CN101168064B - Recombination human pancreatic cancer mucus protein core peptide DNA vaccine - Google Patents
Recombination human pancreatic cancer mucus protein core peptide DNA vaccine Download PDFInfo
- Publication number
- CN101168064B CN101168064B CN 200710047160 CN200710047160A CN101168064B CN 101168064 B CN101168064 B CN 101168064B CN 200710047160 CN200710047160 CN 200710047160 CN 200710047160 A CN200710047160 A CN 200710047160A CN 101168064 B CN101168064 B CN 101168064B
- Authority
- CN
- China
- Prior art keywords
- muc1
- vntr
- sequence
- cell
- pancreatic cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of biology, relating to restructuring DNA molecules of a single replication sequence which is capable of encoding human pancreatic cancer MUC1 and also DNA vaccine inoculation constructed by the DNA molecules and the usage of the vaccine inoculation. The restructuring DNA molecules enable coding genes of the most immunogenic nucleic replication sequences of the MUC1 to be the core. An MCP-I signal peptide gene order is inserted at the amidogen end, simultaneously a promoting eukaryon translating sequence of a KOZAK is inserted in front of an initial code. The restructuring DNA molecules are directionally cloned into a eukaryon expression vector MCS, constructing and restructuring the DNA vaccine inoculation of a mucus core protein peptide of the human pancreatic cancer. After ukaryotic cells transfect the vaccine inoculation of the invention, a VNTR can be expressed in the inner and outer cells. Animal experimental results display that the vaccine inoculation has protective effects against a tumor attack, has treatment effects on a mouse pancreatic cancer of the expression human MUC1, can specifically kill the human pancreatic cancer cells of the expression MUC1, and has the treatment effect on the human pancreatic cancer.
Description
Technical field
The invention belongs to biological technical field, relate to recombination human pancreatic cancer mucus protein core peptide (MUC1) dna vaccination, be specifically related to a kind of recombinant DNA molecules of the human pancreas cancer MUC1 core sequence of encoding, and constructed dna vaccination and uses thereof.
Background technology
Known in this field, the cancer of pancreas prognosis is very poor.The treatment of relevant cancer of pancreas does not still have breakthrough progress so far.Show that according to the relevent statistics 65% Pancreas cancer patients is dead in diagnosis clear and definite later six months, dead in about 90% the patient 1 year.There are some researches show that 90% pancreatic cancer cell all has the expression of MUC1, its polypeptide backbone by extracellular fragment, stride film section (28aa) and born of the same parents' inner segment (72aa) is formed.Extracellular fragment contains 20～125 continuous repetitive sequences, and (variable numbersof tandem repeats, VNTRs), each VNTR forms (GVTSAPDTRPAPGSTAPPAH) by 20 aminoacid.Cause glycosylation incomplete owing to glycosyl transferase activity increases, the APDTR sequence in its continuous repetitive sequence (VNTR) zone is exposed, become the target spot of immunization therapy.
The research report is arranged, and host's inoculation is the vaccine that target antigen makes up with MUC1, can bring out special humoral immunization of MUC1 and cellular immunization, kills and wounds the tumor cell of expressing MUC1.The Goydos synthetic contain 105 amino acid whose MUC1 peptide chains of 5 VNTR, with BCG mixed immunity Pancreas cancer patients, DTH at the MUC1 polypeptide reacts than obviously strengthening before the art, the T Premeabilisation of cells is obviously strengthened the special CTL precursor of MUC1 and is also increased 2-4 doubly in the tumor specimen, shows that the MUC1 polypeptide vaccine can effectively bring out the cell immune response of antigen-specific.
But MUC1 peptide chain vaccine provides exogenous antigen.In vivo, exogenous antigen by antigen presenting cell (APC) catch, pinocytosis or engulf, thereby enter the endosome that is arranged in Cytoplasm, be degraded into antigenic peptides, form antigenic peptides-mhc class ii molecular complex, pass through the fusion of endosome film and cell membrane again with the mhc class ii molecule that imports in the endosome by the γ chain, be expressed in the APC surface, discern mutually with the TCR/CD3 complex of ripe CD4+TH cell, activate the TH cell, thereby excite anti tumor immune response based on specific humoral immunity.The specific cell immunoreaction deficiency is the major defect that the peptide chain vaccine exists.Simultaneously, although VNTR can be by BCR, TCR identification, the MUC1 peptide chain vaccine that single or less VNTR forms when the APC processed, may also be difficult to excite efficient immune because epi-position is difficult to be kept perfectly.
Dna vaccination (DNAvaccine), it is in the nature exogenous antigen encoding gene fragment cloning on eucaryon plasmid DNA expression vector, behind the interior APC of direct inoculation success transfection body, then can produce endogenous antigen at APC, mainly bring out the anti tumor immune response that body produces specific cellular immunity, thereby reach the purpose of prevention and treatment disease based on the MHCI classpath.Cellular immunization is the of paramount importance component of antineoplastic immune, it is simple that dna vaccination has preparation simultaneously, advantages such as stable in properties, thereby be considered to third generation vaccine after attenuation, inactivated vaccine and recombinant vaccine subunit vaccine, main developing direction for following vaccine, especially tumor vaccine.
