field, relate to a kind of Antioncogene vaccine, more particularly, the present invention relates to a kind of Antioncogene vaccine and application thereof that contains rat cell cytochrome p 450 family 26 superfamily A polypeptide 1 (Cyp26a1).
Background technology
China has cancer patient more than 300 ten thousand people now, and the patient who dies from malignant tumour every year is about 1,300,000 people.The malignant tumour case that 1,600,000 to 2,000,000 New Developments are arranged every year, the patient who dies from cancer every year is about 1,300,000, and with 3% speed increase, cause grave danger for China people's life and health, bring huge pressure for society and family, restricted the Sustainable development of China's economy to a certain extent.Mammary cancer is a kind of malignant tumour of the serious harm mankind, especially women's health.Mammary cancer is the modal malignant tumour of women, ranks ten big frequently-occurring tumours, approximately just has a people to suffer from breast cancer among per 100 women.Though the probability that the male sex suffers from breast cancer lacks 100 times than the women, because the ignorance of checking, male breast carcinoma patient's lethality rate is higher.In the U.S., there were 212,600 women to suffer from breast cancer on inspection in 2003,40,200 women die from this disease.More allow the people pay close attention to and what worry is in making a definite diagnosis, to have had 40% patient that transfer has taken place.Mammary cancer has several different histological types, and wherein most (about 90%) originates from breast duct, and modal mammary cancer is infitrating ductal carcinoma; About 5% mammary cancer occurs in lobule of mammary gland, is called lobular carcinoma of breast.In addition, the probability of patient with breast cancer's recurrence also is higher than general crowd.The treatment of mammary cancer comprises operative treatment, radiotherapy, chemotherapy, biotherapy etc., although researcher has been thrown in a large amount of energy to the research of capturing mammary cancer, but in case take place to shift and invade profit, still seem very thorny of the treatment of mammary cancer.
The early 1990s in last century, a series of about the injection foreign gene in vivo the report of induce immune response opened the prelude of dna vaccination research, from then on the new technology of genetic immunization causes the great attention of countries in the world.Because genetic immunization can induce body to produce comprehensive immunne response, have advantages such as safe, reliable, convenient for production again simultaneously, so The World Health Organization's appropriation in 1994 is included dna vaccination in the WHO immune development plan in the whole world.NIH (NIH) in 1994 research work subsidizing to carry out dna vaccination treatment HIV with the specific project expenditure of the biological strategic evolutionary operation(EVOP) of country, nineteen ninety-five has been reported the clinical trial of the first in the world example dna vaccination treatment acquired immune deficiency syndrome (AIDS), and 1998-2007 has reported the phase ii clinical trial and the clinical application report of dna vaccination treatment acquired immune deficiency syndrome (AIDS) in succession.Experiment showed, dna vaccination because the antigen of its coding can be offered by antigen presenting cell by external source and endogenous two kinds of approach, so can evoke comprehensive immune response.The core of dna vaccination technology is exactly foreign gene to be assembled into contain on the eucaryon plasmid expression vector that is necessary expression regulation element, plasmid with reorganization imports in the animal body then, foreign gene is expressed in vivo, thereby activate the immunity system of body, cause immune response.Compare with traditional vaccine, dna vaccination has a lot of advantages, and is relatively stable as vaccine, convenient production, transportation and preservation and cheap; Designability is strong; It is convenient to detect, check.Experiment showed, dna vaccination because the antigen of its coding can be offered by antigen presenting cell by external source and endogenous two kinds of approach, so can evoke comprehensive immune response.Therefore, the dna vaccination technology is used on the study on prevention of infectious diseases such as hepatitis, AIDS, tuberculosis very soon.Dna vaccination was used on the study on prevention of tumour in recent years, had obtained bigger progress, and part tumour dna vaccination has entered clinical experimental stage.The immunity of organism tolerance be can break according to the full-length gene vaccine of tumor associated antigen (Tumor Associated Antigens:TAAs) development,, B cell response and CD4+, CD8+T cell response caused by MHC-I and MHC-II type angtigen presentation approach.Thereby effectively protect body to avoid the invasion and attack of tumour.The investigator is arranged by making up the method for homologous dna vaccine between biology not of the same race, for a new thinking has been opened up in the development of tumor gene vaccine.Potential unfavorable factor of TAAs full length DNA vaccine is exactly the biological action that the TAAs expression in vivo produces.As some TAAs itself is exactly oncoprotein, and they may play effect (Fong et al., 2001 of signaling molecule in the canceration process; Pupa et al., 2005).Zhu etc. (Zhu et al., 2001) as adjuvant, merge SvFv with the avirulent C fragment of tetanus toxin (Frc), make up the Id dna vaccination, found that this vaccine not only has effect to this Id positive tumor, and are equally effective to negative tumour.The antigen MUC 1 that epithelial cancer is relevant is a kind of mucoitin of high glycosylation, and the epithelial cell cancerization in many glands source is relevant with cell excessive secretion MUC1.Immunization experiment shows that the MUC1 gene vaccine has certain result of treatment for the patient of prostate gland and mammary cancer, further clinical estimate (Stevenson et al., 2004) of second phase.The Methionin kinases HER2 (neu of overexpression growth hormone receptor in 20% to 30% mammary cancer and ovarian cancer patient's the cancer cells, c-erbB-1), mouse tumor model experimental results show that the MMTV-HER2 gene vaccine can excite ctl response and antibody at body, further the experiment of immunocyte disappearance shows it only is that vaccine-induced antibody response itself just can effectively be treated tumour (Renard et al., 2003).
Cyp26a1 (retinoic acid hydroxylase) claim P450RA1 again, be Cytochrome P450 (Cyp) family member, although CYP26 has similar structure to the member of Cytochrome P450 family, but any member of it and Cytochrome P450 family does not have homology, therefore is considered to a newcomer of the gene family of Cytochrome P450.Discover Cyp26 vitamin A acid (RA) optionally can be converted into the polar metabolite of RA (4-OH-, 18-OH-, 4-oxo-RA).Cyp26a1 is by regulating RA level and then control retinoid signal.Feed the feed of raising the A that is deficient in vitamin to animal, fetal development occurs unusual, and the embryo accepts too much retinoid signal also can destroy embryo's normal development.Give rat uterus matrix that RA can reduce estrogen-induced and myocyte's hyperplasia.Can infer that thus endogenic retinoid may prevent the generation of reproductive tract tumour.People such as Piamsomboon report that retinoid is clinical has certain effect to reproductive system treatment for cancer and chemoprophylaxis, and the tissue specificity of RA product distributes and their adjusting will help to formulate the treatment for cancer strategy under the effect of sexual hormoue.People such as Warrell find that also RA is effective to the prevention and the treatment of certain cancers.There is the target gene expression that experimental results show that the RA adjusting relevant recently with the anticancer signal of retinoid.People such as Edwin find that in 1999 Cyp26a1 is a key molecule of embryo cells differentiation.