Johnen inoculates the tumor cell (MC38-muc1) that can protect the host not expressed MUC1 behind the mice with total length MUC1 naked DNA and attacks.Aromatization has been built the full gene vaccine of MUC1 and can be induced body to produce the special CTL of MUC1 to reply and antibody response during domestic Yuan, suppresses to express the attack of the EMT6 cell of MUC1.These researchs have all shown to be that the dna vaccination of target antigen has good immunoprotective effect to the tumor of expressing MUC1 with MUC1.Yet, complete antigenic some inhibition epi-positions and other epi-positions, the irrelevant sequence of comprising that the full-length gene vaccine is expressed.The inhibition epi-position can cause the immunosuppressant phenomenon, and the interference of other epi-positions, irrelevant sequence also can reduce immunological effect, and, increased the risk of immune safeties such as autoimmune disease.And only to contain 7-8 amino acid whose single epitope constructed dna vaccine, also have to be difficult to efficiently express, a little less than the antigenicity, be difficult to excite the shortcoming of effective immunological effect.
Therefore, the immunological effect and the immune safety of raising human pancreas cancer MUC1 dna vaccination become problem demanding prompt solution.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of immunological effect and immune safety good recombination human pancreatic cancer mucus protein core peptide DNA vaccine.
The invention provides a kind of recombinant DNA molecules of the human pancreas cancer MUC1 core sequence of encoding.The present invention comprises CTL epi-position and B cell epitope by the reorganization design of dna molecular, improves the immunogenicity of vaccine, does not contain inhibition epi-position and other epi-positions, irrelevant sequence simultaneously, has increased the vaccine immunity safety.
Another object of the present invention is to make up the MUC1 dna vaccination and its purposes is provided with recombinant DNA molecules.
It is the core of recombinant DNA molecules that the present invention takes single VNTR coded sequence.
Described VNTR forms (GVTSAPDTRPAPGSTAPPAH) by 20 aminoacid, forms a stable βZhuan Jiao.The immunogenicity of cancer of pancreas MUC1 is mainly reflected on its polypeptide backbone, and single VNTR has also comprised the antigenic information that all repetitive sequences are cascaded and are contained, and contains B cell epitope and t cell epitope equally.Austin academy according to 20 aminoacid synthetic of VNTR 3 kinds of small peptides: 1. P1-24 (PDTRPAPGSTAPPAHGDTSAPDTR) comprises 20 aminoacid of first VNTR and preceding 4 aminoacid of next VNTR; 2. P1-15 (PDTRPAPGSTAPPAH) contains preceding 15 aminoacid; 3. P16-24 (GDTSAPDTR) contains back 9 aminoacid.The monoclonal antibody BC1 of anti-MUC1, BC2, BC3 all can with P1-24 and P16-24 reaction, and with the P1-15 anergy.And add an alanine at the P1-15 afterbody, responding property of A-P1-15 then.
The single VNTR coded sequence of the core of recombinant DNA molecules provided by the invention (GGT GTC ACC TCGGCC CCG GAC ACC AGG CCG GCC CCG GGC TCC ACC GCC CCC CCA GCC CAC) does not contain inhibition epi-position and other epi-positions; irrelevant sequence; itself can farthest reduce interference and influence to immunological effect; the VNTR polypeptide of Biao Daing does not have side chain simultaneously; can not cause that the autoimmune response disease is (although the lumen of gland face of normal glandular cell also has the expression of a small amount of MUC1 yet; but because the glycoprotein side chain of volume is arranged, make that the VNTR on its polypeptide backbone is protected).
The present invention has introduced endoplasmic reticulum and has inserted signal sequence in recombinant DNA molecules, in the aminoterminal insertion of VNTR coded sequence, bootable VNTR directly enters endoplasmic reticulum and strengthens MHCI class antigen processing approach in translation.The present invention has also introduced short eukaryotic translation sequence in recombinant DNA molecules, can improve the accuracy and the productive rate of VNTR translation.
Described endoplasmic reticulum is inserted signal sequence behaviour monocyte chemoattractant protein (monocyte chemoattractantprotein, MCP) I signal peptide gene sequence: ATG AAG TGG GTA ACC TTT CTC CTC CTC CTCTTC ATC TCC GGT TCT GCC TTT TCT AGG GGT.
Described short eukaryotic translation sequence is KOZAK sequence (GCC GCC ACC), and this sequence can be regulated allosteric to the 40S ribosomal subunit successively, the accurate location of AUG when promoting translation.
The present invention goes into the recombinant DNA molecules directed cloning in the carrier for expression of eukaryon, makes up the MUC1 dna vaccination.Described vaccine is at rotaring redyeing COS 7 cell and carry out the expression that Western blot has detected VNTR.
For estimating the immunogenicity of described dna vaccination,, estimate humoral immunization that vaccine brought out (detecting the specific antibody of the anti-VNTR of serum) and cellular immunization (CTL activity) with the normal C57BL/6 mice of dna vaccination immunity.Can suppress the growth of mice pancreatic cancerous cell of the expressing human MUC1 of follow-up inoculation behind the immanoprotection action experiment confirm vaccine immune mouse of vaccine to cancer of pancreas; And mice earlier the inoculation pancreatic cancer cell inoculate the vaccine therapy growth of tumor and obviously slow down.In addition, the present invention has carried out the in-vitro transfection killing experiments and has estimated the described vaccine lethal effect to the human pancreatic cancer cell of expressing MUC1.
Described carrier for expression of eukaryon is pcDNA3.1/Myc-his (+) A plasmid (commercial), and (ampicillin resistance, Ampr) gene is to be used as selection markers to contain amicillin resistance.Contain in the described resistant gene 2 significantly six oligonucleotide sequences 5 of enhancing gene immune effect '-AACGTT-3 ', this sequence contains the CpG structure, be called immune activation DNA sequence (immuno-stimulatory DNA sequence, ISS), but by non-specific polyclone activation immune cell activated, stimulate to produce IL-12 and IL-18, and promote IFN-γ synthetic then, making inmature T cell development is the TH1 cell.
The mice pancreatic cancerous cell of described expressing human MUC1 is with people's total length MUC1 cDNA sequence transfection mice pancreatic JEG-3 Panc02 (commercial), the stably express strain that filters out, i.e. Panc02-muc.
Described in-vitro transfection killing experiments lapses in maturation process and the dna vaccination immunology in vivo of in-vitro simulated DC, external described vaccine transfection DC is hatched jointly with homology PBMC again, induces out the special CD8 of MUC1
+CTL to be target cell from the body pancreatic cancer cell, estimates the therapeutical effect of vaccine by the CTL killing experiments.
The present invention is undertaken by following technical proposals and step:
1. the design of recombinant DNA molecules and synthetic:
Recombinant DNA molecules is followed successively by KOZAK sequence, hMCP-I signal peptide sequence, VNTR sequence, and two ends are inserted Hind III enzyme restriction enzyme site, EcoRI enzyme restriction enzyme site respectively; Synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and cloned plasmid in pUC18.
2. make up the MUC1 dna vaccination:
PUC18 plasmid PCR product (recombinant DNA molecules), carrier for expression of eukaryon pcDNA3.1/Myc-his (+) A plasmid are carried out double digestion with Hind III enzyme, EcoR I enzyme respectively, form different sticky ends, polymerization by the T4 dna ligase, the recombinant DNA molecules directed cloning is gone in the carrier, make up the MUC1 dna vaccination, i.e. pcDNA3.1-VNTR/Myc-his (+) A recombiant plasmid.
3. in-vitro transfection test
Adopt Cacl
2Method is with pcDNA3.1-VNTR/Myc-his (+) A recombiant plasmid rotaring redyeing COS 7 cell.After the transfection 48 hours, collection culture fluid supernatant, cell lysate supernatant adopt 12%SDS-PAGE to carry out the expression that Western blot detects VNTR, and the result shows that vaccine can efficiently express VNTR inside and outside cell.
4.DNA the immunogenicity experiments of vaccine
The female Mus of 6-8 week C57BL/6 in age, random packet (empty expression vector, normal saline are done contrast), 15 every group.Intramuscular injection inoculation, 4 all booster immunizations behind the initial immunity, booster immunization adopt the ELISA method to detect the specific antibody of the anti-VNTR of serum after 2 weeks; The aseptic mouse spleen of getting, the preparation single cell suspension adopts Calcein AM method to carry out the CTL killing experiments, and the result shows that the vaccine immunity normal mouse can induce the special CTL of MUC1 to reply and antibody response.
5. vaccine is to the immanoprotection action zoopery of cancer of pancreas
With people's total length MUC1 cDNA sequence transfection mice pancreatic JEG-3 Panc02, the stably express strain Panc02-muc that filters out is the mice pancreatic JEG-3 of expressing human MUC1.
The female Mus of 6-8 week C57BL/6 in age is divided 3 groups (empty expression vector Neo, PBS do contrast) at random, the intramuscular injection inoculation, and per 2 all booster immunizations are 1 time behind the initial immunity, and totally 2 times, method is the same.
The 2nd booster immunization, the growth of tumour cell situation is observed in the inoculation back, measures tumor major diameter (a) and tumor (b), according to formula Volume (mm
3)=a * b
2/ 2 calculate gross tumor volume, observe the protective effect of vaccine antagonism tumor challenge.The result shows that in the tumor time of origin evening of vaccine group mice, gross tumor volume is little, and obviously prolong the life cycle of mice, confirms that vaccine antagonism mice pancreatic cancerous cell of the present invention attack has protective effect.
6. vaccine is to the therapeutical effect zoopery of cancer of pancreas
The C57BL/6 mice, left fore oxter subcutaneous plantation pancreatic cancer cell suspension Panc02-muc, random packet group (MUC1 group, MUC1+GM-CSF group, GM-CSF adjuvant matched group, empty expression vector Neo matched group, PBS matched group), inoculate the Panc02 cell respectively and do target cell negative control and vaccination, the growth of tumour cell situation is observed in the inoculation back, measure tumor major diameter (a) and tumor (b), according to formula Volume (mm
3)=a * b
2/ 2 calculate gross tumor volume, observe the therapeutic effect of vaccine.The result shows that after the vaccine group treatment, growth of tumor obviously slows down, and volume is less than matched group, and prolong life cycle, confirms that vaccine of the present invention also has certain therapeutical effect to mouse pancreas cancer.
7. in-vitro transfection killing experiments
The present invention separates PBMC from healthy human peripheral blood, induces and amplify in a large number dendritic cell external with GM-CSF, IL-4, and cultured cell to the 5 days adopts Lipofectamine
TM2000 transfection vaccines (empty expression vector, liposome are done contrast), fluorescence microscope detects DC plasmid transfection situation down after 24 hours, and Western Blot detects the VNTR expression of polypeptides.According to 10: 1 ratios, DC is added homology PBMC hatch jointly, induce out the special CD8 of VNTR
+CTL.Than effector lymphocyte and target cell Capan-2 (expressing MUC1) are added 96 hole U base plates respectively, AsPC-1 (not expressing MUC1) does the target cell negative control and carries out LDH cell toxicant killing experiments by different effect targets.The result shows that MUC1 vaccine in-vitro transfection DC can induce the special CD8 of VNTR
+CTL, but specific killing is expressed the human pancreatic cancer cell of MUC1.