Summary of the invention
The inventor adopts the subtractive hybridization technology (SSH) that suppresses, uterus difference expression gene before and after the screening rat is implanted, and by RT-PCR and Northern blotting experiment confirm: Cyp26a1 gene embryo is a difference expression gene before and after implanting.Clone the cDNA total length of SD rat uterus Cyp26a1 gene first with the RACE method, and logined GenBank (accession number: DQ317305).This gene and construction of eukaryotic expression vector Cyp26a1DNA vaccine are model with mouse 4T1 breast tumor, carry out repeatedly immune anti-tumor experiment, show that the Cyp26a1DNA vaccine has the effect of obvious inhibition tumor growth and migration.
Therefore, the objective of the invention is to, a kind of gene vaccine that is used for the recombinant plasmid of Mammals Antioncogene vaccine and comprises this recombinant plasmid is provided.
Another object of the present invention is, the preparation method and the purposes of above-mentioned recombinant plasmid and gene vaccine is provided.
The objective of the invention is to realize by the following technical solutions.On the one hand, the invention provides a kind of recombinant plasmid that is used for Mammals Antioncogene vaccine, its cDNA fragment and carrier for expression of eukaryon by the Cyp26a1 gene is formed, and carrier for expression of eukaryon is preferably pVAX1.
Preferably, the cDNA fragment of described Cyp26a1 gene is derived from the Spreqne-Dawley rat uterus, and its base sequence is preferably shown in SEQ ID NO.1.
On the other hand, the invention provides the bacterial strain that comprises above-mentioned recombinant plasmid, preferably, described bacterial strain is selected from intestinal bacteria Top10F ' bacterial strain.
The present invention also provides the cell that comprises above-mentioned recombinant plasmid, and preferably, described cell is selected from human cervical carcinoma's (HeLa) cell and people's suede cancer (JEG-3) cell.
Another aspect the invention provides the antineoplastic gene vaccine of a kind of Mammals, wherein comprises above-mentioned recombinant plasmid.
Preferably, described vaccine also comprises adjuvant; More preferably, it is 0.25% PROCAINE HCL, PHARMA GRADE that described adjuvant is selected from percentage concentration, the volume mass proportioning of per injection adjuvant consumption and gene vaccine consumption is 2 (V/ μ l): 1 (W/ μ g), concrete preferred adjuvant consumption and gene vaccine consumption are respectively 100 μ l and 50 μ g.
In addition, the present invention also provides a kind of method for preparing above-mentioned recombinant plasmid, the cDNA fragment of Cyp26a1 gene as described in the base sequence of this method employing shown in SEQ ID NO.2 and SEQ ID NO.3 cloned as primer; Preferably, this method adopts temperature fall reverse transcription-polymerase chain amplification method to clone the cDNA fragment of described Cyp26a1 gene.
Also on the one hand, the invention provides a kind of method of the Cyp26a1 of preparation independent mammary tumor animal model, it is the segmental 4T1 mouse mastopathy cell injection of the cDNA animal's mammary gland position preparation that comprises people source Cyp26a1 gene by use; Preferably, the cDNA fragment of people Cyp26a1 gene as described in the base sequence of this method employing shown in SEQ ID NO.4 and SEQ ID NO.5 cloned as primer.The present invention also provides the Cyp26a1 independent mammary tumor animal model of method for preparing, and preferably, described animal model is a mouse model.
Again on the one hand, the invention provides the purposes of above-mentioned recombinant plasmid in preparation Mammals Antioncogene vaccine, preferably, described Mammals is selected from the people, and described tumour is selected from mammary cancer.
The present invention also provides above-mentioned gene vaccine to prevent and/or treat purposes in the medicine of mammalian milk gland cancer in preparation, and preferably, described Mammals is selected from the people, and described tumour is selected from mammary cancer.In sum, the present invention uses prevention and treatment mammary cancer with the antitumor technology of dna vaccination immunity, thereby provide a kind of dna vaccination that can obviously prevent and/or treat mammary cancer, this vaccine is formed by containing Codocyte cytochrome p 450 family 26 proteic genes of superfamily A polypeptide 1-Cyp26a1 and eucaryon plasmid expression vector pVAX1, the proteic gene of the wherein said Codocyte cytochrome p 450 26 superfamily A polypeptide 1-Cyp26a1 of family is the total length complementary DNA (cDNA) gene of SD rat uterus, and this gene has been logined gene library (Cyp26a1:DQ317305).。Though other eucaryon plasmid expression vectors, vaccine as pCR3.1, pCMV4 vector construction, its expressing quantity is higher than the vaccine of pVAX1 vector construction, immune effect also is better than the vaccine of pVAX1 vector construction, but because preceding two carriers can not be used for the mankind, and the pVAX1 carrier can be directly used in the mankind, approves through U.S. FDA.Research anti-tumor vaccine final purpose is can be directly used in human pVAX1 carrier so chosen for human cytotoxic provides new technology.
The present invention has cloned the cDNA of SD rat uterus total length Cyp26a1 gene first by the RACE method, has successfully made up the pVAX1-Cyp26a1DNA vaccine, and it can efficiently express Cyp26a1 albumen in the HeLa cell.20 μ g pVAX1-Cyp26a1 plasmid DNA are passed through the drug-induced back of ethocaine (bupivacaine-HCI) intramuscular inoculation mouse muscle, mouse can absorb plasmid DNA, and the Cyp26a1 proteantigen of expression coding, the Cyp26a1 that expresses is by the immune system recognition of mouse, the humoral immunization and the cell immune response of simultaneous excitation Cyp26a1 antigen-specific, antibody titers reaches as high as more than 1: 4000, and this immunne response of two types can be at interaction in vitro in HeLa cell and JEG-3 cell, induce it that apoptosis takes place, show that immune response that the pVAX1-Cyp26a1 dna vaccination excites has an antitumor action external.Meanwhile, experimentize with mouse animal Cyp26a1 dependent form 4T1 mammary tumor model and Cyp26a1 independent form 4T1 mammary tumor model, experimental result shows: compare with control group, the Cyp26a1DNA vaccine has the effect of obvious inhibition tumor growth and migration.But the advantage of this vaccine little effect that is dosage obviously, safely, be applicable to all kinds of crowd's mass production and be convenient to accumulating and convenient the use.