Description of drawings:
Fig. 1 is the invention process example used recombination human pancreatic cancer MUC1DNA vaccine pcDNA A3.1-VNTR/Myc-his (+) A plasmid sketch map (a) and sequencer map (b).
Fig. 2 is that MUC1 dna vaccination Western blot detects, wherein,
Fig. 3 is the immunogenicity experiments result of MUC1 dna vaccination,
Wherein, a.: the immune mouse specific CTL kills and wounds; B: the polypeptid specificity that immune mouse CTL replys,
The result shows, the specific killing rate of vaccine group (V group) C57BL/6 mice (H-2b) splenocyte CTL is significantly higher than empty plasmid matched group (D group) and normal saline matched group (a), the CTL killing activity that vaccine induced is that VNTR is specific, CTL only has specific killing to the EL4 that contains target antigen VNTR, with monoclonal antibody VU3C6 (the GVTSAPDTRPAP sequence on the identification VNTR) and the target cell EL4-VNTR of MUC1
+In conjunction with, can suppress CTL killing activity (b).
The immanoprotection action of Fig. 4 MUC1 dna vaccination,
Wherein, a.:Panc02-muc attacks tumor growth situation behind the different immune mouses; B.: tumor growth situation behind the different tumor cells attack MUC1 dna immunization mices; C.:Panc02-muc attacks the different immune mouse survival curves in back; D.: different tumor cells are attacked back MUC1 dna immunization mice survival curve,
The result shows, C57BL/6 mice (H-2
b) with 3 immunity back inoculations 1 * 10 of MUC1 dna vaccination immunity
6Panc02-muc cell, the growth of tumor cell be significantly than matched group slow (a), but inoculate 1 * 10
6The Panc02 cell, the growth of tumor cell is not suppressed (b), shows that vaccine has the special immanoprotection action of MUC1; With gross tumor volume＞1000mm
3As observing terminal point, MUC1 group mice median survival interval is 27 days, organizes 20 days (P＜0.05) (c) greater than Neo group 21 days and PBS; Vaccine group does not then have immanoprotection action for the attack of the mice pancreatic cancerous cell Panc02 that does not express MUC1, and its median survival interval is 15 days (d).
Fig. 5 is the therapeutical effect of MUC1 dna vaccination,
Wherein, a.: tumor growth situation behind the C57BL/6 mice vaccine therapy of inoculation Panc02-muc; B.: inoculate tumor growth situation behind the different tumor cell mice vaccine therapies; C: survival curve behind the C57BL/6 mice vaccine therapy of inoculation Panc02-muc; D.: inoculate survival curve behind the different tumor cell mice vaccine therapies,
The result shows, behind C57BL/6 mice (H-2b) inoculation 1 * 106 Panc02-muc cell the 4th, 9,14 day respectively the intramuscular injection vaccination treat, MUC1 group, the growth of MUC1+GM-CSF group mouse tumor significantly are slower than GM-CSF group, Neo group and PBS group (a); As observing terminal point, MUC1 group, MUC1+GMCSF group mice median survival interval significantly are longer than GM-CSF group, PBS group and Neo group (c) with gross tumor volume＞1000mm3; And the mice of the Panc02 cell of MUC1 is not expressed in inoculation, and GM-CSF is an adjuvant no matter have or not respectively, all fails to suppress the growth (b) of tumor cell behind the vaccine therapy, does not prolong life cycle (d) yet.
Fig. 6 is MUC1 dna vaccination in-vitro transfection killing experiments result,
Wherein, dendritic cell becomes colony growth figure under a:40 * light microscopic; B: mixed lymphocyte reaction result; C: flow cytometer dendritic cell phenotypic evaluation figure; D.: the dendritic cell of transfection luciferase plasmid pcDNA3.1-GFP under the fluorescence microscope; E.: plasmid transfection dendritic cell shape stimulated in vitro is from body T cell proliferation experiment; F: the external CTL that induces is to Capan-2 (MUC1
+, HLA-A2
+) kill and wound; G:MUC1 monoclonal antibody VU 3C6 is to the inhibition of CTL specific killing; H: the external CTL that induces kills and wounds the CTL of different target cells.
The specific embodiment
1. the design of recombinant DNA molecules and synthetic:
The design of recombinant DNA molecules: be followed successively by Hind III enzyme restriction enzyme site, KOZAK sequence (GCCGCCACC), hMCP-I signal peptide sequence (line part), VNTR sequence (italicized item), EcoRI enzyme restriction enzyme site.
5′-AAG CTT GCC GCC ACC
ATG AAG TGG GTA ACC TTT CTC CTC CTC CTC TTC ATC TCC GGT TCT GCC TTT TCT AGG GGT GGT GTC ACC TCG GCC CCG GACACC AGG CCG GCC CCG GGC TCCACC GCC CCC CCA GCC CAC GAA TTC-3′。
Synthesizing of recombinant DNA molecules: synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and cloned plasmid in pUC18.
2. make up the MUC1 dna vaccination:
PUC18 plasmid PCR product (recombinant DNA molecules), carrier for expression of eukaryon pcDNA3.1/Myc-his (+) A plasmid are carried out double digestion with Hind III enzyme, EcoR I enzyme respectively, form different sticky ends, polymerization by the T4 dna ligase, the recombinant DNA molecules directed cloning is gone in the carrier, make up the MUC1 dna vaccination, i.e. pcDNA3.1-VNTR/Myc-his (+) A recombiant plasmid.From transforming dull and stereotyped picking list bacterium colony, enzyme action is identified the positive colony of the about 140bp of back picking endonuclease bamhi size (Bo Ya company) (Fig. 1 a, the b) that check order.
3. in-vitro transfection test
Adopt Cacl
2(configuration contains 5ug GFP plasmid, 2.5M Cacl to method during transfection with pcDNA3.1-VNTR/Myc-his (+) A recombiant plasmid rotaring redyeing COS 7 cell
2With 2 * HBS-Hepes buffer, 5ugpcDNA3.1-VNTR/Myc-his (+) A recombiant plasmid).After the transfection 48 hours, collection culture fluid supernatant, cell lysate supernatant adopt 12%SDS-PAGE to carry out the expression that Western blot detects VNTR, VNTR synthesizes polypeptide (GVTSAPDTRPAPGSTAPPAH, the Shenzhen Hanyu Bioengineering Co.Ltd, purity is 97.5%, and molecular weight is 1887) positive contrast, one anti-is mouse-anti people MUC1 monoclonal antibody VU 3C6, the GVTSAPDTRPAP sequence of the VNTR polypeptide of identification MUC1,1: 500.
The swimming lane 1,2 that Fig. 2 shows is represented the COS7 cell lysate and the culture fluid supernatant of pcDNA3.1-VNTR/Myc-his (+) A recombiant plasmid transfection respectively, and the result shows the expression that VNTR is all arranged inside and outside the cell, in born of the same parents.And the COS7 cell of blank carrier pcDNA3.1/Myc-his (+) A plasmid transfection ( swimming lane 3,4, negative control) does not all have the expression of VNTR inside and outside born of the same parents.Because the VNTR of expression of recombinant plasmid contains signal peptide and epi-positions such as Myc, His, the VNTR polypeptide of molecular weight ratio synthetic (swimming lane 5, positive control) is obviously big.
4.DNA the immunogenicity experiments of vaccine
The female Mus of 6-8 week C57BL/6 in age, body weight 19-25 gram (must triumphant laboratory animal company limited available from west, Shanghai pul one) is divided into 3 groups (empty expression vector, normal saline are done contrast), 15 every group at random.Intramuscular injection inoculation: earlier with after the anesthesia of 0.75% pentobarbital sodium 120-180 μ l abdominal cavity mice (50 μ g/g body weight), with 100u insulin syringe (available from BD company) in bilateral tibialis anterior inserting needle, slowly inject plasmid DNA normal saline solution, every side 50 μ g/50ul.4 all booster immunizations behind the initial immunity, method is the same.Booster immunization is got blood 0.5ml after 2 weeks in angular vein, separation of serum adopts the ELISA method to detect the specific antibody of the anti-VNTR of serum; Aseptic treating excess syndrome is tested mouse spleen, and the preparation single cell suspension adopts Calcein AM method to carry out the CTL killing experiments: with the gamma-rays deactivation homology of 3000rad not the immune mouse spleen cell suspension be prepared into irritation cell.Ratio adding irritation cell with 20: 1 in splenocyte was cultivated 6 days altogether, added mIL-2 20U/ml, and anti-CD28 0.1ug/ml keeps.Stimulate the after effect cell with Ficoll separating medium purification, resuspended there not to be phenol red culture fluid.With 37 ℃ of water-bath labelling exponential phase target cells of 5～10uM calcein AM (available from U.S. Molecular Probe company) (EL4-VNTR
+, EL4-VNTR
-), Hank ' s liquid is washed the back, and phenol red culture fluid is resuspended does not make the good target cell of labelling to have.In 96 hole U base plates by imitating target than E/T:80: added gradient dilution effector lymphocyte and the good target cell of labelling in 1,40: 1,20: 1,10: 1.If (add the 50mM sodium borate, 0.1%TritonX-100 is PH9.0) with spontaneous release group (adding no phenol red culture fluid) for release group fully.
Hatch and centrifugal after 6 hours supernatant is transferred to 96 new hole flat undersides, the fluorescence measurement instrument detects the fluorescent value (FI) of supernatant, and exciting light is 485nm, and emission light is 538nm.CTL specific killing rate (%)=(experimental group FI value-spontaneous release group FI value)/(maximum release group FI value-spontaneous release group FI value) * 100%.