This dna vaccination can cause CTL as therapeutic vaccine, yet the effect of treatment tumour is but as one wishes not to the utmost sometimes, discover that further the CTL effect generally occurs in the early stage of tumor growth, the CTL effect of antigen stimulation is strengthened to greatly when tumour is grown fast, late in the tumour, the cytotoxic T cell effect begins forfeiture gradually, so the vaccine therapy of infantile tumour can be obtained curative effect preferably by the ctl response of strengthening, but since the CD8+T cell function weaken and tumour to immune escape, the gene vaccine result of treatment of late tumor is just had a greatly reduced quality.This shows, when adopting gene vaccine treatment tumour, the curative effect of infantile tumour is better than curative effect to late tumor.
This shows that the advantage of pVAX1-Cyp26a1DNA vaccine provided by the present invention is: (1) required immunity amount is less, and 20～100 micrograms get final product; (2) dna vaccination can efficiently excite humoral immunization and cellular immunization simultaneously, and is stronger than other immune means immune efficacy; (3) has the effect of effective inhibition tumor growth and migration; (4) genetic immunization safety, long-acting (immunological competence has long memory), stable, simple operation are beneficial to industrialization production.
Description of drawings
Fig. 1 is the total RNA electrophorogram in pregnant rat uterus in the embodiment of the invention 1.
Fig. 2 is by the cDNA fragment of Cyp26a1 gene and the recombinant plasmid spectrogram of carrier for expression of eukaryon pVAX1 structure in the embodiment of the invention 1.
Fig. 3 is the distribution expression pattern that RT-PCR analyzes CYP26A 1 gene in the embodiment of the invention 4, and wherein A is the uterus, and B is an ovary, and C is a hypothalamus, and D is a hypophysis, and E is the heart, and F is a liver, and G is a spleen, and H is a lung, and I is a kidney, and J is a stomach, and K is a small intestine, and L is a large intestine.
Embodiment
Below the invention will be further described by specific embodiment.Should be understood that following examples only are used to illustrate the present invention, and be not used in the scope of the present invention that limits.
In following each embodiment, related extraction cell total rna, enzyme cut, the concrete operation method of plasmid purification, RT-RCR method, ELISA method, cell transfecting equimolecular biological means and technology sees also works such as " fine works molecular biology experiment guide " F. Ao Sibai, Yan Ziying etc. translate, Science Press, 1998.
In following each embodiment, employed laboratory animal Spreqne-Dawley rat and BaLb/c mouse are all available from Beijing Vital River Experimental Animals Technology Co., Ltd..
Embodiment 1: make up the pVAX1-Cyp26a1 dna vaccination
Present embodiment is the structure of pVAX1-Cyp26a1DNA vaccine provided by the present invention, and concrete steps are as follows:
(1) animal tissues draws materials: optional maturation, the Spreqne-Dawley female rats of body weight 220-260g mates mating in oestrus and male rat (1: 1), gets pregnant D3-D6 uterus.(2) extraction of the total RNA of gravid uterus and purifying: adopt the Promega RNAgents of company
Total RNA extraction system (Total RNA Isolation System kit) is extracted the total RNA of gravid uterus, concrete operations are as follows: the uterine cancer cell of collecting is extracted total tissue RNA with the TRIzol single stage method, the about 50-100mg of 1mlTRIzol and uterine cancer cell) adds in the homogenate pipe of sterilization, change the Eppendoff pipe after the homogenate fragmentation over to, add the 0.2ml chloroform, shake 15sec, room temperature leaves standstill 2-3min, 12, the centrifugal 15min of 000rpm (4 ℃) gets supernatant to managing with new Eppendoff, add the 0.5ml Virahol, mixing gently, room temperature leaves standstill 10min, and 12, the centrifugal 10min of 000rpm (4 ℃), abandon supernatant, add 1ml 75% ethanol washing and precipitating, 7, the centrifugal 5min of 500rpm (4 ℃) (this step repeats twice), the vacuum seasoning, the water 500 μ L that add nuclease free (Nuclease-free) dissolve-80 ℃ of preservations.Get 5 μ L sample solutions and carry out 1.5% denaturing formaldehyde sepharose (containing 0.2 μ g/mLEB) electrophoresis detection (Jian-Jun Chang, Jing-Pian Peng
*YingYang, Jing-LingWang, LiXu (2006) Study on the Anti-fertility Effects of the Plasmid DNA Vaccine Expressing Partial brLDH-C4.Reprod.131:183-192).Gel band analytical results as shown in Figure 1, the amount that shows 28S rRNA in the prepared sample is about two times of 18S rRNA, this result has proved the integrity of total RNA.
(3) design Auele Specific Primer: the Auele Specific Primer (having restriction enzyme site) that designs amplification total length Cyp26a1 gene according to the highly conserved sequence of people, mouse Cyp26a1 gene order beginning codon and terminator codon outer end:
Upstream primer (SEQ ID NO.3):
5′-CG
A?AGC?TT(HindIII)A?TGG?GGC?TCC?CGG?CGC?TGC?T-3′；
Downstream primer (SEQ ID NO.4):
5′-CG
CTC?GAG(XhoI)TCAGATATCTCCCTGGAAGTGG-3′。
(4) clone Cyp26a1 gene: adopt temperature fall reverse transcription-polymerase chain amplification method (TDRT-PCR, Touch-down reverse transcription polymerase chain reaction) the cDNA sequence of clonal expansion rat uterus Cyp26a1 gene, concrete operations are as follows: the reaction system of 50 μ L is by AMV/Tf1 5 * reaction buffer of 10 μ L, the dNTPs mixture of 2mM, the MgSO of 2mM
4, the upstream primer of 1 μ M and upstream primer, the AMV reversed transcriptive enzyme (promega product, article No. M1701 is available from the flat science and technology limited Company in pool, Beijing) of 0.1U/ μ L, the RNasin of 0.1U/ μ L
The total RNA of nucleic acid inhibitor (available from Sigma company), 2 μ g and the water of nuclease free are formed.Reverse transcription reaction is at 48 ℃ of reaction 45min, then at 95 ℃ of deactivation 5min, Tf1 archaeal dna polymerase (the promega product that adds 0.1U/ μ L, available from Beijing Yili Fine Chemicals Co., Ltd.), in the landing TD-PCR amplification stage, denaturation temperature is 94 ℃ of 1min, and annealing temperature is from beginning 65 ℃ of 1min, speed with 1 ℃ of per two circulation decline is reduced to 55 ℃ of 1min, and elongating temperature is 68 ℃ of 1.5min; Continuing 15 circulations of amplification with 58 ℃ of annealing temperatures at last.Extend 10min at 72 ℃ at last.