The result shows, after the external special stimulation of the synthetic peptide of VNTR was cultivated 6 days, the specific killing rate of pcDNA3.1-VNTR/Myc-his (+) A plasmid mice immunized splenocyte CTL was significantly higher than empty plasmid matched group (P＜0.01) and normal saline matched group (P＜0.01).Three groups kill rate is respectively 10.0% ± 2.6%, 2.6% ± 0.2%, 1.5% ± 0.1% during E/T=10/1, kill rate is respectively 14.9% ± 1.3%, 4.0% ± 0.4%, 3.1% ± 0.3% during E/T=20/1, kill rate is respectively 28.8% ± 2.6%, 7.1% ± 0.6%, 5.9% ± 0.5% during E/T=40/1, and kill rate is that 34.8% ± 3.1%, 9.2% ± 0.8%, 6.1% ± 0.6% (Fig. 3 a) during E/T=80/1.Show that pcDNA3.1-VNTR/Myc-his (+) A plasmid mice immunized has produced significant CTL and replied.And this CTL to reply be that the recombinant DNA molecules of cloning in expression vector induces.Fig. 3 b shows: the target cell EL4-VNTR of the specific CTL that recombiant plasmid induces to hatching through the synthetic peptide of VNTR
+Lethal effect is obvious, and the target cell EL4-VNTR-of hatching without the synthetic peptide of VNTR is killed and wounded lower (P＜0.01).Show that the CTL killing activity that recombiant plasmid induces is that VNTR is specific.And prior monoclonal antibody VU3C6 and target cell EL4-VNTR with MUC1
+Effect, then can suppress CTL killing activity (P＜0.01), show that VU3C6 (the GVTSAPDTRPAP sequence on the identification VNTR) has sealed the VNTR target position on the target cell, thereby reduce CTL specific recognition target cell, make lethal effect reduce, show that further the CTL killing activity is that VNTR is specific.
(2323.5ug/ml ± 238.3ug/ml) is higher than empty plasmid matched group (1895.8ug/ml ± 532.7ug/ml) and normal saline matched group (1736.4ug/ml ± 141.9ug/ml) to the anti-VNTR antibody of pcDNA3.1-VNTR/Myc-his (+) A plasmid mice immunized serum equipotent concentration, difference is (P＜0.01, table 1) very significantly.Can bring out the antibody response that the host produces anti-VNTR after showing the recombinant DNA molecules immune mouse that is inserted in pcDNA3.1-VNTR/Myc-his (+) the A recombiant plasmid.
5. vaccine is to the immanoprotection action zoopery of cancer of pancreas
With people's total length MUC1 cDNA sequence transfection mice pancreatic JEG-3 Panc02, the stably express strain Panc02-muc that filters out is the mice pancreatic JEG-3 of expressing human MUC1.
The female Mus of 6-8 week C57BL/6 in age is divided into 3 groups (empty expression vector Neo, PBS do contrast), 15 every group at random.Intramuscular injection inoculation: with after the anesthesia of 0.75% pentobarbital sodium 120-180 μ l abdominal cavity mice (50 μ g/g body weight), in bilateral tibialis anterior inserting needle, slowly inject the plasmid DNA normal saline solution, every side 50 μ g/50ul earlier with insulin syringe.Per 2 all booster immunizations are 1 time behind the initial immunity, and totally 2 times, method is the same.
1 week behind the 2nd booster immunization is in mice left fore oxter subcutaneous vaccination Panc02-muc 1 * 10
6/ 100 μ l, other has 15 vaccine group mouse inoculation Panc02 cells 1 * 10
6/ 100 μ l.As the target cell negative control.The growth of tumour cell situation is observed every day in the inoculation back, measures tumor major diameter (a) and tumor (b) once, according to formula Volume (mm
3)=a * b
2/ 2 calculate gross tumor volume, observe the protective effect of vaccine antagonism tumor challenge.
The result shows, C57BL/6 mice (H-2
b) with 3 immunity back inoculations 1 * 10 of MUC1 dna vaccination immunity
6Panc02-muc cell, the growth of tumor cell (vaccine group) significantly are slower than Neo group and PBS group (Fig. 4 a), MUC1 group gross tumor volume (818.75 ± 87.5mm in the time of the 24th day
3) significantly less than Neo group (1868.8 ± 187.5mm
3) and PBS group (1781.3 ± 150mm
3) (P＜0.05); But if vaccine group is inoculation 1 * 10
6Panc02 cell, the growth of tumor cell are not suppressed (Fig. 4 b), show that vaccine has the special immanoprotection action of MUC1.
With gross tumor volume＞1000mm
3As observing terminal point, the result shows that MUC1 group mice median survival interval is 27 days, greater than Neo group 21 days and 20 days (P＜0.05) (Fig. 4 c) of PBS group, can specific prevention mice after the immunity of prompting MUC1 dna vaccination be subjected to expressing the attack of the mice pancreatic cancerous cell Panc02-muc of MUC1.Vaccine group does not then have immanoprotection action for the attack of the mice pancreatic cancerous cell Panc02 that does not express MUC1, and its median survival interval is 15 days (Fig. 4 d).
6. vaccine is to the therapeutical effect zoopery of cancer of pancreas
75 C57BL/6 mices, left fore oxter subcutaneous plantation pancreatic cancer cell suspension Panc02-muc, 5 groups of random packet (MUC1 group, MUC1+GM-CSF group, GM-CSF adjuvant matched group, empty expression vector Neo matched group, PBS matched group), preceding two groups respectively have 15 mouse inoculation Panc02 cells to do the target cell negative control in addition.Behind the tumor cell the 4th, 9,14 day respectively intramuscular injection vaccination 100 μ g/100 μ l treat.The growth of tumour cell situation is observed every day in the inoculation back, measures tumor major diameter (a) and tumor (b) once, according to formula Volume (mm
3)=a * b
2/ 2 calculate gross tumor volume, observe the therapeutic effect of vaccine.
The result shows, C57BL/6 mice (H-2
b) inoculation 1 * 10
6Behind the Panc02-muc cell the 4th, 9,14 day respectively intramuscular injection vaccination 100 μ g/100 μ l treat.In the time of the 25th day, MUC1 group, MUC1+GMCSF group mouse tumor growth volume are respectively 1252.5 ± 150.0mm
3, 847.5 ± 120.0mm
3, significantly less than GM-CSF group (1777.5 ± 135.0mm
3), Neo organizes (2017.5 ± 115.0mm
3) and PBS group (2227.5 ± 225.0mm
3) (Fig. 5 a) for (P＜0.05) group.With gross tumor volume＞1000mm
3As observing terminal point, the result shows that MUC1 group, MUC1+GMCSF group mice median survival interval were respectively 31 days, 24 days, significantly be longer than GM-CSF group, PBS group and Neo group median survival interval 21 days (Fig. 5 c), after showing the treatment of MUC1 dna vaccination, the CTL of the antigen-specific that induces can suppress the growth of already present tumor cell Panc02-muc in the body; With GM-CSF is the therapeutical effect that adjuvant can strengthen vaccine.
And the mice of the Panc02 cell of MUC1 is not expressed in inoculation, and GM-CSF is an adjuvant no matter have or not respectively, all fails to suppress the growth (Fig. 5 b) of tumor cell behind the vaccine therapy, and do not prolong (Fig. 5 d) life cycle yet, and the therapeutical effect that shows vaccine is that MUC1 is special.
7. in-vitro transfection killing experiments
The present invention separates PBMC from healthy human peripheral blood, induces and amplifies dendritic cell in a large number with GM-CSF, IL-4 external, and it is irregular to be cultured under the inverted microscope on the 5th visible dendritic cell form, and (Fig. 6 a) for the more raised structures in surface; Be cultured to flow cytometer on the 7th and carry out phenotypic evaluation, the specific surface marker CD86 of dendritic cell, HLADR, CD209 etc. obviously raise; And CD3/CD8
+, CD3/CD4
+, CD14
+Cell obviously reduces, DC cell trend ripe (Fig. 6 c, table 2); Be cultured to adding TNF α on the 5th and can urge the DC cell maturation, cultivate, have the extremely strong effect of stimulating proliferation (Fig. 6 b) with the Allogeneic T lymphocyte is mixed.
Cultured cell to the 5 days is collected the DC that cultivates, and adopts Lipofectamine
TM2000 transfection vaccines (empty expression vector, liposome are done contrast), after 24 hours fluorescence microscope down can observe transfection the dendritic cell of luciferase plasmid pcDNA3.1-GFP excite green fluorescence (Fig. 6 d).Simultaneously, transfection the DC of vaccine stimulate the lymphopoietic effect of Allogeneic T to strengthen (Fig. 6 e).According to 10: 1 ratios, DC is added homology PBMC hatch jointly, induce out the special CD8 of VNTR
+CTL.
Than effector lymphocyte and target cell Capan-2 (expressing MUC1) are added 96 hole U base plates respectively, AsPC-1 (not expressing MUC1) did the target cell negative control, according to CytoTox 96 according to 10: 1,40: 1,80: 1 effect targets
(CytoTox 96 for Non-Radioactive Cytotoxicity Assay
On-radiation cytotoxicity analysis test kit) carries out LDH cell toxicant killing experiments.The result shows that the CTL that the DC of vaccine group transfection stimulates proliferation can specificly kill and wound the target cell Capan-2 (HLA-A2 of expressing human MUC1
+), imitating the target ratio is 80: 1,40: 1,10: 1 o'clock, its specific killing rate is respectively 71.47% ± 9.62%, 39.86% ± 8.41%, 19.28% ± 0.28%; Apparently higher than empty expression vector matched group (19.64% ± 4.24%, 6.56% ± 8.03%, 2.49% ± 4.31%) and liposome matched group (11.57% ± 5.12%, 1.98% ± 5.48%, 0) (Fig. 6 f).Because the GVTSAPDTRPAP epi-position of VU3C6 monoclonal antibody identification VNTR, with it in conjunction with after can suppress CTL to the identification of Capan2 cell with kill and wound, imitating the target ratio is 80: 1,40: 1,10: 1 o'clock, CTL specific killing rate is respectively 13.64% ± 9.38%, 9.81% ± 0.64%, 3.29% ± 4.43%, significantly, show that killing and wounding of CTL is that MUC1 is specific less than the lethal effect (Fig. 6 g) of CTL to the Capan2 cell that do not seal the MUC1 epi-position.And for the target cell AsPC-1 (HLA-A2 of expressing human MUC1
-), vaccine group induce CTL do not have tangible lethal effect, imitating the target ratio is 80: 1,40: 1,10: 1 o'clock, and CTL specific killing rate is respectively 24.18% ± 2.63%, 8.06% ± 3.23%, 2.86% ± 3.24% (Fig. 6 h), and pointing out this lethal effect is that HLA-A2 is restrictive.
Table 1 be the anti-VNTR antibody of immune serum equipotent concentration (ug/ml ,-x ± s, n=15).
Table 2 is dendritic cell phenotypes of flow cytometer before and after cultivating.