(5) reclaim purifying Cyp26a1 cDNA:, utilize Wizard with the Cyp26a1 gene fragment of amplification
PCR Preps.DNA resin purification system reagent box (promega product, article No. A7170 is available from Beijing Yili Fine Chemicals Co., Ltd.) reclaims purifying;
The present invention clones the full-length gene of rat uterus Cyp26a1 first, and its sequence is shown in SEQ IDNO.2, and its GeneBank accession number is DQ317305.
(6) make up the pVAX1-Cyp26a1DNA vaccine: with the cDNA fragment of Cyp26a1 full length amino acid encoding sequence pVAX1 eukaryon expression plasmid (the Invitrogen product of recombinating, available from the leading Science and Technology Ltd. in Central Plains, Beijing) in, be built into pVAX1-Cyp26a1 DNA recombinant plasmid, concrete plasmid map is seen Fig. 2, the recombinant plasmid that is built into is antineoplastic pVAX1-Cyp26a1 gene vaccine.Above-mentioned concrete operation is as follows: with ligation liquid transformed competence colibacillus cell Top10F ' (available from the Beijing Quanshijin Biotechnology Co., Ltd), get bacterium liquid coating LB (the containing penbritin) flat board that 80 μ L transform, in 37 ℃ of overnight incubation; The picking positive colony shakes bacterium and spends the night, and extracts plasmid DNA with the alkali cracking method, i.e. the pVAX1-Cyp26a1 of present embodiment carries out enzyme and cuts and identify and positive recombinant plasmid is checked order; To have forward inserted the recombinant clone plasmid of Cyp26a1 amplified production exogenous dna fragment purified after, utilize cloned plasmids sequencing primer M13 reverse primer (reverseprimer) and T7 promoter primer (promoter primer) (available from Beijing AudioCodes biotechnology limited liability company) to the two-way order-checking of Cyp26a1 amplified production, its nucleotide sequence is shown in SEQ ID NO.1.Through enzyme cut and check order identify correct after, extract pVAX1-Cyp26a1 in a large number according to the big upgrading grain of Qiagen test kit operation steps, utilize ultraviolet spectrophotometer (U.S. Beckman, model DU530) quantitative.
Embodiment 2: make up pEGFP-Cyp26a1 eukaryon expression plasmid and Cyp26a1 independent mammary tumor model
Present embodiment is the structure of pEGFP-Cyp26a1 eukaryon expression plasmid and Cyp26a1 independent mammary tumor model, and concrete steps are as follows:
(1) animal tissues draws materials: choose 2-3 month miscarriage placenta (taking from the birth control outpatient service of north doctor three institutes) of people's gestation.
(2) extraction of the total RNA of gravid uterus and purifying: adopt the Promega RNAgents of company
Total RNA Isolation System kit extracts the total RNA of pregnant placenta, concrete operations are as follows: the placenta tissue of collecting is extracted total tissue RNA with the TRIzol single stage method, the about 50-100mg of 1ml TRIzol and placenta tissue) adds in the homogenate pipe of sterilization, change the Eppdoff pipe after the homogenate fragmentation over to, add the 0.2ml chloroform, shake 15sec, room temperature leaves standstill 2-3min, 12, the centrifugal 15min of 000rpm (4 ℃) gets supernatant to managing with new Eppdoff, add the 0.5ml Virahol, mixing gently, room temperature leaves standstill 10min, and 12, the centrifugal 10min of 000rpm (4 ℃), abandon supernatant, add 1ml 75% ethanol washing and precipitating, 7, the centrifugal 5min of 500rpm (4 ℃), the vacuum seasoning adds Nuclease-free water 500 μ L dissolving ,-80 ℃ of preservations.Get 5 μ L sample solutions and carry out the integrity of 1.5% denaturing formaldehyde sepharose (containing EB0.2 μ g/mL) the total RNA of electrophoresis detection.
(3) design Auele Specific Primer: according to people Cyp26a1 gene order (GeneBank number: the NP_000774) Auele Specific Primer (having restriction enzyme site) of the highly conserved sequence design amplification total length people Cyp26a1 gene of beginning codon and terminator codon outer end:
The Auele Specific Primer (having restriction enzyme site Kpn1) of human cloning placenta tissue Cyp26a1 gene:
Upstream primer (SEQ ID NO.5):
5′-CGG
GGTACC(Kpn1)ATGGGGCTCCCGGCGCT-3′；
Downstream primer (SEQ ID NO.6):
5′-TCATCAG
GGTACC(Kpn1)CCATGGAAATGG-3′。
(4) clone Cyp26a1 gene: adopt TDRT-PCR clonal expansion human placenta Cyp26a1 gene cDNA sequence, concrete operations are as follows: the reaction system of 50 μ L is by AMV/Tf1 5 * reaction buffer of 10 μ L, the dNTPs mixture of 2mM, the MgSO of 2mM
4, 1 μ M upstream primer and upstream primer, the RNasin of the AMV reversed transcriptive enzyme of 0.1U/ μ L, 0.1U μ L
The total RNA of ribonuclease inhibitor (Ribonuclease Inhibitor), 2 μ g and the water of nuclease free are formed.Reverse transcription reaction then at 95 ℃ of deactivation 5min, adds the Tf1 archaeal dna polymerase of 0.1U/ μ L at 48 ℃ of reaction 45min, denaturation temperature is 94 ℃ of 1min, annealing temperature is reduced to 55 ℃ of 1min from beginning 65 ℃ of 1min with the speed of 1 ℃ of per two circulation decline, and elongating temperature is 69 ℃ of 1.5min; Continuing 20 circulations of amplification with 58 ℃ of min of annealing temperature at last.Extend 10min at 72 ℃ at last.
(5) reclaim purifying Cyp26a1 cDNA:, utilize Wizard with the Cyp26a1 gene fragment of amplification
PCR Preps.DNA resin purification system reagent box reclaims purifying;
(6) make up the pEGFP-Cyp26a1 eukaryon expression plasmid: the cDNA fragment of people Cyp26a1 full length amino acid encoding sequence is recombinated among the carrier for expression of eukaryon pEGFP (available from BD Biosciences company) that has the green fluorescent protein mark, be built into the pEGFP-Cyp26a1 eukaryon expression plasmid, concrete operations are as follows: with ligation liquid transformed competence colibacillus cell Top10F ', get bacterium liquid coating LB (the containing penbritin) flat board that 80 μ L transform, in 37 ℃ of overnight incubation; The picking positive colony shakes bacterium and spends the night, and carries out enzyme and cuts and identify and positive recombinant plasmid is checked order; Selection has forward to insert the recombinant clone plasmid of exogenous dna fragment; The correct bacterium of checking order shakes bacterium once more, does not have the intracellular toxin test kit with Qiagen and extracts, and obtains the pEGFP-Cyp26a1 plasmid, and is standby.
(7) make up Cyp26a1 independent mammary tumor model: with the pEGFP-Cyp26a1 eukaryon expression plasmid through liposome transfection 4T1 mouse mastopathy cell (available from the biological product ATCC of collecting center of USS), concrete operations are as follows: cover with mouse breast cancer 4T1 cell and use the DMEM nutrient solution of serum-free to wash twice in 24 orifice plates, 2 μ g pEGFP-Cyp26a1 (containing people Cyp26a1 gene) plasmid is joined in the DMEM nutrient solution of 100 μ L serum-frees mixing gently, 5 μ L liposomes join in the DMEM nutrient solution of 95 μ L serum-frees mixing gently.Put 37 ℃, 5%CO
2Cultivate after 12 hours, change and add the complete culture solution that contains 10% foetal calf serum and continue to cultivate after 12 hours cell, after 24 hours, in nutrient solution, add 800 μ g/mLG418 with cultivating in tryptic digestion to the 60 millimeter culture dish.After about 2 weeks, in culture dish, form cell colony; Add 1mL0.25% trypsinase+0.04%EDTA Digestive system.Treat the cell complete digestion become unicellular after, stop digestion.Behind the 500g eccentric cell 5 minutes, cell sieved the agglomerating cell of elimination with 100/200 order cell.The unicellular resuspended one-tenth single cell suspension of DMEM of using after the filtration; Selected by flow cytometry apoptosis 488nm positive cells.Unicellular with normal 4T1 as the sorting negative control; The positive cell that sub-elects continues to cultivate, after the repeated screening three times, cell flow cytometer (FACS-vantage/Diva is available from U.S. BD Biosciences) and fluorescent microscope (Nikon 80i Microscope System, cold CCDSPOT, Japanese Nikon) purity assay.Fluorescent microscope and flow cytometer showed prove that Cyp26a1-EGFP express cell purity is 97.8%; 1 * 10
5The mammary gland position of containing the 4T1 cell subcutaneous injection BALB/C mice of people Cyp26a1 gene, the 9th day tumor formation rate 100% (N=20) set up and finished people Cyp26a1 independent mammary tumor model.
Embodiment 3: the external transient expression of pVAX1-Cyp26a1 dna vaccination detects
Present embodiment is the pVAX1-Cyp26a1 dna vaccination constructed to embodiment 1, carries out the detection of the outer transient expression of external source Cyp26a1 genosome, is divided into Cyp26a1 gene in vitro mRNA and expresses and the external protein expression two portions of external source Cyp26a1.
(1) Cyp26a1 gene in vitro mRNA expresses:
Set up the external transient expression system of HeLa cell (human cervical carcinoma cell) and JEG-3 cell (people's suede cancer cells) (available from basis institute of Beijing consonance medical university cell centre), concrete operations are as follows: the pVAX1-Cyp26a1 eukaryon expression plasmid is through liposome transfection, respectively at 24 hours, 48 hours, 72 hours collection nutrient solutions, extract the transfection HeLa cell of collection and total RNA of JEG-3 cell, adopt RT-PCR methods analyst Cyp26a1 gene in vitro to express.The RT-PCR method amplifies Cyp26a1 cDNA band, the result shows that the PVAX1-Cyp26a1 eukaryon expression plasmid is in HeLa cell and JEG-3 cells in vitro transient expression system, can efficiently express on the mRNA level, the expression amount of mRNA level exceeds 172% than empty plasmid control group.
(2) the external protein expression of external source Cyp26a1:
Adopt the method for Western-Blotting to detect transfection HeLa cell and the proteic expression of JEG-3 cell Cyp26a1, concrete operations are as follows: get HeLa cell and JEG-3 cell behind the transfection 48h: in Tissue Culture Flask or culture plate with PBS or physiological saline washed cell 2 times, add lysis liquid RIPA buffer (available from Beijing Puli's lema gene technology limited liability company), shake ice bath 10-15min.Scrape cell with cell, change in the EP pipe, detect protein content with the Bio-ford method.Get 50 μ g total proteins and add sample buffer on the albumen, boil sex change 5min, carry out SDS-PAGE, concentrating the peptization degree is 5%, separation gel solubility is 12.%, wet method forwards on the nitrocellulose filter, 5% bovine serum albumin sealing 1 hour, the washing film adds mouse-anti Cyp26a1 antibody (available from Acris Germany company), after carry out the washing film three times, drip anti-mouse two anti-(the Jackson products of HRP mark on the film, available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing), ECL colour developing, darkroom X-ray film developing.X-ray sheet HP scanner gray scale scanning.The result confirms that pVAX1-Cyp26a1 can express the Cyp26a1 albumen of biologically active in Hela cell JEG-3 cells in vitro transient expression system, and is higher by 165% than empty plasmid control group.
Embodiment 4: the expression in vivo of pVAX1-Cyp26a1 dna vaccination detects
Present embodiment is the pVAX1-Cyp26a1 DNA gene vaccine constructed to embodiment 1, and the expression in vivo that carries out external source Cyp26a1 gene detects, and concrete operations are as follows:
BALB/C mice is divided into 3 groups at random, and 20 every group, experimental group is intramuscular injection 50 μ gpVAX1-Cyp26a1DNA vaccines; Control group 1 intramuscular injection 50 μ g pVAX1 empty plasmid DNA; Control group 2 intramuscular injection 100 μ L physiological saline.Muscle, liver and the spleen tissue of the 3rd week back collection pVAX1-Cyp26a1 immune balb/c mice extract total tissue RNA, and RT-PCR detects external source Cyp26a1 gene at the intravital expression of BALB/C mice.Detected result shows, behind the pVAX1-Cyp26a1DNA vaccine immunity in muscle, liver and the spleen tissue cell Cyp26a1 on the mRNA level, express, the result is as shown in Figure 2.
Embodiment 5: pVAX1-Cyp26a1 dna vaccination humoral immunoresponse(HI) detects
Present embodiment detects the BALB/C mice humoral immunoresponse(HI) for using the constructed pVAX1-Cyp26a1 dna vaccination of embodiment 1, and concrete operations are as follows:
Uterogolbin with the BALB/C mice purifying in 2 μ g/ holes is made envelope antigen (96 orifice plate), and 4 ℃ of bags are spent the night, and PBST washes plate, 5min/ time, washes the sealing of 1%BSA+5% skim-milk, 37 ℃, 2h altogether 3 times; PBST washes plate, 5min/ time, wash 1 time, with the dilution proportion of the serum of pVAX1-Cyp26a1DNA vaccine immune BALB/C mouse after three weeks by 1: 100 to 1: 6000, anti-as one, with the serum of the intramuscular injection 50 μ g pVAX1 empty plasmid DNA of the normal serum (not injecting any plasmid) of 1: 100 times of dilution and 1: 100 times of dilution respectively in contrast, 37 ℃, 2h; PBST washes plate, and 5min/ time, wash altogether 5 times, add two anti-(the anti-mouse IgG of HRP mark is available from U.S. Santa Cruz companies) of suitably dilution, 37 ℃, 1h; PBST washes plate, 5min/ time, washes altogether 5 times; Add TMB reagent colour development (R﹠amp; D Systems Inc, USA); Add 1M H
2SO
4Termination reaction is with reading plate device (Bio-Rad) in the A450 reading numerical values.Detected result shows that body can produce high titre antibody (antibody titers＞1: 5000) behind the pVAX1-Cyp26a1DNA vaccine immunity.
Embodiment 6: pVAX1-Cyp26a1 dna vaccination cellullar immunologic response detects
Present embodiment detects the BALB/C mice cellullar immunologic response for using the constructed pVAX1-Cyp26a1 dna vaccination of embodiment 1, comprises that T lymphocyte cytotoxicity detects and NK cytoactive detection two portions.
(1) T lymphocyte cytotoxicity (CTL) detects:
BALB/C mice is divided into 3 groups at random, and 10 every group, experimental group is intramuscular injection 50 μ gpVAX1-Cyp26a1 dna vaccinations; Control group 1 is intramuscular injection 50 μ g pVAX1 empty plasmid DNA; Control group 2 is intramuscular injection 100 μ L physiological saline.The T lymphocyte of mouse is respectively organized in the back collection of the 3rd week, detect effector cell (Effector cells) as CTL and press the Cyp26a1 albumen of about 5 * 106 cells/mL and 2 μ g/mL purifying as the differential stimulus antigenic action, in CTL nutrient solution (1640-10%FBS) (available from GIBCO company) in 37 ℃, 5%CO
2Cultivate 5d altogether, the BSA negative control.(E: T) ratio was from 50: 1,25: 1 to 12.5: 1, and behind the effect 6h, every hole adds the CellTiter 96 of 20 μ L for Effector: Target
The single solution cell proliferation detection reagent (available from promega company) of Aqueous, 37 ℃, 5%CO
2The middle 4h that cultivates reads sample OD490nm absorption value.The result shows that the pVAX1-Cyp26a1 dna vaccination is induced the cellullar immunologic response that has produced antigen-specific in the BALB/c mouse body.
(2) natural killer (NK) cytoactive detects:
The effector cell (Effector cells) that the lymphocyte single cell suspension of not doing the sensitization processing of experimental group, control group 1 and control group 2 detects as the NK cytoactive adjusts concentration to 2 * 10 with the 1640-5%FBS nutrient solution that contains 50 μ M 2-ME
6Cell/mL, the JEG-3 cell is as target cell (Target cells).The JEG-3 cell of 50 μ L and the spleen lymphocyte of 50 μ L be at 37 ℃, 5%CO
2Middle effect 18h establishes 50: 1,25: 1 to 12.5: 1 Effector: Target (E: T) ratio.Similarly, add CellTiter 96 again
After the single solution cell proliferation of Aqueous detection reagent is cultivated 4h, read sample OD490nm absorption value.Detected result shows that body can produce immunization of cell after the immunity of pVAX1-Cyp26a1 dna vaccination, and promptly the NK cytoactive has the specific killing active function.
Embodiment 7: the pVAX1-Cyp26a1 dna vaccination anti-tumor experiment that exsomatizes
Present embodiment adopts the TUNEL experiment for using the constructed pVAX1-Cyp26a1 dna vaccination of embodiment 1, and anti-tumor experiment exsomatizes.
The 5th week was got the T lymphocyte behind the pVAX1-Cyp26a1DNA vaccine immune mouse, and the anticancer experiment in vitro that carries out of immune serum, found that the T lymphocyte of pVAX1-Cyp26a1 dna vaccination immune mouse can apoptosis take place at external attack human breast cancer cell MDA-MB-231, human breast cancer cell MCF7 cell (available from basis institute of Beijing consonance medical university cell centre); The serum of immune mouse also can act on MDA-MB-231 and MCF7 simultaneously, induces MDA-MB-231 and MCF7 cell generation apoptosis.
This shows, the pVAX1-Cyp26a1 dna vaccination not only can excite mouse to produce corresponding humoral immunization and cell immune response, and this two types the immunne response that excites all can act on human breast cancer cell MDA-MB-231 and MCF7, induce it that apoptosis takes place, the effect of kill tumor cell is promptly arranged.
Embodiment 8: the experiment of pVAX1-Cyp26a1 dna vaccination anti-tumor in vivo
Present embodiment carries out the anti-tumor in vivo experiment for using the constructed pVAX1-Cyp26a1 dna vaccination of embodiment 1 to BALB/C mice.
(1) anti-tumor in vivo experiment-I
Available from 80 of the female BALB/C mice of Beijing Vital River Experimental Animals Technology Co., Ltd., animal rearing under manually operated condition, raising temperature about 25 ℃, 12h:12h periodicity of illumination, free choice feeding, drinking-water.80 are divided into 4 groups at random, and 20 every group, 24h injects 100 μ L immunological adjuvants, 0.25% PROCAINE HCL, PHARMA GRADE before the immune plasmid, alcohol partly sterilised before the injection, 3 injections of inoculation position leg muscle.
The 1st group: do not inoculate any material, the proteic 4T1 mouse mastopathy cells of Cyp26a1 are expressed in 3 week back injections, i.e. embodiment 2 " the people Cyp26a1 independent mammary tumor model " set up.
The 2nd group: inoculate 100 μ l physiological saline, the proteic 4T1 mouse mastopathy cell of Cyp26a1 is expressed in the back injection of 3 weeks.
The 3rd group: inoculate 100 μ l pVAX1 empty plasmids, the proteic 4T1 mouse mastopathy cell of Cyp26a1 is expressed in the back injection of 3 weeks
The 4th group: inoculate 100 μ l pVAX1-Cyp26a1DNA vaccines, the proteic 4T1 mouse mastopathy cell of Cyp26a1 is expressed in the back injection of 3 weeks.
Check the mouse tumor growing state after 10 days.
Observations is as follows:
The 1st group, the 2nd group, the 3rd group
Check after 10 days that 60 mouse all have tumor mass, its size is 7-11mm
3
Check after 20 days, 60 mouse, 58 have only tumor mass, and its size is 15-18mm
3, 2 dead mouses wherein, other mouse hair pine is dredged wan.
Check after 30 days, 58 mouse, 39 have only tumor mass, its big or small 17-26mm
3, 19 dead mouses wherein, other mouse hair pine is dredged wan
Check after 50 days, 39 mouse, 2 have only tumor mass, and its size is 25-38mm
3, wherein 37 mouse are all dead.
Above-mentioned dead mouse lung is all found the metastatic tumour piece.
The 4th group
Check after 10 days, 20 mouse, 5 have only tumor mass, and its size is 3-6mm
3
Check after 20 days, 20 mouse, 8 have only tumor mass, and its size is 4-10mm
3, other 12 mouse are all healthy.
Check after 30 days, 20 mouse, 9 have only tumor mass, and its size is 11-24mm
3, 4 dead mouses wherein.
Check after 50 days, 16 mouse, 6 have only tumor mass, and its size is 12-28mm
3, other 10 mouse are still healthy.
Above-mentioned dead mouse lung is all found the metastatic tumour piece.
Compare with in contrast the 1st, 2 and 3 group, the 4th winding kind pVAX1-Cyp26a1 dna vaccination has tangible anti-Cyp26a1 dependent form mammary cancer effect (p＜0.01), has clinical value.
(2) anti-tumor in vivo experiment-II
Available from 60 of the female BALB/C mice of Beijing Vital River Experimental Animals Technology Co., Ltd., animal rearing under manually operated condition, raising temperature about 25 ℃, 12h:12h periodicity of illumination, free choice feeding, drinking-water.60 are divided into 4 groups at random, and 15 every group, 24h injects 100 μ L immunological adjuvants, 0.25% PROCAINE HCL, PHARMA GRADE before the immune plasmid, alcohol partly sterilised before the injection, 3 injections of inoculation position leg muscle.
The 1st group: do not inoculate any material, the proteic 4T1 mouse mastopathy cell of Cyp26a1 is expressed in the back injection of 3 weeks.
The 2nd group: inoculate 100 μ l physiological saline, the proteic 4T1 mouse mastopathy cell of Cyp26a1 is expressed in the back injection of 3 weeks.
The 3rd group: inoculate 100 μ l pVAX1 empty plasmids, the proteic 4T1 mouse mastopathy cell of Cyp26a1 is expressed in the back injection of 3 weeks
The 4th group: inoculate 100 μ l pVAX1-Cyp26a1DNA vaccines, the proteic 4T1 mouse mastopathy cell of Cyp26a1 is expressed in the back injection of 3 weeks.
Check the mouse tumor growing state after 10 days.
Observations is as follows:
The 1st group, the 2nd group, the 3rd group
Check after 10 days that 45 mouse are all found tumor mass, its size is 6.5-13mm
3
Check after 20 days, 45 mouse, 41 have only tumor mass, and its size is 14-18mm
3, 4 dead mouses wherein, other mouse hair pine is dredged wan.
Check after 30 days, 41 mouse, 24 have only tumor mass, and its size is 19-28mm
3, 17 dead mouses wherein, other mouse hair pine is dredged wan.
Check after 50 days, 24 mouse, 3 have only tumor mass, and its size is 27-36mm
3, 21 dead mouses wherein.
Above-mentioned dead mouse lung is all found the metastatic tumour piece.
The 4th group
Check after 10 days, 15 mouse, 3 have only tumor mass, its big or small 4-7mm
3
Check after 20 days, 15 mouse, 5 have only tumor mass, and its size is 6-14mm
3, other 10 mouse are all healthy.
Check after 30 days, 15 mouse, 3 have only tumor mass, and its size is 9-26mm
3, 3 dead mouses wherein.
Check after 50 days, 12 mouse, 4 have only tumor mass, and its size is 15-33mm
3, 1 dead mouse wherein, other 7 mouse are still healthy.
Above-mentioned dead mouse lung is all found the metastatic tumour piece
Repeat anti-tumor in vivo embodiment, result and embodiment-I are similar, show that the effect of the anti-Cyp26a1 dependent form of pVAX1-Cyp26a1 dna vaccination tool mammary cancer is stable, reliable.
Embodiment 10: the safety evaluation of pVAX1-Cyp26a1 dna vaccination
The constructed pVAX1-Cyp26a1 gene vaccine of present embodiment assessment embodiment 1 is to the security of immune animal, and concrete operations are as follows:
Detect 45 BALB/C mice of various dose (20 μ g-300 μ g) pVAX1-Cyp26a1 dna vaccination immunity, none is only dead after 180 days; Carrying out the toxicity behind the various dose pVAX1-Cyp26a1 dna vaccination injection animal and the observation of conventional physical signs measures, utilize histochemical method, (concrete grammar is referring to " practical immunocytochemistry and making nucleic acid molecular hybridization technology " Cai Wenqin, Wang uncle Yun chief editor, Sichuan science tech publishing house, 1994) determine animal tissues's pathological change after the vaccinate: respective organization organs such as brain, the heart, liver, spleen, lung, kidney, uterus and muscle tissue are carried out frozen section, at microscope (Nikon 80i Microscope System; Cold CCD SPOT, Japanese Nikon) observe down, do not find that there is pathological change in animal tissues.
Sequence table
＜110〉Institute of Zoology, Academia Sinica
＜120〉antitumor recombinant plasmid, gene vaccine and its production and use
<130>DIC09110032
<160>5
<170>PatentIn?version?3.3
<210>1
<211>1766
<212>DNA
＜213〉SD rat Cyp26a1cDNA
<400>1
acgcgggggc?gagggcggcg?gcggcaggtg?gcgcgggagg?cttgctgcgt?gccatggggc 60
tcccggcgct?gctggccagt?gctctctgca?ccttcgtgct?gccgctgctg?ctcttcctgg 120
cggcgctcaa?gctctgggac?ctgtactgtg?tgagcagccg?cgatcgcagc?tgcgctctcc 180
ccttgccccc?gggtaccatg?ggcttcccat?tctttgggga?aacattgcag?atggtgctgc 240
agcggaggaa?gtttctgcag?atgaagcgca?ggaaatacgg?cttcatctac?aagacgcatc 300
tgtttgggcg?gcccacggtg?cgagtgatgg?gcgcggataa?tgtgcggcgc?atcttgctgg 360
gggagcaccg?gttggtgtca?gtgcactggc?ctgcttcggt?gcgcaccatc?ctgggcgccg 420
gctgcctctc?caacctgcat?gattcctcgc?acaagcagcg?aaagaaggtg?attatgcagg 480
ccttcaaccg?agaggcgctt?cagtgctacg?tgccagtgat?tgctgaagaa?gtgagcggtt 540
gtctggagca?gtggctaagc?tgcggcgagc?gcggcctcct?ggtctacccc?gaggtgaagc 600
gcctcatgtt?ccgcatcgcc?atgcgcatcc?tgctgggctg?cgagccgggt?ccagcgggcg 660
gcggggaaga?cgagcagcag?ctagtggagg?ctttcgagga?gatgacccgc?aatctcttct 720
ctctccccat?tgacgtgccc?tttagcgggc?tgtaccgggg?cgtgaaggcg?cggaacctta 780
tccacgcgcg?catcgaggag?aacattcggg?ccaagatccg?ccggcttcag?gccgcagagc 840
cggatgcggg?ctgcaaggac?gcactgcagc?tcttgattga?gcactcatgg?gagagaggag 900
agaggctgga?tatgcaggca?ctaaaacaat?cgtccacaga?gctcctcttt?ggtggccatg 960
aaactacagc?cagtgcagcc?acatcactga?tcacctacct?aggactctac?ccacatgtcc 1020
tccaaaaagt?tcgagaagag?ataaagagca?agggcttact?ttgcaagagc?catcacgagg 1080
acaagttaga?catggaaact?ttggaacagc?tcaaatacat?tgggtgtgtt?attaaggaga 1140
cccttcgatt?gaatcctccg?gttccgggag?ggtttcgggt?ggctctgaag?acttttgagc 1200
tgaacggtta?ccagattccc?aaggggtgga?atgttattta?cagtatctgt?gacacccatg 1260
acgtggcaga?cagcttcact?aacaaggagg?agtttaatcc?cgaccgattt?acatcgcttc 1320
atccagagga?cacctccagg?ttcagtttca?ttccatttgg?aggaggcctt?cggagctgcg 1380
taggcaaaga?gtttgcaaaa?attcttctta?agatatttac?cgtggagctg?gccagacgtt 1440
gtgactggca?gctgctaaat?ggacctccta?caatgaagac?aagccccacc?ttctaccctg 1500
tggacaatct?tcctgcaaga?ttcacccact?tccagggaga?tatctgacag?ctatttcagt 1560
tcttggactc?atttgaagtg?tacattgttt?ttttttttta?aatagtgtca?tgttgccttt 1620
atttaatttc?taaatgtata?gtataatatt?tatatgtctc?tactacagcc?ccatggtctt 1680
taaatattaa?aataatgaat?ttgtatgatt?tcccaataaa?gtaaaatttt?aaagtgtaaa 1740
aaaaaaaaaa?aaaaaaaaaa?aaaaaa 1766
<210>2
<211>28
<212>DNA
＜213〉artificial sequence
<400>2
cgaagcttat?ggggctcccg?gcgctgct 28
<210>3
<211>30
<212>DNA
＜213〉artificial sequence
<400>3
cgctcgagtc?agatatctcc?ctggaagtgg 30
<210>4
<211>26
<212>DNA
＜213〉artificial sequence
<400>4
cggggtacca?tggggctccc?ggcgct 26
<210>5
<211>25
<212>DNA
＜213〉artificial sequence
<400>5
tcatcagggt?accccatgga?aatgg 25
Claims (10)
1. recombinant plasmid that is used for Mammals Antioncogene vaccine, its cDNA fragment and carrier for expression of eukaryon by the Cyp26a1 gene is formed, and carrier for expression of eukaryon is preferably pVAX1.
2. recombinant plasmid according to claim 1 is characterized in that, the cDNA fragment of described Cyp26a1 gene is derived from the Spreqne-Dawley rat uterus, and its base sequence is preferably shown in SEQ IDNO.1.
3. the bacterial strain that comprises claim 1 or 2 described recombinant plasmids; Preferably, described bacterial strain is selected from intestinal bacteria Top10F ' bacterial strain.
4. the cell that comprises claim 1 or 2 described recombinant plasmids; Preferably, described cell is selected from human cervical carcinoma cell and people's suede cancer cells.
5. a Mammals Antioncogene vaccine wherein comprises claim 1 or 2 described recombinant plasmids.
6. gene vaccine according to claim 5 is characterized in that described vaccine also comprises adjuvant; Preferably, to be selected from percentage concentration be 0.25% PROCAINE HCL, PHARMA GRADE to described adjuvant.
7. a method for preparing claim 1 or 2 described recombinant plasmids is characterized in that, the cDNA fragment of Cyp26a1 gene as described in the base sequence of this method employing shown in SEQ ID NO.2 and SEQ ID NO.3 cloned as primer; Preferably, this method adopts temperature fall reverse transcription-polymerase chain amplification method to clone the cDNA fragment of described Cyp26a1 gene.
8. method for preparing Cyp26a1 independent mammary tumor animal model, it is the segmental 4T1 mouse mastopathy cell injection of the cDNA animal's mammary gland position preparation that comprises people source Cyp26a1 gene by use; Preferably, the cDNA fragment of people source Cyp26a1 gene as described in the base sequence of this method employing shown in SEQ ID NO.4 and SEQ ID NO.5 cloned as primer; More preferably, described animal is a mouse.
9. claim 1 or the 2 described recombinant plasmids purposes in preparation Mammals Antioncogene vaccine; Preferably, described Mammals is selected from the people, and described tumour is selected from mammary cancer.
10. claim 5 or 6 described gene vaccines prevent and/or treat purposes in the medicine of mammal tumor in preparation; Preferably, described Mammals is selected from the people, and described tumour is selected from mammary cancer.
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| 《分子科学学报》 20071231 苏循瑞 小鼠早期胚胎肢体发育中的视黄酸诱导的视黄酸代谢 第390-393页 1-10 第23卷, 第6期 * |
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| BING-CHEN HAN等: "Retinoic Acid-Metabolizing Enzyme Cytochrome P450 26a1 (Cyp26a1) Is Essential for Implantation: Functional Study of Its Role in Early Pregnancy", 《JOURNAL OF CELLULAR PHYSIOLOGY》 * |
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