Table 1
| Group | Antibody concentration | The P value |
| V group NS group D group | 2323.5±238.3 *# 1736.4±141.9# 1895.8±532.7 | *＝0.000，#＝0.002 #＝0.231 |
*: compare with the NS group; #: compare with the D group
Table 2
| Before the cultivation | After the cultivation | The P value | |
| CD3/CD8 CD3/CD4 CD14 CD86 HLA-DR CD209 | 15.06％±6.67％ 21.39％±7.47％ 17.24％±5.65％ 20.37％±9.32％ 19.73％±6.97％ 0.56％±0.22％ | 3.11％±2.05％ 9.12％±4.99％ 1.36％±1.3％ 95.94％±4.31％ 92.97％±4.56％ 62.28％±16.50％ | 0.000 0.004 0.001 0.000 0.000 0.003 |
Claims (3)
1. the purposes of recombination human pancreatic cancer mucus protein core peptide DNA vaccine in the medicine of the mouse pancreas cancer of preparation treatment or prevention expressing human MUC1, wherein said recombination human pancreatic cancer mucus protein core peptide DNA vaccine is that recombinant DNA molecules and the carrier for expression of eukaryon by coding human pancreas cancer MUC1 core sequence constitutes, the recombinant DNA molecules of described coding human pancreas cancer MUC1 core sequence is to be core with single VNTR coded sequence, its nucleotide sequence: GGT GTC ACC TCG GCC CCG GAC ACC AGG CCG GCCCCG GGC TCC ACC GCC CCC CCA GCC CAC; Described carrier for expression of eukaryon is pcDNA3.1/Myc-his (+) A plasmid.
2. by the described purposes of claim 1, it is characterized in that the recombinant DNA molecules of described coding human pancreas cancer MUC1 core sequence inserts endoplasmic reticulum insertion signal sequence MCP I signal peptide gene sequence successively at the aminoterminal of VNTR: ATG AAG TGG GTA ACC TTT CTC CTC CTC CTC TTC ATC TCCGGT TCT GCC TTT TCT AGG GGT.
3. by the described purposes of claim 1, it is characterized in that the recombinant DNA molecules of described coding human pancreas cancer MUC1 core sequence inserts short eukaryotic translation sequence KOZAK sequence successively at the aminoterminal of VNTR: GCCGCC ACC.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710047160 CN101168064B (en) | 2007-10-17 | 2007-10-17 | Recombination human pancreatic cancer mucus protein core peptide DNA vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710047160 CN101168064B (en) | 2007-10-17 | 2007-10-17 | Recombination human pancreatic cancer mucus protein core peptide DNA vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101168064A CN101168064A (en) | 2008-04-30 |
| CN101168064B true CN101168064B (en) | 2010-06-30 |
Family
ID=39388917
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200710047160 Expired - Fee Related CN101168064B (en) | 2007-10-17 | 2007-10-17 | Recombination human pancreatic cancer mucus protein core peptide DNA vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101168064B (en) |
-
2007
- 2007-10-17 CN CN 200710047160 patent/CN101168064B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 吴文川.胰腺癌MUC1-VNTR核酸疫苗的实验研究.复旦大学博士学位论文.2004,材料与方法 部分. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101168064A (en) | 2008-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US12059459B2 (en) | Therapeutic anticancer neoepitope vaccine | |
| JP5998370B2 (en) | Methods and compositions for eliciting an immune response against EGFRVIII | |
| CN109152830A (en) | core/shell structure platform for immunotherapy | |
| CN104736553B (en) | Cell-penetrating peptides | |
| CN101918544B (en) | Method for increasing immunoreactivity | |
| Zhang et al. | LAH4 enhances CD8+ T cell immunity of protein/peptide-based vaccines | |
| CN101146550A (en) | Methods to elicit, enhance and sustain immune responses against MHC class I-restricted epitopes, for prophylactic or therapeutic purposes | |
| CN108472350A (en) | New epitope RNA cancer vaccines | |
| EP1487482B1 (en) | Dna vaccine against proliferating endothelial cells and methods of use thereof | |
| Lode et al. | Tyrosine hydroxylase‐based DNA‐vaccination is effective against murine neuroblastoma | |
| CN114181935B (en) | Self-assembled DNA tetrahedron and peptide vaccine delivery system | |
| CN107952069A (en) | Recombinant vaccine and its application | |
| CN110408634A (en) | A kind of nonconformity Listeria vaccine and anti-tumor immune response method | |
| US20230072079A1 (en) | Nucleic acid vaccination using neo-epitope encoding constructs | |
| CN107286244A (en) | Anti-immunity checkpoint PD-L1 and PD-L2 tumor vaccines | |
| CN101380465A (en) | Construction method of Beta-alexin-2-tumor vaccine and uses thereof | |
| CN101096680A (en) | NDA vaccine eucaryon expression carrier and application in preparation of gene vaccine | |
| CN101168064B (en) | Recombination human pancreatic cancer mucus protein core peptide DNA vaccine | |
| CN107281474A (en) | Strengthen the New Immunity Strategy and immune composition of anti tumor immune response | |
| ES2546301T3 (en) | Composition containing Leishmania Lip2a | |
| EP0689596B1 (en) | Stimulation of immune response by viral protein | |
| CN107722118A (en) | The application of peptide vaccine and minigene vaccine based on FAP α CTL epitope peptides | |
| CN101897965A (en) | Hepatitis B virus dendritic cell therapeutic vaccine and preparation method thereof | |
| CN101361983B (en) | Lung cancer DNA plasmid vaccine and preparation method thereof | |
| CN101824424A (en) | DNA vaccine based on B7-2-PE40 exotoxin fusion gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100630 Termination date: 20171017 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |